key: cord- -bcdjwpd authors: tsegaye, theodros solomon; pöhlmann, stefan title: the multiple facets of hiv attachment to dendritic cell lectins date: - - journal: cell microbiol doi: . /j. - . . .x sha: doc_id: cord_uid: bcdjwpd entry of enveloped viruses into host cells depends on the interactions of viral surface proteins with cell surface receptors. many enveloped viruses maximize the efficiency of receptor engagement by first binding to attachment‐promoting factors, which concentrate virions on target cells and thus increase the likelihood of subsequent receptor engagement. cellular lectins can recognize glycans on viral surface proteins and mediate viral uptake into immune cells for subsequent antigen presentation. paradoxically, many viral and non‐viral pathogens target lectins to attach to immune cells and to subvert cellular functions to promote their spread. thus, it has been proposed that attachment of hiv to the dendritic cell lectin dc‐sign enables the virus to hijack cellular transport processes to ensure its transmission to adjacent t cells. however, recent studies show that the consequences of viral capture by immune cell lectins can be diverse, and can entail negative and positive regulation of viral spread. here, we will describe key concepts proposed for the role of lectins in hiv attachment to host cells, and we will discuss recent findings in this rapidly evolving area of research. viruses need to access host cells to propagate and spread. the plasma membrane constitutes a physical barrier to infection, and enveloped viruses evolved specialized surface proteins (also termed envelope-or glycoproteins) to overcome this obstacle. these proteins mediate binding of viruses to host cells and subsequent fusion of the viral and a limiting host cell membrane, which allows the delivery of viral nucleic acid and protein into the host cell cytoplasm (harrison, ) . the first essential step in the entry cascade is the cognate binding of viral envelope proteins to components of the host cell surface, termed viral receptors (harrison, ) . receptor binding can limit infection efficiency, for example when receptor expression levels and/or receptor affinity of the viral envelope protein are low (bannert et al., ) . to circumvent this limitation, viruses can also engage cellular attachment factors, which promote viral binding to the cell surface and thus increase the possibility of successful receptor engagement and infectious entry. for instance, hiv can incorporate cellular proteins into its envelope, which interact with binding partners on target cells and thereby augment viral attachment and entry (cantin et al., ) . dendritic cells are professional antigen presenting cells, which can initiate primary and stimulate memory immune responses. immature dendritic cells are particularly adept in antigen capture, while mature dendritic cells efficiently present antigen to t cells. the major dendritic cell subsets in human blood are myeloid and plasmacytoid dendritic cells, which can produce high amounts of il- and ifn-a respectively (wu and kewalramani, ) . dendritic cells of myeloid origin also line body surfaces and attachment of hiv to these cells in the anogenital mucosa might play a prominent role in the sexual transmission of hiv, the major route of viral spread, as discussed below. initial evidence for an important role of myeloid dendritic cells in hiv transmission came from cell culture studies demonstrating that mature, myeloid dendritic cells isolated from blood boost hiv infection of cocultured t cells without becoming infected (trans-infection) (cameron et al., ) . subsequent studies extended these findings to monocytederived immature and mature dendritic cells (reviewed by wu and kewalramani, ) , model systems for myeloid dendritic cells in blood and tissues. a molecular basis for hiv trans-infection by immature dendritic cells was provided by geijtenbeek and colleagues, who showed that these cells express the lectin dc-sign (for dendritic cellspecific intercellular adhesion molecule -grabbing nonintegrin), and that dc-sign binds to glycans on hiv env and facilitates trans-infection of adjacent t cells (geijtenbeek et al., ) . based on these findings, and taking into account the natural ability of dendritic cells to migrate from the periphery into lymphoid tissue, it was proposed that sexually transmitted hiv hijacks submucosal dendritic cells via dc-sign to promote its dissemination, a concept referred to as the trojan horse model (geijtenbeek et al., ) . reports that other viruses and nonviral pathogens exploit dc-sign on dendritic cells to augment their spread (table ) suggested that the trojan horse model might be paradigmatic for the host invasion by a broad spectrum of pathogens. recent work challenged important features of this model, but also demonstrated new mechanisms how pathogens can target dc-sign to propagate. in the present review, we will introduce potential consequences of hiv interactions with dc-sign and other immune cell lectins and we will discuss recent developments in the field. our focus will be on hiv, since important mechanisms underlying dc-sign-dependent augmentation of pathogen spread have been established with this virus, and apply to other viruses as well. dc-sign is expressed by monocyte-derived dendritic cells (mddcs) and by dendritic cells in mucosal and lym-phoid tissues, although concerns have been raised that dc-sign-positive cells found in some tissues might indeed be macrophages (granelli-piperno et al., ; gurney et al., ) . in addition, expression of dc-sign outside the macrophage/dendritic cell lineage has been noted; for instance a subset of b cells expresses dc-sign (rappocciolo et al., a) . dc-sign is a type ii transmembrane protein, in which the following domains have been identified: a cytoplasmic domain, a transmembrane domain, a neck-region and a lectin domain. the cytoplasmic domain contains motifs involved in receptor internalization and signalling, while the neck region facilitates dc-sign tetramerization, which is required for highaffinity ligand binding (serrano-gomez et al., ; tabarani et al., ) . finally, the lectin domain, which requires calcium for its structural integrity (c-type), binds to high-mannose and fucose residues on pathogens and cellular proteins (guo et al., ) . trans-infection was reported to depend on dc-signmediated binding and cellular uptake of hiv into dendritic cells (geijtenbeek et al., ; kwon et al., ) , followed by intracellular transport of virions to sites of dendritic cell-t cell contact, termed infectious synapses (mcdonald et al., ) (fig. ) . at the t cell site of the infectious synapse cd and coreceptor are concentrated (mcdonald et al., ) , resulting in the establishment of a microenvironment ideally suited for hiv trans-infection. collectively, these results suggest that hiv exploits mechanisms normally used for antigen presentation by dendritic cells to increase its infectivity for adjacent t cells. in the following, we will review recent work examining key features of this concept. initial work suggested that dc-sign was required for efficient mddc-mediated hiv trans-infection (geijtenbeek et al., ) . albeit this finding is controversial (gummuluru et al., ; boggiano et al., ) , a number of reports indicate that blockade of dc-sign by either antibodies, carbohydrates (baribaud et al., ; wu et al., ; wang et al., a) or rnai (arrighi et al., a,b) indeed diminishes hiv trans-infection (gurney et al., ) . in addition, dc-sign promotes hiv dissemination by migratory cells from cervical explants (hu et al., ) . however, the indicated role of dc-sign in hiv trans-infection by dendritic cells is not universal. thus, different types of dendritic cells employ different c-type lectins, dc-sign, mannose receptor (mr) and langerin, as well as cd for binding to hiv env, and dendritic cell maturation shifts env capture from lectins to cd (turville et al., ) . accordingly, dc-sign is believed to contribute to hiv trans-infection mediated by immature but not by mature dendritic cells (izquierdo-useros et al., ; wang et al., a) , although one study reached a different conclusion (baribaud et al., ) , and coexpression of cd was found to diminish dc-sign-mediated trans-infection, possibly by facilitating uptake and transport of hiv into late endosomal compartments (wang et al., b) . the list of lectins involved in hiv attachment to dendritic cells was recently expanded to include the c-type lectin dcir (dendritic cell immunoreceptor), but the relative contributions of dc-sign and dcir to hiv trans-infection remain to be established (lambert et al., ) . finally, hiv attachment to dendritic cells can also proceed in a lectin-and cd -independent fashion (de witte et al., a; hatch et al., ; izquierdo-useros et al., ). thus, dendritic cells have multiple means to capture hiv, and the prevalent mode of viral binding depends on the origin and the maturation status of the cells. kwon and colleagues found that hiv transfer to t cells by dc-sign-positive cells was dependent on lectinmediated viral internalization into low ph compartments (kwon et al., ) , in which viral infectivity was preserved (geijtenbeek et al., ; kwon et al., ) ( fig. ). an ll motif in the cytoplasmic tail of dc-sign facilitates ligand endocytosis by dc-sign (engering et al., ) . however, a contribution of the ll motif to hiv transmission by dc-sign expressing cell lines was not observed by a subsequent study (burleigh et al., ) , and the role of a low ph compartment in hiv trans-infection has been questioned (nobile et al., ; wang et al., a) . the former observation is in agreement with work demonstrating that dc-sign contributes to hiv uptake into dendritic cells, but targets the majority of internalized virions for degradation and mhc presentation (moris et al., ; (fig. ) . the identification of lsp as a cellular binding partner of the cytoplasmic tail of dc-sign further supports such a scenario (smith et al., ) . thus, normal expression of lsp diverts hiv into the proteasome of dendritic cells, while lsp knockdown increases trans-infection (smith et al., ) . regardless of the role of dc-sign, it is undisputed that hiv is efficiently taken up into dendritic cells and important differences between immature and mature dendritic cells have been noted (frank et al., ; wang et al., a) . thus, virions taken up into mature dendritic cells accumulate in large endocytic compartments, which resemble structures seen in macrophages upon hiv uptake by macropinocytosis (wang et al., a) . in comparison, virions associated with immature dendritic cells are mostly found close to the cell surface, with only a few virions being present in intracellular vesicles, which exhibit a clathrin-coat (wang et al., a) . notably, the ability to traffic hiv into deep intracellular compartments was found to correlate with protection of virus from proteases, and inhibitors of intracellular trafficking and cytoskeleton integrity were shown to inhibit trans-infection (wang et al., a; , in agreement with the proposal that hiv traffics intracellularly, via a tetraspaninsorting pathway, to infectious synapses (garcia et al., ) . in contrast, a separate study postulated that virions reach infectious synapses exclusively by transport on the cell surface instead of travelling along intracellular routes (cavrois et al., ) . in agreement with this scenario, it was reported that hiv is routed towards the infectious synapse in a surface accessible, intracellular compartment (yu et al., ) . irrespective of the route of hiv trafficking, there is ample evidence that hiv capture does not preserve viral infectivity (turville et al., ; nobile et al., ; burleigh et al., ; wang et al., a) , with the initially postulated conservation of viral infectivity likely being due to productive infection of the transmitting cells (nobile et al., ; burleigh et al., ) . collectively, hiv trans-infection driven by dendritic cells is short lived (hours) and dc-sign on immature dendritic cells contributes to this process in at least two ways: dc-sign promotes capture and potentially uptake of virions subsequently transferred to t cells. in addition, dc-sign seems to stimulate the formation of infectious synapses by a so far incompletely understood mechanism (arrighi et al., a) . recent studies revealed a novel facet of hiv interactions with dc-sign on dendritic cells, the modulation of the immune response. it was demonstrated that binding of hiv to mddcs induces erk-dependent signal transduction, which correlates with production of the immunosuppressive cytokine il- and compromised maturation of dendritic cells (shan et al., ) . these effects were dependent on appropriate env glycosylation and on c-type lectin expression, and a role of dc-sign was postulated (shan et al., ) . removal of mannose-rich glycans from env improved immunogenicity of the protein (banerjee et al., ) , further pointing towards a role of mannose-specific lectins in immune responses shaped by dendritic cells. in agreement with these findings, hiv binding to dc-sign on dendritic cells was demonstrated to induce signalling via the rho guanine nucleotide-exchange factor larg and the small gtpase rhoa, which results in aberrant dendritic cell maturation (hodges et al., ) . thus, the cells fail to upregulate cd and mhc ii but readily form infectious synapses with t cells (hodges et al., ) , indicating that hiv signalling via dc-sign compromises the immune function of dendritic cells and simultaneously primes the cells for trans-infection. a separate study showed that binding of hiv to dc-sign induces signals via a multi-protein complex, including the kinase raf- , which modulates tlr-induced cytokine production by regulating acetylation of the nfkb subunit p (gringhuis et al., ) . interestingly, activation of raf- was dependent on larg and rhoa and on the carbohydrate profile of the pathogen bound to dc-sign. thus, hiv and mycobacterium tuberculosis, which bound to dc-sign via high-mannose residues, activated raf- signalling and induced production of a cytokine profile different from that triggered by helicobacter pylori, which was due to recognition of fucose containing structures and did not involve raf- activation (gringhuis et al., ) (figs and ) . finally, signalling via tlr and dc-sign was required for nfkb-dependent recruitment of the transcription factor ptef-b to the viral promoter, and thus for the generation of full-length hiv transcripts in dendritic cells -a prerequisite for productive infection (gringhuis et al., ) (fig. ) . however, hiv infection of dendritic cells is inefficient compared with t cells and macrophages (reviewed by wu and kewalramani, ) , and a contribution of this mechanism to viral spread in vivo remains to be established. in sum, dc-sign has signalling capacity, which can be exploited by pathogens to modulate immune responses and to establish productive infection of dendritic cells and adjacent target cells. langerhans cells are located in the top layer of the mucosa and are most likely the first cell type to come in contact with sexually transmitted hiv. langerhans cells are dc-sign-negative but express cd and the c-type lectin langerin (soilleux and coleman, ) . pioneer work by de witte and colleagues provided evidence that langerin constitutes a defence mechanism against hiv invasion (de witte et al., b) . thus, langerin binds to env and targets bound virions into birbeck granules, an intracellular compartment specific to langerhans cells, where the virus is degraded (de witte et al., b) . counter-intuitively, however, c-type lectins were reported to play a minor role in hiv entry into vaginal langerhans cells (hladik et al., ) , and examination of skin explants inoculated with hiv indicated hiv infection of langerhans cells, but not other types of dendritic cells, and transfer of virus from langerhans cells to t cells (kawamura et al., ) . the latter observations suggest that infection of langerhans cells might be an important early event in hiv transmission. such a scenario can be reconciled with the findings of de witte and colleagues, when taking into account that the barrier imposed by fig. . hiv induces signalling via dc-sign, which promotes dendritic cell infection and release of immunosuppressive cytokines. triggering tlr- or tlr- induces nfkb activation. concomitant engagement of dc-sign by hiv triggers raf- -dependent signalling, which results in phosphorylation of ser of the nfkb subunit p . phosphorylation of ser in turn induces acetylation of lysines in p , which increases and prolongs transcription of the il- gene. il- inhibits the th -mediated response and incapacitates the antigen presentation capabilities of dendritic cells. induction of tlr- signalling by hiv activates nfkb and allows synthesis of short hiv transcripts. parallel triggering of dc-sign signalling by hiv induces phosphorylation of the p subunit of nfkb at ser . this phosphorylation event allows recruitment of the transcription-elongation factor ptef-b to the hiv promoter, which phosphorylates rna polymerase ii at ser . phosphorylation increases processivity of the enzyme and thus allows synthesis of full-length hiv transcripts. langerin can be overcome by high doses of virus (de witte et al., b) and that tnf-a, produced upon genital coinfections, like candida albicans, might increase permissiveness of langerhans cells to hiv infection (de jong et al., b) -hypotheses that should be tested in animal models. dc-sign is targeted by different viruses which all contain envelope proteins with an appropriate glycan signature. three major consequences of viral engagement of dc-sign have been described: first, dc-sign can function as a bona fide entry receptor. in this case, expression of dc-sign is sufficient to render cells susceptible to infection. such a scenario has been suggested, e.g. for dendritic cell infection by human herpesvirus (hhv- ) (rappocciolo et al., ) , the causative agent of karposi sarcoma and for ebolavirus infection of t cells (alvarez et al., ) . however, it is technically challenging to discriminate between entry mediated solely by dc-sign (receptor function) and entry augmented by dc-sign but facilitated by a coexpressed receptor, a process termed cis-infection (lee et al., ) . in fact, dc-sign most likely functions as an attachment factor in the context of ebolavirus entry into lymphoid cells (marzi et al., ) . second, dc-sign might augment cis-infection, as described above. dc-sign-driven cis-infection has been established for dengue virus infection of dendritic cells, which are important early targets of this pathogen (lozach et al., ) . polymorphisms in the dc-sign gene were shown to impact disease development, suggesting that cis-infection might facilitate viral spread in patients (sakuntabhai et al., ) . third, dc-sign can promote trans-infection of target cells. while this route has first been established for hiv, trans-infection of several viruses by dc-sign has been demonstrated, including measles virus (de witte et al., ) and ebolavirus (alvarez et al., ) , and might promote transmission of these pathogens. whether dc-sign engagement by viruses other than hiv also modulates cytokine release and thus impacts the establishment of an immune response remains to be determined. detailed information on the processes ensuing exposure of anogenital mucosa to sexually transmitted hiv is indispensable to understand viral dissemination and pathogenesis and to develop effective antiviral strategies. there is continued and well founded belief that dendritic cells play an important role in the establishment of hiv infection. however, the idea of the role of dc-sign in hiv interactions with dendritic cells has changed considerably (fig. ) . it has become clear that dendritic cells have several means to bind and transfer hiv to cells, with c-type lectins being one of them. in addition, an appreciable protection of hiv from antiviral agents upon attachment to dendritic cells is under discussion, and the concept of long-term storage and subsequent regurgitation of infectious hiv by dendritic cells had to be abandoned. on the other hand, dc-signmediated hiv trans-infection, although found to be a short-lived process, and cis-infection might promote viral amplification in the genital mucosal, which could be critical for the establishment of the primary infection. conversely, langerin on langerhans cells was discovered to target hiv for degradation, suggesting a barrier function of this lectin. finally, intriguing new findings revealed a signalling capacity of dc-sign (and other lectins), which is exploited by hiv to compromise immune defences and to promote its spread. vaccine development should therefore encompass the optimization of the glycan profiles of candidate substances, and new strategies for immunotherapy can be envisioned. ultimately, key concepts need to be evaluated in improved cell culture systems and animal models for sexual transmission of hiv. an important but often overlooked parameter determining outcome and significance of such experiments is the source of the virus. glycosylation of hiv depends on the producer cell type, and viruses generated in macrophages are unlikely to be detected efficiently by c-type lectins on mucosal dendritic cells . consequently, these viruses might overcome the mucosal barrier with different efficiency and potentially by employing different strategies compared with viruses generated in t cells -and similar considerations apply to most if not all other dc-sign ligands. c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-signmediated infectious synapse formation enhances x hiv- transmission from dendritic cells to t cells lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells enzymatic removal of mannose moieties can increase the immune response to hiv- gp in vivo the level of cd expression limits infection of primary rhesus monkey macrophages by a t-tropic simian immunodeficiency virus and macrophagetropic human immunodeficiency viruses quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells dc-sign interacts with mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the pakistani population helicobacter pylori modulates the t helper cell /t helper cell balance through phase-variable interaction between lipopolysaccharide and dc-sign dendritic cell-mediated trans-enhancement of human immunodeficiency virus type infectivity is independent of dc-sign infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells the c-type lectin dc-sign (cd ) is an antigen-uptake receptor for candida albicans on dendritic cells dendritic cells exposed to human immunodeficiency virus type- transmit a vigorous cytopathic infection to cd + t cells plunder and stowaways: incorporation of cellular proteins by enveloped viruses in vitro derived dendritic cells trans-infect cd t cells primarily with surface-bound hiv- virions dc-sign facilitates fusion of dendritic cells with human t-cell leukemia virus type -infected cells dendritic cell (dc)-specific intercellular adhesion molecule (icam- )-grabbing nonintegrin (dc-sign, cd ), a c-type surface lectin in human dcs, is a receptor for leishmania amastigotes sequence and expression of a membrane-associated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp the dendritic cellspecific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x the dendritic cell-specific adhesion receptor dc-sign internalizes antigen for presentation to t cells infectious and whole inactivated simian immunodeficiency viruses interact similarly with primate dendritic cells (dcs): differential intracellular fate of virions in mature and immature dcs hiv- trafficking to the dendritic cell-t-cell infectious synapse uses a pathway of tetraspanin sorting to the immunological synapse l-sign (cd l) is a liver-specific capture receptor for hepatitis c virus dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells mycobacteria target dc-sign to suppress dendritic cell function dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin/ cd is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction c-type lectin dc-sign modulates toll-like receptor signaling via raf- kinase-dependent acetylation of transcription factor nf-kappab carbohydrate-specific signaling through the dc-sign signalosome tailors immunity to mycobacterium tuberculosis, hiv- and helicobacter pylori hiv- exploits innate signaling by tlr and dc-sign for productive infection of dendritic cells binding of human immunodeficiency virus type to immature dendritic cells can occur independently of dc-sign and mannose binding c-type lectin receptors via a cholesterol-dependent pathway structural basis for distinct ligandbinding and targeting properties of the receptors dc-sign and dc-signr binding and transfer of human immunodeficiency virus by dc-sign+ cells in human rectal mucosa human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection viral membrane fusion glycosphingolipid composition of human immunodeficiency virus type (hiv- ) particles is a crucial determinant for dendritic cell-mediated hiv- trans-infection initial events in establishing vaginal entry and infection by human immunodeficiency virus type- activation of the lectin dc-sign induces an immature dendritic cell phenotype triggering rho-gtpase activity required for hiv- replication blockade of attachment and fusion receptors inhibits hiv- infection of human cervical tissue maturation of blood-derived dendritic cells enhances human immunodeficiency virus type capture and transmission capture and transfer of hiv- particles by mature dendritic cells converges with the exosome-dissemination pathway dendritic cells mediate herpes simplex virus infection and transmission through the c-type lectin dc-sign tnf-alpha and tlr agonists increase susceptibility to hiv- transmission by human langerhans cells ex vivo significant virus replication in langerhans cells following application of hiv to abraded skin: relevance to occupational transmission of hiv dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses dc-sign specifically recognizes streptococcus pneumoniae serotypes and dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection the c-type lectin surface receptor dcir acts as a new attachment factor for hiv- in dendritic cells and contributes to trans-and cis-infection pathways cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd and a coreceptor differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e dendritic cellspecific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals recruitment of hiv and its receptors to dendritic cell-t cell junctions dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of ifn-alpha and tnfalpha dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus analysis of the interaction of ebola virus glycoprotein with dc-sign (dendritic cellspecific intercellular adhesion molecule -grabbing nonintegrin) and its homologue dc-signr dc-sign promotes exogenous mhc-i-restricted hiv- antigen presentation dendritic cells and hiv-specific cd + t cells: hiv antigen presentation, t-cell activation, and viral transfer dendritic-cellspecific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquitocell-derived dengue viruses covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes hepatitis c virus glycoproteins interact with dc-sign and dc-signr dc-sign on b lymphocytes is required for transmission of hiv- to t lymphocytes dc-sign is a receptor for human herpesvirus on dendritic cells and macrophages human herpesvirus infects and replicates in primary cultures of activated b lymphocytes through dc-sign a variant in the cd promoter is associated with severity of dengue disease structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam -grabbing non-integrin (cd ) on the cell surface hiv- gp mannoses induce immunosuppressive responses from dendritic cells leukocyte-specific protein interacts with dc-sign and mediates transport of hiv to the proteasome in dendritic cells langerhans cells and the cells of langerhans cell histiocytosis do not express dc-sign dc-sign neck domain is a ph-sensor controlling oligomerization: saxs and hydrodynamic studies of extracellular domain dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells diversity of receptors binding hiv on dendritic cell subsets immunodeficiency virus uptake, turnover, and -phase transfer in human dendritic cells functionally distinct transmission of human immunodeficiency virus type mediated by immature and mature dendritic cells cd coexpression regulates dc-signmediated transmission of human immunodeficiency virus type dc-sign mediates avian h n influenza virus infection in cis and in trans macropinocytosis and cytoskeleton contribute to dendritic cell-mediated hiv- transmission to cd + t cells measles virus targets dc-sign to enhance dendritic cell infection syndecan- is a dendritic cell-specific attachment receptor for hiv- langerin is a natural barrier to hiv- transmission by langerhans cells dc-sign and cd have distinct roles in transmission of measles virus from dendritic cells to t-lymphocytes dendritic-cell interactions with hiv: infection and viral dissemination functional evaluation of dc-sign monoclonal antibodies reveals dc-sign interactions with icam- do not promote human immunodeficiency virus type transmission ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells we thank tf schulz for support and the bmbf (grant ki ) and dfg (sfb ) for funding. we apologize to all colleagues whose work could not be cited because of space constraints. key: cord- - wok n authors: sainz, juan; lupiáñez, carmen belén; segura-catena, juana; vazquez, lourdes; ríos, rafael; oyonarte, salvador; hemminki, kari; försti, asta; jurado, manuel title: dectin- and dc-sign polymorphisms associated with invasive pulmonary aspergillosis infection date: - - journal: plos one doi: . /journal.pone. sha: doc_id: cord_uid: wok n the recognition of pathogen-derived structures by c-type lectins and the chemotactic activity mediated by the ccl /ccr axis are critical steps in determining the host immune response to fungi. the present study was designed to investigate whether the presence of single nucleotide polymorphisms (snps) within dc-sign, dectin- , dectin- , ccl and ccr genes influence the risk of developing invasive pulmonary aspergillosis (ipa). twenty-seven snps were selected using a hybrid functional/tagging approach and genotyped in haematological patients, fifty-seven of them diagnosed with proven or probable ipa according to the eortc/msg criteria. association analysis revealed that carriers of the dectin- (rs t/t) and dectin- (rs g/g) genotypes and dc-sign (rs g), dc-sign (rs t), dc-sign ( a) and dc-sign ( c) alleles had a significantly increased risk of ipa infection (or = . %ci . – . ; or = . %ci . – . ; or = . %ci . – . ; or = . %ci . – . ; or = . %ci . – . and or = . %ci . – . , respectively). there was also a significantly increased frequency of galactomannan positivity among patients carrying the dectin- (rs _t) allele and dectin- (rs _g/g) genotype. in addition, healthy individuals with this latter genotype showed a significantly decreased level of dectin- mrna expression compared to c-allele carriers, suggesting a role of the dectin- (rs ) polymorphism in determining the levels of dectin- and, consequently, the level of susceptibility to ipa infection. snp-snp interaction (epistasis) analysis revealed significant interactions models including snps in dectin- , dectin- , ccl and ccr genes, with synergistic genetic effects. although these results need to be further validated in larger cohorts, they suggest that dectin- , dc-sign, dectin- , ccl and ccr genetic variants influence the risk of ipa infection and might be useful in developing a risk-adapted prophylaxis. invasive pulmonary aspergillosis (ipa) is a life-threatening infection caused mainly by aspergillus fumigatus, an opportunistic fungal pathogen that frequently colonizes respiratory tracts and rapidly spreads to blood vessels and tissues [ ] . the incidence of ipa infection has increased in the last years due to the use of immunosuppressive and immunomodulatory drugs and is still causing significant morbidity and mortality worldwide, especially in immunosuppressed and hematopoietic stem cell transplantation (hsct) patients [ ] . early recognition of a. fumigatus by myeloid leukocytes is a crucial for down-stream immune response and conidia clearance and, therefore, in the development and progression of ipa infection [ ] . myeloid leukocytes are activated by patternrecognition receptors (prrs), which directly recognize fungal cell wall structures and pathogen-associated molecular patterns (pamps) [ ] . among the prrs expressed in neutrophils, pulmonary macrophages and dendritic cells, c-type lectin family members such as dc-sign (dendritic cell-specific intercellular adhesion molecule- -grabbing non-integrin), dectin- (dendritic cell-associated c-type lectin- ) and dectin- (dendritic cellassociated c-type lectin- ) seem to be important in the effectiveness of innate immune response against a. fumigatus [ , ] . recent studies have reported that the c-type lectins mediate a. fumigatus recognition, capture and internalization [ ] and that their binding to fungal cell wall carbohydrates is highly specific and selective [ ] . dc-sign and dectin- recognize b-( - )-glucans and galactomannans (gm) in the cell wall of a. fumigatus [ , ] while dectin- interacts with a-mannans [ ] . however, numerous studies have shown that c-type lectins are not only involved in the recognition of fungal pathogens but also in the induction of antifungal th and th immune responses [ , ] . although the mechanisms underlying dc-sign-mediated immune response are still highly speculative, it has been suggested that dc-sign promotes dendritic cell (dc) migration and t-cell activation through the icam- binding [ , ] and modulates tlr signalling by targeting raf- , which regulates nfkb p activation and, consequently the production of pro-inflammatory cytokines [ ] . likewise, dectin- and dectin- induce sykdependent canonical and noncanonical nfkb pathways [ , ] promoting the production of some pro-inflammatory cytokines (il a, il b, il and il ), chemokines (ccl /mcp- , ccl / mip- , cxcl /mip- and cxcl ) [ ] and the il -mediated neutrophil recruitment [ ] . it has also been described that dectin- and dectin- are able to work in collaboration with tlrs (mainly tlr , tlr and tlr ) modulating immune responses through the production of il , il p and tnf [ , ] . these findings support the hypothesis that c-type lectins, cytokines or chemokines are important mediators during infection with a. fumigatus. several polymorphisms in human prrs [ ] [ ] [ ] [ ] [ ] [ ] as well as in some cytokines [ ] [ ] [ ] , chemokines [ ] and their receptors [ , ] have hitherto been associated with an increased risk of invasive aspergillosis in susceptible hosts. based on these observations, the objective of the present study was to investigate the role of tagging and potentially functional single-nucleotide polymorphisms (snps) located within the dc-sign, dectin- , dectin- , mcp- /ccl and ccr genes on ipa susceptibility. all participants enrolled were caucasian and recruited in the university hospital virgen de las nieves (granada, spain) or in the university hospital of salamanca (salamanca, spain). all determinations and genetic analyses in hematological patients were performed with fully informed written consent, and anonymity of the data was guaranteed. the study protocol was approved by the ethical review committee of virgen de las nieves university hospital, granada, spain. the population included hematological patients recruited between january and january . all hematological patients in this study received a prolonged chemotherapy treatment or underwent hsct and were therefore considered susceptible to develop ipa infection. demographic information and clinical data were obtained by detailed review of hospital records. data were gathered on: site of infection; host factor criteria (severe neutropenia for . days, persistent fever for . h refractory to appropriate broad-spectrum antibacterial treatment, signs and symptoms indicating graft-versus-host disease [gvhd], corticoid therapy [. . mg/kg per day], and invasive fungal infection during a previous episode of neutropenia), microbiological criteria (positive result for aspergillus antigen in $ consecutive blood samples when considering an index of . or in only sample when the index was higher than . ), and clinical criteria of lower respiratory tract infection [major criteria: any of the following new infiltrates on computed tomography (ct) imaging: halo sign, aircrescent sign, or cavity within area of consolidation; minor criteria: cough, thoracic pain, hemoptysis, pathologic pulmonary sound, and radiological evidence suggestive of invasive infection]. laboratory data were also recorded. proven and probable ipa was diagnosed based on the updated criteria ( ) reported by the european organization for research and treatment of cancer/invasive fungal infections cooperative group (eortc/ ificg) [ ] . serum gm detection has been shown to be a useful test for the early diagnosis and follow-up of ipa and is now included in ipa diagnosis criteria [ ] . in the present study, serum gm antigen was determined twice weekly during the hospital stay and at each outpatient visit until the end of their immunosuppressant or chemotherapeutic treatment. serum gm concentrations were determined by platelia aspergillus elisa kit (bio-rad, marnes-la-coquette, france) according to the manufacturer's instructions. this commercial kit has proven to offer good sensitivity to detect gm [ ] , and gm concentration was found to correlate with the fungal tissue burden [ , ] . a test sample was classified as positive when the optical density ratio was $ . in two consecutive positive samples or . . in a unique serum sample. a careful review of concomitant treatments (piperacillin-tazobactam or amoxicillin-clavulonic acid) in each patient was necessary to detect possible false-positive gm determinations. likewise, tests were performed on the same day to avoid sample contamination and to ensure accuracy of results. twenty-seven tagging/functional snps within dc-sign, dectin- , dectin- , ccl and ccr were selected to genotype the entire panel of individuals ( table ). the aim of the snp tagging was to identify a set of snps that efficiently tags all the known snps while the functional approach was used to determine the net impact of potentially functional variants within dc-sign, dectin- , dectin- , ccl and ccr genes on ipa risk. tagging snps were selected using haploview tagger program (http://www.broad.mit.edu/ mpg/haploview/; http://www.broad.mit.edu/mpg/tagger/) and a pairwise tagging with a minimum r of . . in this selection we forced the inclusion of the dc-sign rs , ccl rs and ccl rs polymorphisms as their functionality has been suggested [ ] [ ] [ ] . genomic dna was extracted from peripheral blood mononuclear cells (pbmcs) qiagen mini kit (qiagen, valencia, ca, usa). genotyping of dc-sign, dectin- , dectin- , ccl and ccr polymorphisms was carried out using kaspar assays (kbiosciences, hoddesdon, hertfordshire, uk) in a well plate format (applied biosystems, foster city, ca, usa) where hematological patient samples were randomly distributed. kaspar reactions were performed using kaspar assay mix (containing probes) and kaspar kit containing reaction mix and mgcl ( mm). pcr conditions were: denaturation at uc for min, cycles of denaturation at uc for sec, annealing at uc for sec and elongation at uc for sec and cycles of denaturation at uc for sec, annealing at uc for sec and elongation at uc for sec. recycling conditions were uc for sec, annealing and elongation at uc for sec. pcr products were analyzed with the abi prism ht detection system using the sds . software (applied biosystems). for internal quality control, about % of samples were randomly selected and included as duplicate. concordance between the original and the duplicate samples for the snps analyzed was $ . %. call rates for all snps were $ . % with the exception of the dectin- rs snp with a call rate of . %. the hardy-weinberg equilibrium (hwe) tests were performed in the control group (non-ipa patients) by a standard observedexpected chi-square (x ) test at each polymorphic site (http://ihg . helmholtz-muenchen.de/cgi-bin/hw/hwa .pl). unconditional logistic regression was used to assess the main effects of the genetic polymorphisms on ipa risk using co-dominant, dominant and recessive inheritance models. for each snp, the more common allele in the control group was assigned as the reference category. all analyses were adjusted for age, gender, hematological malignancy and established risk factors for ipa infection (hsct, neutropenia, gvhd and corticoid therapy use) and were conducted using the statistical software ssps (version . , spss inc., chicago, usa). all tests were considered to be statistically significant with a p value of , . . adjustment for multiple testing was carried out following a conservative threshold for statistical significance, based on a revised version of the bonferroni method: we calculated for each gene the ''number of effective independent variables'' (m eff ) by use of the snp spectral decomposition approach (http://gump.qimr. edu.au/general/dalen/snpspdsuperlite/) [ ] . we obtained a study-wise m eff value by adding up the gene m eff 's. snptool (http://www.dkfz.de/de/molgen_epidemiology/tools/ snptool.html) [ ] and the haploview v . software were used for ld blocks reconstruction and haplotype association statistics. block structures were determined according to the method of gabriel et al. [ ] . we used the web-based tool fastsnp [ ] available at http:// fastsnp.ibms.sinica.edu.tw for predicting the functional significance of the snps associated with ipa infection. fastsnp utilizes information from another online program polyphen (http://www. bork.embl-heidelberg.de/polyphen/) and from four different web resources (tfsearch, esefinder, rescue-ese and fas-ess) to determine whether snps are located at exonic splicing regulatory sites, cause a non-conservative amino acid change or whether they alter the transcription factor-binding site of a gene (for instance, acting as intronic enhancer). the score was given by this tool on the basis of levels of risk with a ranking of (no effect), (very low), (low), (medium), (high), or (very high). whole blood samples from healthy donors were collected into paxgene rna tubes and stored at uc until use. total rnas were extracted using a paxgene blood rna isolation kit (preanalytix) and reverse transcribed to cdna using quantitect reverse transcription kit (catalog number: ; qiagen). real-time quantitative pcr was carried out using an abi prismh ht sequence detection system (applied biosystems) according to manufacturer's instructions. briefly, ml of the cdna were loaded in a pcr reaction containing . ml of quantitect sybr green pcr master mix with an appropriate concentration of mgcl , . ml of primers (hs_cle-c a_ _sg quantitect primer assay, catalog number: qt ; geneglobe, qiagen) and ml of rnase-free water. pcr cycling conditions were as follows: uc for minutes, followed by cycles of denaturation at uc for seconds combined with annealing at uc for seconds and extension at uc for seconds. all samples were run in duplicate. relative quantification of dectin- mrna expression was calculated with the ddct method. we obtained the fold changes in gene expression normalized to an internal control gene (gapdh, hs_gapdh_ _sg quantitect primer assay, catalog number: qt ; qiagen) and relative to one calibrator (first- we also analyzed high-order interactions between snps using the multifactor dimensionality reduction (mdr) constructive induction algorithm. a detailed explanation on the mdr method has been described elsewhere [ , ] . snps in ld (cut-off of r , . ) were excluded from the mdr analysis. cross-validation and permutation testing were used to identify the best models. all possible two-, three-and four-way snp interactions were tested using -fold cross-validation and the exhaustive search. the model with the highest testing balanced accuracy (ta) and cross validation consistency (cvc) was selected as ''best model''. statistical significance was evaluated using a . -fold permutation test to compare observed testing balanced accuracies with those expected under the null hypothesis of no association (using the mdr permutation testing module . . alpha). mdr results were considered statistically significant at the . level. finally, interactions were visualized by constructing an interaction dendrogram according to the method described by moore et al [ ] . mdr software and mdr permutation testing module are open-source and freely available from http://www.epistasis.org. population characteristics are described in table . compared to non-ipa, ipa cases were more likely to have cough and pathologic pulmonary sound (p, . and . , respectively) and presented more often pathological chest radiographies and ct scans (p, . ). established risk factors for ipa infection such as hsct, neutropenia, gvhd and corticoid therapy use were homogenously distributed between ipa and non-ipa patient groups. fifty-seven patients were diagnosed with proven or probable ipa infection according to the revised eortc/msg criteria ( ). the association of risk of ipa infection with the individual snps in the c-type lectin and chemokine genes is shown in the table s . all analyzed polymorphisms fulfilled hardy-weinberg expectations for the control group (non-ipa patients). several polymorphisms were found to be associated with ipa infection ( table ) table and table s ). after correction for multiple testing using snpspd (number of independent marker loci, ; p = . / = . ), none of the snps retained significance although dectin- rs showed significance when the carrier status was analyzed (cc+cg vs. gg, p = . ; table ). in order to assess the degree to which the selected snps and the positivity of gm correlated, we also performed lineal regression analysis. in the whole population, assays were performed in duplicate and were considered as positive ( . %). we found a significantly higher percentage of positive gm among patients carrying the dectin- rs _t allele and among those patients bearing the dectin- rs _g/g genotype suggesting a role of these polymorphisms in determining a defective recognition and clearance of aspergillus conidia (figure ). no correlation was observed for the polymorphisms within dc-sign that were associated with the infection. we subsequently assessed whether snps associated with ipa infection showed capacity to change putative transcription factor binding sites using fastsnp. the predictive functional analysis suggested an intronic enhancer function for the dectin- rs snp due to its location in transcription factor cdxa (caudal type homeo-box transcription factor ) binding site (gaaagac; score - ). these data suggest a central role of the rs in the susceptibility to ipa infection. none of the remaining snps associated with ipa infection was predicted to affect transcription factor binding sites or splicing or to introduce a damaging amino acid change. to explore the potential consequences of dectin- rs snp, dectin- mrna expression was measured in healthy donors and was correlated with genotypes. our results showed that subjects harbouring gg genotype showed a significantly decreased level of dectin- mrna expression compared to c-allele carriers (p, . ; figure ). these results further supported our hypothesis suggesting that dectin- rs _g allele may disrupt binding sites for potential transcription factors. the mdr analysis of the snps tested revealed statistically significant interactions. two snps (rs and rs ) were excluded from the mdr analysis as they were in ld with other snps using a cut-off value of r , . in our sample set. the best interaction models selected by the turf filter algorithm along with its testing accuracy and cross-validation consistency are shown in table . the overall best model with the highest crossvalidation consistency (cvc) consisted of a model that included the ccl rs , dectin- rs , ccr rs and dectin- rs snps. this model had a significant testing accuracy of . (permutation p, . ) and a cross-validation consistency of / . of note is that two snps showing genetic interaction in this model were not significantly associated with an increased risk of ipa infection in the univariate analysis (ccr rs and dectin- rs ). the best two-locus model consisted of the dectin- rs and dc-sign rs snps, two variants that showed also association in the single-locus analysis. this model had a testing accuracy of . and a cross-validation consistency of / . this model was not any more significant after . -fold permutation testing. however, the entropy based information gain calculated for this pair of snps indicated strong synergy, which may be interpreted as the two snps acting together to increase the risk of ipa infection. the best three-locus model included the ccl rs , dectin- rs and dectin- rs snps. in this model, testing accuracy raised to . (permutation p = . ) whereas the cross-validation consistency was of / . figure illustrates an interaction dendogram that summarizes the estimates of interactions. the marked differences in susceptibility to ipa infection among hematological patients (with or without allo-hsct) suggest that the effective immune response against aspergillus is determined by both environmental and host genetic factors [ , ] . studies on genetic polymorphisms in genes coding for components of the innate immunity have supported this hypothesis [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . in this report, we studied the influence of the tagging and potentially functional polymorphisms of dc-sign, dectin- , dectin- , ccl /mcp- and ccr in susceptibility to ipa infection in a spanish population. polymorphisms in these genes have been reported to influence a number of infectious diseases including hiv- [ ] [ ] [ ] , htlv- [ ] , cmv [ ] , tuberculosis [ ] [ ] [ ] [ ] , hcv [ , ] , hbv [ ] , dengue [ ] and sars [ ] among others, revealing their potential role in host defense against pathogens. we found that polymorphisms in dectin- (rs and rs ) and dc-sign (rs , rs , rs and rs ) were associated with an increased risk to develop ipa infection, which points towards their critical involvement in the pathogenesis of this invasive fungal infection. the highest risk of ipa infection was found for carriers of the dectin- rs _t/t and dectin- rs _g/g genotypes and the dc-sign rs _g allele. patients carrying these genotypes/alleles had from to times increased risk of ipa infection. additionally, patients carrying the dc-sign rs _t , dc-sign _a and dc-sign rs _c alleles showed a -fold increased risk in comparison with patients harboring the wild-type allele. although it was not statistically significant, we also found that the dc-sign rs snp may be associated with a reduced risk of ipa infection. interestingly, this latter result was in agreement with our previously reported findings using the former eortc/msg classification criteria, [ ] . the apparent effect of these snps on ipa susceptibility persisted even after adjustment for age, gender, hematological malignancy and several known risk factors (hsct, neutropenia, gvhd and corticoid therapy use), indicating that dectin- and dc-sign variants contribute independently to the risk of infection. another interesting finding of this study was the significantly greater positive gm percentage of patients carrying the dectin- rs _t allele than those with the wild-type allele. additionally, patients harboring the g/g genotype for the dectin- rs snp showed an increased percentage of positive gm compared to those carrying the c allele (c/c+c/g). no differences were found when positive gm determinations were correlated with dc-sign polymorphisms. these data along with the remarkable degree of association of dectin- and dc-sign variants with risk of ipa infection provides a compelling evidence for a critical role for these prrs in immune response to ipa infection. in this regard, numerous studies have shown that dectin- and dc-sign are not only involved in the recognition of fungal pathogens but also in the induction of anti-fungal th and th immune responses [ , ] . mezger et al. also demonstrated that dectin- is involved in the induction of several pro-inflamatory cytokines, chemokines and immune receptors [ ] while werner et al. showed that dectin- is also regulating th -mediated immune response in the lungs [ ] . furthermore, dennehy and brown suggested a role of dectin- mediating its own signaling, as well as synergizing with tlrs to trigger nfkb-mediated immune response against fungal pathogens [ ] . although it is now well recognized that snps in genes modulating immune response are likely to be determinants of host susceptibility to fungal infections, so far, little is known regarding the biological significance of these variants. in order to shed light into the potential functionality of dectin- (rs and rs ) and dc-sign (rs , rs , rs , rs and rs ) variants, we investigated whether they were involved in disruption of a binding site for critical transcription factors that might influence transcription level of these genes. our predictive analysis showed that the carriage of the c allele for the dectin- rs snp creates a putative binding site for cdxa, a relatively unknown transcription factor, which might be involved in the control of dectin- gene expression. to assess whether the dectin- rs polymorphism might be associated with a decreased expression of dectin- , we correlated dectin- mrna expression levels with dectin- rs genotypes. interestingly, we observed that individuals harbouring the gg genotype showed a relatively lower expression than those carrying the c allele (cc+gc). these results further supported our hypothesis suggesting that dectin- rs snp may have an effect on the dectin- -mediated recognition of aspergillus conidia and subsequent immune responses. however, this predicted change in transcription activity is only suggestive at this stage and will need further validation using in vitro functional assays. several lines of evidence point to the relevance of epistatic effects in the etiology of complex diseases but, up to now, no studies have been carried out to analyze the presence of snp-snp interactions in ipa infection. for this reason, we decide to assess interactions among genetic polymorphisms within dc-sign, dectin- , dectin- , ccl and ccr , genes and the risk of ipa infection. the mdr approach used in this study was able to determine two multilocus combinations associated with high risk to develop ipa infection. of the interactions identified, mdr indicated that the type of interaction in the two significant models was synergistic. these results support the hypothesis that multiple snp-snp interactions may play a role in determining the risk of ipa infection. this hypothesis is biologically plausible since the immune system would warrant prevention of fungal infection even when some genetic variants were present. recent population-based studies have even led to the identification of several snps involved in the early recognition of aspergillus and associated them with an increased risk for invasive fungal infection. it has previously been suggested that snps within c-type lectin genes are associated with fungal infections. platinga et al. ( ) and cunha et al. ( ) suggested that patients carrying the y x (rs ) polymorphism in the dectin- gene were more likely to be colonized with aspergillus and candida species [ , ] , compared with those harboring the wild-type allele. however, these results were not replicated in a very recent study [ ] . in our study, such findings were neither evidenced even when hsct and non-hsct patients were analyzed separately (data not shown). this puzzling finding suggests that, although intronic polymorphisms within dectin- and dc-sign are indeed themselves a strong indication that these genes play an important role in the susceptibility to ipa infection, we cannot rule out the figure . interaction dendrogram generated by the mdr software. the interaction dendrogram was used to confirm, visualize, and interpret the interaction model. the mdr analysis was performed by using the open-source mdr software package. the colors used depict the degree of synergy, ranging from red (highest information gain) to blue (highest information redundancy). note that the interaction between dectin- (rs ) and dc-sign (rs ) snps showed the highest degree of synergy (gain of information). doi: . /journal.pone. .g possibility that these snps are part of a bigger haplotype containing important other genetic variants in the neighboring genes. in any case, because all these population-based studies have been conducted using relatively small cohorts, additional studies in larger set of patients are needed to definitely establish the role of these variants in the susceptibility to invasive fungal infection. in summary, this study provides evidence of association between dectin- and dc-sign polymorphisms and the risk of ipa infection. by the inclusion of functional prediction analyses, the correlation of genotypes with positive gm determinations and dectin- mrna expression levels, the study strongly supported the role of dectin- gene variants in determining susceptibility to ipa infection. epistatic analyses also suggested the presence of a gene-gene interaction involving dectin- with ccl and ccr variants to determine ipa infection. despite all these evidences, additional studies using larger cohorts will be necessary to confirm the effect of these polymorphisms on the susceptibility to ipa infection. table s associations of polymorphisms involved in the phagocyte-immune related response against aspergil-lus. models adjusted for age, gender, hematological malignancy, hsct, neutropenia (defined as absolute neutrophil count , cells/mm for a period of more than days), gvhd and corticoid therapy use (. . mg/kg/day). { assuming a recessive model of inheritance. abbreviations: or, odds ratio; ci, confidence interval. differences in samples numbers are due to failures in genotyping. (doc) the clinical spectrum of pulmonary aspergillosis invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors phagocytosis of aspergillus fumigatus conidia by murine macrophages involves recognition by the dectin- beta-glucan receptor and toll-like receptor mechanisms of phagocytosis in macrophages signalling through c-type lectin receptors: shaping immune responses the betaglucan receptor dectin- recognizes specific morphologies of aspergillus fumigatus dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin mediates binding and internalization of aspergillus fumigatus conidia by dendritic cells and macrophages differential highaffinity interaction of dectin- with natural or synthetic glucans is dependent upon primary structure and is influenced by polymer chain length and sidechain branching structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr dectin- recognition of alpha-mannans and induction of th cell differentiation is essential for host defense against candida albicans dectin- directs t helper cell differentiation by controlling noncanonical nf-kappab activation through raf- and syk identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dendritic cells transport conidia and hyphae of aspergillus fumigatus from the airways to the draining lymph nodes and initiate disparate th responses to the fungus innate signaling by the ctype lectin dc-sign dictates immune responses syk-and card -dependent coupling of innate immunity to the induction of t helper cells that produce interleukin dectin- and dectin- in innate immunity against fungi pattern recognition: recent insights from dectin- proinflammatory response of immature human dendritic cells is mediated by dectin- after exposure to aspergillus fumigatus germ tubes dectin- y x polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient-and donor-dependent mechanisms of antifungal immunity tolllike receptor polymorphisms and aspergillosis in stem-cell transplantation polymorphisms in toll-like receptor genes and susceptibility to pulmonary aspergillosis tlr and tlr polymorphisms are associated with susceptibility to invasive aspergillosis after allogeneic stem cell transplantation influence of polymorphisms in innate immunity genes on susceptibility to invasive aspergillosis after stem cell transplantation the y x stop codon polymorphism in the human beta-glucan receptor dectin- and susceptibility to invasive aspergillosis il gene cluster polymorphisms and its haplotypes may predict the risk to develop invasive pulmonary aspergillosis and modulate c-reactive protein level interleukin- promoter polymorphism as risk factor to develop invasive pulmonary aspergillosis protective role of interleukin- promoter gene polymorphism in the pathogenesis of invasive pulmonary aspergillosis after allogeneic stem cell transplantation polymorphisms in the chemokine (c-x-c motif) ligand are associated with invasive aspergillosis after allogeneic stem-cell transplantation and influence cxcl expression in monocyte-derived dendritic cells tnfr mrna expression level and tnfr gene polymorphisms are predictive markers for susceptibility to develop invasive pulmonary aspergillosis variable number of tandem repeats of tnf receptor type promoter as genetic biomarker of susceptibility to develop invasive pulmonary aspergillosis revised definitions of invasive fungal disease from the european organization for research and treatment of cancer/invasive fungal infections cooperative group and the national institute of allergy and infectious diseases mycoses study group (eortc/msg) consensus group detection of galactomannan antigenemia by enzyme immunoassay for the diagnosis of invasive aspergillosis: variables that affect performance prospective clinical evaluation of lower cut-offs for galactomannan detection in adult neutropenic cancer patients and haematological stem cell transplant recipients use of circulating galactomannan screening for early diagnosis of invasive aspergillosis in allogeneic stem cell transplant recipients a variant in the cd promoter is associated with severity of dengue disease a novel polymorphism in the mcp- gene regulatory region that influences mcp- expression ccl polymorphisms are associated with serum monocyte chemoattractant protein- levels and myocardial infarction in the framingham heart study accounting for decay of linkage disequilibrium in haplotype inference and missing-data imputation snp_tools: a compact tool package for analysis and conversion of genotype data for ms-excel the structure of haplotype blocks in the human genome fastsnp: an always up-to-date and extendable service for snp function analysis and prioritization power of multifactor dimensionality reduction for detecting gene-gene interactions in the presence of genotyping error, missing data, phenocopy, and genetic heterogeneity computational analysis of gene-gene interactions using multifactor dimensionality reduction genetic susceptibility to aspergillus fumigatus infections toll-like receptor polymorphisms and aspergillosis plasminogen alleles influence susceptibility to invasive aspergillosis association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection contrasting genetic influence of ccr and ccr variants on hiv- infection and disease progression san francisco city cohort (sfcc), alive study rantes - g delays and dc-sign - c enhances aids progression in hiv type -infected japanese hemophiliacs dc-sign (cd ) gene promoter polymorphisms in a brazilian population and their association with human t-cell lymphotropic virus type infection investigation of promoter variations in dendritic cell-specific icam -grabbing non-integrin (dc-sign) (cd ) and their relevance for human cytomegalovirus reactivation and disease after allogeneic stem-cell transplantation cd genetic polymorphism and tuberculosis disease joint effect of mcp- genotype gg and mmp- genotype g/ g increases the likelihood of developing pulmonary tuberculosis in bcg-vaccinated individuals a functional promoter polymorphism in monocyte chemoattractant protein- is associated with increased susceptibility to pulmonary tuberculosis promoter variation in the dc-sign-encoding gene cd is associated with tuberculosis variant in cd promoter is associated with severity of liver disease in chronic hepatitis c virus infection a novel mcp- gene polymorphism is associated with hepatic mcp- expression and severity of hcv-related liver disease association of common promoter polymorphisms of mcp with hepatitis b virus clearance cd (dc-sign) - a.g promoter polymorphism and severe acute respiratory syndrome in hong kong chinese association between genetic polymorphism in the promotor region of cd and propensity to develop invasive pulmonary aspergillosis neutrophils produce il- a in a dectin- and il- dependent manner during invasive fungal infection the role of the beta-glucan receptor dectin- in control of fungal infection early stop polymorphism in human dectin- is associated with increased candida colonization in hematopoietic stem cell transplant recipients we thank all participants in this study and rosa cano (laboratory technician) for sample collection and technical support. key: cord- -goil gjr authors: dos santos, Ália; hadjivasiliou, andreas; ossa, felipe; lim, novandy k.; turgut, aylin; taylor, maureen e.; drickamer, kurt title: oligomerization domains in the glycan‐binding receptors dc‐sign and dc‐signr: sequence variation and stability differences date: - - journal: protein sci doi: . /pro. sha: doc_id: cord_uid: goil gjr human dendritic cell‐specific intercellular adhesion molecule‐ grabbing nonintegrin, dc‐sign, and the sinusoidal endothelial cell receptor dc‐signr or l‐sign, are closely related sugar‐binding receptors. dc‐sign acts both as a pathogen‐binding endocytic receptor and as a cell adhesion molecule, while dc‐signr has only the pathogen‐binding function. in addition to differences in the sugar‐binding properties of the carbohydrate‐recognition domains in the two receptors, there are sequence differences in the adjacent neck domains, which are coiled‐coil tetramerization domains comprised largely of ‐amino acid repeat units. a series of model polypeptides consisting of uniform repeat units have been characterized by gel filtration, differential scanning calorimetry and circular dichroism. the results demonstrate that two features characterize repeat units which form more stable tetramers: a leucine reside in the first position of the heptad pattern of hydrophobic residues that pack on the inside of the coiled coil and an arginine residue on the surface of the coiled coil that forms a salt bridge with a glutamic acid residue in the same polypeptide chain. in dc‐signr from all primates, very stable repeat units predominate, so the carbohydrate‐recognition domains must be held relatively closely together. in contrast, stable repeat units are found only near the membrane in dc‐sign. the presence of residues that disrupt tetramer formation in repeat units near the carbohydrate‐recognition domains of dc‐sign would allow these domains to splay further apart. thus, the neck domains of dc‐sign and dc‐signr can contribute to the different functions of these receptors by presenting the sugar‐binding sites in different contexts. human dc-sign, the dendritic cell-specific intercellular adhesion molecule- grabbing nonintegrin, plays a dual role as an adhesion receptor for t cells and as a pathogen-binding receptor. , the cell adhesion function is probably associated with the ability of the receptor to bind the lewis x trisaccharide found on cell surface molecules, including icam- . pathogens bound by the receptor include viruses and fungi, which bear high mannose oligosaccharides, as well as parasites that are rich in the lewis x epitope. the dual binding specificity can be explained at least in part by accommodation of both lewis x and high mannose oligosaccharides in the binding site of the c-type carbohydrate recognition domain (crd) that forms the ligand-binding portion of the receptor. in contrast, the closely related human receptor dc-signr shows more restricted binding to the high mannose oligosaccharides, allowing it to interact with viral pathogens. this receptor is also designated l-sign, reflecting its expression on sinusoidal endothelial cells of lymph nodes and liver, as well as on specific cell types in the placenta. dc-sign and dc-signr are similar in overall organization, consisting of tetramers that facilitate binding to multivalent targets, such as viral and cell surfaces. in both proteins, clusters of c-terminal crds are projected from the cell surface by extended neck domains that consists largely of multiple amino acid repeat units (fig. ) . information about the mechanisms by which the crds in dc-sign and dc-signr are held together in oligomers is of interest because differences in the geometry of binding site clustering is believed to underlie differences in the ability of human immunodeficiency virus (hiv), west nile virus, and other viruses to bind and infect cells expressing these receptors. [ ] [ ] [ ] recent studies with mannose-capped quantum dots have provided further evidence that the arrangement of the binding sites must be different in the two receptors. while dc-sign exists as a single form with seven full repeat units and one partial repeat unit, polymorphisms in the gene for dc-signr result in forms that differ in the number of neck repeat units and this variation is also correlated with differences in susceptibility to hiv infection. to facilitate analysis of the oligomer formation by dc-sign and dc-signr, a strategy for purification of the neck domains has been developed, based on addition of the sequence gly-his-his at the cterminus of the neck domain at the point where the crd would normally be attached [ fig. (b) ]. when the neck domain oligomerizes, the resulting cluster of histidine residues allows purification of the expressed polypeptides on immobilized nickel resin. using this approach, the neck domain of each receptor has been shown to function as an oligomerization domain that can form tetramers in the absence of the flanking membrane anchor and crd. the structure of the repeat units in the neck domain of dc-signr has been deduced from an unusual crystal form in which the unit cell comprises a single repeat unit in the context of a fragment containing seven repeat units. each repeat unit consists of a short n-terminal nonhelical region that includes a proline residue, followed by a coiled-coil region. a heptad pattern of leucine, isoleucine and valine residues in the helical region mediates packing of the helices in a -stranded coiledcoil in the neck domain tetramer. although similar in organization, the neck domains of dc-sign and dc-signr differ in important respects. for instance, the neck domain of dc-signr is much more stable to thermal denaturation than the neck domain of dc-sign. these differences presumably reflect the effects of differences in the sequences in the repeat units in the two receptors. however, the way that individual differences in the neck domain repeat units affect the structure and stability of the neck domain has not been defined. force measurements of the extracellular portions of both dc-sign and dc-signr are consistent with the presence of the -Å -long amino acid repeat units in an extended structure. , however, no detailed structural information for the neck domain of dc-sign is available. the goal of the present studies was to analyze the properties of engineered versions of the neck domains from dc-sign and dc-signr in order to understand the effects of sequence variations in the neck domains, both between the two proteins and between the different polymorphic forms of dc-signr. the analysis suggests that differences in key residues can result in differences in the assembly and organization of the extracellular portions of the receptors. two different crystal structures of the c-terminal portion of dc-signr suggest that there are only limited interactions between the crds in the oligomer. because similar crystals of dc-sign have not been obtained, an alternative approach was used to confirm that the presence of the crds has little influence on formation of tetramers in dc-sign. in these experiments, the ability of the isolated neck domain to interact with the full extracellular portion of the receptor was investigated. if there were significant interactions between the crds that stabilize the tetramers, it would be expected that these additional interactions would favor formation of homooligomers of the fragment representing the full extracellular portion of the receptor rather than mixed oligomers containing the neck domain fragments and the full extracellular fragment. when the purified neck domain and the extracellular fragment of dc-sign were mixed, denatured and allowed to renature, fractionation of the resulting renatured protein on a mannose-sepharose affinity column results in both fragments being retained on the column and eluted with edta. because the neck domain cannot bind to mannose-sepharose on its own, the results represent purification of mixed oligomers that contain both the neck domain and the full extracellular fragment (fig. ) . the fact that the two different polypeptides associate in an apparently random fashion is consistent with the interpretation that the formation of tetramers of dc-sign is independent of interactions between the crds and is dictated by the sequences of the neck domain. some information about the effects of variations in the sequences of the neck repeat units can be derived from earlier studies. the n-terminal portions of the extracellular portions of dc-sign and dc-signr consist of a short region not related to the -amino acid repeat units, followed by a half repeat unit that is significantly divergent from the other repeat units. in previous studies, truncation of the neck domain of dc-signr to remove the nonrepeat region at the n-terminus plus the partial repeat unit at this end resulted in no change in the stability of the neck domain, since the observed denaturation temperature measured by differential scanning calorimetry remained at c. , similar truncation of the neck domain of dc-sign results in a -repeat fragment that remains a tetramer at room temperature and the denaturation temperature of c measured by calorimetry is very close to the value of c for the full length neck domain previously reported (data not shown). thus, for both dc-sign and dc-signr, the stability of the tetramers is largely a function of the full repeat units at the c-terminus of the neck domain. the most divergent sequence is the c-terminal repeat unit of dc-signr, adjacent to the crd [ fig. (c,d) ]. the crystal structure of the neck domain did not reveal a contribution of the variant residues to the electron density of the average repeat unit, suggesting that the c-terminal repeat unit is not ordered like the other repeat units in the crystal. in the structure of the crd of dc-signr with a truncated portion of the neck domain, the four copies of the c-terminal repeat unit were found to be splayed apart. a key feature of this c-terminal repeat unit is a phenylalanine residue at position . in all other copies of the repeat unit, this position is occupied by a valine residue, which is one of the residues that forms part of the heptad motif of hydrophobic residues characteristic of coiled-coil structures. the valine side chain is able to pack into the coiled coil, while the phenylalanine side chain is too large to be accommodated. thus, the variation in this final repeat unit is associated with the special functions of this part of the neck domain in positioning the crds at a broad spacing. aside from this unusual c-terminal repeat unit, a key point of variation between dc-sign and dc-signr is at position of the repeat units, which forms the first residue of the heptad pattern in the primary structure and the beginning of the a helix in the crystal structure. in all of the repeat units visible in the crystal structure of the dc-signr neck domain, corresponding to repeat units through , this position is occupied by a leucine residue, which packs into the hydrophobic core of the coiled coil [ fig. (e) ]. however, this leucine residue is present only in repeat units through of dc-sign. to assess the effect of the residue at position on the structure and stability of the neck domain, an engineered neck domain consisting of seven repeat units, each containing a leucine residue at position , was prepared. gel filtration revealed that this version of the neck domain forms a stable tetramer at room temperature since it elutes at the same position as a natural fragment of the neck domain of dc-signr containing seven repeat units, which has been characterized as a tetramer [ fig. (a) ]. analysis by differential scanning calorimetry showed that the uniform neck domain is actually more stable than the equivalent portion of the natural dc-signr neck domain, with a thermal denaturation temperature of c compared to c for the natural neck domain [ fig. (b) ]. one reason for the lower stability of the natural neck domain would be the presence of the potentially destabilizing c-terminal repeat unit. in addition, the uniform neck domain contains an arginine residue at each position , while this residue is glutamine or glutamic acid in several of the repeat units in the natural neck domain. in the crystal structure of the neck domain, the arginine residue is on the outer surface of the bundle of helices and forms a salt bridge with a conserved glutamic acid side chain at position in the same polypeptide [ fig. (e) ]. such an i,i salt bridge hydrogen bond would contribute to stability of the repeat units. , together, these results define a prototype sequence of a neck repeat unit with very high stability, in which leucine is present at position , packed on the inside of the coiled coil and arginine is present at position making a salt bridge on the outside of bundle of helices. in addition to differences in stability reflecting minor variations in the sequences of the repeat units in the neck domains of dc-sign and dc-signr, differences in the properties of the neck domains could arise from differences in the number of repeat units in polymorphic forms of dc-signr. to compare these factors, multiple versions of the neck domain were created with to . copies of the amino acid repeat unit containing leucine at position and arginine at position . changes in the stability of the neck domain oligomers were demonstrated by gel filtration analysis, which reveals a single peak of tetramers for the longer constructs, but the appearance of increasing amounts of monomer for shorter fragments containing and repeat units, while a fragment containing only three repeat units appears entirely as monomer [ fig. (a) ]. similar trends in stability were also observed by differential scanning calorimetry, in which a transition occurs at lower temperatures for the shorter constructs and is entirely absent for the version with three repeat units [ fig. (b) ]. these experiments are performed at high concentrations that would favor tetramer formation, which makes it possible to observe unfolding of the tetramer in all cases except for the fragment with three repeat units. this shortest version thus shows no evidence of tetramer formation in either the gel filtration or calorimetry experiments. fig. (d) ], indicating that the repeat units have the same conformation as seen in the crystal structure. however, when the number of repeat units drops below , there is a change in the ratio of the signals at and nm. values of this ratio higher than are associated with small differences in the helical pitch resulting from packing in coiled-coil structures. the expected changes are best characterized for parallel -and -stranded coiled-coils, but formation of the parallel -helical bundle would be expected to have a similar effect. the slight decrease in the intensity of the cd signal per repeat unit for the neck domain with four repeat units is consistent with the gel filtration results showing that coiledcoil tetramer is in equilibrium with a monomeric structure at the concentration used for gel filtration and cd analysis. the higher molar concentrations of the smaller fragments required to obtain good quality spectra would favor tetramer formation, so the results probably slightly under-estimate the effect of partial dissociation for the fragment with repeat units. in contrast, the fragment with only three repeat units shows a more significant loss of helical structure and a reduced ratio of the signals at and nm, which is consistent with the conclusion that this fragment does not form stable tetramers containing coiled coils. taken together, these results indicate that a fully stable coiled-coil structure requires the presence of at least five repeat units. a leucine residue is present at position only in repeat units through of dc-sign, with position occupied by a glutamine residue in most of the remaining repeat units, although a methionine residue is present in repeat unit . to assess the effect of this variation at position on the structure and stability of the neck domain, further engineered versions of the domain containing repeat units with either glutamine or methionine at position in each repeat unit were prepared. in contrast to the homogeneous neck domain containing leucine at position of each repeat unit, otherwise identical neck domain in which glutamine is present at this position in each repeat unit fail to form stable oligomers. at room temperature, the allglutamine version of the neck domain elutes as a monomer on gel filtration [ fig. (a) ]. this neck domain does not yield a clear denaturation transition by differential scanning calorimetry, which is consistent with the absence of stable oligomers (data not shown). circular dichroism analysis revealed the presence of a helical structure, but the molar ellipticity is reduced compared to the neck domain containing leucine residues at position of the repeat units [ fig. (b) ]. the all-glutamine version of the neck domain shows a ratio of signals at compared to nm comparable to that of the all-leucine neck domain. these results suggest that the all-glutamine version transiently forms coiled coils under the static conditions of the circular dichroism experiment, but that these oligomers are not stable enough to be observed in the gel filtration experiment. an analog of the neck domain containing methionine residues at position of each repeat unit also did not show a defined denaturation peak by calorimetry (data not shown) and has even more diminished helical content [ fig. (b) ]. in this case, the ratio of molar ellipticity at nm compared to nm is reduced, suggesting that helix formation is not accompanied by formation of coiled coils like those seen in the other forms of the neck domain. the oligomeric state assessed by gel filtration is also the combined results of the gel filtration, calorimetry and circular dichroism studies suggest that the presence of glutamine at position of the neck domain repeat unit is compatible with formation of the tetrameric coiled coil, so that a repeat unit containing this sequence can have the same conformation as a repeat unit with leucine at position , although the glutamine form is less stable. in contrast, a methionine residue at this position disrupts the conformation. a final unusual feature of the dc-sign neck domain is a tryptophan residue at position in the fourth repeat unit. this substitution has little effect on the behavior of the neck domain, as shown by the fact that substitution of the first portion of the neck domain from dc-signr, including an arginine residue at this position, for the corresponding portion of dc-sign results in removal of the tryptophan residue with no detectable effect on the stability of the neck domain. dc-sign and its homologs are subject to rapid genetic change. the -amino acid repeat units of the neck domain are present only in primates. a very divergent set of eight homologous genes is found in mice. based on the roles of some of the key amino acids in the neck domain described here, comparison of the dc-sign and dc-signr genes in the primates provides some insights into the potential functional effects of these genetic variations (fig. ) . some of the distinct structural features conserved in the dc-signr genes are the presence of the stabilizing leucine residue at position of all but the first and last repeat units and the presence of the unusual phenylalanine residue at position in the repeat unit adjacent to the crd, although there is variation in the number of repeat units. in contrast, the dc-sign sequences show greater diversity, although the more stable repeat units, with the leucine at position and arginine at position , are generally found nearer to the membrane, while repeat units at the c-terminal end, towards the crd, mostly contain glutamine at position . the destabilizing methionine residue is present at position in only one or two repeat units in the middle of the neck domain. conservation of these patterns, in spite of variation in the number of repeat units and the specific sequences, suggests that these features are selected because they confer different functional properties in the two receptors. the previously described crystal structure of the neck domain of dc-signr is based on an average from the different types of repeat units present. each of these repeat units has a leucine residue at position , although they vary at position . the results can be assembled into a model for the full extracellular portion of dc-signr. it has not been possible to obtain crystals of the forms of the neck domains with glutamine or methionine residues at position of the repeat units, or for the natural neck domain of dc-sign containing a mixture of residues at position of different repeat units, so the alternative strategy described here was to use spectroscopy, calorimetry, and gel filtration to suggest ways that sequence variation alters the structure from that observed for the repeat units with leucine residues at position . based on this combination of information, it is concluded that repeat units with leucine residues at position and arginine residues at position form particularly stable tetrameric coiled coils. changing the arginine residue at position results in loss of the salt bridge with the conserved glutamic acid residue at position , which causes some destabilization, while introduction of alternative residues at position is more destabilizing. the summary of the sequences of dc-sign and dc-signr molecules shown in figure reveals that relatively few repeat units are stabilized by the presence of both a leucine residue at position and an arginine residue at position , but a high proportion of the repeat units contain one or the other of these residues. appropriate residues at either one of these two positions may be alternative ways of achieving stability of the coiled coil. the loss of stability resulting from the presence of a glutamine residue at position is somewhat surprising, since favorable packing and hydrogen bonding of glutamine residues has been observed in trimeric coiled coils and the glutamine side chain can be accommodated sterically in the interior of the tetrameric coiled coil. the circular dichroism results confirm that the presence of glutamine is compatible with the coiled coil structure, with the decreased signal reflecting the loss of stability so that the tetramer is dissociated more of the time. it may be significant that the glutamine residue at position would be the first residue of the coiled coil, following the disruption caused by the preceding proline and charged residues. the loss of hydrophobic interactions may be of greater significance in this context, leading to destabilization. in contrast, the presence of a methionine residue at position leads to more significant disruption of the structure of the neck domain, which may be explained based on steric constraints resulting from the longer, linear side chain that would not readily pack inside the coiled coil. loss of stability and the potential disruption to the helical structure resulting from the presence of a methionine residue in the middle repeat unit probably explains why the neck domain of dc-sign is resistant to crystallization. for dc-signr, a key finding to emerge from these studies is the observation that the repeat units are mostly stabilized by leucine residues at position and arginine residues at position . the presence of a phenylalanine residue at position in the repeat unit adjacent to the crd appears to cause a flaring apart of the crds, but the remainder of the neck domain is a stable tetrameric coiled coil. this arrangement of very stable repeat units in the neck domain, which is conserved in all of the dc-signr proteins in the primates, may be necessary to ensure that the neck domain remains tetrameric even though the final repeat units are splayed apart by the presence of the phenylalanine residue. the studies reported here suggest that the presence of stabilizing residues at positions and of the repeat units allows for the formation of stable tetramers even for shorter versions of the neck domain present in some individuals, which result from common genetic polymorphisms in the human population. , however, the results also define a minimal length for the neck domain to be fully stable, even in the presence of stabilizing residues within the repeat units. the very stable structure of the coiled coil ensures that the spacing between the crds in dc-signr is relatively fixed, although the exact orientation of the crds would be potentially flexible because of the flaring apart caused by the unusual repeat unit adjacent to the crd. in contrast to dc-signr, only the repeat units at the membrane-proximal end of the neck domain in dc-sign are stabilized by leucine residues at position . the presence of potentially less stable repeat units in the half of the dc-sign neck domain further from the membrane, and the presence of repeat units containing the potentially disruptive methionine residues at position in a repeat unit in the middle of the neck domain, raises the possibility that the cterminal portion of the neck domain might dissociate some of the time, so that the extracellular domain would be able to accommodate different spacings of sugar residues, as observed in recent studies with mannose-coated particles. high affinity binding of lattices of sugars by multivalent receptors requires both multivalency and appropriate spacing and orientation of sugar-binding sites, , so this arrangement would potentially allow the cluster of crds in the tetramer of dc-sign to bind clusters of ligands at a greater range of spacings. dos santos et al. some differences in the interactions of dc-sign and dc-signr with glycans on pathogens may result from differences in the local sugarbinding characteristics of the crds. for example, only dc-sign binds to parasite glycoproteins because this binding results from recognition of the lewis x epitope, which is bound by dc-sign but not dc-signr. , however, it is more difficult to explain differential binding of pathogens that bear high mannose oligosaccharides, as in the case of west nile virus, which binds better to dc-signr than to dc-sign, based on the interaction of individual oligosaccharides with the binding sites in the crds. it seems more likely that such differences result from different capacities of the receptors to accommodate various spacings of glycan epitopes resulting from the distinct properties of the neck domains described here. procedures for expression of the extracellular portions of human dc-sign and dc-signr in escherichia coli followed by purification, employing affinity chromatography on mannose-sepharose, were as previously described. wild type and mutated versions of the neck domain bearing -histidine tags were purified by chromatography on immobilized ni followed by anion exchange chromatography. , in each case, the ' bamh site allows insertion into the expression vector pt t so that the neck polypeptide is translated when a ribosome restarts following termination of a truncated phage t gene protein. vectors encoding uniform neck domain repeat units with leucine at position were created starting from the cdna for the dc-signr neck. synthetic oligonucleotides were used to replace the ' sequence encoding the n-terminus of repeat unit and the ' sequence encoding the c-terminus of repeat unit , to create a dna sequence encoding a block of repeat units with identical amino acid sequences and a c-terminal -histidine tag [ fig. (a) ]. fragments encoding various numbers of repeat units from the ' or ' ends of this construct were created by partial digestion with bglii followed by digestion with the flanking enzymes bamhi or ecori, respectively. pairs of ' and ' fragments were ligated to generate sequences encoding to repeat units. the resulting bamh to ecor fragments were inserted into the t expression vector as for the wild type neck domains. a similar strategy was used to generate a dna sequence encoding three repeat units with glutamine at position , by substituting synthetic oligonucleotides for the portions of the dc-sign cdna encoding the ' end of repeat unit and the ' end of repeat unit [ fig. (b) ]. the bglii partial digestion strategy was used to expand the number of repeat units to and then to . for creation of neck domain polypeptides containing methionine residues at position , three sets of synthetic oligonucleotides were assembled to generate a dna fragment encoding two repeat units [ fig. (c) ]. partial bglii digestion was repeated three times to expand the number of encoded repeat units first to , then to and finally to repeat units. analytical affinity chromatography on mannose-sepharose was performed using loading buffer consisting of . m nacl, mm tris-cl, ph . , and mm cacl and elution buffer containing of . m nacl, mm tris-cl, ph . , and . mm edta. sds-polyacrylamide gel electrophoresis was performed in the buffer system of laemmli. gel filtration analysis employed a cm superdex s column eluted with mm nacl, mm tris-cl, ph . , . mm edta. the positions of marker proteins are indicated by the stokes radius: cytochrome c, Å ; bovine erythrocyte carbonic anhydrase, . Å ; bovine serum albumin, . Å ; yeast alcohol dehydrogenase, . Å ; b-amylase, Å ; e. coli bgalactosidase, Å ; and thyroglobulin, Å . protein concentrations were determined by ninhydrin assay following alkaline hydrolysis. differential scanning calorimetry samples for calorimetry were dialyzed extensively against mm nacl, hepes, ph . , . mm cacl , degassed under vacuum and introduced into the sample loop of a nano-iii calorimeter from calorimetry sciences corporation. repeated equilibration scans from to c were used to remove any remaining dissolved gas. scans were performed at min . protein concentrations used were based on those previously employed for the native neck polypeptides, since these give signals that can be quantified accurately. for experiments in which different proteins were compared, protein concentrations varied by less than three-fold. proteins were prepared in mm nacl, mm tris-cl, ph . , mm cacl . analysis was performed on ll samples in a -mm cuvette in a chirascan spectropolarimeter from applied photophysics, with data collected for sec at . nm intervals followed by averaging of scans. comparisons were made between proteins within a three-fold range of concentrations. in test experiments over this concentration range, the ratio of tetramers to monomer observed on gel filtration changed by less than . -fold for the fragments that undergo some dissociation into monomers, such as the version containing four repeat units with leucine at position . c-type lectin dc-sign: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity c-type lectins on dendritic cells: key modulators for the induction of immune responses structural basis for distinct ligand-binding and targeting properties of the receptors dc-sign and dc-signr dc-sign and l-sign: the signs for infection segmented helical structure of the neck region of the glycan-binding receptor dc-signr hiv- transmission by dendritic cell-specific icam- -grabbing nonintegrin (dc-sign) is regulated by determinants in the carbohydrate recognition domain that are absent in liver/lymph node-sign (l-sign) west nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection differential use of the c-type lectins l-sign and dc-sign for phlebovirus endocytosis compact, polyvalent mannose quantum dots as sensitive, ratiometric fret probes for multivalent protein-ligand interactions repeat-region polymophisms in the gene for the dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin-related molecule: effects on hiv- susceptibility autonomous tetramerization domains in the glycan-binding receptors dc-sign and dc-signr geometry and adhesion of extracellular domains of dc-signr neck length variants analyzed by force-distance measurements binding site geometry and flexibility in dc-sign demonstrated with surface force measurements extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr lys salt bridges in short peptides of de novo design surface salt bridges stabilize the gcn leucine zipper comparison of antiparallel and parallel two-stranded a-helical coiled coils: design, synthesis and characterization the evolutionary history of the cd (dc-sign) family in humans and non-human primates novel mouse homologs of human dc-sign: widely divergent biochemical properties of the complete set of mouse dc-sign-related proteins crystal structure and biophysical properties of a complex between the n-terminal snare region of snap and syntaxin a impact of polymorphisms in the dc-signr neck domain on the interactions with pathogens polymorphisms in the c-type lectin genes cluster in chromosome and predisposition to severe acute respiratory syndrome coronavirus (sars-cov) infection effects of clustered epitopes in multivalent ligand-receptor interactions identification of novel contributions to high-affinity glycoprotein-receptor interactions using engineered ligands van die i ( ) molecular basis of the differences in binding properties of the highly related c-type lectins dc-sign and l-sign to lewisx trisaccharide and schistosoma mansoni egg antigens a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr: subunit organisation and binding to multivalent ligands primary structure and functional expression from complementary dna of a human interleukin- receptor antagonist characterization studies on a new lectin found in seed of vicia ervilia cleavage of structural proteins during the assembly of the head of bacteriophage t detection of peptides by chemical methods the authors declare no conflicts of interest. key: cord- - etvgoxc authors: ellis, christine title: ferrets date: - - journal: saunders manual of small animal practice doi: . /b - - - / - sha: doc_id: cord_uid: etvgoxc nan m clinical techniques restraint • most pet ferrets are gentle, tractable, and are easy to restrain without assistance. often only minimal restraint is needed when performing a physical examination. some ferrets may be lightly restrained on the examination table. others will need to be restrained in a firmer manner. • tractable ferrets can be lightly restrained on an examination or treatment table by placing one hand under the chest and lifting slightly. • energetic ferrets may be restrained by scruffing ( fig. - ). use one hand to grasp the skin over the back of the neck and lift the ferret up, suspending all the limbs. stroke the abdomen with a downward motion to relax the ferret. the ferret's back may be supported with the other hand, or the ferret may then be reclined along the forearm of the arm used to scruff the ferret. most ferrets will become very relaxed, although some young ferrets and some females may resist. • firm restraint is often required when administering vaccinations or when performing treatment procedures. control the head by scruffing, or by cupping the back of the ferret's neck and placing the thumb and fingers along the caudal border of the mandibles. place the other hand over the pelvis to restrain the hindquarters on the table top with the hind legs underneath the body; do not pull the legs back. • aggressive ferrets, such as nursing females, kits, or ferrets raised with little human contact, are uncommon. restrain these ferrets by the scruff of the neck, using the techniques previously described. avoid using leather gloves, which are awkward. use sedation if necessary. there are several suitable sites for blood collection in ferrets: • • cephalic or lateral saphenous venipuncture may be used to obtain small amounts of blood (< . ml) for a packed cell volume (pcv), blood glucose, complete blood cell count (cbc), or serum biochemistry analysis. • the jugular vein, cranial vena cava, cephalic vein, or ventral tail artery may be used to collect larger volumes of blood. • use the jugular vein to collect blood for transfusion. christine ellis figure - . "scruffing" a ferret for restraint. • sedation: collection of blood from the jugular veins, cranial vena cava, or ventral tail artery may require sedation and/or the assistance of two people for restraint. sedation is rarely required for venipuncture of cephalic, lateral saphenous, or jugular veins. • if necessary, clip the hair over the venipuncture site to see the vein. • the normal hematocrit of ferrets is high; draw three times as much blood as the volume of plasma or serum required. (see tables - and - for blood values reported in normal ferrets.) • the pcv, red blood cell count (rbc), hemoglobin, white blood cell count (wbc), and plasma proteins often rapidly decrease after induction of isoflurane anesthesia. cephalic vein • collect blood from the cephalic vein in ferrets using the same restraint technique described for dogs and cats. • use an insulin syringe with a -gauge needle or a cc tuberculin syringe with a -gauge needle to collect volumes of blood up to . ml. larger volumes of blood may be collected with a cc syringe and a -gauge needle. • alternatively, place a -gauge needle in the vein and collect blood directly from the hub into small blood collection tubes. ‡these white blood cell counts are higher than those currently seen in clinical practice. at our laboratories, the normal white blood cell count is - ¥ /ml, and most are - ¥ /ml. adapted with permission from lee ej, moore we, fryer hc, minocha hc: hematological and serum chemistry profiles of ferrets (mustela putorius furo). lab anim : - , ; and thornton pc, wright pa, sacra pj, goodier tew: the ferret, mustela putorius furo, as a new species in toxicology. lab anim : - , . copyrights and lateral saphenous vein • the lateral saphenous vein runs diagonally across the lateral surface of the hindleg, just proximal to the hock. • use an insulin syringe with a -gauge needle or a -ml syringe with a -gauge needle to collect small blood samples (< . ml). • several methods are described for jugular venipuncture: • the ferret may be placed in sternal recumbency at the edge of the table. extend the head dorsally with the front legs held down, out of the path of the venipuncturist. • alternatively, wrap the ferret in a towel with the front legs drawn back along the thorax, leaving the head and neck extended from the towel. position the ferret in dorsal recumbency, and extend the head and neck by scruffing. • restrain the ferret in the same manner described for the cranial vena cava (see below). • ferrets that struggle should be sedated for this procedure. • use a -ml or -ml syringe with a -to -gauge needle for sample collection. • this procedure is referred to as cranial vena cava venipuncture, but, in reality, blood is collected from the jugular vein as it passes into the thoracic cavity at the thoracic inlet. m key point blood collection from the cranial vena cava requires complete immobilization of the ferret; otherwise, do not attempt the procedure. use sedation or the help of two assistants for restraint. • do not use this site if intrathoracic disease (e.g., mediastinal mass, mega-esophagus) or coagulopathy is suspected. . place the ferret in dorsal recumbency. one assistant restrains the head and neck in extension while holding the forelegs alongside the thorax. a second assistant restrains the hindquarters without pulling the rear legs back. precise positioning facilitates the procedure. . palpate the manubrium and locate the "notch" on either side where the manubrium and the first rib meet. . insert a -ml syringe with a -gauge, / -inch needle at either notch and direct the needle at a shallow angle (< degrees) along an imaginary line running from the notch toward the opposite rear leg. . insert the needle to the hub and gently aspirate while withdrawing the needle. . if the ferret struggles, abort the procedure, and do not make a second attempt until the ferret is quiet. ferrets that struggle persistently should be sedated for this procedure. • venipuncture at this site may be painful. . scruff the ferret and place it in dorsal recumbency (wrapping it in a towel may help with restraint), or anesthetize the ferret and place it in dorsal recumbency. . prepare the site aseptically. . use a -or -cc syringe and a -to -gauge needle for sample collection. . insert the needle on the ventral midline of the tail at -degree angle toward the body approximately to cm from the anus. . advance the needle to the bone; withdraw it slowly while applying a slight vacuum to the syringe. . apply direct pressure to the site for several minutes after the needle is withdrawn. a detailed discussion of the techniques used for blood collection for transfusion and blood transfusion is presented in the hematopoietic system section in this chapter. • use standard radiographic techniques (see chapter ), including sedation for correct positioning and to limit exposure of the technician, as well as high detail radiographic film and cassettes. • when interpreting films, it helps to think of the ferret as an elongated cat. • the kidneys are relatively short (about two lumbar vertebrae in length). • splenomegaly is a common radiographic finding. • for barium-contrast radiography of the gastrointestinal (gi) tract, give ml/kg of % barium solution po via syringe feeding or lavage tube. most ferrets will accept syringe feeding of barium. normal gi transit time is about to hours. • echocardiography may be used to evaluate the heart in ferrets with suspected cardiac disease (see "cardiovascular disease"). • other uses of ultrasound in ferrets include investigation of intra-abdominal or intra-thoracic masses, organomegaly, paraurethral cysts, or prostatic cysts. • the indications, guidelines, and techniques for bone marrow sampling are the same as those described for dogs and cats. (see chapter ) • preferred sites include the proximal femur and humerus. the iliac crest may be used for sample collection as well, but can be a difficult site to access. • sedation is required for bone marrow aspiration. use a -or -gauge spinal needle with stylet for sample collection. fine-needle aspiration of the spleen has been performed successfully in ferrets and is a rapid means of evaluating splenic cytology. in ferrets, the only contraindication is suspected hemangiosarcoma of the spleen. sedation is rarely necessary, but is recommended if the ferret persistently struggles. . two assistants are recommended for restraint. place the ferret in dorsal recumbency. one assistant should restrain the head and neck by scruffing the ferret with one hand. the other hand is used to restrain the forelimbs. a second assistant restrains the hind limbs by placing one hand around the pelvis. . palpate the spleen and position it against the left lateral or ventral body wall. . clip and prepare the site aseptically. . insert a -gauge needle attached to a -ml syringe to the hub at a perpendicular angle to the skin and aspirate from the spleen. . when a small amount of bloody fluid is visualized in the needle hub, withdraw the needle and prepare slides routinely for cytology. • urine may be collected by cystocentesis using the same technique described for the cat. • a -or -gauge needle on a -or -cc syringe may be used for sample collection. • anesthesia is recommended if the ferret is difficult to restrain. m key point sedation is often required for placement of a butterfly or indwelling intravenous (iv) catheter, or for small-volume iv therapy. • for small-volume iv therapy ( . - . ml), use an insulin syringe. the cephalic or lateral saphenous veins are the preferred sites for injection. sedation may not always be required for a single injection. • a -gauge butterfly catheter may be used to administer larger, single-dose volumes into the cephalic vein. • an indwelling catheter can be placed in the cephalic, lateral saphenous, or jugular vein. sedation is usually required. • flush indwelling catheters with small volumes of heparinized saline solution to maintain catheter patency. • peripheral indwelling catheters can be placed rapidly and are useful in emergency situations and for surgery. • restrain the ferret by scruffing. some ferrets may require two people for restraint. • sc injections may be given in the loose skin over the shoulders. • im injections may be given in the quadriceps, the semimembranosus-semitendonosus muscles of the hind limbs, or in the expaxial muscles of the lower back. • limit the volume of the im injections, due to the small muscle mass of the ferret. • fluids may be administered sc, iv, or io, depending on the needs of the patient. • the daily fluid requirement for ferrets has not been reported but can be estimated at to ml/kg/ day. adjust for dehydration and fluid loss. • administer sc fluids over the dorsal shoulder and thoracic region. • iv or io fluid therapy is used for a wide range of medical and surgical situations, and is recommended for ferrets that are > % dehydrated. • iv or io fluids must be administered with an infusion pump. fluids may be given as a continuous infusion, may be administered by continuous infusion, or may be given in to bolus doses over a -hour period. • ferrets may require the addition of dextrose to fluids because hypoglycemia is common. • oral medications are most easily given to ferrets in liquid form. • if possible, compound medications formulated in tablet or capsule form into liquid suspensions, or crush and mix them with a sweet-tasting substance such as nutri-cal (evsco pharmaceuticals) feline hairball laxative, or fruit-flavored syrup, and administer by syringe. • ferrets suffering from insulinoma should not be given sugar-based treats or medications if at all possible. hide medications in fatty acid supplements, vegetable oil, whipping cream, or meat baby food. • supplemental feeding is important in the management of anorectic or critically ill ferrets, and in the treatment and prevention of hypoglycemic episodes associated with insulinoma. • most ferrets can be force-fed dietary supplements by syringe. once they acquire a taste for a given supplement, it may be possible to offer it in a bowl. • feed ferrets as much food as they will take comfortably ( - ml) to times daily. • foods useful for force-feeding include the following: meat baby foods, slurried cat or ferret food, and science diet a/dliquid soy-based formulas (e.g. deliver . , mead johnson nutritionals) may be added to the mixture to increase the calorie content and improve palatability. • use of all medications is considered off-label for the ferret; there are no approved drugs available for ferrets in the united states. • several exotic animal formularies are commercially available that include drug dosage information on ferrets. • when dosing information is not available, use feline dosages with the following exceptions: • chloramphenicol: ( mg/kg) bid, iv, sc, im, or po. • aspirin: ( - mg/kg) bid-tid po (canine dosage). • many ferrets become lethargic when placed on enalapril (enacard, merck agvet) for cardiac disease. start with a very low dose ( . - . mg/kg) q h po. some ferrets cannot tolerate more than every-otherday therapy. • ivermectin ( . mg/kg) q d po for heartworm prevention; ( . mg/kg) once to weeks after adulticide treatment as a heartworm microfilaricide; ( . - . mg/kg) po, sc, repeat in days for sarcoptic mites; ( mg/kg) instill half the calculated dose into each ear and repeat in days for ear mites. a detailed discussion of the techniques used for blood collection for transfusion and blood transfusionis presented in the hematopoietic system section in this chapter. • sedation or isoflurane anesthesia facilitates urinary catheterization. the procedure may be difficult in ferrets with urethral disease or urethral calculi. . position in ventral recumbency with the hindquarters elevated and use a tomcat catheter or a . -fr. feeding tube with or without a stylet. . the urethral orifice is located on the ventral floor of the vaginal vault. catheterize the urethra blindly or after identification using a vaginal speculum. • the catheter may only pass part way into the urethra (often to the pelvic flexure). this may be sufficient to allow retrograde flushing of urethral calculi into the bladder, or to empty the bladder of a ferret with urethral obstruction secondary to prostatic enlargement. m key point isoflurane administered by face mask is the most convenient method to immobilize a ferret for procedures such as venipuncture and radiography. induction and recovery are rapid. doses for parenteral agents used in ferrets are listed in table - . • acepromazine is useful for sedation. • butorphanol tartrate has been used at sc, im, iv, but can cause very profound sedation in some ferrets. • ketamine alone does not produce effective muscle relaxation. use in combination with acepromazine for minor surgical procedures, or with diazepam for more complicated procedures. • medetomidine is not analgesic; animals will respond to painful stimuli. use with an analgesic agent. this agent is reversible (atapamazol). • tiletamine-zolazepam (telazol, fort dodge) gives variable muscle relaxation but is useful for immobilization for procedures such as venipuncture, radiography, and electrocardiography. recovery may be prolonged. • xylazine may cause bradycardia or vomiting, and is not recommended for use. • premedicate with parenteral agents followed by facemask induction. alternatively ferrets may be given • isoflurane is the inhalant anesthetic most commonly used in small mammal practice. sevoflurane is used in some practices as well. pharmacokinetic and pharmacologic isoflurane and sevoflurane are very similar; sevoflurane smells better and is better tolerated during face-mask induction. • isoflurane does not provide analgesia. administer an analgesic pre-or intra-anesthesia if a painful procedure is to be performed. analgesic agents commonly used in ferrets include buprenorphene ( . - . mg/kg q - h sc, im, iv), butorphanol ( . - . mg/kg q - h sc, im), carprofen ( mg/kg q - po), and flunixin meglumine ( . - . mg/kg q - h im, iv). • intubate ferrets to facilitate intermittent positive pressure ventilation (ippv). a . to . mm endotracheal tube usually is suitable. • the same planes of anesthetic depth reported for dogs and cats occur in the ferret. • follow basic principles of anesthesia for small animals, (see chapter ) and provide supplemental heat during surgery; administer iv fluids (isotonic electrolyte solutions supplemented with . - % dextrose) during long procedures and for insulinoma surgery. canine distemper and influenza are the two most common viral diseases of the ferret. influenza is zoonotic between humans and ferrets, and is typically passed from humans to ferrets. canine distemper is % fatal in the ferret, making distemper vaccination imperative. • the canine distemper virus (cdv) is a paramyxovirus. transmission occurs through direct contact with infected animals of any species, and through contact with fomites such as in shoes or clothing. • the incubation period for cdv in the ferret is typically to days; however, incubation for some strains of cdv may take up to days. (for discussion of cdv in dogs, see chapter ). • early in the disease the only clinical sign may be a mild unilateral or bilateral conjunctivitis. • pyrexia (> °c), anorexia, and profuse mucopurulent naso-ocular discharge develop as the disease progresses. • vaccinate all ferrets in the household or facility. currently only two vaccines are approved for use in ferrets: purevax (merial, athens, ga), and fervac-d (united vaccines, inc., madison, wi). give ml sc, using the following schedule: • if the dam is vaccinated: vaccinate kits at weeks of age and repeat vaccination every to weeks until the kits are weeks of age. • if the dam is unvaccinated: vaccinate the kits at weeks of age and repeat every to weeks until the kits are weeks of age. • revaccinate annually. sources claim that immunity lasts for years; however, outbreaks have been known to occur months after vaccination. • use of serum titers as a method to evaluate an animal's current immunological status is unsubstantiated. • quarantine new ferrets and canines for weeks before exposure to other resident animals. vaccinate new animals immediately after acquisition at the beginning of the quarantine period. • use of galaxy d (schering-plough animal health co., omaha, ne) for distemper vaccination has been described. use of this product in ferrets is extra-label. this product has proved effective in preventing canine distemper in young ferrets; however, duration of immunity is unknown. • do not use cdv vaccines that contain canine parvovirus, adenovirus, or other viruses. it is not necessary to vaccinate for leptospirosis unless there is exposure to wild rodents. the influenza virus is an orthomyxovirus. ferrets are susceptible to influenza a and b; this is the only documented zoonotic disease of the ferret. human-to-ferret transmission is more common than ferret-to-human transmission. transmission occurs by direct contact with naso-ocular discharges, and via inhalation of aerosolized droplets. • the incubation period is typically to days postexposure. • the clinical course of the disease is typically to days. m key point influenza typically causes only mild illness and discomfort, and is usually self-limiting in an otherwise healthy animal. • clinical signs may include any combination of the following: • sneezing with a clear, serous nasal discharge. • mild conjunctivitis with serous ocular discharge. crusting around the eyes may occur rarely. • a nonproductive cough that may be loud and paroxysmal, and often occurs more frequently at night. • diarrhea. vomiting may occur in rare cases. • partial to total anorexia, listlessness, and fever. • pneumonia, severe illness, or death may occur in neonates, geriatric patients, and in ferrets with concurrent diseases such as lymphosarcoma or insulinoma. • ferrets with underlying immunosuppressive disorders, especially lymphosarcoma, may develop repeated or cyclic episodes of influenza. rule out lymphosarcoma by performing a complete blood cell count (cbc), bone marrow biopsy/cytology, or a peripheral lymph node biopsy (see lymphoma in this chapter). diagnosis is based primarily on the clinical signs, history, and physical examination. • the history often indicates recent exposure to a human or another ferret with influenza or signs of upper respiratory tract disease. • the overall physical condition often remains good, although slight or moderate dehydration may be present if the animal is not eating or drinking normal amounts. • differential diagnoses include the very early stages of canine distemper, gi rotavirus infection, and lymphosarcoma. if mucopurulent nasal or ocular discharge is noted, consider early cdv or a secondary bacterial infection. • supportive care generally is sufficient. • encourage the ferret to eat and drink. offer to tablespoonfuls of hill's science diet a/d or strained meat baby food bid-qid if the animal refuses the regular diet. • if indicated, give an oral electrolyte solution that is palatable to ferrets. • if sneezing or coughing is excessive and interferes with eating or sleeping, give an antihistamine such as chlorpheniramine ( . - . mg/kg) bid-tid po, or diphenhydramine ( . - . mg/kg) bid-tid po. nasal solutions containing phenylephrine may be used to relieve nasal congestion. • antibiotics are not necessary unless secondary bacterial infection is present. • antiviral medications such as amantadine ( mg/kg) bid po (symmetrel, endo pharmaceuticals, chadds ford, pa) may be useful in the treatment of ferrets with influenza. zanamivir ( . mg/kg) once intranasally (relenza, glaxosmithkline, research triangle park, nc) has been shown experimentally to prevent influenza infection. good hygiene is the key to prevention. • discuss the zoonotic potential with the client. advise clients to wash their hands frequently, and to avoid holding the ferret near the face. • in the veterinary hospital, do not allow influenzainfected personnel to handle ferrets, especially if the animal is a neonate, a geriatric patient, or a patient debilitated by serious disease. • vaccination is not recommended; only short-term immunity results, and the wide variation of the influenza virus makes appropriate vaccination difficult. rabies is caused by a rhabdovirus that results in fatal disease in ferrets. it is transmitted via contact with an infected animal's saliva (see chapter ). this is a zoonotic disease; however, there has never been a report of ferret-to-human rabies transmission. experimentally, the incubation period is to days. m key point very little is known about rabies in naturally infected ferrets. • it is known that ferrets may become naturally infected; however, there is some question as to how easily they can contract the disease and the length of the incubation period. information about clinical signs is derived primarily from literature associated with laboratory-infected ferrets. signs are variable and include: • behavioral abnormalities that range from anxiety and hyperactivity to lethargy. • neurologic signs such as ascending paralysis, ataxia, hyperparesthesia, and posterior paresis. diagnosis is based on clinical signs, and/or a history of known or potential exposure to rabies. • history may include a recent bite wound or exposure to a rabid animal. • the ferret may be unvaccinated; however, development of rabies in vaccinated individuals has occurred in other animal species. • differential diagnoses include aleutian disease, botulism, brain hypoxia from severe seizures, cns neoplasia, insulinoma, intervertebral disc disease, and viral or bacterial encephalitis. • postmortem laboratory testing of brain tissue (fluorescent antibody staining [fas] or virus isolation) confirms the diagnosis (see chapter ). • there is no treatment for rabies. • euthanize the suspect animal to protect humans and other animals in its environment. submit animal for postmortem fas testing of brain tissue. m key point it has not been demonstrated that ferrets are carriers of rabies. many public health facilities now recognize and accept the -day quarantine period for ferrets; however, in some states, unvaccinated ferrets involved in biting incidents will be euthanized and submitted for rabies testing. it is important to be familiar with state and local laws regarding vaccination requirements and the laws following a biting incident. m key point the compendium of animal rabies prevention and control recommends that ferrets be confined and observed for days following human exposure. if signs compatible with rabies develop, the animal should be euthanized and protocols for rabies testing should be followed. vaccinated ferrets exposed to a potentially rabid animal should be revaccinated and quarantined for days. euthanize any unvaccinated ferret exposed to a rabid animal. • vaccination is the only prevention, and is mandatory in some states. • imrab (rhone merieux) is an inactivated rabies vaccine that is currently the only rabies vaccine approved for use in ferrets. administer at a dose of ml sc. • vaccinate initially at months of age. revaccinate annually. aleutian disease (adv) is caused by a parvovirus that affects both mink and ferrets. transmission occurs by direct contact or via contact with fomites contaminated with any infected body fluid, including blood. adv produces a progressive immune-mediated disease accompanied by the deposition of antigen-antibody complexes in multiple organs of the body. the virus is prevalent in the ferret population, but the percentage of ferrets that develop clinical illness is low. in one survey of ferrets, % were serologically positive, but only two animals developed clinically active disease. some ferrets may be asymptomatic carriers, while others may have natural immunity to the disease. m key point the clinical signs of aleutian disease are extremely variable, and the incubation period can be as short as day or as long as to days. • ataxia, mild incoordination, posterior paresis, or tremors may be the initial presenting signs. initially ferrets often continue to eat, and appear bright and alert. as the disease progresses, paresis progresses to the forelimbs, and wasting develops that may continue for weeks or months. • anorexia, lethargy, melena, and urinary incontinence are seen in later stages of the disease. • a slow wasting disease existing without neurologic signs may also occur. • diagnosis may based on the history, clinical signs, physical examination findings, the presence of high serum total protein and hypergammaglobulinemia, and a positive adv test. diagnosis is confirmed with histopathology. • differential diagnoses include bacterial or viral encephalitis, cns neoplasia, canine distemper, lymphosarcoma, gastric foreign body, tuberculosis, intervertebral disc disease, systemic mycoses, and rabies (in cases with behavioral changes and sudden paralysis). • exposure history is often not helpful because of the prevalence of asymptomatic carriers. • high serum total protein may be present. serum protein electrophoresis may demonstrate hypergammaglobulinemia (> % of the total serum protein). • blood samples may be submitted for counterimmunoelectrophoresis testing (united vaccines, inc., madison, we) or enzyme-linked immunosorbent assay (elisa) testing (avecon diagnostics, bath, pa). an in-house saliva sample kit is available as well (avecon diagnostics, bath, pa). • histopathology demonstrates lymphocytic plasmacytic infiltration and perivascular cuffing in many organ systems. the kidneys, liver, lymph nodes, and spleen are often affected. m key point there is no effective treatment for aleutian disease. provide supportive care and do not allow contact between clinically ill animals and healthy ferrets. euthanasia is usually indicated only for clinically affected animals. should not be euthanized because they may never become clinically ill. infected ferrets may remain asymptomatic for life, but can remain persistently infected. other ferrets may develop nonpersistent, self-limiting disease and fully recover. • administration of corticosteroid therapy and supportive care may prolong the life of some ferrets with clinically active disease. breeding colonies • breeding colonies should be closed. new animals should be adv tested and quarantined prior to introduction. • test all resident ferrets and remove serologically positive animals from the population. • adv-negative animals should be retested in months, before adding them to the colony, due to the potentially long incubation period. • it is not necessary to test a pet ferret unless it has been exposed to a clinically ill animal. • it is not necessary to euthanize a clinically normal, non-breeding adv-positive ferret or remove it from contact with other pet ferrets. advise the client, however, that there is a slight possibility that the pet may develop clinical illness. • do not house ferrets in close proximity to mink. • retest adv-positive animals in months since some animals may eventually eliminate the virus and become negative. rotavirus causes gastrointestinal infection and a bright green or yellowish-green diarrhea. rotavirus is described in the "gastrointestinal system" section in this chapter. lymphosarcoma is common in the ferret, and is discussed in the "neoplasia" section in this chapter. staphylococcus, streptococcus, escherichia coli, and other common bacteria from the environment can be introduced through penetrating wounds, punctures, abrasions, contact with mucous membranes, and by inhalation or ingestion. • abscessation is an uncommon form of bacterial infection in ferrets. • an abscess may occur in any part of the body, including the anal glands, mammary tissue, mouth, mucous membranes, reproductive tract, respiratory tract, subcutis, and prostatic tissue. • body temperature may be £ °c if bacterial sepsis is present. • bacterial dermatitis causes thickened, irritated areas of skin. affected ferrets may lick and chew these areas until they become denuded and ulcerated. • bacterial conjunctivitis causes a thick mucopurulent ocular discharge and swelling of the conjunctiva; corneal ulcerations may be present. • bacterial pneumonia causes lethargy, fever, anorexia, and dyspnea, and is often accompanied by mucopurulent nasal discharge and coughing. • bacterial mastitis occurs primarily in the lactating jill, and is accompanied by depression, fever, and anorexia. one or more mammary glands are swollen, discolored, and warm to the touch. • bacterial metritis may or may not cause a vaginal discharge; depression, fever, and partial or total anorexia are often present. • bacterial vaginitis causes a thick mucopurulent yellow-to-green vaginal discharge with little odor. fever is usually absent, and the animal does not appear clinically ill. • presumptive diagnosis is based on clinical signs, physical examination, demonstration of bacteria on routine cytology, results of bacterial culture, and sensitivity of the affected sites. the total wbc may demonstrate a marked leukocytosis (> , ). • treatment should consist of appropriate antibiotic therapy based on culture and sensitivity results, and surgical drainage or excision of the affected tissue when appropriate. • begin treatment with a broad spectrum antibiotic pending the results of culture and sensitivity testing, or when obtaining a culture is not feasible. • provide supportive treatment as needed, such as fluid therapy and nutritional support. • lance and thoroughly flush with an antiseptic solution. keep the area open and flush twice daily until healing occurs by second intention. • administer oral antibiotics until signs of infection are gone and a healthy bed of granulation tissue is present. see mastitis in the reproductive disease section in this chapter. see uterine infection in the reproductive disease section in this chapter. • if possible, perform a tracheal wash and submit samples for cytology, bacterial culture, and sensitivity testing. • if pleural effusion is evident on radiography, perform thoracocentesis. submit samples for cytology, bacterial culture, and sensitivity testing. • start oral broad-spectrum antibiotic therapy immediately, pending culture and sensitivity results. if pleural effusion is present, consider treating with a combination of clindamycin and cephalosporins (use cat dosages). • use of bronchodilating agents and/or nebulization therapy may be beneficial treatment modalities as well. • treat conjunctivitis with a broad-spectrum ophthalmic ointment. • perform a fluorescent corneal staining test to rule out corneal ulcers. (see chapter ) • submit samples for bacterial culture and sensitivity testing. • begin treatment with broad-spectrum oral antibiotics. modify treatment based on culture sensitivity results. • instruct owners to hot pack the affected area to minutes bid-tid. • treat until infection and swelling resolve, then perform anal sacculectomy. • continue antibiotic treatment to days postoperatively. • campylobacter spp. typically causes gi disease (see "gastrointestinal system"). • salmonella spp. may rarely cause gastroenteritis in ferrets (see "salmonella" within "gastrointestinal system"). • botulism is a rarely encountered disease in the domestic ferret caused by the ingestion of food contaminated with the clostridium botulinum toxin. c. botulinum is commonly found in the soil. • uncooked food or food contaminated with soil can be the source of the infection. • clinical cases of tuberculosis in the ferret are reported infrequently; however, ferrets are susceptible to bovine, avian, and human mycobacterium spp. infections. • the disease can be transmitted by ingestion of contaminated meat (poultry or meat), unpasteurized milk, or food contaminated by the droppings of infected wild or pet birds (see chapter for information about tuberculosis in dogs and cats). • clinical signs include chronic weight loss, and diarrhea that is unresponsive to treatment. vomiting may occur as well in some cases. • diagnosis is based on history, clinical signs, and the exclusion of other diseases; it is confirmed by intestinal biopsy. histopathologically granulomatous inflammation and acid-fast bacteria are identified. infection may also be confirmed by culturing the organism, and with polymerase chain reaction (pcr) testing. • because of the zoonotic potential of this disease, treatment is not recommended. affected animals should be euthanized. dermatophytosis is rare in the ferret and is typically caused by microsporum canis and trichophyton mentagrophytes. dermatophytes are transmitted by direct contact with infected animals or contaminated bedding, caging, and fomites. m key point ferrets are usually not carriers of these organisms. clinical disease is typically selflimiting. the most common source of infection of the pet ferret is the household cat. • young, debilitated, or geriatric ferrets are the most commonly affected. • lesions are consistent with those described in other species (see chapter ). alopecia with erythema, inflammation, hyperkeratosis, superficial crusting, lichenification, and erythema are present. pruritis is common and may lead to self-trauma and secondary pyoderma. • diagnosis is based on the identification of the fungal agent on skin scrapings, fungal culture, or a positive wood's light examination (m. canis). (see chapter ). • dermatophytosis is often self-limiting and may resolve without therapy. however, due to the zoonotic potential, treatment is recommended. • topical treatment includes the use of keratolytic shampoos, and/or lime sulfur dips. (see chapter ). • oral therapy consists of the administration of grisofulvin ( mg/kg) po sid for to days. perform a cbc every days while the ferret is receiving treatment. • disinfect the home by steam cleaning, the application of dilute ( : ) bleach or chlorhexidine solutions, and vacuuming thoroughly to remove infectious spores. dispose of the vacuum cleaner bag after vacuuming is complete. a thorough cleaning of the heat ducts and air conditioner/heater filters is also recommended. systemic mycoses are rare in the ferret; however blastomycosis, histoplasmosis, cryptococcosis, coccidioidomycosis, and aspergillosis have been reported. • consider these infections in the differential diagnosis of any systemic disease that is refractory to treatment and involves wasting, granulomatous lesions, persistent or recurring draining wound tracts, and respiratory tract disease. • diagnosis is based on the histopathological or cytological demonstration of the fungal organism in biopsies or aspirates (see chapter ). • complement fixation and precipitation tests have been used with variable success. • treatment is the same as described for the dog and cat (see chapter ). primary disease of the spleen is uncommon. splenomegaly is often a common incidental finding in a healthy adult ferret, or it may occur in association with a wide variety of disease conditions. perform a complete medical evaluation in all splenomegaly cases. • splenomegaly may be a normal or incidental finding in some patients. • pathological causes of splenomegaly may include chronic immune stimulation, erythroid bone marrow insufficiency, extra-medullary hematopoiesis (emh), hypersplenism, heart disease, and neoplasia. • splenomegaly can occur concurrently with adrenal gland disease and insulinoma, but it is usually an incidental finding. • extramedulllary hematopoiesis (emh) may cause enlargement of the spleen. the etiology of emh is unclear; compensation for myeloid insufficiency and chronic immune stimulation have been suggested as causes. ferrets with emh typically do not show evidence of hematological abnormalities. grossly the spleen has a normal shape and color, but it appears enlarged. • hypersplenism may cause enlargement of the spleen, but is rare in the ferret. • destruction of one or more blood cell lines by the splenic reticuloendothelial system occurs; affected ferrets will have anemia, leukopenia, thrombocytopenia, or pancytopenia. • bone marrow may be normal or hyperplastic in affected patients. • lymphoma is the most common neoplasia of the ferret spleen. hemangioma or hemangiosarcoma may occur as well. • when splenic lymphoma is present the spleen typically has irregular borders and a nodular texture. white or tan nodules may be noted grossly on the surface of the spleen and in the parenchyma. metastasis may be present. • splenic torsion and abscessation are rare in the ferret. • the normal ferret spleen measures approximately cm ¥ cm ¥ cm, and may be palpated in the left cranial abdominal quadrant. the texture of the spleen should be slightly firm, smooth, and the edges should be sharp. • an enlarged spleen is often noted on abdominal palpation as a firm, elongated smooth mass extending down the left side of the ferret abdomen, or crossing diagonally across the ventral abdomen from the left cranial abdominal quadrant to the caudal right abdominal quadrant. • abdominal distention may occur. • occasionally the spleen is so large and pendulous that the ferret can barely lift its abdomen off the ground. • abdominal discomfort due to splenomegaly appears to be uncommon in ferrets. • perform a cbc, platelet count, serum biochemical analysis, bone marrow cytology, and whole body radiography. • diagnosis of hypersplenism is based on the presence of one or more cytopenias, normal to hypercellular bone marrow cytology/biopsy, and the absence of blood loss, infection, or neoplasia. • obtain whole body radiographs to delineate the borders of the spleen and to rule out other abnormalities such as cardiomegaly or hepatomegaly that may contribute to splenomegaly. • splenic aspiration or biopsy may be performed. perform fine-needle aspiration of the spleen using a -gauge needle (see "clinical techniques"). do not perform splenic aspiration if hemangiosarcoma is suspected. • perform an abdominal ultrasound to evaluate the spleen. when the splenic parenchyma appears irregular, an ultrasound-guided biopsy or fine-needle aspiration may be performed. • perform a splenic biopsy during abdominal exploratory surgery, particularly if the spleen is irregular or discolored. treatment depends on the primary disease condition. usually splenectomy is not necessary. m key point indications for splenectomy are the same as for other species and include hypersplenism, splenitis, splenic abscess, torsion, rupture, neoplasia, and discomfort caused by excessive splenomegaly. • to perform a splenectomy, follow the surgical guidelines for splenectomy in dogs and cats (see chapter ). • anemia may result after splenectomy; the decision to perform splenectomy should be made cautiously, and with consideration to the health of the ferret as a whole. • administer antibiotics and fluid therapy pre-and postoperatively. • monitor asymptomatic ferrets with splenomegaly with periodic physical examination, cbc evaluation, imaging, and splenic aspiration. the clinical approach to anemia in ferrets is the same as for other species. anemias are classified as regenera-tive or nonregenerative; treatment is directed at the specific cause. there are many causes of anemia in ferrets; decreased erythropoiesis, destruction of red blood cells, and blood loss contribute to anemia. • nonregenerative anemia (normocytic, normochromic, nonregenerative anemia) occurs when bone marrow hematopoiesis is disrupted. bone marrow cytology of affected ferrets may appear normal. • decreased erythropoiesis may be caused by chronic metabolic disease (renal, hepatic), chronic inflammation, hyperestrogenism, bone marrow suppression, and neoplasia. • anemia of chronic disease can occur whenever long-term illness is present and is caused by decreased erythrocyte survival, decreased availability of iron, or a decreased response to the anemia treatment. • anemia associated with chronic inflammation is mediated by sustained inflammatory cytokine release. • hyperestrogenism may cause nonregenerative anemia due to estrogen-induced bone marrow suppression. unspayed female ferrets, female ferrets with ovarian remnants, or hyperestrogenism associated with chronic adrenal disease may contribute to this syndrome. • myeloid and leukemic neoplasias can cause suppression of bone marrow erythropoiesis due to replacement of normal bone marrow by neoplastic or fibrotic changes. • erythrocyte destruction may cause anemia; causes include immune-mediated disease, toxins, parasitism, or septicemia. • idiopathic immune-mediated hemolytic anemia, and immune-mediated hemolysis secondary to viral disease or blood parasites have not been reported in the ferret. • drug-induced hemolysis and heavy metal toxicosis (including zinc) are potential causes of hemolytic anemia. • anemia secondary to blood loss may be secondary to trauma, hemostatic disorders, bleeding lesions, and parasitism. • bleeding lesions may be internal or external. • bleeding ulcers may lead to anemia, and may be associated with h. mustelae gastritis, gastrointestinal foreign body, or chronic use of ulcerogenic drugs. • parasitism is uncommon in the ferret. coccidiosis in the young ferret or severe flea infestation may cause anemia. • hemostatic disorders include thrombocytopenia associated with estrogen toxicity, rodenticide poisoning, and liver disease. • clinical signs include weakness, pallor, lethargy, and inappetence. jaundice may be seen if hemolysis is present. • a soft systolic murmur is common in anemic ferrets. • a swollen vulva is present in ferrets with persistent estrus, an ovarian remnant, and in some cases of adrenal gland disease. hair loss may also be present on the shoulders and flanks. • ferrets with estrogen toxicity may have signs of thrombocytopenia such as petechiae, ecchymoses, and melena. • melena may be noted if gi bleeding is present. • palpate the spleen. splenomegaly may be caused by hypersplenism and subsequent anemia. • check carefully for fleas. perform a fecal examination. • diagnosis is based on the medical history, the physical examination findings, and a complete diagnostic work-up that includes a cbc, reticulocyte count, serum biochemical analysis, whole-body radiographs, and bone marrow cytology if indicated. • obtain a careful history regarding possible blood loss, toxicity, and foreign body ingestion. determine the duration of vulvar swelling (if present). • characterize the anemia based on rbc parameters and hemoglobin concentration. • the normal hematocrit for the ferret is % to %, higher than that of other animals. the erythrocyte count is higher as well; erythrocyte counts as high as . ¥ cells/µl have been reported. • the normal reticulocyte count may be as high as %. reticulocyte counts greater than % are indicative of a regenerative bone marrow response. • regenerative anemia is often the result of blood loss or hemolysis. • perform bone marrow aspiration (see clinical techniques), particularly if the anemia is nonregenerative, to identify infiltrative processes and assess the morphology of rbc precursors. bone marrow cytology from animals affected with anemia of chronic disease may be normal. bone marrow cytology is also indicated in ferrets with nonregenerative anemia that is unresponsive to treatment after to days. • obtain blood for blood lead concentration if lead poisoning is suspected. • whole-body radiographs are indicated to rule out abdominal neoplasia, gi foreign body, and thoracic neoplasia. a gi contrast study may be helpful to rule out the presence of gi ulcers. ultrasound may be helpful based on the rule outs established. m key point anemia in an intact female ferret with a swollen vulva for more than weeks most likely is due to estrogen toxicity. the objectives of treatment are to treat both the anemia and the underlying cause. • general supportive care includes oxygen therapy, subcutaneous fluids, and nutritional supplementation. • specific supportive care includes whole blood transfusion, iron dextran therapy, and the administration of erythropoietin. • oral iron supplements may be administered to replenish whole-body iron stores. • erythropoietin may be used to treat ferrets with nonregenerative anemia. administer u/kg three times per week until the pcv is stable, then administer to times a week. continue to monitor the pcv, and titrate the dose as needed. • indications for blood transfusion include the clinical status of the patient, a low packed-cell volume (pcv) of < %, the specific cause of the anemia, and the potential for continued blood loss. • ferrets lack specific blood types; transfusion reactions are rare in the ferret; up to three transfusions from the same donor and transfusions from multiple donors are considered safe. • the normal blood volume of an adult ferret may be calculated as % of the body weight. • the ideal value of the pcv post-transfusion would be within the normal reference range; a more likely goal is % to % higher than the pre-transfusion pcv. for dosage guidelines, see chapter . • before transfusion, administer a rapid-acting corticosteroid, such as dexamethasone sodium phosphate ( - mg/kg once iv) or prednisolone sodium succinate ( mg/kg once iv), as a slow bolus infusion, and administer an antihistamine such as diphenhydramine ( . - mg/kg iv, im, sc) to the recipient ferret. • the normal blood volume of ferrets has not been reported, but is estimated to be % to % of the total body weight (approximately ml/kg). twenty percent of the estimated blood volume (approximately ml) may be collected from healthy ferrets. • the jugular vein is the preferred site for the collection of large volumes of blood for transfusion. • sedate the donor ferret and place it in dorsal recumbency. • use a butterfly catheter to collect the blood into a syringe containing an anticoagulant such as sodium citrate ( . ml citrate per . ml blood) or acid-citratedextran (acd) ( ml per ml blood collected). • transfer the blood immediately to the recipient. blood should be filtered as it is transfused to the recipient. • administer fresh blood transfusions through an indwelling catheter or via a butterfly catheter into the cephalic or jugular vein. if a vein is inaccessible, administer into the peritoneal cavity or via the intraosseous route into the proximal femur. • whole blood is commercially available from marshall farms (marshall pet products inc., wolcott, ny). • hemoglobin substitutes such as oxyglobin (bioprure corporation, cambridge, ma) ( - ml/kg) iv or io over hours may be used if whole blood is unavailable. • administer oxyglobin slowly in normovolemic patients and in patients with renal disease, heart disease, or when the risk of pulmonary edema is present. • stop bleeding (internal or external). • correct the underlying cause of gi bleeding, including medical therapy for gi ulceration (see "gastrointestinal system"), surgery to remove gi foreign body, and antibiotics and supportive care for enteritis/colitis. • for anemia of chronic disease, treat the underlying primary disease process. • address metabolic disease (renal, hepatic) if present. • to correct estrogen toxicity in the intact female, terminate estrus (see below) and provide supportive care until the bone marrow is functional. broad-spectrum antibiotic therapy is important for the control of sepsis in leukopenic patients. estrogen toxicity associated with ovarian remnants is treated by surgical removal of the remnants when the ferret is stable enough for surgery. estrogen toxicity associated with adrenal gland disease is treated by adrenalectomy. preoperative care is the same as that described for the intact female (see below). (for diagnosis and treatment of adrenal tumors see "adrenal gland disease.") • treat fleas with any product that is safe for use in cats (see chapter ). • treat lead poisoning following the same protocols recommended for cats (see chapter ). • anemia secondary to neoplasia is associated with a poor prognosis. some cases of lymphoma may respond to treatment (see "neoplasia"). • administer human chorionic gonadotropin (hcg) in a single injection of iu (or usp) im. repeat this dose in to weeks if vulvar swelling has not diminished. • alternatively, give gonadotropin-releasing hormone (gnrh) at a dose of mg im or sc; repeat in weeks if necessary. • gnrh and hcg are effective only after the th day of estrus. bone marrow toxicity is not immediately reversible with termination of estrus; the pcv continues to fall for days to weeks. • monitor the pcv as a useful guide to therapy and prognosis: • pcv > %-the prognosis is good and termination of estrus is the only therapy required. • pcv % to %-the prognosis is guarded because the pcv level can decrease further after termination of estrus. • pcv < %-the prognosis is poor and aggressive supportive care is indicated, including multiple blood transfusions until bone marrow function is restored. • some causes of anemia in ferrets can be prevented. educate owners about proper husbandry techniques to avoid flea infestation, foreign body ingestion, and trauma. m key point to prevent estrogen toxicity, spay all female ferrets not used for breeding. insulinoma (pancreatic beta-cell tumor) is one of the most common neoplasias of the ferret. disease is most common in ferrets over years of age, and results in progressive, cyclic, or persistent hypoglycemia. • the incidence of insulinoma is typically higher in ferrets in the united states than in ferrets in other countries. the cause(s) are unknown. • possible etiologies include a limited genetic pool and diet. ferrets in the united states are typically fed processed foods containing large amounts of cereal grains. ferrets in other countries are typically fed a more natural diet consisting of meats and whole prey items. • early signs may be subtle and transient. cyclic or progressive episodes of profuse hypersalivation and pawing at the mouth (which is indicative of nausea), lethargy, depression, "stargazing," and posterior paresis may be seen during periods of hypoglycemia. • as the disease progresses, or during periods of inadequate food intake, symptoms become more pronounced and may progress to stupor or coma. seizures may occur. • splenomegaly is a common, unassociated finding on physical examination (see "splenomegaly" under "hematopoietic system" in this chapter). • adrenal disease is often identified concurrently in many ferrets with insulinoma (see "adrenal neoplasia" in this chapter). m key point base a presumptive diagnosis of insulinoma on the history, clinical signs, and repeated evidence of hypoglycemia in the presence of normal or elevated blood insulin levels. make a definitive diagnosis via surgical removal and histopathology of a pancreatic tumor or biopsy. • differential diagnoses include hepatic disease, sepsis, starvation, and laboratory error. • a carefully monitored fast of to hours is sufficient. • if necessary, obtain several samples over a period of several days. • normal fasting serum glucose concentration is to mg/dl. ferrets with insulinoma often have a fasting serum glucose of to mg/dl. ferrets with fasting serum glucose between to mg/dl are considered suspect and should be monitored. • do not fast the animal for more than hours. discontinue fast if signs of hypoglycemia occur. prolonged fasting may lead to collapse, coma, or seizures. feed the ferret a high-protein and high-fat meal as soon as possible after collection of blood. • measurement of blood insulin concentration is not consistently reliable in the ferret. • false positive results may occur if liver disease, nonislet cell tumors, or sepsis are present. • blood insulin concentrations greater than pmol/ liter (or mu/ml) are considered elevated in ferrets. however, if the ferret is severely hypoglycemic at the time of sample collection, the blood insulin value may be normal because insulin and glucose are in a constant dynamic state. • the serum biochemical profile is typically normal except for the presence of hypoglycemia. • the cbc is typically normal. • a slight elevation in alanine aminotransferase (alt) and aspartate aminotransferase (ast) may be noted. the cause is unknown and may incidental, or may indicate hepatic lipidosis due to chronic hypoglycemia or some hepatic pathology. m key point insulinoma is a progressive disease in ferrets. educate owners how to recognize the signs of hypoglycemia and how to manage hypoglycemia at home (see "medical therapy" and "hypoglycemia episodes"). • treatment options include medial therapy and/or surgical therapy. • medical therapy will need to be adjusted as the disease progresses. • surgery is used as a management tool and is not curative. insulinoma is a progressive disease, even after surgical intervention. medial therapy is often effective in controlling symptoms associated with insulinoma for to months. frequent feeding is the first step in treatment. add prednisone and diazoxide as clinical signs and hypoglycemia worsens. • feed high-quality protein and high-fat meals frequently, especially after exercise or a long sleep. avoid foods containing sugar or excessive carbohydrates (except to treat hypoglycemic episodes); these foods cause short term hyperglycemia followed by a period of hypoglycemia to hours later. • to tablespoons of hill's science diet a/d or meatbased baby foods may be given twice daily and as needed. • chromium has been anecdotally reported to stabilize blood glucose and insulin levels in humans. brewer's yeast, which is a rich source of chromium, has been beneficial in some ferrets with insulinoma. surgical therapy is the treatment of choice even though surgical removal or debulking of pancreatic tumors or partial pancreatectomy is palliative and provides only temporary remission of signs ( to months). • follow canine preoperative protocols (see chapter ). fast the ferret for only to hours preoperatively to avoid hypoglycemia. • administer iv or io isotonic fluids containing dextrose ( . - . %) to hours preoperatively if possible, during surgery, and continue postoperatively until the ferret is stable and is eating and drinking. • evaluate the blood glucose concentration pre-, intra-, and postoperatively if possible. • see chapter for information about the surgical removal of insulinoma. m key point perform a complete abdominal exploratory; insulinomas can metastasize to the regional lymph nodes, liver, and spleen (uncommon). concurrent adrenal tumors (see adrenal gland disease) are common. • if the spleen is enlarged and appears irregular or mottled, consider performing a complete or partial splenectomy and submit for histopathology. • postoperatively monitor the blood glucose concentration bid-tid until the ferret is discharged from the hospital. many ferrets become euglycemic immediately after surgery. some ferrets may remain hypoglycemic. rarely, ferrets may become transiently hyperglycemic after surgery. • most ferrets will require resumption of medical therapy to months after surgery. some ferrets will need medical therapy immediately postoperatively. • monitor blood glucose levels for to days after surgery and at to day intervals. • iatrogenic pancreatitis is rarely a problem in ferrets; however, as a precaution, withhold food and water for hours postoperatively; give . % to . % dextrose iv during this period. monitor blood glucose to times daily. • transient diabetes mellitus may occur postoperatively. hyperglycemia and glucosuria may be present for to days postoperatively; generally no treatment is required. • histopathologic examination of the pancreatic mass may reveal hyperplasia, adenoma, and/or adenocarcinoma of the pancreatic beta cells, even within a single tissue specimen. • the prognosis is guarded, but with surgery and medical treatment, ferrets have had a good quality of life for more than year after diagnosis of insulinoma. the median survival time was months (range, . - months) in one study of ferrets treated with surgery, medical management, or both. adrenal tumors are common in ferrets, and occur with approximately the same frequency as insulinomas. adrenal neoplasia and insulinomas often appear concurrently. adrenal tumors have been identified in ferrets as young as year of age, although they typically occur in ferrets > years of age. • adrenal gland disease in ferrets is not cushing's disease. excessive sex steroids, not corticosteroids, are produced by a hyperplastic or neoplastic adrenal gland. • the etiology is unknown. possible causes of adrenal disease in ferrets include early neutering, genetic factors, and lack of exposure to normal seasonal photoperiods. the incidence of adrenal neoplasia is higher in ferrets in the united states. in the u.s., ferrets are typically neutered at weeks of age, and are housed indoors under artificial light cycles. ferrets in europe and australia are typically housed outside, and are not neutered until months of age. • adrenal neoplasia in ferrets causes a variety of clinical signs, and appears to be the result of excessive secretion of estrogens and androgens, not cortisol. pituitary-dependent hyperadrenocorticism has not been documented in ferrets. • adrenal tumors most commonly arise from the adrenocortical tissue. common histopathological findings include hyperplasia, adenoma, and adenocarcinoma. • signs include progressive, bilaterally symmetric alopecia, usually starting at the tail base and progressing cranially. hair loss often starts in the early spring or fall. there may be a history of alopecia and spontaneous hair regrowth as well. • pruritis often is reported, along with excessive dryness of the skin and small excoriations. thinning of the skin is common. • an enlarged vulva, mimicking estrus, may be the only clinical sign in spayed females. mucoid or mucopurulent vulvar discharge may be noted. castrated males may exhibit territorial marking and sexual behaviors, and may develop the strong body odor and oily hair coats of intact males. mammary hyperplasia can occur in either sex. • male ferrets may present with partial to complete urinary obstruction. persistent elevation of adrenalderived androgenic hormones may cause development of prostatic hypertrophy, prostatic cysts, or periurethral cysts, which cause narrowing of the urethra. affected ferrets may present with stranguria, dysuria, azotemia, and severe metabolic derangement. male ferrets that are described as straining to urinate should be treated as an emergency (see "urinary system"). • atrophy of abdominal musculature and mobilization of fat to the ventral abdomen, leading to a pendulous appearance, may be seen. • atrophy of hind limb musculature and rear limb paresis can occur. • polyuria/polydipsia is uncommon but has been reported. • collapse, anemia, and petechiation resembling estrogen toxicity have been described in male and female ferrets with chronic or advanced adrenal disease (see hematopoietic system). • enlarged adrenal glands may occasionally be noted on the physical examination. the left adrenal gland is easier to palpate than the right. • radiographs are not typically helpful in confirming this disease. ultrasonography may be useful for identification of adrenalmegaly. • cbc is typically unremarkable unless estrogen toxicity-like anemia is present. the serum chemistry profile is typically within normal limits unless insulinoma is present. m key point a history of symmetric truncal hair loss suggests the diagnosis. differential diagnosis includes seasonal alopecia, which typically appears in the spring or fall, affects only the tail, and resolves after several weeks. • female ferrets often present with a swollen vulva. differential diagnoses include an intact female ferret, a female ferret with an ovarian remnant, and seasonal alopecia. perform a serum steroid panel or administer human chorionic gonadotropin ( iu) im to determine if a female ferret is unspayed or has an ovarian remnant. • a plasma steroid hormone assay may be used to support the diagnosis. elevated plasma concentration of estradiol, androstenedione, and/or -hydroxyprogesterone is a reliable indicator of adrenal gland disease (see table - ). a hormone panel is commercially available through the clinical endocrinology laboratory of the department of comparative medicine at the university of tennessee. • the adrenocorticotropic hormone (acth) stimulation test and the low-dose dexa-methasone suppression test are not useful in ferrets. ferrets with adrenal gland disease do not produce abnormal concentrations of cortisol, and adrenal gland disease in the ferret appears to be independent of acth. urine cortisol/creatinine ratio does not appear to be a specific indicator of adrenal gland disease. • perform exploratory surgery to confirm the diagnosis. • adrenal tumors can be managed medically or surgically. surgical management is preferred and recommended. • medical treatment may cause clinical signs to regress, but will not stop growth of the adrenal tumor. the goal of medical treatment is to decrease or eliminate the clinical signs of adrenal gland disease. medical therapy will not stop or prevent the growth of an existing tumor, and should be reserved for ferrets that are poor surgical candidates, ferrets with inoperable bilateral adrenal tumors, or ferrets with recurrent adrenal gland disease. • medical treatments described in the literature include mitotane, ketoconazole, androgen receptor blockers, aromatase inhibitors, and gonadotropinreleasing hormone analogs. • gonadotropin-releasing hormone analogs. there are two general types of gnrh analogs: gnrh agonists and gnrh antagonists. to date only gnrh agonists such as leuprolide acetate (lupron depot, tap pharmaceuticals inc., lake forest, il) have been used to control the signs of adrenal disease in the ferret. of the medical treatments described, anecdotal reports suggest that leuprolide acetate has been most effective in alleviating dermal and urogenital signs of adrenal disease. administer the month depot formulation of leuprolide acetate at a dose of ( µg/kg) im every days. • androgen receptor blockers theoretically block the actions of androgens at the receptor site, and decrease or reverse the signs of adrenal gland disease. in human medicine these drugs are used to treat men with prostatic carcinoma or prostatic hyperplasia. flutamide (eulexin, schering corporation, kenilworth, nj) and bicalutamide (casodex, astrazeneca pharmaceuticals lp, wilmington, de) have been used, primarily in male ferrets. results are variable. • aromatase inhibitors such as anastraozole (arimidex, astrazeneca pharmaceuticals lp) inhibit aromatase, an enzyme involved in estrogen production. some ferrets show decreased evidence of adrenal gland disease symptoms when treated with this drug. • mitotane ( ,p'-ddd) (lysodren, bristol-myers squibb oncology, princeton, nj) is rarely effective in ferrets with adrenal gland disease, presumably because ferrets do not develop pituitary-dependent hyperadrenocorticism. if clinical signs do resolve, they will often recur as soon as the mitotane therapy is withdrawn. m key point perform a fasting blood glucose test before starting mitotane therapy. do not use mitotane if blood glucose is low (indicative of concomitant insulinoma). mitotane causes a decrease in endogenous cortisol production; if insulinoma is present, serum glucose levels also may fall, causing a hypoglycemic crisis. • give mitotane ( mg/kg) po q h for days, then q h until clinical signs start to resolve. at that time decrease to q h until signs are fully resolved, then maintain the ferret on mg/kg once q - d as necessary. • mitotane must be compounded in -mg aliquots in # capsules. capsules must be administered intact. have owners coat the capsules with vegetable oil, push into the back of the throat, and follow with a palatable liquid or blenderized cat food to promote swallowing. • the most common side effect of mitotane is hypoglycemia. teach owners to recognize the signs of hypoglycemia, and have prednisone available at home. if side effects occur, discontinue mitotane and administer prednisone ( . - . mg) po. • if continuation of mitotane therapy is desired after a hypoglycemic crisis, administer concomitantly with prednisone (see insulinoma). • ketoconazole is not effective in the treatment of adrenal disease in the ferret. follow the adrenalectomy preoperative protocol described for dogs (see chapter ). • fast the ferret to hours preoperatively. place an indwelling iv or io catheter preoperatively, and administer fluids pre-, intra-, and postoperatively. if insulinoma is present concurrently treat and monitor appropriately. • perform a ventral midline laparotomy. palpate and visualize both adrenal glands carefully. normal adrenal glands are to mm ¥ - mm in size, are pale pink in color, and are typically surrounded by fat. • the left adrenal gland lies in a fat pad cranial to the left kidney. the right adrenal gland is located cranio-medial to the right kidney under the caudate liver lobe adjacent to the vena cava. it may be necessary to transect the hepatorenal ligament to fully visualize and palpate this gland. • adrenal changes may be subtle, especially in younger ferrets and because the adrenal glands are surrounded by fat. visual changes, such as dark circular lesions and small raised cysts, may be present instead of gross enlargement. • one or both adrenal glands may be affected. if only the left adrenal gland is affected, removal is relatively straightforward. if the right adrenal gland is affected, removal can be difficult because of the gland's proximity to the vena cava and liver (see chapter ). • if both adrenal glands are affected, remove the left adrenal gland and debulk the right adrenal gland. bilateral adrenalectomy has been described in the ferret, but should be done with caution. monitor postoperatively for development of acute adrenal hypocorticism. if acute ahc develops, treat as described for dogs (see chapter ). m key point always perform a complete abdominal exploratory. observe and palpate the pancreas at surgery for insulinomas, which often are found concurrently with adrenal neoplasia. • monitor fasting serum glucose levels every to days during mitotane therapy and after adrenalectomy, even if no pancreatic nodules were evident during surgery. • the prognosis following successful surgery is good. a full resolution of clinical signs can be expected in many cases. • recurrent or continued symptoms of adrenal gland disease may be associated with development of a tumor on the remaining adrenal gland, or recurrence of an adrenal tumor due to metastasis (which is rare). • even without treatment, ferrets may survive up to years or longer after diagnosis, although the hair loss is generally progressive. • potential sequelae to chronic adrenal gland disease include prostatic disease, bone marrow suppression, or mechanical interference with the vena cava (right adrenal gland). pheochromocytomas are adrenal tumors that arise from the adrenal medulla and produce excessive amounts of catecholamines. pheochromocytomas have been reported in ferrets, but are rare. treatment of choice is surgical removal of the affected gland. lymphosarcoma (lymphoma) is common in ferrets of all ages, and is similar in presentation to the disease in cats and dogs (see chapter ). three presentations may occur in the ferret and include lymphosarcoma, lymphocytic, and lymphoblastic forms. • a viral etiology has been hypothesized. clinical signs are variable, depending on the form of lymphoma present and the organ system involved. • lymphosarcoma: solid tissue tumors are present in the organs or lymph nodes. • lymphocytic lymphoma: adult ferrets are most commonly affected. the course and survival time can be long. peripheral lymphadenopathy is typically present and metastasis to visceral organs may occur. the neoplastic cell identified on cytology or histopathology is a mature, well-differentiated lymphocyte. • lymphoblastic lymphoma: young ferrets are most commonly affected. leukemia and neoplasia in visceral organs occur early in the course of this form of the disease. large immature lymphocytes are noted on cytology or histopathology. • other forms: cutaneous lymphoma may occur in the ferret. • clinical signs that may accompany any form of lymphoma include: • inappetence, lethargy, splenomegaly, and weight loss despite a normal appetite • dyspnea, tachypnea, and exercise intolerance • peripheral lymphadenopathy and/or abnormal cbc • acute collapse, often with pyrexia • fever of unknown origin • cutaneous masses • chronic diarrhea and/or rectal prolapse • some ferrets are asymptomatic; lymphoma may be an incidental finding during evaluation for another medical problem. • lymphoma tends to be a more acute, fulminant disease in younger animals. the method of diagnosis depends on the organ system involved. • obtain a thorough history and physical examination. • perform a cbc, platelet count, and a serum biochemistry profile. if the ferret is anemic, perform a reticulocyte count. • often the cbc and differential wbc counts are not diagnostic for lymphoma. the cbc may be normal or may reveal an absolute or relative lymphocytosis. anemia, leukopenia, and thrombocytopenia may be seen. abnormal lymphocytes may occasionally appear in the differential count. • persistent absolute lymphocyte counts greater than or a relative lymphocytosis (> %) are considered suspicious; repeat the cbc in to weeks and perform a bone marrow biopsy and/or lymph node biopsy if the cbc results are repeatable or if lymphadenopathy is present. • the serum chemistry profile may disclose elevated liver enzymes if the liver is involved; paraneoplastic syndromes are uncommon in the ferret. • perform thoracic and abdominal radiography and ultrasonography to evaluate for intra-thoracic and intra-abdominal masses. • perform fine-needle aspiration or biopsy of affected tissues for histological and cytological examination. fine-needle aspiration of the spleen is usually inconclusive. • lymph node biopsy is often the most helpful diagnostic tool for diagnosis of lymphoma. if possible, biopsy an enlarged lymph node. when lymphadenopathy is not present, biopsy the popliteal lymphnode. the popliteal lymph node is the most accessible peripheral node for biopsy. avoid biopsy of intra-abdominal lymph nodes if possible. • perform bone marrow aspiration to identify infiltration by neoplastic cells and the disease (see "clinical techniques"). • if the spleen is involved, perform a splenectomy (see chapter ) to reduce the overall tumor load. chemotherapy for lymphoma may be successful (approximately % remission rate). in general, protocols have been adapted from feline medicine (see chapters and ). • success of chemotherapy may be affected by the age of the ferret, concurrent disease (e.g., adrenal gland disease, insulinoma), concurrent medication, inappropriate use of and resistance to chemotherapeutic agents (ferrets treated with prednisone prior to chemotherapy), and the type of lymphoma present. • ferrets with bone marrow involvement or with solid tumors involving organs typically have a poor prognosis. • longer periods of remission tend to occur in individuals with adult onset or lymphocytic lymphoma. • iv chemotherapeutic agents are given via butterfly catheter or small-gauge needle with the ferret under sedation; face-mask administration of isoflurane is the most convenient and rapid method of sedation. • one chemotherapy protocol is outlined in table - . m key point monitor the cbc weekly. if the wbc falls below wbc/ml, or the rbc falls below ¥ /ml discontinue vincristine for week or more until the wbc count increases to at least wbc/ml. • palliative therapy may be attempted by administering oral prednisone ( . mg/kg) po q h. • supportive care is important (see "nutritional support for insulinoma"). • consider referral to an oncologist if experience with chemotherapeutic agents is limited. • chordoma: chordomas are tumors that arise from notochord remnants. tumors occur most often at the tip of the tail, but may occur in the cervical region as well. • git: gastric adenocarcinoma. • other tumors reported in ferrets include chondroma, chondrosarcoma, fibroma, fibrosarcoma, hepatic adenocarcinoma, hemangioma, hemangiosarcoma, mast cell tumor, mesothelioma, osteoma, osteosarcoma, schwannoma, squamous cell carcinoma, thymoma, and renal and pancreatic carcinomas. ferrets may experience dramatic seasonal changes in the haircoat triggered by photoperiod changes. this change is most apparent in the intact animal. if one is unfamiliar with these changes, normal coat changes may be interpreted incorrectly as a medical problem. m key point individual animals may exhibit different patterns of coat change each successive year. • a normal, diffuse, gradual thinning of the coat typically occurs in the spring when the photoperiod is increasing and continues through the summer. the coat typically becomes shorter and darker at this time, and the face mask may appear or disappear. focal alopecia should not be present. • some ferrets may experience a dramatic -day loss of the undercoat. • a normal, but dramatic, loss of body weight (up to %) may occur at this time as well. • hair growth will reverse in the fall and winter. coats typically become longer, thicker, and lighter. body weight may change (up to %) as well. • females in estrus and males "in season" may show an even more marked hair loss but should not have areas of alopecia. • males typically lose hair in the inguinal area because of constant rubbing to mark territory; the mid-and caudal abdomen is often wet with urine. • neutered ferrets or ferrets kept under artificial lighting conditions often experience no coat changes. • neutering or spaying may cause temporary, diffuse alopecia hair thinning postoperatively, particularly if the animal was reproductively active at the time of surgery. the preoperative color pattern may not return. • at any time of the year, regrowth of hair that has been shaved for medical procedures is slow. this is particularly true in the winter and summer when no active hair growth is occurring. • hair regrowth (regardless of the cause of alopecia) is often preceded by a blue to purple discoloration of the skin that can alarm the owner. this discoloration is caused by new hairs growing through the dermis, and is most noticeable on the abdomen and face. • intact jills may exhibit a bluish discoloration of the skin during estrus. if ovariohysterectomy is performed while a jill is in estrus, this discoloration may occur approximately days postoperatively. • pseudonails associated with hyperkeratosis of the footpads may occur in ferrets older than years of age that are housed on carpet or linoleum surfaces. trim pseudonails as necessary. rub a small amount of petroleum jelly or vitamin e oil into the pads daily to help prevent lesions. cutaneous bacterial infections in ferrets are typically manifested as abscesses or as a diffuse, ulcerative pyoderma. • abscesses may develop secondary to puncture wounds, bites, or may develop in the inguinal fat after traumatic injury (e.g., being stepped on). for diagnosis and treatment of abscesses, see "infectious diseases." ulcerative pyoderma is the second most commonly encountered form of bacterial dermatitis in the ferret. • various bacteria can cause ulcerative pyoderma. the most common agents are staphylococcus and streptococcus spp. • focal alopecia with diffusely hyperemic, thickened, ulcerated skin may occur over any area of the body. • perform a cutaneous punch biopsy (see chapter ) to rule out diffuse cutaneous mast cell tumor, which may have a similar gross appearance. • perform bacterial culture and sensitivity testing. • administer systemic antibiotics based on culture and sensitivity testing. antibiotics effective in the treatment of pyoderma in ferrets often include amoxicillin-clavulanate (clavamox, smithkline) ( - mg/kg) q h po and cephalosporins (use feline dosages). • topical treatments include twice-weekly cleansing with an antibacterial shampoo containing chlorhexidine or benzoyl peroxide. daily application of an antibacterial cream may be beneficial if the lesion is small and localized. dermatologic lesions are quite prominent with cdv infection in ferrets. • dermatologic signs typically begin with hyperemia around the lips, chin, eyes, and sometimes the inguinal area. with time, crusts and skin thickening may appear. • hyperkeratosis of the foot pads occurs as the disease progresses. • see infectious diseases in this chapter for a detailed discussion of cdv in ferrets; also see chapter for a discussion of cdv in dogs. • microsporum canis and trichophyton mentagrophytes are the most common causes of superficial mycotic infections in the ferret. • see "infectious diseases" in this chapter for diagnosis and treatment. • flea infestation may be encountered in pet ferrets. clinical signs are similar to those seen in cats (see chapter ). • flea shampoos, dips, or powders containing pyrethrin may be used. products containing lindane or organophosphates are not recommended for use in the ferret. • imidacloprid (advantage, bayer corporation, shawnee mission, ks) ( . ml) topically every weeks has been reported to be effective. no adverse effects have been noted. this drug may be used in conjunction with lufenuron. • lufenuron (program, norvartis animal health, greensboro, nc) ( mg) po every weeks has been anecdotally reported to be effective. advise clients that there is a -to -week period before flea numbers are observed to decline. • fipronil (frontline, merial ltd., iselin, nj). half the cat dose has been anecdotally reported to be effective. this drug may be used in conjunction with lufenuron. • selamectin (revolution, pfizer, new york, ny). administration of the cat dosage has been anecdotally reported to be effective. • flea collars are not recommended because they come off easily and small pieces can be ingested. • treat the environment for fleas. ear mite infection in ferrets is caused by otodectes cyanotis, the same parasite that infects cats and dogs. • ferrets rarely exhibit pruritis, even with heavy mite infestation. • • when chronic ear mite infestation is present, lichenification and a bluish pigment may appear on the inner surface of the pinnae. these changes are caused by a response to chronic irritation, and usually regress after treatment. • rarely, o. cyanotis may colonize other parts of the body. • examine all ferrets for ear mites; the incidence of infestation is high in some populations. • mites in the ear canal can often be visualized using an otoscope; however, otoscopic examination is often difficult because of the uncooperative nature of the patient and small size of the ear canal. • tresaderm (merck agvet, rahway, nj) may be used to treat ear mites in ferrets. administer drops in each ear q h for days, stop for days, then repeat. this medication has been reported to be effective in treatment of ear mites in the ferret. • selamectin may be used for treatment of ear mites in the ferret. use at the dose described for treatment of fleas. • bathe the ferret within to hours after treatment. wash all bedding and treat all other potential hosts in the household (see chapter ). • topical treatments may not be effective due to the narrow size of the ear canal, and patient resistance to treatment. • persistent infections may be due to the presence of ear mites on the body, or failure to deliver the topical agent effectively. in such cases, parenteral administration of ivermectin ( . mg/kg) sc every to days for treatments may be necessary. do not use topical and parenteral ivermectin together. • lesions are typically confined to the feet, which become hyperemic, swollen, and intensely pruritic. crusting often occurs around the nails, and in severe cases the nails may slough. • generalized alopecia, accompanied by intense pruritis, occurs rarely. • a positive diagnosis is based on clinical signs, exclusion of differential diagnoses, and positive skin scrapings obtained from several sites (false negative results do occur). • a common differential diagnosis is contact allergy. similar lesions have been observed in ferrets housed on plastic-floor cages. these lesions resolve when the cage bottom is changed to wire or wood. • advise clients of the zoonotic potential of this parasite. • treatment may need to be based on differential diagnoses; mites may be difficult to identify on skin scrapings. • administer ivermectin ( . mg/kg) sc every weeks for three treatments. • lime sulfur dips may be used instead of ivermectin. dip ferrets in % lime sulfur every days until signs have resolved for weeks. • wash all bedding and treat all potential contact hosts in the household. demodicosis is rare in the ferret. • otitis externa has been associated with demodicosis. this may be the only presenting sign. • localized alopecia accompanied by pruritis may occur. • mites may be identified on routine skin scrapings and examination of ear canal debris. • treatment can be difficult. use ivermectin at the daily dose described for dogs (see chapter ). • do not use mitotane. the etiology of tail alopecia in the ferret is unknown but is suspected to be caused by hormonal fluctuations because the disease responds to changes in the photoperiod. hair loss occurs most commonly at the time of the fall molt, when the photoperiod is becoming shorter, but may be seen any time of year under artificial lighting conditions. hair regrowth usually occurs in - weeks. the same pattern of alopecia is not always repeated annually. • hair loss, ranging from diffuse hair thinning to complete alopecia, occurs from the base of the tail to the tip. m key point alopecia occurs only on the tail. if alopecia extends to the body, suspect another form of endocrine disease, such as adrenal gland disease. • comedones and a brown, waxy scale may accompany the alopecia. • diagnosis is based on clinical signs. • differential diagnoses include the early stages of adrenal gland disease; however, hair loss on the body typically occurs as well when this condition is present. • no treatment is necessary. hair regrowth will occur when the photoperiod changes. • artificially lengthening the photoperiod may speed hair regrowth, although not reliably. • if the tail exhibits excessive amounts of waxy scaling or comedones, clean the tail weekly with a mild shampoo. alopecia may be seen in intact females that have been in estrus for month or longer. • bilaterally symmetrical hair loss over the shoulders and flanks, which eventually progresses to involve the entire body. hairs epilate easily, and the underlying skin appears normal. • a grossly enlarged vulva indicates a state of estrus. be aware that the ferret also may be anemic and thrombocytopenic (see "anemia"). • diagnosis is based on clinical signs in an intact female. • perform an ovariohysterectomy (see chapter ) if the ferret is stable enough for the procedure, or induce ovulation with hcg (see "termination of estrus"; "anemia"). • hair regrowth will recur rapidly after surgery or ovulation; however, changes in hair length, color, or thickness are common. • bilateral, symmetrical alopecia is a common sign of adrenal disease in the ferret (see "adrenal gland disease"). • hypothyroidism has not been documented in ferrets. neoplasia of the skin is the third most common neoplasia reported in the ferret and commonly occurs in ferrets year of age and older. complete removal of skin masses using wide surgical excision followed by histopathology is recommended. mast cell tumors are the most common skin masses encountered and are typically benign in the ferret. • individual tumors typically appear as slightly raised, flat, button-like cutaneous masses ranging in size from to mm. the tumors are often tan in color or may be hyperemic with a dark flaky crust. tumors may also appear as raised, ulcerated areas, or as diffuse areas of erythema and crusting. pruritis may be present at the site. • mast cell tumors have occasionally been associated with diffuse or generalized areas of alopecia that resolve with surgical removal of the tumor. • metastasis is rare but has been reported in the lung and gallbladder (see chapter for information about mast cell tumors in dogs and cats). • these tumors may also be referred to as haral cell tumors or sebaceous adenomas and are common in the ferret. • tumors may appear as wart-like, ulcerated, or cystic masses ranging in size from . to cm. • excision is usually curative. recurrence is rare, and metastasis is not reported. • other, less common neoplasms of the skin and subcutaneous tissues include: basal cell carcinoma, basi-squamo-sebaceous carcinoma, hemangioma, histiocytoma, leiomyosarcoma, lymphoma, myxosarcoma, neurofibrosarcoma, perianal gland adenocarcinoma, sebaceous gland adenocarcinoma, and squamous cell carcinoma. • adenocarcinomas often metastasize to regional lymph nodes, liver, and lungs. • diagnosis, treatment, and prognosis for these tumors in ferrets are the same as for dogs and cats (see chapter ). • cardiac auscultation is centered more caudally in the thorax than are auscultations in cats. • the heart extends from the sixth rib to the caudal border of the seventh or eighth rib (compared with cats, where it extends from the second to the sixth rib). • the heart rate averages to beats per minute. • a pronounced sinus arrhythmia and pronounced bradycardia are common during auscultation. • cardiac disease is relatively common in the ferret. quality of life and long-term prognosis for ferrets with cardiac disease depends on the type and severity of cardiac disease present, and the initial response to treatment. many ferrets do well for months on the appropriate medications. • ferrets appear to compensate well for early cardiac insufficiency, perhaps because a slight decrease in activity is not readily apparent to owners. • ferrets with congestive heart failure (chf) may present with clinical signs that resemble symptoms associated with other disease entities, such as anorexia, ascites, coughing, dehydration, dyspnea, exercise intolerance, generalized weakness, hindlimb weakness, hypothermia, lethargy, tachypnea, and weight loss. • pale or cyanotic mucus membranes and a prolonged capillary refill time (crt) may be noted on physical examination. • jugular pulses may be present when right-sided chf is present. • femoral pulses may be weak, irregular, or normal. • ascites, hepatomegaly, or splenomegaly may be noted on abdominal palpation. • murmurs may be noted on auscultation, and are typically associated with valvular insufficiency. • history and physical examination findings are important in the diagnosis of heart disease. • perform a complete physical examination, including auscultation of the heart, and evaluation of the capillary refill time. observe for tachypnea or dyspnea and auscult the lungs. palpate the abdomen and examine for ascites. m key point proceed with further testing only if the ferret is stable. otherwise, administer furosemide and oxygen therapy. • diagnosis requires information obtained by radiography, ecg, and echocardiography. • obtain whole-body radiographs. the cardiac silhouette typically appears enlarged and globoid in shape with rounded right and left ventricles. ascites, hepatomegaly, pleural effusion, and pulmonary edema may be present as well. • evaluate a cbc, serum biochemical profile, and urinalysis to determine if azotemia, electrolyte abnormalities, or other systemic diseases are present. perform a heartworm test if the history is supportive for potential exposure. • if thoracic or abdominal effusion is present, perform thoraco-or abdominocentesis and submit fluid for cytologic examination. perform centesis as described for cats; take into consideration the relatively caudal position of the heart in ferrets. sedation is usually necessary. a modified transudate is typically associated with chf. • perform standard six-lead electrocardiography (ecg) if possible (table - lists normal ferret ecg parameters). sedation may be necessary. electrocardiography may reveal atrial premature contractions, atrial tachycardia, atrial fibrillation, ventricular premature contractions, and ventricular tachycardia. m key point sedation with isoflurane is recommended when necessary. sedation with ketamine or a ketamine-diazepam combination raises the heart rate. the heart rate tends to decrease with ketamine-xylazine sedation; therefore, avoid using xylazine in ferrets with suspected cardiac disease. • echocardiography is the most useful diagnostic tool in the ferret. the same echocardiographic changes observed in the dog and cat are seen in the ferret. (table - ) . m key point treatment of acute chf should focus on improving oxygenation and reducing cardiac preload and afterload. • place the ferret in an oxygen-rich environment. administer supportive care such as subcutaneous fluids (e.g. . % saline and . % dextrose), and provide nutritional support for ferrets that are anorexic. • administer diuretics such as furosemide ( - mg/kg) im or iv bid-tid. • nitroglycerin % ointment may be applied to the skin in the axilla, inguinal area, or on a hairless body surface. • angiotensin-converting enzyme (ace) inhibitors may be given to reduce afterload and preload. give enalapril (enacard, merck agvet division) ( . mg/kg) po q h, then titrate up to q h if possible. ace inhibitors may cause hypotension in ferrets, titrate to effect. • when diuretics and ace inhibitors are used together it is important to monitor for azotemia. • perform thoracocentesis or abdominocentesis when indicated. • monitor body weight, crt, heart rate and rhythm, hydration status, mucous membrane color, respiratory rate, respiratory effort, bun, creatinine, and serum electrolytes. m key point chronic therapy typically includes the use of ace inhibitors, and diuretics with the addition of digoxin in ferrets with dilated cardiomyopathy. whenever possible, try to titrate the diuretic dose to the lowest possible dose without recurrence of pleural effusion or pulmonary edema. • administer digoxin elixir ( . mg/kg) po sid-bid to ferrets with dilated cardiomyopathy. • side effects associated with digoxin include anorexia, arrhythmias, diarrhea, lethargy, and vomiting. • serum digoxin levels should be monitored every to weeks. normal values have not been published for the ferret; reference values for dogs and cats are used for interpretation. • use of antiarrhythmic drugs such as atenolol or diltiazem is not well documented in the ferrets, but may be useful in the treatment of ferrets with hypertrophic cardiomyopathy. • salt-free diets may be beneficial; however, they are often unpalatable to ferrets. instruct the owner to avoid feeding snacks, treats, or food items with a highsalt content. • management includes periodic reevaluation of heart rate and rhythm, serum electrolytes, and renal values. radiographs should be used to monitor for the development of pulmonary edema or changes in the cardiac silhouette. ecg and echocardiography should be repeated periodically as well. cardiomyopathy may occur in ferrets years of age or older. dilated (congestive) and hypertrophic forms can occur; the dilated form is more common. the cause of dilated cardiomyopathy (dcm) in the ferret is unknown. • abdominal enlargement secondary to ascites, anorexia, dyspnea, lethargy, and weight loss are often noted. • ascites, heart murmur, pale mucous membranes, tachycardia, and weakness may be noted on physical examination. • moist rales and increased respiratory sounds may be noted when pulmonary edema is present. • pleural effusion may be present, and may cause an increased inspiratory effort. the heart may sound muffled on auscultation. • coughing generally is not noted. • see the chf section in this chapter. • treatment is the same as that described for chf. • taurine supplementation does not appear to have any effect on dcm in the ferret. • see the chf section in this chapter. the cause of hypertrophic cardiomyopathy (hcm) is unknown. • clinical signs may be compatible with those described for chf or dcm (see above). • other clinical signs are similar to those described for the cat, and include acute onset of congestive heart failure and/or sudden death. • follow the same guidelines described for dcm. • include hcm on the rule-out list when evidence of cardiac disease is noted on the physical examination or diagnostic evaluation. • radiographs may not be beneficial in the diagnosis of hcm. • echocardiography should be used for definitive diagnosis. • treatment should be aimed toward alleviating signs of chf and improving the diastolic efficiency of the left ventricle. • administer beta-adrenergic blocking drugs such as atenolol ( . - . mg) po sid. titrate to effect. • administer calcium channel blockers such as diltiazem ( . - . mg) po bid. titrate to effect. • diuretics are indicated if symptoms of chf are present (see above). • follow-up is the same as that described for chf and dcm. natural and experimental heartworm infections have been reported in ferrets (see chapter ). the clinical presentation of heartworm disease typically resembles that of cats; however, the life cycle of dirofilaria immitis in ferrets is similar to the life cycle present in the dog. reported adult worm burdens range from to . the presence of only one adult worm in the heart can be lethal. • heartworm disease is caused by the canine heartworm dirofilaria immitis, a filarial nematode that is transmitted via mosquitoes. • ferrets that are housed outdoors in endemic areas are at greatest risk of infection; however, ferrets kept indoors also can become infected. • clinical signs include coughing, dyspnea, hepatomegaly, inappetence, lethargy, melena, weakness, and symptoms associated with right-sided chf (pulmonary edema, pleural effusion, ascites). sudden death due to pulmonary artery obstruction may also occur. • microfilaria may be present in the blood of approximately % of infected ferrets. • diagnosis is based on the history, clinical signs, heartworm test results, radiographs, and echocardiography. • if the history is compatible with cardiac failure, inquire about possible mosquito exposure. • physical examination findings resemble those of heart failure (see above). m key point minimize stress in ferrets suspected of heartworm disease. if symptoms of congestive heart failure are present, delay further diagnostic evaluation until the patient is stabilized (see "treatment of congestive heart failure"). • obtain whole body radiographs. thoracic changes may include cardiomegaly with enlargement of the right atrium, caudal vena cava, and right ventricle. pleural edema and pleural effusion may be present as well. radiographic changes in the peripheral pulmonary arteries are not typically noted because the worms tend to reside in the right side of the heart and in the main pulmonary artery. abdominal changes often include hepatomegaly, splenomegaly, and ascites. • if possible, draw blood for the modified knott's test for microfilaria. microfilaria are identified in approximately % of infected ferrets. • submit blood for an enzyme-linked immunosorbent assay (elisa) for dirofilaria antigen. antigen is produced by adult female heartworms; there is a potential for false negative test results in ferrets with low worm burdens. a commercial assay (snap heartworm antigen test kit; idexx laboratories inc., portland, me) has been used to detect heartworm infection in the ferret. • perform a cbc, serum biochemical profile, and urinalysis to rule out the presence of other systemic diseases. • if pleural or abdominal effusion is present, submit fluid for cytology. a modified transudate is typically noted when chf is present. • echocardiography may be used to visualize heartworm(s) in the pulmonary artery, right ventricle, and right atrium; dilation of the right ventricle and right atrium may be assessed as well. doppler echocardiography may be used to evaluate the patient for the presence of pulmonary hypertension. • treatment of heartworm disease in ferrets is difficult. success is dependent on early diagnosis, diligent supportive care, and long-term antithrombotic therapy in conjunction with adulticide therapy. • if signs of chf are present, treat this first, and stabilize the patient (see the chf section). • if the patient is symptomatic and microfilaremia positive: • administer microfilaricidal therapy: ivermectin ( µg/kg) sc every days until clinical signs and microfilaremia resolve. • follow with adulticide therapy: melarsomine (immiticide, rhone merieux, athens, ga) using a two-stage protocol: stage : administer a single dose of melarsomine ( . mg/kg) im. stage : month later, administer two injections of melarsomine ( . mg/kg) im given hours apart. • transient swelling at the site of injection is common. • administer prednisone ( . mg/kg) po sid-tid during adulticide treatment and for as long as clinical signs persist. • if pleural effusion is present administer diuretics (see the chf section). • cage confinement is important for to weeks after treatment. • perform a post-treatment elisa for heartworm antigen months after adulticide therapy. repeat every days if results are positive. most ferrets become seronegative months after treatment. • begin heartworm prevention month after adulticide treatment. • if ferrets are microfilaria negative, administer adulticide therapy as described above. m key point because of the high mortality associated with heartworm disease, recommend preventive therapy for all ferrets in heartworm-endemic areas. • ivermectin may be given as preventive therapy beginning month before and continuing months after mosquito season. liquid ivermectin % may be diluted in propylene glycol ( . ml ivermectin in ml propylene glycol) and administered at a dose of ( . ml/kg) po every month. this solution must be stored in an amber glass bottle out of sunlight. feline heartguard (merck agvet) may be administered using the dose appropriate for a -to -lb cat. • if possible, house all ferrets in endemic areas within structures with mosquito-proof screening. • follow the same recommended guidelines for heartworm prevention in dogs and cats. valvular heart disease may occur in ferrets > years of age. • clinical signs depend on the severity of the underlying disease process. • mitral regurgitation may be ausculted as a systolic murmur in the left apical region. • tricuspid regurgitation is ausculted in the right parasternal region. • dyspnea and moist rales may be noted on auscultation of the lungs if chf is present. • obtain thoracic radiographs to evaluate the size of the heart and to determine if chf is present. pulmonary edema typically appears as a mixed alveolar and interstitial pattern in the caudodorsal lung lobes. • electrocardiography (ecg) may be normal or may demonstrate evidence of atrial arrhythmias. • echocardiography typically demonstrates thickening of affected valves and atrial enlargement. • doppler echocardiography may be used to identify and quantify the degree of regurgitation present. aortic regurgitation is often noted in ferrets and is considered an incidental finding. • treatment is recommended if chf is present, or if cardiac enlargement is significant (see the chf section). myocarditis occurs when the myocardium is infiltrated with inflammatory cells, resulting in the development of reduced myocardial function, arrhythmias, and replacement of the normal myocardial tissue with fibrous tissue. • causes include sepsis, systemic vasculitis, parasitic, bacterial or viral infection, and autoimmune disorders. • aleutian disease can cause fibrinoid necrosis and mononuclear cell infiltration of the arterioles of the heart. • antemortem diagnosis is difficult. • suspect myocarditis if arrhythmia and/or acute myocardial dysfunction is noted in association with multisystemic illness. • definitive diagnosis is made by histopathological evaluation of affected myocardial tissue. • treatment should be directed at identifying and treating the underlying systemic disease. • cardiovascular support should be provided and may include the use of diuretics or antiarrhythmic drugs (see the chf section). as clinical experience with pet ferrets increases, other types of cardiac disease are likely to be recognized. third-degree heart block (of unknown etiology) and various forms of valvular disease, including mitral and tricuspid insufficiency and endocarditis, have been seen in ferrets. • the approach to these conditions in ferrets is the same as for other companion animals; use the drug dosages given previously for cardiac myopathies. • the permanent teeth erupt between and days of age. • the dental formula is (i / , c / , pm / , m / ). • the third upper premolar (carnassial tooth) has three roots. the second lower molar has one root. all other premolars and molars have two roots. • the ferret is an obligate carnivore with a simple stomach, short intestinal tract, no cecum or ileocolic valve, and a short colon. • the duodenum terminates at the jejunoileum; there is no gross anatomic distinction between the jejunum and the ileum. • the junction of the jejunoileum and the colon is determined by evaluating the pattern of anastomosis between the jejunal artery and the ileocolic artery. • gi transit time is approximately to hours. • the anal sacs are located between the external and internal anal sphincter muscles at and o'clock. the ducts are located near the mucocutaneous junction. • the exact nutritional requirements of the ferret have not been determined. • the diet of the ferret must contain predominantly animal protein and fat. • due to the short digestive tract and rapid gi transit time, the ferret requires a concentrated maintenance diet high in protein ( - %) and fat ( - %), and low in fiber. the protein quality should be % to % digestible. • breeding ferrets and kits may require diets higher in protein and fat. • meat, poultry, meat and poultry meals, and other animal-based proteins should appear first, then several more times on the food ingredient list. • complex carbohydrates (starch, fiber) are not readily digested by the ferret. high-fiber diets can induce a relative protein-calorie deficiency; the ferret cannot eat enough of a low-density food to meet its high maintenance requirements. • premium cat foods and ferret diets typically meet the ferret's nutritional requirements for growth and reproduction. • treats and supplements should not exceed more than % of the daily diet. acceptable treats include meat baby foods, and moist cat or ferret diets. high-sugar or carbohydrate treats should be limited, especially if insulinoma is present. • fatty acid supplements should be given in measured amounts (a few drops per day). administration of large quantities of fatty acid supplements may reduce the intake of the balanced diet. • canine diets should not be fed to ferrets; the protein, fat, and carbohydrate content is not appropriate, and the diets often contain high percentages of grain and vegetable matter. • the long-term effect of formulated dry and canned diets on the long-term health of ferrets is controversial among some practitioners. • some practitioners feel that feeding commercial diets containing large quantities of plant-based ingredients contributes to the development of eosinophilic gastroenteritis, inflammatory bowel disease, insulinoma, urolithiasis, and general untriftiness. for example, most ferrets in the united states are fed dry kibbled diets, and the incidence of insulinoma is high. many ferrets in europe and australia are fed whole prey items (e.g., a "natural diet), and the incidence of insulinoma is low. • a correlation between diet and the development of certain diseases in ferrets is hypothetical at this time; however, this controversy demonstrates the need for longer-term diet studies in the ferret. • the canine teeth are often worn or broken at the tips due to biting and gnawing. • broken canine teeth typically are not painful unless the dental pulp is exposed. • dental tartar and periodontal disease are common in ferrets over years of age. • soft, moist diets may predispose ferrets to the development of dental disease. • tartar typically accumulates first on the second and third upper premolars. • dental abscesses are not common, but may be noted, even in young ferrets. • follow the basic medical and surgical treatment principles described for dental diseases in dogs and cats (see chapter ). • ferrets have five major pairs of salivary glands: the parotid, submandibular, sublingual, molar, and zygomatic. • salivary mucocele occurs secondary to trauma or infection of a salivary gland. • salivary mucocele typically presents as a soft to firm swelling in the region of the orbit, oral commissure, or mandibular lymph node. aspiration of the swelling often yields a clear to serosanguinous or mucinous fluid; microscopic examination demonstrates amorphous debris and occasional rbcs. • treatment of choice is surgical excision of the affected gland (see chapter ). • advise clients that recurrence is possible. megaesophagus is rare in ferrets. • the etiology of megaesophagus in ferrets is unknown (see list of possible causes in dogs in chapter ). • clinical signs resemble those described for the dog and include: lethargy, anorexia, dysphagia, coughing, choking, dyspnea, weight loss, and regurgitation. • clients may indicate that the ferret vomits up large boluses of food. • diagnosis may be based on clinical signs and radiographic evidence of megaesophagus. • obtain thoracic radiographs. the esophagus is often dilated and filled with air in the cervical and thoracic regions. food may be present within the lumen of the esophagus. • perform a barium contrast study to delineate the esophageal mucosa and to identify potential mural lesions, strictures, or obstructions. • aspiration pneumonia may be visible radiographically. • follow canine treatment protocols. the prognosis is poor; response to therapy is usually not successful. • gi promotility agents such as metoclopramide • administer antibiotics if indicated for aspiration pneumonia. • supportive care includes feeding high-calorie, highprotein slurried diets to times per day, and elevating the ferret for to minutes immediately after feeding. • ferrets, like other carnivores, are able to vomit. • differential diagnoses to consider for vomiting include esophageal and gastroenteric disorders (see below). • ferrets often demonstrate symptoms associated with nausea or vomiting when gastroenteritis, gi disease, gastric ulcers, helicobacter mustelae gastritis, or gi foreign bodies are present. hypoglycemia may cause signs of nausea as well (see discussion of insulinoma in this chapter). • signs of nausea include hypersalivation and pawing at the mouth. • ferrets may demonstrate bruxism (grinding of the teeth) when abdominal discomfort is present. • gi parasites are uncommon in the ferret. coccidiosis and giardiasis are occasionally seen. nematodiasis is rare. • routine fecal testing is still recommended, especially in young animals and ferrets with diarrhea or rectal prolapse. • young ferrets with coccidiosis may have diarrhea and may be severely dehydrated. • cryptosporidiosis may occur in ferrets, but typically does not result in clinical disease. the zoonotic potential is unknown; however, it may be prudent to warn immunosuppressed owners of the potential for zoonosis. • treat with appropriate anthelmentics following the protocols and dosages used for cats (see chapter ). • obtain fasting whole-body radiographs to help rule out the presence of a gi foreign body or trichobezoar. • a barium study may be used to demonstrate gi ulceration. • exploratory laparotomy/gastrotomy is often required for a definitive diagnosis. • diagnosis of h. mustelae gastritis is often a diagnosis of exclusion. definitive diagnosis requires the finding of organisms along typical histological lesions on gastric biopsy specimens. • debilitated, anorexic ferrets may require hospitalization for supportive care. • if the patient is vomiting, withhold food for to hours. administer iv fluids containing dextrose, and monitor for signs of hypoglycemia. when vomiting has resolved begin to offer small, bland meals. • feed small meals of a bland, moist diet tid-qid (see diet recommendations in "insulinoma" section). avoid feeding high-fiber dry foods. • administer broad spectrum antibiotics if the ferret is debilitated. • administer a gastric protectant. options include: • gi obstruction caused by foreign body ingestion or hairballs is one of the most common problems in pet ferrets. • foreign bodies typically occur in ferrets younger than year of age; trichobezoars (hairballs) are common in ferrets older than years of age. m key point suspect the presence of a gi foreign body in any young ferret presented for anorexia, even if no vomiting is reported. • rubber and foam objects are the most common foreign bodies. obstruction with a hairball (older ferrets), cloth, or plant material also may occur. • lethargy, partial or total anorexia, hypersalivation, bruxism, pawing at the mouth, weight loss, and diarrhea are the most common clinical signs of gi foreign body. hindlimb weakness, dehydration, and melena may be noted as well. • vomiting is uncommon; however, if the ferret is vomiting, be suspicious that a gi foreign body may be present. • diagnosis is based on the history, physical examination findings, radiographs, or exploratory laparotomy. • history: identify possible types or causes of foreign body ingestion. ask the owners if hairball preventative is used routinely. • physical examination: large gastric foreign bodies are often palpable. small foreign bodies in the small intestine may be associated with localized pain. • obtain fasting ( - hours) plain whole body radiographs. radiographs may reveal segmental ileus, and marked gaseous distention of the stomach and/or bowel. occasionally a foreign body or trichobezoar can be identified. • obtain a gi barium contrast study to identify small foreign bodies and to rule out gi ulceration. • perform a cbc and serum biochemical panel to rule out hepatic lipidosis and other systemic diseases. surgical removal is the treatment of choice. if the ferret is debilitated, begin supportive therapy, and perform surgery as soon as possible. • surgery: follow routine preoperative, operative, and postoperative procedures for gastrotomy or enterotomy (see chapters and ). ferret tissues are more delicate than those of a puppy or kitten of equivalent weight. use - or - suture material to close the gi tract. • perform gastric biopsy to rule-out underlying h. mustelae infection, and other gi diseases. perform biopsy of the liver. • evaluate the entire abdominal cavity prior to closure. older ferrets often have concurrent diseases such as insulinoma or adrenal gland disease. • the prognosis following gastrotomy is good with prompt therapy. m key point instruct owners to "ferret proof" the house if ferrets are allowed to roam. in particular, restrict access to rubber toys and rubber objects. • to prevent trichobezoars, administer a feline hairball laxative product ( - cm) po to times per week. epizootic catarrhal enteritis (ece, "green slime disease") is a highly infectious diarrheal disease that first appeared in . the etiological agent is thought to be a coronavirus. ece can spread rapidly through a ferret population, often affecting % of ferrets within hours. histological examination of intestinal biopsy samples reveals lymphocytic enteritis with villous atrophy and blunting, and degeneration of the apical epithelium. • the history often includes recent exposure of an older ferret to a new or young ferret that appears healthy. often within hours the older ferret becomes anorexic and lethargic. • four clinical syndromes are typically seen: . ece may cause relatively mild diarrhea that lasts several days in young ferrets with no underlying disease. . ece may cause severe diarrhea lasting for several days that may be followed by an acute onset of severe bloody diarrhea in older ferrets or ferrets with concomitant disease. anemia may develop as a sequelae. . a wasting disease with abnormal stools that have the appearance of bird-seed or of being grainy. these stools may develop in ferrets that initially appear to have recovered from the diarrheal phase. . voluminous green, watery diarrhea and occasional vomiting followed by chronic wasting may occur in some ferrets. • the clinical course of disease can be prolonged in some ferrets, and may last weeks to months. affected ferrets typically appear to recover, but continue to have persistent, intermittent diarrhea. • the disease may recur in previously affected ferrets; an asymptomatic carrier state appears to be possible. • do not house ferrets that have had ece with ferrets that have not had the disease. • rotavirus has been associated with several outbreaks of diarrhea and high mortality in ferret kits to weeks of age; it is often referred to as "ferret kit disease." • rotavirus also causes diarrhea in the young of several other species, including humans, cattle, swine, sheep, and rats. • in adult ferrets, rotavirus infection is rarely fatal, but may cause bright green mucoid diarrhea that lasts for several days. • there is no readily available antemortem test for the rotavirus infection; rotavirus particles can be identified in feces by electron microscopy. • treatment consists of supportive care. administer fluids, antibiotics, and nutritional support. salmonellosis is rare in the ferret, and is typically associated with exposure to contaminated raw meat and meat by-products. salmonella typhimurium, s. newport, and s. choleraesuis may be associated with clinical disease. • clinical signs include anorexia, lethargy, fever, and diarrhea (usually bloody). conjunctivitis and anemia have also been reported. • diagnosis is based on clinical signs and a positive fecal culture. multiple fecal samples must be collected, and selective media is used for culture. • treatment includes aggressive supportive care and antibiotic therapy. • ferrets may be presented in shock. iv fluids and administration of rapidly acting intravenous corticosteroids may be necessary for treatment of these patients. • other details of salmonellosis, including its public health significance, are discussed elsewhere in this text. eosinophilic gastroenteritis (ege) is an inflammatory bowel disease that occurs in ferrets and other animals. no specific etiological agent has been identified in the ferret, but food allergy is implicated in humans and other animals. • chronic diarrhea with or without mucus or blood, and weight loss are the most common signs. inappetence, intermittent vomiting, and skin lesions may be seen as well. • on physical examination, the mesenteric lymph nodes may be enlarged and the intestines may feel thickened. • a marked peripheral eosinophilia is often present on the cbc differential. • presumptive diagnosis is based on history, clinical signs, physical examination findings, the presence of a peripheral eosinophilia, and/or the presence of eosinophils on fecal cytology. • definitive diagnosis is based on histological examination of intestinal biopsy specimens. mild to extensive eosinophilic infiltration of the mucosa, submucosa, and muscularis of the stomach and small intestines are noted. focal eosinophilic granulomas maybe identified in the mesenteric lymph nodes. • treatment is similar to that described for treatment of dogs and cats. • begin corticosteroid therapy with prednisone ( . - . mg/kg) po sid every days. perform a recheck examination and cbc weeks after the last dose. if the ferret has improved clinically and the peripheral eosinophilia is resolving, decrease the prednisone dose by % every days, and recheck again. continue the prednisone taper at -week intervals until the ferret is tapered to the lowest possible dose, or withdrawn from the steroids altogether. • although food allergy has not been identified as a definitive etiological cause of ege, changing the ferret to a hypoallergenic diet, such as a feline lamb and rice-based diet may be helpful in resolution of signs. • there have been reports of ferrets with peripheral eosinophilia (up to %), and erythema and crusting of the feet, ears, and face. histological lesions in biopsy specimens from affected skin were consistent with allergic dermatitis. these ferrets were treated with corticosteroids, and did respond to treatment. one also responded to diet change. inflammatory bowel disease (ibd) can occur in the ferret. the etiology is unknown; dietary factors, hypersensitivity reactions, or an immune-mediated cause have been considered. • clinical signs can be subtle and include diarrhea; nausea; occasional vomiting; soft, malformed stools that resemble bird seed; and weight loss. these signs often resemble ece, ege, and helicobacter gastroenteritis. • affected ferrets are often young or middle-aged adults. • elevation of liver enzymes and serum globulins may be noted on serum biochemistry analysis. • lymphocytosis may be noted on the cbc. • diagnosis is based on the history, clinical signs, and diagnostic work-up. • definitive diagnosis is made by histological examination of gastric and intestinal biopsy samples. mild to severe lymphoplasmacytic gastritis and enteritis are noted on histopathology. • administer corticosteroids such as prednisone ( . - . mg/kg) po sid every days initially, and taper in a manner similar to that described for ege. some ferrets respond poorly to steroid therapy. • azathioprine (imuran, prometheus laboratories, san diego, ca) ( . mg/kg) po q - h may be used as an alternative to steroid treatment. • hypoallergenic diets may offer some benefit. proliferative bowel disease (pbd) in ferrets was first reported in , and is similar to the pbd that occurs in swine and hamsters. pbd was a commonly encountered disease in the late s and early s, but is relatively uncommon now. pbd is caused by lawsonia intracellularis, an intracellular bacteria that cannot be propagated by routine culture methods. • this disease affects primarily young ferrets to months of age. • acute and chronic forms of the disease can occur. • diarrhea is present and often contains mucus and blood. defecations are frequent and small; ferrets often cry out when they defecate. the rectum may be partially prolapsed. • other signs include lethargy, depression, inappetence, weight loss, dehydration, and pyrexia. • neurologic signs such as ataxia and muscle tremors may be present. • the intestines may feel firm or thickened on abdominal palpation. • a tentative diagnosis of proliferative bowel disease is based on clinical signs and physical examination. definitive diagnosis requires intestinal or colonic biopsy, but this rarely is warranted because response to therapy usually is good if initiated early. • a polymerase chain reaction (pcr) assay specific for the swine isolate, and an indirect fluorescent antibody test (ifa) are available. • necropsy lesions include gross thickening and discoloration of the small intestine and/or colon. ridges of proliferative tissue that are distinct from normal adjacent tissues are present on the mucosal surface. • histological examination of biopsy samples or necropsy specimens typically demonstrate epithelial proliferation, hypertrophy of the muscularis, and infiltration of the bowel wall with monocytic or granulocytic inflammatory cells. silver-stained tissues reveal intracellular, comma-shaped organisms in crypt epithelial cells. glandular hyperplasia consisting of irregular, branching proliferative glands that lack goblet cells, and necrotic debris may be identified in the crypts. severe glandular hyperplasia may resemble neoplasia and can metastasize. • treat mild cases on an outpatient basis. • hospitalization for supportive care (fluid therapy, nutritional support) may be necessary when severe disease is present. • administer chloramphenicol ( mg/kg) q h, po, iv, im, or sc as the drug of choice. treat for at least weeks; longer therapy often is necessary to prevent relapse. • metronidazole ( mg/kg) q h, po may be effective. • the prognosis is good with timely therapy. • some ferrets improve temporarily and then relapse at the end of the treatment period. use a long-term course of antibiotic therapy in these animals. • rectal prolapse is usually a disease of young ferrets, and is often associated with diarrhea. • possible causes of rectal prolapse include colitis, diarrhea, gi parasitism (e.g., coccidiosis), pbd, and other diseases that may cause straining or diarrhea. • other differentials include gi lymphoma, benign intestinal polyps, and postoperative complications of anal gland removal. • perform direct fecal and fecal flotation tests to screen for parasites. • medical treatment is similar to that described for other species. administer anthelmentics and antibiotics when indicated. • the prolapse may resolve without surgical intervention when the underlying disease process is resolved. • surgical correction is usually unnecessary (see below). • if indicated, perform a biopsy of the prolapsed tissue to rule out lymphoma. • flush the prolapsed tissues with sterile saline and replace them into the rectum. • place a purse-string suture in the anus with a small opening to allow passage of feces. keep the pursestring suture in place for to days. • in ferrets with chronic prolapse, surgery may be necessary to reduce the size of the anal opening. excise a small triangular wedge of anal mucosa and routinely close the defect by suturing. alternatively, consider abdominal exploratory surgery and colopexy (see chapter ) • clinical signs and physical examination findings in ferrets with an abscessed anal sac are the same as those described in dogs and cats. • the recommended treatments include antibiotic therapy, lancing and drainage of the abscess, or surgical removal of both anal sacs (see chapter ). anal sacculectomy is performed as a treatment for anal sac abscesses, or to decrease the musky "ferret" odor. for odor reduction, neutering should be performed simultaneously because the apocrine, perianal, sebaceous, and scent glands in the skin are under hormonal control and contribute to the overall musky odor. some clinicians believe that neutering is sufficient to decrease odor and that routine anal sacculectomy should be discouraged. . grasp the anal sac duct and hold it closed with mosquito forceps. make a circumferential skin incision around the duct opening. . apply gentle caudal traction to the anal sac, and use a scalpel blade or gauze to tease away the surrounding fascia. . leave the surgical sites open, and allow to heal by second intention. . make small, arc-like incisions just lateral to the duct openings. . dissect the subcutaneous tissues bluntly to reveal the neck of the anal sac; grasp the opening and hold it closed with mosquito forceps. . dissect the sac free of surrounding tissues, using gentle traction. . do not suture the incisions. ferrets reach sexual maturity during the first breeding season after birth. the breeding season runs from march to august under natural lighting conditions. • the opening of the prepuce is located just caudal to the umbilical area. • males (hobs) have a j-shaped os penis. • during the breeding season (march-august), testicle size is twice that noted in the fall and winter months. • prostatic tissue is located at the base of the urinary bladder and surrounds the urethra. prostatic disease associated with adrenal gland disease may occur in middle-aged and geriatric male ferrets (see "adrenal gland disease" and "prostatic disease"). • female ferrets (jills) are seasonally polyestrous and are induced ovulators. ovulation typically occurs to hours after mating. • the vulva is located in the perineal region ventral to the anus. in non-estrous females the vulva is small, and looks like a slit; during estrus (or when adrenal gland disease is present), the vulva becomes swollen and is easily visualized. • if mating is unsuccessful, pseudopregnancy results and lasts to days. • approximately % of females remain in estrus if they are not bred. the resultant prolonged elevation of serum estrogens can cause bone marrow toxicity and pancytopenia (see the discussion of anemia under "hematopoietic system" in this chapter). • submit blood for a cbc and platelet count if the ferret has been in estrus for more than days. • termination of estrus is recommended (see "termination of estrus in the hematopoietic".) • most pet male ferrets in the united states have already been neutered prior to weeks of age. • castrate intact male ferrets at to months of age in order to reduce aggressive behavior and odor. • castration is performed using techniques similar to those used in cats (see chapter ). • make an incision in the scrotum over each testicle. • most pet female ferrets in the united states have already been spayed prior to weeks of age. • spaying intact female ferrets is recommended to prevent estrogen-induced bone marrow hypoplasia. • ovariohysterectomy is similar to the procedure performed in cats (see chapter ). • the ventral midline incision is made approximately cm caudal to the umbilicus, and may be extended as necessary. • the uterus is bicornuate, and is located dorsal to the bladder. • ovarian vasculature may be difficult to locate due to the large amount of body fat typically present in this region. • pyometra and metritis are uncommon in pet ferrets in the united states because they are usually spayed prior to being sold as pets. • clinical signs may include anorexia, lethargy, pyrexia, and vulvar discharge. polyuria and polydipsia are not usually noted. • persistent estrus may predispose ferrets to pyometra. • preoperatively perform a cbc and a serum biochemical analysis to rule out estrogen-induced bone marrow hypoplasia (see "anemia" in "hematopoietic system"). • provide appropriate supportive care pre-and postoperatively. • perform ovariohysterectomy when the patient is stable (see chapter ). • start the ferret on broad-spectrum antibiotic therapy preoperatively, and continue postoperatively for to days. use broad-spectrum antibiotics. organisms commonly associated with pyometra include staphylococcus spp, streptococcus spp, corynebacterium spp, and e. coli. • vulvar swelling is an external sign of estrus in female ferrets. • in a spayed female, a swollen vulva indicates a remnant of ovarian tissue, or another source of estrogens and estrogen precursors such as adrenal gland disease (see "adrenal gland disease"). • ovarian remnants typically induce signs of estrus in ferrets younger than years of age. • administer hcg ( iu) im. vulvar swelling should subside if an ovarian remnant is present. if no changes occur, adrenal gland disease is probably the cause of the clinical signs. • perform exploratory laparotomy to remove the ovarian remnant (see chapter ). evaluate for uterine remnants and adrenal gland disease as well. • preoperatively evaluate a cbc to rule out estrogeninduced bone marrow hypoplasia. • pregnancy toxemia is a potentially life-threatening condition that occurs in late pregnancy. primiparous females are most commonly affected. • the disease results in high mortality of jills and kits. • toxemia can be induced if an accidental fast occurs in the last week of gestation. • pregnancy toxemia may also develop in primiparous jills that are carrying large litters due to nutritional compromise induced by the size of the gravid uterus and the resultant reduced capacity of the stomach. • advise owners that pregnant jills must have access to food and water al lib during pregnancy. • suspect pregnancy toxemia if acute lethargy develops in the last week of gestation. other clinical sings include dehydration, melena, hypoglycemia, ketonuria, and azotemia. • affected ferrets usually are presented in an acute state of shock. • treatment includes aggressive supportive care including iv or io fluids containing dextrose. perform an immediate cesarean section (see chapter ). • postoperative care includes continued supportive care, including frequent feedings of high-calorie critical care diets. • jills that survive pregnancy toxemia often do not produce milk. kits can be difficult to hand rear; if a foster jill is not available, attempts may be made to hand rear the kits using a kitten milk replacer (see below). kits born before days of gestation often do not survive. • the prognosis is usually poor, even with aggressive treatment. • do not breed females with a history of mastitis. • abrasions to the mammary tissue and nipples can cause mastitis. prevent trauma from occurring by providing a large nest box opening with smooth edges that allows the jill to pass through easily. • mastitis may be acute or chronic. • acute mastitis typically occurs immediately after whelping or during the third week of lactation. • affected glands appear swollen, firm, red to purple in color, and are painful. gangrene can develop within hours of clinical signs. • treatment must be aggressive. administer broadspectrum antibiotics, and apply hot packs to the affected area to times per day for days. debride necrotic tissue if present. provide supportive care and analgesic therapy. • submit a sample for bacterial culture and sensitivity testing. modify antibiotic therapy based on test results. • if there is no clinical response to medical therapy in days, or if gangrene rapidly develops, consider surgical removal of the affected mammary tissue. because of the potential for severe toxicity and life-threatening disease, do not delay surgery if gangrene is already present, or if there is no improvement with medical therapy. • if the jill continues to lactate, leave the kits with her. supplement feed the kits with a kitten milk replacer if necessary. do not foster the kits with another jill because this may result in mastitis in the foster jill. • ingestion of infected milk may cause gastroenteritis in the kits; kits may need to be treated with antibiotics as well. • chronic mastitis is often difficult to diagnose. the affected jill often appears normal, while the kits lose weight or fail to thrive. • mammary glands appear firm but are not painful or discolored; often the glands are presumed to be full of milk. • affected mammary glands become scarred and are no longer functional. affected jills should be culled from the breeding program. • hand-rearing kits from birth is difficult. prognosis is poor for survival. • it may be necessary to provide supplemental feeding for kits if the jill's milk production is reduced, or if the litter size is large. • whenever possible, foster kits with another lactating jill. most jills will accept kits of any size or age. • kits require a milk supplement that contains a high fat content ( %). kitten milk replacers mixed with cream may be used. • feed kits as much as they will eat times per day with a dropper or small pet nurser. • begin to mix solid food with the enriched milk replacer when kits are weeks of age. this mixture may be offered in a shallow dish or bowl. • kits may be weaned onto a solid diet at to weeks of age. feline or ferret growth diets are recommended. • the right kidney lies cranial to the left kidney. the cranial end of the right kidney often lies under the caudate lobe of the liver. • the bladder is small, and can hold up to ml of urine. • male ferrets have a small prostate gland that surrounds the urethra at the base of the bladder. • urinalysis: • the normal urine ph is . for ferrets on a meatbased diet. • normal values for urine-specific gravity have not been reported. • there is evidence that proteinuria may be normal in ferrets ( - mg/dl in males; - mg/dl in females) and that bilirubinuria can occur in the absence of liver disease. renal disease in not common in ferrets, but may occur. • clinical signs are similar to those described in other animals, and include ataxia, bruxism, halitosis, hindlimb weakness, inappetence, melena, mucus membrane ulceration, polyuria/polydipsia, vomiting, and weight loss. • physical examination findings may include cachexia, dehydration, irregularity in the shape and size of the kidneys, pale mucous membranes, and oral ulceration. • diagnosis is based on clinical signs, physical examination, and cbc, serum biochemical analysis, and urinalysis results. m key point hyperphosphatemia, hypocalcemia, and high bun may be noted on serum biochemical analysis. serum creatinine concentration is often normal or only moderately elevated. • treatment should address the underlying cause, if possible. • nonspecific treatment includes fluid therapy, nutritional supportive care, and antibiotic therapy based on culture and sensitivity when indicated. • prognosis is guarded, depending on laboratory findings and response to treatment. unilateral or bilateral renal cysts are relatively common in ferrets (see chapter for a description of this disease in dogs and cats). the condition is usually an incidental finding in middle-aged and older ferrets, although clinical signs associated with this condition can occur at any age. • the cause of renal cysts in the ferret is unknown. • heredity does not appear to be a factor. renal cysts are not associated with hepatic or biliary cysts. • renal cysts typically present as one or more smooth masses on the surface of the kidney. on abdominal palpation affected kidneys feel smoothly enlarged or irregular. • polycystic disease is unusual in the ferret. when present, affected kidneys appear rough and irregular; multiple cysts are often distributed throughout the renal tissue. cysts may be present in other organs as well. • usually there are no clinical signs associated with renal cysts. • rarely, there may be enough disruption of normal renal parenchyma to lead to renal failure, and subsequent clinical signs. • palpate the kidneys for irregular shape. • perform a cbc, serum biochemical profile, and urinalysis. • abdominal radiography usually is not helpful unless the kidneys are very irregular. intraosseous fluid therapy in small exotic animals exotic animal formulary biology and diseases of the ferret. philadelphia: lea & febiger ferret husbandry, medicine and surgery essentials of pet ferrets: a guide for practitioners ferrets, rabbits, and rodents: clinical medicine and surgery ferrets for dummies. indianapolis • perform an abdominal ultrasound to detect renal cysts, to evaluate renal architecture, and to rule out other conditions such as renal neoplasia.• intravenous pyelography or nuclear scintigraphy may be used to evaluate renal function.• renal cysts may be an incidental finding during abdominal surgery. • there is no specific treatment for renal cysts. no treatment is necessary in asymptomatic animals.• monitor affected ferrets by periodic abdominal palpation, serum biochemical profile, urinalysis, and ultrasound, if indicated.• if an affected kidney becomes very large, consider unilateral nephrectomy (if the opposite kidney is functional) (see chapter ).• symptomatic ferrets may be managed using the same supportive care methods used in dogs and cats with chronic renal failure.• the prognosis is grave for ferrets in renal failure. • hydronephrosis is uncommon in ferrets. iatrogenic hydronephrosis may occur as the result of inadvertent ligation of a ureter during ovariohysterectomy (see chapter for information about hydronephrosis in dogs and cats). • bacterial cystitis without urinary calculi is rare in pet ferrets. follow treatment protocols for cystitis in dogs (see chapter ). urinary calculi was a common cause of stranguria in ferrets at one time; improvement in the quality of ferret diets has decreased the incidence of calculi. calculi are usually composed of calcium oxalate or struvite (magnesium ammonium phosphate hexahydrate). cysteine calculi also have been reported. • the cause of urinary calculi is unknown; however, diet is believed to be a factor.• diets containing plant proteins or poor quality meatbased proteins may be associated with the development of urinary calculi. urolithiasis is uncommon in ferrets maintained on a high-quality feline or ferret diet containing high-quality animal-based proteins.• other factors may include urinary tract infection, metabolic, genetic, and congenital factors. clinical signs depend on the location of the urolith(s) and may include dysuria, stranguria, hematuria, persis-tent wetness in the perineal region, and frequent licking of the perineum.• urethral calculi may cause obstruction in both male and female ferrets. • ferrets with urethral obstruction often strain and cry as they attempt to urinate. • if complete obstruction is present ferrets often appear lethargic and anorexic, and may not demonstrate obvious signs of dysuria. • palpate the bladder to identify cystic calculi. the urinary bladder wall may be thickened; in ferrets with urethral obstruction, the bladder is distended and firm.• obtain abdominal radiographs to confirm the presence of radiopaque urinary calculi. cysteine calculi are not radiopaque and require contrast radiography or ultrasonography for diagnosis.m key point small stones located at the base of the os penis can be very hard to identify.• renal calculi may be an incidental finding on whole body radiographs, or may be associated with renal failure. m key point urethral obstruction is an emergency.severe metabolic derangement, coma, and death may occur if urethral obstruction is not diagnosed and treated quickly.• stabilize non-obstructed ferrets by providing supportive care, fluids, analgesics, and antibiotics (if indicated) prior to performing cystotomy to remove the urolith(s).• cystic calculi may be removed surgically via cystotomy; the procedure is similar to that used in cats and dogs (see chapter ). close the bladder wall with - or - absorbable sutures.• submit a urolith sample for analysis and bacterial culture/sensitivity testing.• administer antibiotics for a minimum of to days. use results of follow-up urinalysis, and urine culture/sensitivity testing to determine when to discontinue antibiotic therapy.• begin conversion to a high-quality-animal, proteinbased feline or ferret diet. urinary acidifies are not usually necessary once the ferret is on a high-quality animal, protein-based diet, since this diet alone will cause the urine to be acidic.• feline calculi-dissolving diets and preventative diets may be offered to ferrets; however, many ferrets do not find these diets palatable. • renal calculi can often be managed medically by administering antibiotic therapy and changing the diet. m key point the bladder is very fragile in ferrets.handle ferrets with obstruction gently to avoid bladder rupture.• urinary obstruction in the male ferret can be difficult to manage. catheter placement is challenging due to the small size of the urethra and the j-shaped os penis. (see urinary catheterization in the techniques section of this chapter.)• to facilitate placement of the urinary catheter, empty the bladder via cystocentesis prior to catheterization. submit urine samples for urinalysis and bacterial culture/sensitivity testing.• use either a ferret urinary catheter (slippery samferret urinary catheter, cook veterinary products), a standard tom cat catheter, or a . -fr red rubber catheter for catheterization.• inhalant anesthesia with isoflurane or sevoflurane is strongly recommended to facilitate catheter placement.• if the urinary catheter placement is not successful, consider emergency cystotomy, and attempt to perform anterograde flushing of the urethra via the cystotomy site.• perineal urethrostomy may be considered if cystotomy is unsuccessful (see chapter ). • feed a high-quality, animal protein-based feline or ferret diet. prostatic disease and subsequent urethral obstruction is a potentially life-threatening condition of middle-aged and geriatric male ferrets. this condition typically occurs in association with adrenal gland disease. • prostatic disease and prostatic cyst formation are presumed to be the effect of excessive androgens on the prostate. excessive androgen production occurs with adrenal gland disease.• squamous metaplasia of prostatic glandular epithelium occurs and may subsequently lead to the development of cysts ranging in size from to cm or larger. secondary bacterial infection and abscessation may occur.• prostatic abscesses associated with transitional cell tumor of the bladder, prostatic seminoma, and pro-static carcinoma have also been reported in the ferret, but are rare. • clinical signs associated with prostatic disease may include symptoms associated with a urinary tract infection, urethral obstruction, or urinary incontinence.• signs of adrenal gland disease are often present (see "adrenal gland disease"). • on physical examination, a large, firm, often painful caudal abdominal mass is usually palpable. with careful palpation, this mass is found to be bilobed, representing the urinary bladder and a cystic structure. ferrets with mild to moderate prostatic disease may appear to have a normal-sized prostate on abdominal palpation, yet are still symptomatic. it is important to remember that adrenal gland disease is usually the cause of prostatic disease. perform a complete diagnostic work-up that includes whole-body radiography, cbc, serum biochemistry analysis, and urinalysis. a plasma steroid hormone assay, and abdominal ultrasound may be indicated as well.• obtain abdominal radiographs; prostatic enlargement or prostatic cysts appear as mass lesions dorsal to the bladder.• perform abdominal exploratory surgery for a definitive diagnosis. • address urethral obstruction if present (see "urolithiasis").• manage medically until the ferret is stable for adrenalectomy and surgical drainage of the cysts.• medical management includes maintenance of urinary catheterization for several days, administration of fluids, antibiotic therapy, anti-inflammatory and analgesic therapy, and nutritional support as needed.• consider administration of an androgen receptor blocker (see "adrenal gland disease").• consider administration of leuprolide acetate -day depot formulation (lupron depot, bristol-myers-squibb oncology, princeton, nj) ( ug/kg) im; prostatic tissue shrinkage may occur within hours in some individuals. some ferrets have been maintained successfully on monthly injections of this drug, although results are highly variable.• perform adrenalectomy and drainage of the cysts.large cysts may require debulking. m key point omental pull-through procedures and marsupialization have been described as means of prostatic abscess management in the ferret. these procedures should be used with some caution. prostatic abscesses and prostatic cysts can be difficult to differentiate from paraurethral cysts. paraurethral cysts communicate with the urethra or bladder neck. consider performing contrast radiography to determine if there is communication between the cyst/abscess and the bladder prior to performing these procedures.• administer postoperative antibiotic therapy for a minimum of to days, along with androgen receptor blockers or leuprolide acetate.• base the decision to discontinue antibiotic therapy and androgen receptor blocker/leuprolide acetate therapy by monitoring changes on physical examination, follow-up radiography, and follow-up urinalysis. • the long-term prognosis is good if prostatic changes regress, and if subsequent adrenal gland disease does not occur in the remaining adrenal gland.• some ferrets may need to be maintained on androgen receptor blockers or leuprolide acetate indefinitely. paraurethral cysts are thin-walled single or multiple cysts present on the dorsal aspect of the bladder and proximal urethra. these cysts appear to also be associated with adrenal gland disease and can cause urethral obstruction.it is important to differentiate between prostatic cysts and paraurethral cysts when planning the surgical protocol. • paraurethral cysts have been reported in male and female ferrets.• clinical signs are similar to those described for prostatic disease, and include symptoms associated with a urinary tract infection, urethral obstruction, urinary incontinence.• clinical signs of adrenal gland disease are usually present (see "adrenal gland disease"). • a large, firm caudal abdominal mass is often palpable dorsal to the bladder, just cranial to the pelvic inlet. • perform a complete diagnostic work-up that includes whole-body radiography, cbc, serum biochemical analysis, and urinalysis.• because adrenal gland disease is usually the underlying etiology, consider performing a plasma steroid hormone assay.• radiographically, paraurethral cysts appear as mass lesions dorsal to the bladder.• ultrasonography may be useful in evaluation of the paraurethral cysts and adrenal glands. • surgical drainage and debulking of the cysts is the treatment of choice.• marsupialization is an alternative, but may lead to a formation of a permanent cystotomy.• do not perform an omental pull-through procedure. key: cord- -ody u n authors: loh, so hee; park, jin-yeon; cho, eun hee; nah, seung-yeol; kang, young-sun title: animal lectins: potential receptors for ginseng polysaccharides date: - - journal: j ginseng res doi: . /j.jgr. . . sha: doc_id: cord_uid: ody u n panax ginseng meyer, belonging to the genus panax of the family araliaceae, is known for its human immune system-related effects, such as immune-boosting effects. ginseng polysaccharides (gps) are the responsible ingredient of ginseng in immunomodulation, and are classified as acidic and neutral gps. although gps participate in various immune reactions including the stimulation of immune cells and production of cytokines, the precise function of gps together with its potential receptor(s) and their signal transduction pathways have remained largely unknown. animal lectins are carbohydrate-binding proteins that are highly specific for sugar moieties. among many different biological functions in vivo, animal lectins especially play important roles in the immune system by recognizing carbohydrates that are found exclusively on pathogens or that are inaccessible on host cells. this review summarizes the immunological activities of gps and the diverse roles of animal lectins in the immune system, suggesting the possibility of animal lectins as the potential receptor candidates of gps and giving insights into the development of gps as therapeutic biomaterials for many immunological diseases. panax ginseng meyer is a well-known medicinal plant in the world. the ginseng is a deciduous perennial belonging to the family araliaceae and genus panax. the genus name of ginseng, panax, is derived from the greek pan (all) akos (cure), meaning "cure-all" or "all healing," which describes the traditional belief that ginseng has properties to heal all aspects of the body. the name ginseng comes from the chinese words "jen sheng," meaning "man-herb," because of the humanoid shape of the root or rhizome of the plant, which is the part of the plant most commonly used for extraction [ , ] . there are about different species of ginseng which have being identified all over the world. among them, the most commonly used species of ginseng are asian ginseng (p. ginseng meyer, renshen) and american ginseng (panax quinquefolius l., xiyangshen) which all belong to the panax genus of the araliaceae family [ ] . asian ginseng has been used for thousands of years as a tonic to improve overall health, restore the body to balance, help the body to heal itself, and reduce stress [ ] , and american ginseng has been used by native americans for at least hundreds of years [ , ] . ginseng is prepared and used in several ways as fresh ginseng (sliced and eaten, or brewed in a tea), white ginseng (peeled and dried), red ginseng (peeled, steamed, and dried), extract (tincture or boiled extract), powder, tea, tablet, or capsule [ , ] . it has been reported that ginseng exhibits a wide range of beneficial pharmacological effects including immunomodulation, antitumor, antioxidation, antidepression, hypoglycemic, inhibition of gastric lesions, attenuation of leptininduced cardiac hypertrophy, heart protection against ischemia and reperfusion injury, prevention of glucose-induced oxidative stress, prevention of diabetic nephropathy, retinopathy, and cardiomyopathy [ e ]. this broad spectrum of biological activity of ginseng has originated from its multiple bioactive components, namely ginsenosides, polysaccharides (pss), peptides, polyacetylenic alcohols, and gintonin [ e ]. ginsenosides were considered to be responsible for most of ginseng's pharmacological effects. however, recent studies indicate that ginseng polysaccharides (gps), one of the active components of ginseng [ ] , also possess a wide range of biological and pharmaceutical activities, including immune-modulation, antitumor, antiadhesive, antioxidant and hypoglycemic activities [ , ] . especially, gps are known for their immunostimulatory effects [ , , ] and a major contributor to the bioactivity of herbal medicines, providing great potential applications in food, pharmaceuticals, and other industries. therefore, gps were extensively studied for their constituents and chemical structures. gps are biopolymers formed from a complex chain of monosaccharides rich in l-arabinose, d-galactose, l-rhamnose, d-galacturonic acid, d-glucuronic acid, and d-galactosyl residue linked together through glycosidic bonds, resulting in complex macromolecular architectures [ , , ] . their molecular weights range from da to , , da [ ] , which contributes to their diverse physicochemical properties and biological activities [ , , , ] . gps include acidic and neutral pss. the pharmacological effects of gps, including immunomodulation, can be attributed to these acidic and neutral ps components [ ] . while the acidic gps contain different amounts of uronic acids and neutral sugars [ , ] , the neutral pss mainly contain different ratios of neutral sugar residues [ ] . so far, the studies about american gps have mainly been centered on acidic pss, resulting in relatively limited research that explores neutral pss. however, researchers also have interest in neutral pss of american gps, because neutral pss are also one of the important active components in the american ginseng roots. the pss from ginseng roots have many bioactivities, such as immunomodulation, antitumor, and hypoglycemic activities [ , ] , and contain % neutral starch-like pss, % arabinogalactans, and % pectins [ ] . similarly, the pss from ginseng leaves are also bioactive, and contain about % arabinogalactans and % pectins. gps enable enhancement of the production of cytokines and reactive oxygen species by stimulating macrophages [ , ] . in a recent study, gp was shown to stimulate dendritic cells (dcs) resulting in enhanced production of interferon-g (ifn-g) [ ] . it was reported that acidic gps promoted the production of cytotoxic cells against tumors and stimulated macrophages to produce helper types and (th and th ) cytokines [ , ] . an acidic gp from p. ginseng has been shown to display immunomodulatory effects either in an immunostimulatory or in an immunosuppressive manner, depending on timing of treatments and disease environments [ ] . this acidic gp was also shown to modulate the antioxidant defense systems such as superoxide dismutase and glutathione peroxidase enzymes, probably via inducing regulatory cytokines [ , ] . therefore, acidic gps have been considered as the major bioactive species for immune modulations. tomoda et al [ ] reported that two acidic pss of p. ginseng enhance the phagocytic activity of macrophages, and sonoda et al [ ] found that an acidic gp of p. ginseng was a potent inducer of interleukin- (il- ) production by human monocytes and thp- cells. shin et al [ ] reported that an acidic ps of p. ginseng shows immune modulatory activities via macrophage no production. recently, lemmon et al [ ] reported that the immunostimulatory effects of acidic gps of p. quinquefolius are mediated by ps with a molecular weight higher than kda. it was reported that acidic gps promoted the production of cytotoxic cells against tumors and stimulated macrophages to produce helper types and (th and th ) cytokines [ , ] . intravenous pretreatment of gp attenuated the production of serum proinflammatory and antiinflammatory cytokines after septic bacterial infection [ ] . in addition, ginsan, an acidic gp from of p. ginseng, is a wellknown medicinal herb and has been shown to have critical effects on immune cells, which shows an immunomodulatory acidic gp from p. ginseng [ ] . kim et al [ ] showed that ginsan induces th cell and macrophage cytokines. ginsan enhances the production of cytokines and reactive oxygen species by macrophages [ ] and stimulates the phagocytic activity of macrophages [ ] . also, ginsan induces the maturation of dcs [ ] , profoundly enhancing the production of il- , il- , and tumor necrosis factor alpha (tnf-a) by dcs and showed that ginsan may modulate dc function by altering cytokine levels [ ] . for neutral gps, it was reported that neutral gps of p. ginseng stimulate the proliferation of lymphocytes, increase the cytotoxicity of natural killer cell, enhance the phagocytosis and no production by macrophages, and increase the level of tnf-a in serum [ , ] . due to these results, many scientists have considered both acidic and neutral gps as stimulators in the immune system. x. zhang et al and kim et al reported that both acidic and neutral gps of p. ginseng (asian ginseng) may stimulate b cells, t cells and macrophages [ , ] . in addition, they considered the relation of acidic and neutral gps as the supporter, in which neutral gps help the enhancement of immunostimulatory effects of acidic gps. in fact, w. ni et al reported that neutral gps of p. ginseng enhance macrophage production of no [ ] . on the contrary to immunostimulatory effects of gps, recent studies showed that gps also suppress the proinflammatory responses. recently, it was reported that a novel neutral ps (ppqn, . kda) was isolated from american ginseng roots and could suppress inflammation by inhibiting the secretion of inflammatoryrelated mediator nitric oxide (no) and cytokines (tnf-a, il- , and il- b) compared to lipopolysaccharide (lps) treatment, implicating the therapeutic implications of ppqn in inflammatoryrelated diseases like tumors, atherosclerosis, and so on [ ] . as an example, one study reported that gps inhibit immunological responses associated with collagen-induced arthritis [ ] . other studies also suggest that cvt-e , a poly-furanosyl-pyranosyl polysaccharide-rich herbal and unique extract product of the root of american ginseng (p. quinquefolium), suppresses the inflammatory immune responses, reducing the activation of neutrophils [ ] , inducing the production of il- , ifn-g, tnf-a, and il- in spleen [ , , ] , and increasing the proliferation of splenic b lymphocytes, bone marrow, and natural killer cells. intravenous pretreatment of gp attenuated the production of serum proinflammatory and antiinflammatory cytokines after septic bacterial infection [ ] . also, this intravenous pretreatment of gps in mice enhances macrophage-mediated bactericidal activity by reducing the number of staphylococcus aureus which is present in the spleen, kidney, and blood and exerts a protective effect against infected septic mice by suppressing early acute inflammation [ , ] . in addition, recent studies reported that pretreatment with gp suppressed acute inflammatory responses at an early phase resulting in the enhancement of antimicrobial activities and protection of mice from staphylococcus aureus-induced sepsis as an antiinflammatory function [ , ] . as an example, cvt-e has been shown to be effective for preventing acute respiratory illness caused by influenza and respiratory syncytial virus [ , ] . another study revealed that intranasal administration of gps showed a protective effect on influenza viral infection by lowering the levels of inflammatory cytokines (il- ) and lung viral titers [ ] . because gps were reported to significantly increase the viability of peritoneal macrophage cells [ ] and ginseng was shown to inhibit degradation of long-lived proteins and to stimulate protein synthesis similar to polypeptide growth factors [ ] , it was suggested that maintaining the cell viability under the condition of viral infection-induced stress might be an another alternative mechanism for the protective effects of gp. it was reported that the recognition and binding of plant pss by toll-like receptor (tlr ) leads to the recruitment of various cytoplasmic toll/il- receptor (tir) domain-containing adaptors such as myeloid differentiation factor (myd ), tir domaincontaining adaptor protein (tirap), and tir (toll/interleukin- receptor)-domain-containing adapter-inducing interferon-b (trif)related adaptor molecule (tram) [ ] . it was also shown that the expression of tlrs including tlr , tlr , and the adaptor molecule myd is significantly reduced in murine macrophages by gp pretreatment in vitro, which were increased in murine macrophages with the stimulation of s. aureus [ , ] . on the contrary, friedl et al [ ] and lemmon et al [ ] showed that american gp extracts may mediate the immunostimulatory effect by the inducible nitric oxide synthase (inos), mitogen-activated protein kinase (mapk) kinases such as p , extracellular signal-regulated kinases / (erk / ), phosphoinositide -kinase (pi k), and nuclear factor kappa b (nf-kb) signaling pathways [ , ] . therefore, these results suggest that gps might be associated with the ability of the extract's ps fractions to bind to tlr receptor and upregulate or downregulate tlr receptor expression, which triggers or inhibits the intracellular signaling cascades and the production of proinflammatory mediators under basal or lps endotoxic conditions, respectively. animal lectins are carbohydrate-binding proteins which are highly variable in their amino acid sequences, widely distributed in microorganisms, viruses, animals and higher plants with different functions, structures, tissue localizations, and carbohydratebinding specificities [ ] . animal lectins were discovered before plant lectins, although many were not recognized as carbohydrate binding proteins for many years after first being reported [ ] . although plant and animal lectins do not have homologous primary structures, they have similar preferential binding to carbohydrates [ ] . animal lectins are neither immune origin nor catalyst, in contrast to antibodies or enzymes, and are able to detect or bind complex carbohydrate structures specifically through the carbohydrate-recognition domain (crd) [ , ] . each animal lectin possess its own crd which has an identical sequence motif of to amino acid residues and four cysteines that is thoroughly conserved and form two disulfide bonds [ , ] . animal lectin activity is found in association with an astonishing diversity of primary structures [ ] . at least structural families are known to exist and bind structures other than carbohydrates via protein-protein, protein-lipid or protein-nucleic acid interactions [ ] . their roles in glycol-recognition systems include complement activation, cell recognition, cell adhesion, cell migration, cell signaling, and morphogenesis. moreover, animal lectins are able to take part in defense mechanisms, importantly by recognizing a carbohydrate of pathogens [ , ] . animal lectins are means of attachment to various cells or viruses via the surface carbohydrate types of the cells to be attached [ ] . the function in recognition or cell surface interaction of animal lectins has been implicated in direct first-line defense against pathogens [ ] . for example, the mannose specific receptor, presented on the surface of macrophages, can bind to the infectious organisms which expose mannose-containing glycans on their surface, enabling them to ingest and kill the foreign organisms [ ] . in addition, animal lectins are involved in cell trafficking, immune regulation, and prevention of autoimmunity [ ] . animal lectins are classified into four families based on structure or function. these are the c-type (calcium-requiring) lectins, p-type (mannose- -phosphate binding) lectins, s-type (galectins) lectins, and i-type (immunoglobulinlike) lectins ( fig. ) [ ] . within each family, they have similar sequences and structural properties [ ] . recently, other lectin types have been found, including m-type, l-type, chitinase-like, and ftype lectins. in this review, four traditional families of animal lectins are introduced (table ). c-type lectins are endocytic receptors which are mostly expressed by macrophages, dcs, and some endothelial cells. c-type lectins require ca þ for activity and have common sequence motif of invariable and highly conserved amino acid residues [ , ] . as they have multi crds, c-type lectins are able to recognize a wide range of carbohydrate-based ligands from endogenous molecules to conserved structures found in bacteria, fungi, virusinfected cells, and parasites called pathogen-associated molecular patterns [ ] . after recognition, c-type lectins subsequently participate in the uptake for degradation in order to facilitate direct elimination by macrophages or antigen presentation by dcs and macrophages in major histocompatibility complex (mhc) molecules at the cell surface, resulting in stimulating the adaptive immune system [ , ] . for example, c-type lectins can recognize diverse bacterial pathogens and induce cytokine production and th responses in antibacterial immunity [ ] , and are critical in systemic infections with pathogens like cryptococcus neoformans in antifungal immunity of th effector cells [ ] . in addition, they are involved in clearance, homeostasis, and immunomodulation [ ] . many c-type lectins play primary roles in immunity. c-type lectins include dectins, dc-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign), sign-r , mannose receptor (mr), collectin (mbl, sp-a, sp-d) and selectins (l-, p-, e-) ( table ). dectin- specifically binds to b- , and b- , linked glucans of fungi, plant cell walls, and bacteria, including candida albicans, saccharomyces cerevisiae, coccidioides posadasii, and pneumocystis carinii, but cannot recognize monosaccharides or glucans with other linkages. however, c. neoformans, histoplasma capsulatum, and aspergillus fumigatus are not targeted by dectin- , in spite of the presence of b-glucans in their cell wall [ , ] . dectin- plays a primary role in inducing proinflammatory mediators like tnf-a in response to fungal pathogens. also, dectin- contains an immunotyrosine activation motif within its cytoplasmic tail, helping tlr signaling pathway by interaction with the immunotyrosine activation motif of dectin- [ ] . signaling by dectin- regulates various cellular responses including phagocytosis and the production of inflammatory cytokines such as ifns, il- , il- , and il- [ ] . dectin- expressed on dcs and macrophages can recognize n-glycans of the surface of tumor cells, following nuclear translocation, and the induction of several genes such as inam, which is known to induce tumor killing by nk cells by hemophilic interactions. dc-sign was originally identified as a receptor for intercellular adhesion molecule- (icam- ) that induces dc-mediated t-cell proliferation [ ] . it was subsequently unveiled to bind icam- on vascular endothelial cells, regulating dcs migration through interaction of n-linked high mannose structures consisting of from five to nine terminal mannose units [ ] . dc-sign, which is expressed by dcs, decidual and alveolar macrophages facilitates high-affinity binding to high mannose oligosaccharides through tetramerization [ ] . mannose-dependent interactions demonstrate the ability of dc-sign to bind human immunodeficiency virus (hiv) and various pathogens, including mycobacterium tuberculosis, c. albicans, leishmania mexicana, a. fumigatus, helicobacter pylori, and schistosoma mansoni [ ] . for instance, dc-sign recognizes mannosylated lipoarabinomannan, which is a mannose-capped glycolipid found in the cell wall of m. tuberculosis. this interaction can induce the secretion of the immunosuppressive cytokine, il- , from dcs which expresses dc-sign [ ] . dc-sign expression is mostly induced by il- , and is downregulated by ifn-g, tgfb, and dexamethasone [ , ] . the murine dc-sign homologues were reported to help to identify the roles of dc-sign in infection and inflammation and to play important roles in bacterial, fungal, and parasitic infections. there are seven mouse genes in the mouse dc-sign locus, containing sign-r - and sign-r , , and a pseudogene, sign-r [ , ] . the mrna of three sign-r genes encode type ii transmembrane proteins (sign-r , amino acids; sign-r , amino acids; sign-r , amino acids), but sign-r gene only encodes a crd without a cytosolic domain and a transmembrane domain (sign-r , amino acids) [ ] . amino acid sequence similarities between the crd of human dc-sign and the murine homologues are % for sign-r , % for sign-r , % for sign-r , and % for sign-r [ ] . sign-r , a murine homologous transmembrane of the dc-sign, is expressed by splenic marginal zone macrophages and peritoneal macrophages [ , ] . similar to dc-sign, sign-r is essential for the recognition and clearance of streptococcus pneumoniae-derived capsular pss. moreover, it can also recognize c. albicans, m. tuberculosis, s. pneumoniae cps, hiv, and yeast derived zymosan particles in a mannose inhibitable manner [ ] . sign-r directly binds to c q and dominantly regulates the immunoglobulinindependent classical complement pathway for c deposition of blood borne s. pneumoniae [ ] . in sign-r deficient mice, c deposition is abolished and innate resistance against pneumococci is reduced [ , ] . also, sign-r interacts specifically with , sialylated fc fragments of immunoglobulins, resulting in the antiinflammatory activity of intravenous immunoglobulin, which has been widely used to treat autoimmune diseases including immune mr (cd ) is described in myeloid cells and functions as a viral recognition receptor on the cell membrane for yeast, bacteria, hiv, and a wide variety of pathogens, such as c. albicans, leishmania donovani, p. carinii, klebsiella pneumonia, trypanosoma cruzi, m. tuberculosis [ e ] and capsular pss of s. pneumoniae through a mannose-type crd and pathogen-associated high mannose structures [ ] . mr which is mainly expressed in immune cells induces uptake and presents mannosylated antigens such as lipoarabinomannan on mhc class ii of metallophilic macrophages, resulting in influencing immune responses [ ] . for instance, the interaction between mr and hepatitis b virus (hbv) surface antigen (hbsag) enhances viral uptake by dcs, resulting in the impairment in the function of dcs and the ineffective antiviral response of chronic hbv [ ] . the recognition of viral surface glycoproteins by mr is also beneficial to influenza virus [ ] and hiv [ ] invasion into host cells. in addition, mr is able to mediate the clearance of endogenous inflammatory glycoproteins bearing ligands of the mannose-type crd [ ] . the expression of mr is upregulated by cytokines like il- , il- , and il- , but ifn induces a downregulatory effect to mr [ ] . selectins are cell adhesion molecules and have three groups, including e-selectin (endothelial) and p-selectin (platelet) on endothelium, and l-selectin (leukocyte) on leukocytes [ ] . selectins play roles in leukocyte recruitment from the bloodstream into sites of inflammation [ , ] . the recruitment of leukocytes proceeds initially by attachment leading to the rolling of leukocytes along endothelial vasculature via selectin-carbohydrate interaction. e-selectin (m.w. kda) is expressed by endothelial cells after stimulation with activators like tnf-a, il- , or bacterial lipopolysaccharide [ ] . these cytokines also upregulate the expression of p-selectin (m.w. kda), which is expressed by endothelial cells and platelets [ ] . also, p-selectin in released to the cell surface from storage vesicles in endothelial cells and platelets minutes after stimulation by a number of activators, such as thrombin or histamine [ ] . l-selectin (m.w. e kda) is expressed by leukocytes and aids in the homing of leukocytes. l-selectin has high expression on naïve t lymphocytes but, once t lymphocytes are activated, the expression of l-selectin is low or lacking [ ] . e-, p-, and l-selectin are composed of an n-terminal c-type lectin crd, an epidermal growth factor-like subunit, a number of short consensus repeat units, a membrane spanning region, and a c-terminal cytoplasmic tail [ ] . there is approximately % homology for analogous selectin crds across species, and w % homology between different selectins within a species [ ] . . . . p-type lectins p-type lectins are intracellular transmembrane glycoproteins with specificity for mannose- -phosphate (m p) to identify and route lysosomal enzymes to the lysosomal compartment (mpr signal) and they have two groups. one is the e kda cationdependent m p receptor (cd-mpr) which requires ca þ for activity and contains single extracellular domain, followed by a single transmembrane domain [ ] . the other is the e kda table a summary of the c-type lectin receptors dealt with in this review insulin-like growth factor ii/cation-independent m p receptor (igf-ii/ci-mpr) which does not require cation for activity and has a large extracellular domain containing two high-affinity binding sites [ ] . the crds of p-lectins for m p are located in the extracellular domain of cd-mpr and in extracellular repeats and with high affinity for igfii/ci-mpr [ ] . they are similar both in size and in sequence to the repeating units that consisted of short nterminal extracellular domain and c-terminal cytoplasmic tail [ ] . their function is the intracellular targeting of lysosomal enzymes (lysosomal hydrolases) in the trans-golgi network vertebrate animals and delivering them to prelysosomal compartments [ ] . also, the c-terminal cytoplasmic tail of the receptor which targets amino acid sequence plays a role in recognizing signal for transport to the endosomal compartment [ ] . igf-ii/ci-mpr has the capacity for endocytosis of ligands from the cell surface and serves to turn over igf by endocytosis, but not cd-mpr [ , ] . s-lectins, galectins (from to ), consist of globular galectintype crds which are specific for b-galactoside ligands and have conserved cysteine residues [ ] . they are found predominantly in mammals, but not in plants [ ] . s-lectins have a relatively simple structure and share a highly homologous domain named the scarbohydrate recognition domain (s-crd) [ ] . s-lectins mostly contain multiple sugar-binding sites, as the presence of two type of s-crd in a single polypeptide or its dimer [ ] . the function of slectins may be to crosslink n-acetyllactosamine-containing structures found at cell surfaces or in the extracellular matrix [ ] . s-lectins are conserved in unrelated organisms including frogs, birds, and mammals, meaning that b-galactoside binding to lectin may be important biologically [ ] . mammalian s-lectins are conserved in eight residues of the s-crds and involved in growth regulation, cell adhesion, cell migration, and immune responses [ ] . for example, galectin- mediates cell adhesion and apoptosis, and regulates cellular proliferation. galectin- mediates cell adhesion, regulates inflammation, pre-mrna splicing, and protects against induced apoptosis. galectin- and galectin- have a function in cellÀcell and cellÀextracellular matrix crosslinking and galectin- is involved in maturation and erythrocyte adhesion [ , ] . . . . i-type lectins i-type lectins are members of the immunoglobulin (ig) superfamily [ ] . they share the structural motif, the ig fold, with ig-like domains consisted of similar two planes of b-pleated sheets [ ] . the b-sheets are established about e amino acids, crosslinked by a disulfide bond and contain several hydrogen-bonded antiparallel chains [ ] . i-type lectins are classified into two domains according to the number and arrangement of b-strands present in the domains [ ] . one is the amino terminal, extracellular domain, which is similar to the variable region (v-type domain) of igg and is necessary for sialic acid-dependent binding [ ] . the other is the constant region (c-type domain) of igg that has various forms from to [ ] . i-type lectins function as not only cell adhesion molecules but also growth factor receptors and extracellular matrix molecules [ ] . the major subclasses of i-type lectins are the sialic acid-binding immunoglobulin superfamily lectins (siglecs) which contain an homologous n-terminal v-type domain with the sialic acid binding site and variable numbers of c-type domains [ ] . v-type domain and adjacent c-type domains of siglecs have conserved cysteine residues, resulting in formation of conventional intrasheet and interdomain disulfide bonds [ ] . the c-terminal cytoplasmic tail of most siglecs has immunoreceptor tyrosine-based motifs in the intracellular domain for signaling events [ ] . sialoadhesin found on the surface of macrophages is a member of the siglecs family [ ] . it contains a v-type domain and c-type domains [ ] . the n-terminal v-type domain of sialoadhesin enables binding of sialic acid in the ligands of neutrophils, monocytes, nk cells, b cells, and cytotoxic t cells with sialoadhesin [ ] . the diverse roles of botanical pss have been reported. the antitumor effect of botanical pss was first known more than years ago when it was found that pss could alleviate cancer in cancer patients [ ] . for example, lentinan from lentinus edodes and schizophyllan from schizophyllum commune have antitumor activities and have been used clinically for cancer therapy [ ] . through several experiments, it was suggested that the antitumor effects of botanical pss might be due to potentiation of the response of precursor t cells and macrophages to cytokines produced by lymphocytes after specific recognition of tumor cells [ ] . also, botanical pss of mushroom are known to stimulate natural killer cells, t cells, b cells, and macrophage-dependent immune system responses [ ] . in addition, arabinogalactans of botanical pss possess complement fixation activity and induce chemotaxis of human macrophages, t cells, and nk cells [ ] . although the roles of botanical pss have been continuously identified, the mechanism of action is not clear yet, because the receptors of botanical pss remain unknown. however, many scientists have proposed that various pattern recognition receptors (prrs) might be receptors for botanical pss. for example, shao et al [ ] suggested that the receptor of ps from the roots of astragalus membranaceus, a medicinal herb, might be mr, tlr , b-glucan receptor, etc. schepetkin and quinn [ ] also introduced prrs including tlr , mr, and dectin- as potential receptors of ps polymers. especially, it was reported that specific glycans of botanical pss act as immune stimulating agents and effective t cell immune adjuvants [ , ] . therefore, it was suggested that animal lectins are strong candidates for receptors of botanical pss among various prrs, because animal lectins are specialized in recognizing various pss [ ] . with extensive research for gps, the roles and chemical composition of gps have gradually been discovered. for instance, the gps of p. ginseng, the most common ginseng, are composed of sugars including mannose of . w . % by weight, glucose of . w . % by weight, galactose of . w . % by weight, and arabinose of . w . % by weight [ ] . in addition, cvt-e , an aqueous extract of the roots of north american ginseng, is composed of % poly-furanosyl-pyranosyl-saccharides including rhamnose, glucose, galacturonic acid, galactose, and arabinose [ ] . therefore, some of the animal lectins might recognize these glycan structures of gps, because there are many animal lectins to recognize glycan structures of galactose, glucose, rhamnose, and mannose [ e , , , , , ] . this could be speculated from the extensive research of specific receptors for botanical pss in the above section. in particular, clustering of animal lectins might enhance the recognition of gps, since clustering of simple binding sites in oligomers of the animal lectin polypeptides dramatically increases the affinity for diverse ps structures [ ] . for example, byeon et al [ ] expected dectin- as a receptor of red ginseng acidic ps, which are known to interact with ps fractions such as bglucan and zymosan [ , ] . gps which are isolated from a variety of traditional medicinal ginsengs involved in various innate immune responses, such as the production of cytokines and maturation of dcs in vivo and in vitro, show potential to be immunomodulators with wide applications [ e ]. in addition, most of them are relatively nontoxic and do not cause significant side effects, which are major problems of immunomodulatory bacterial pss and synthetic compounds. thus, gps are becoming ideal candidates for therapeutics for collageninduced arthritis, inflammation, and inflammatory-related diseases like tumors, atherosclerosis, and so on [ , , , , ] . although the roles of gps are being continuously identified, the detailed mechanisms of actions are not clear yet, because the receptors of gps remain largely unknown. therefore, it is tempting to speculate that animal lectins could be strong candidates of receptors for gps. to prove this possibility, extensive researches for the specific structures of gps and the interaction between gps and animal lectins is required in the near future. by unraveling receptors of gps in vivo, it is possible to specifically understand the detailed mechanism for the immunological activities of gps in the immune system, giving insights into the development of gps as therapeutic biomaterials for many immunological diseases. the authors declare no conflicts of interest. inflammation, cancer, and targets of ginseng recent methodology in the phytochemical analysis of ginseng structural and antiinflammatory characterization of a novel neutral polysaccharide from north american ginseng (panax quinquefolius) total fractionation and analysis of polysaccharides from leaves of panax ginseng inflammation and native american medicine: the role of botanicals bioactivity-guided identification and cell signaling technology to delineate the immunomodulatory effects of panax ginseng on human promonocytic u cells effect of cvt-e (cold-fx) versus a ginsenoside extract on systemic and gut-associated immune function immunomodulating activities of polysaccharides isolated from panax ginseng cytoprotective activity of pectic polysaccharides from the root of panax ginseng extractable polysaccharides of panax quinquefolius l. (north american ginseng) root stimulate tnfalpha production by alveolar macrophages ginseng pharmacology: multiple constituents and multiple actions efficacy and safety of ginseng newly identified compounds from ginseng, is novel lysophosphatidic acids-protein complexes and activates g protein-coupled lysophosphatidic acid receptors with high affinity panax ginsengea non-organ-specific cancer preventive? structure and biological activities of the polysaccharides from the leaves, roots and fruits of panax ginseng c.a. meyer: an overview stimulation of nitric oxide synthesis by the aqueous extract of panax ginseng root in raw . cells red ginseng acidic polysaccharide (rgap) in combination with ifn-gamma results in enhanced macrophage function through activation of the nf-kappab pathway bioactive polysaccharides from plants a proprietary extract from north american ginseng (panax quinquefolium) enhances il- and ifngamma productions in murine spleen cells induced by con-a total fractionation and characterization of the water-soluble polysaccharides isolated from panax ginseng ca meyer antitumor activities and immunomodulatory effects of ginseng neutral polysaccharides in combination with -fluorouracil pectin-like acidic polysaccharide from panax ginseng with selective antiadhesive activity against pathogenic bacteria induction of secretory and tumoricidal activities in peritoneal macrophages by ginsan immunostimulating effects of acidic polysaccharides extract of panax ginseng on macrophage function immunomodulatory activity of ginsan, a polysaccharide of panax ginseng, on dendritic cells acidic polysaccharide from panax ginseng, ginsan, induces th cell and macrophage cytokines and generates lak cells in synergy with ril- activation of multiple effector pathways of immune system by the antineoplastic immunostimulator acidic polysaccharide ginsan isolated from panax ginseng protective effect of ginseng polysaccharides on influenza viral infection high molecular weight polysaccharides are key immunomodulators in north american ginseng extracts: characterization of the ginseng genetic signature in primary human immune cells characterization of two novel polysaccharides having immunological activities from the root of panax ginseng stimulation of interleukin- production by acidic polysaccharides from the root of panax ginseng enhancement of antitumor effects of paclitaxel (taxol) in combination with red ginseng acidic polysaccharide (rgap) the immunomodulator ginsan induces resistance to experimental sepsis by inhibiting tolllike receptor-mediated inflammatory signals cytotoxic effects of bilberry extract on mcf -gfp-tubulin breast cancer cells study on antitumor and immunomodulating activities of polysaccharide fractions from panax ginseng: comparison of effects of neutral and acidic polysaccharide fraction effect of ginseng polysaccharide on tnf-alpha and ifngamma produced by enteric mucosal lymphocytes in collagen induced arthritic rats inhibition of neutrophil respiratory burst and degranulation responses by cvt-e , the main active ingredient in cold-fx immunomodulating activity of cvt-e , a proprietary extract from north american ginseng (panax quinquefolium) protection of staphylococcus aureusinfected septic mice by suppression of early acute inflammation and enhanced antimicrobial activity by ginsan a placebo-controlled trial of a proprietary extract of north american ginseng (cvt-e ) to prevent acute respiratory illness in institutionalized older adults ginseng extract inhibits protein degradation and stimulates protein synthesis in human fibroblasts toll-like receptors and innate immunity distribution and function to plant lectins animal lectins: a historical introduction and overview a glycobiology review: carbohydrates, lectins and implications in cancer therapeutics lectins: carbohydrate-specific proteins that mediate cellular recognition structure and function of mammalian carbohydrate-lectin interactions a novel c-type lectin regulating cell growth, cell adhesion and cell differentiation of the multipotent epithelium in budding tunicates insight of lectinsea review biology of animal lectins animal lectins the c-type lectin receptor clecsf (clec d) is expressed by myeloid cells and triggers cellular activation through syk kinase how c-type lectins detect pathogens dual function of c-type lectin-like receptors in the immune system c-type lectins in immunity: recent developments pattern recognition receptors: doubling up for the innate immune response divergent roles for c-type lectins expressed by cells of the innate immune system c-type lectins and phagocytosis dc-sign (cd ) expression is il- dependent and is negatively regulated by ifn, tgf-beta, and anti-inflammatory agents c-type lectins for infection widely divergent biochemical properties of the complete set of mouse dc-signrelated proteins five mouse homologues of the human dendritic cell c-type lectin, dc-sign a dominant complement fixation pathway for pneumococcal polysaccharides initiated by sign-r interacting with c q sign-r contributes to protection against lethal pneumococcal infection in mice identification of a receptor required for the anti-inflammatory activity of ivig macrophage mannosyl fucosyl receptor: its role in invasion of virulent and avirulent l. donovani promastigotes relationships among capsular structure, phagocytosis, and mouse virulence in klebsiella pneumoniae trypanosoma cruzi amastigote adhesion to macrophages is facilitated by the mannose receptor recognition of bacterial capsular polysaccharides and lipopolysaccharides by the macrophage mannose receptor ligand recognition by antigen-presenting cell c-type lectin receptors the mannose receptor acts as hepatitis b virus surface antigen receptor mediating interaction with intrahepatic dendritic cells involvement of the mannose receptor in infection of macrophages by influenza virus oligomerization of the macrophage mannose receptor enhances gp -mediated binding of hiv- new structural insights into lectin-type proteins of the immune system traffic signals for lymphocyte recirculation and leukocyte emigration: the multistep paradigm selectins and their ligands: current concepts and controversies the two mannose -phosphate receptors transport distinct complements of lysosomal proteins lectins as defence molecules in vertebrates and invertebrates lectin-like proteins in model organisms: implications for evolution of carbohydrate-binding activity a year in the life of the immunoglobulin superfamily i-type lectins the treatment of malignant tumors by bacterial toxins as developed by the late william b. coley, m.d., reviewed in the light of modern research t-cell oriented immunopotentiator: its experimental and clinical applications and possible mechanism of immune modulation medicinal mushrooms as a source of antitumor and immunomodulating polysaccharides structural and immunological studies of a pectin and a pectic arabinogalactan from vernonia kotschyana sch a study on the immune receptors for polysaccharides from the roots of astragalus membranaceus, a chinese medicinal herb botanical polysaccharides: macrophage immunomodulation and therapeutic potential mushroom immunomodulators: unique molecules with unlimited applications immunomodulatory dietary polysaccharides: a systematic review of the literature specific medicinal plant polysaccharides effectively enhance the potency of a dcbased vaccine against mouse mammary tumor metastasis in-sung jung, investors; korea institute of radiological & medical sciences, assignee. composition comprising polysaccharide extracted from panax ginseng preventing and treating liver diseases. us patent diversity in recognition of glycans by f-type lectins and galectins: molecular, structural, and biophysical aspects structural basis of lectin-carbohydrate recognition the beta-glucan receptor dectin- functions together with tlr to mediate macrophage activation by mycobacteria molecular mechanism of macrophage activation by red ginseng acidic polysaccharide from korean red ginseng this paper was supported by konkuk university in . key: cord- -rppsmirp authors: carroll, maria v.; sim, robert b.; bigi, fabiana; jäkel, anne; antrobus, robin; mitchell, daniel a. title: identification of four novel dc-sign ligands on mycobacterium bovis bcg date: - - journal: protein & cell doi: . /s - - - sha: doc_id: cord_uid: rppsmirp dendritic-cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign; cd ) has an important role in mediating adherence of mycobacteria species, including m. tuberculosis and m. bovis bcg to human dendritic cells and macrophages, in which these bacteria can survive intracellularly. dc-sign is a c-type lectin, and interactions with mycobacterial cells are believed to occur via mannosylated structures on the mycobacterial surface. recent studies suggest more varied modes of binding to multiple mycobacterial ligands. here we identify, by affinity chromatography and mass-spectrometry, four novel ligands of m. bovis bcg that bind to dc-sign. the novel ligands are chaperone protein dnak, kda chaperonin- (cpn . ), glyceraldehyde- phosphate dehydrogenase (gapdh) and lipoprotein lprg. other published work strongly suggests that these are on the cell surface. of these ligands, lprg appears to bind dc-sign via typical proteinglycan interactions, but dnak and cpn . binding do not show evidence of carbohydrate-dependent interactions. lprg was also identified as a ligand for dc-signr (l-sign; cd ) and the m. tuberculosis orthologue of lprg has been found previously to interact with human toll-like receptor . collectively, these findings offer new targets for combating mycobacterial adhesion and within-host survival, and reinforce the role of dcsign as an important host ligand in mycobacterial infection. tuberculosis is the world's most prevalent infectious disease affecting a third of the global human population. the causative agent of tuberculosis, mycobacterium tuberculosis, avoids the destructive capacity of the host immune system by residing inside the phagosome of host mononuclear phagocytes (armstrong and hart, ; clemens and horwitz, ; sturgill-koszycki et al., ) . many studies have shown that m. tuberculosis, m. paratuberculosis and m. bovis bcg can bind to dendritic-cell-specific intercellular adhesion molecule- -grabbing non-integrin (dc-sign/cd ) to promote entry into human dendritic cells (dcs) and alveolar macrophages maeda et al., ; tailleux et al., ; pitarque et al., ; appelmelk et al., ) . a recent study indicates that a mutation of dc-sign causing lower expression is protective against tuberculosisinduced lung cavitation (vannberg et al., ) . dc-sign is a kda type ii transmembrane protein that consists of a carbohydrate recognition domain, neck domain, transmembrane domain and cytoplasmic tail. it is expressed mainly on dcs and on selected macrophage populations including alveolar macrophages (geijtenbeek et al., a; lee et al., ; maeda et al., ) . dc-sign is a calcium-dependent lectin and has a high affinity for mannosylated surfaces, forming tetrameric complexes when binding to high mannose glycoproteins, such as hiv gp (geijtenbeek et al., b; feinberg et al., ; mitchell et al., ; appelmelk et al., ) . dc-sign has been shown to bind lipolysaccharide le x and mannose structures found on bacteria, such as helicobacter pylori, klebsiella pneumonia and m. tuberculosis geijtenbeek et al., ; tailleux et al., ; van kooyk and geijtenbeek, ) . using purified cell wall components from mycobacteria, dc-sign was shown to bind lipoarabinomannan (lam) structures from m. tuberculosis, m. bovis and m. bovis bcg, all of which express mannose-capped lam (manlam). however, lam purified from m. smegmatis did not bind dc-sign, since it expresses uncapped lam, so-called aralam. similarly, lam from m. avium bound poorly to dc-sign since it expresses single mannose residue attachments and thus presents lower mannoside density maeda et al., ) . manlam was therefore believed to be the major ligand on m. tuberculosis for binding to dc-sign (maeda et al., ; tailleux et al., ) . however, later studies showed that removal of the mannose-cap in experiments using whole bacteria did not appear to have a dramatic effect on dc-sign binding. the faster growing mycobacteria such as m. smegmatis or m. avium could also bind dc-sign despite not having the mannose caps, suggesting that other components in the mycobacterial cell wall were also binding dc-sign. mannosylated lipoproteins found on the cell surface of mycobacteria such as kda lipoprotein lpqh/rv and a kda lipoprotein were shown to contribute to the binding of dc-sign to the bacteria (pitarque et al., ; appelmelk et al., ) . these studies have revealed that the binding interaction of dc-sign to m. tuberculosis is more complicated than originally perceived, and suggests that there may be more potential dc-sign ligands present on m. tuberculosis. in this study we set out to demonstrate dc-sign binding to m. bovis bcg as a model organism for m. tuberculosis. we explored the binding characteristics of dc-sign to whole m. bovis bcg and also observed the binding characteristics of a closely related protein, dc-signr (dc-sign-related/l-sign/cd ) to the mycobacterium. dc-signr shares % amino acid sequence identity with dc-sign (soilleux et al., ) . using affinity chromatography, we purified and identified four novel dc-sign binding ligands of m. bovis bcg: chaperone protein dnak (dnak), kda chaperonin- (cpn . ), glyceraldehyde- phosphate dehydrogenase (gapdh) and lipoprotein lprg. we set out first to confirm the binding of dc-sign to whole m. bovis bcg, using lung surfactant protein a (sp-a) and bsa as positive and negative controls respectively. we also compared the binding of dc-sign to that of dc-signr. by flow cytometry, we found that the binding of dc-sign and dc-signr to whole m. bovis bcg is dose-dependent ( fig. ) , reaching a maximum at a protein input of about μg per × cells ( fig. a) . sp-a also binds dosedependently, while bsa does not bind. binding of dc-sign and dc-signr are predominantly ca + -dependent, as binding is reduced by~ % in the presence of edta ( fig. b ) compared with binding in mm cacl . binding of sp-a appears less dependent on ca + ions, as binding is reduced < % in edta. mannose ( mm) inhibits the binding of dc-sign and sp-a by less than %, while binding of dc-signr is reduced by about % (fig. b ). these findings are compatible with the view that dc-sign, dc-signr and sp-a are all likely to be binding to several bacterial ligands and the results with mannose and edta suggest more than one mode of binding. for dc-signr, the results are consistent with its binding mainly (~ %) via its calcium-dependent carbohydrate binding site. for dc-sign and sp-a, a much smaller proportion ( %- %) of binding may be mediated via these sites, and other binding occurs via ca + -independent sites, and also via ca + dependent sites that do not constitute the canonical carbohydrate binding site. similar diversity for modes of binding of sp-a to viable and apoptotic mammalian cells has been observed previously (jäkel et al., a, b, c) . to identify macromolecules on the mycobacterial cell surface to which dc-sign is binding, m. bovis bcg lysates were passed through a dc-sign affinity chromatography column. bound proteins were eluted with buffer containing edta. the eluted proteins were then concentrated and resolved by sds-page. from the gel (fig. ) four visible bands can be seen at , , and kda. as a control m. bovis bcg lysates were passed through a control column made of underivatised sepharose in the same way. no protein was detected in the eluted fractions of the control column, indicating no non-specific binding interactions (not shown). the , , and kda bands were cut from the gel and analyzed by maldi-tof tryptic peptide fingerprinting mass spectrometry, and database searches carried out against both ncibr and swissprot. the bands were identified as chaperone protein dnak, kda chaperonin (cpn . ), glyceraldehyde- -phosphate dehydrogenase (gadph) and lipoprotein lprg, respectively ( table ). all of these have the same protein sequence in m. tuberculosis as in m. bovis bcg (table ) . two other minor candidates, ctp synthase and atp synthase beta subunit (table ) were not considered further. dnak and cpn . are collectively known as heat shock proteins or chaperone proteins. cpn . generated the highest protein score, with nine peptide sequences matched. these peptide sequences cover . % of the protein sequence (table ). the protein ran at~ kda on a sds-page gel and was calculated to have a mass of , da from the amino acid sequence ( fig. and table ). the second highest protein score was for dnak. this protein band produced four matching peptides sequences which contribute . % sequence coverage. it ran at~ kda on sds-page and had a calculated mass from the amino acid sequence of , da ( fig. and table ) . toward the c-terminal of cpn . , there is one possible nlinked glycosylation site at n as (fig. ) . this potential n-linked glycosylation site occurs in one of the cpn . peptides identified during mass spectrometry. this indicates that the site was not occupied by an oligosaccharide otherwise the peptide molecular mass would have been affected and unidentifiable during analysis. the site may be partially occupied indicating that there may be another population of this protein with an n-linked glycan present at n . however, the form of this protein identified after capture by the affinity column was not glycosylated at this position, and it is therefore very unlikely that dc-sign binds to this ligand via its ca + -dependent lectin activity. similarly, no potential n-linked glycosylation sites for dnak were found ( fig. ) , suggesting that it also is not bound to dc-sign via nglycans. from the current literature it is unknown whether these proteins undergo any o-linked glycosylation, but use of in silico o-glycosylation prediction tools available at the expasy (expert protein analysis system) proteomics server (http://expasy.org/tools/; gasteiger et al., ) indicates no predicted o-glycosylation in either protein. a recent study (hickey et al., ) showed that dnak is located at the cell-surface of m. tuberculosis. there are no published data on the localization of cpn . , but a related protein, cpn . was also shown to be on the cell surface of m. tuberculosis, and has a role in the adherence of m. tuberculosis to macrophages (hickey et al., ). cpn . and cpn . show % amino acid sequence identity (kong et al., ) . hickey et al. ( ) showed that macrophages formed specific interactions with m. tuberculosis, which could be inhibited by pre-incubation with increasing concentrations of cpn . or by blocking surface localized cpn . with f(ab') antibody. this was supported by showing that purified cpn . could bind to the surface of macrophages. although dnak was also shown to be located at the mycobacterial cellsurface, hickey et al. ( ) could not show consistent binding via dnak to macrophages using antibodies to block the reaction. this may have been due to a lack of appropriate anti-dnak antibodies. in listeria monocytogenes, dnak has been shown to facilitate phagocytosis of the pathogen into macrophages (hanawa et al., ) . the same authors observed that wild type bacteria were endocytosed more than dnak knockouts. once inside the macrophage dnak was shown not to be essential for multiplication within the cell although it was necessary for cell entry. studies looking at the pathogenic role of the dnak and its co-chaperone dnaj, in salmonella enterica serovar typhimurium revealed that they are both essential for internalising the bacteria within epithelial cells and survival within macrophages (takaya et al., ) . cpn . and cpn . are potent immunomodulatory proteins in the host. cpn . has been shown to be a more potent activator of stimulatory proinflammatory cytokines (friedland et al., ; lewthwaite et al., ; hu et al., ) . despite chaperones being more commonly known as cytosolic proteins, many pathogenic bacteria express these proteins at the cell-surface possibly to promote attachment to host cells and mediate internalization. cpn proteins have been reported to demonstrate these functions in helicobacter pylori, clostridium difficile, hemophilus ducreyi and salmonella enterica serovar typhimurium (yamaguchi et al., ; frisk et al., ; hennequin et al., ) . here we demonstrate that cpn . can also interact with dc-sign and propose that this could aid the entry of mycobacterial cells into dc or macrophage. gapdh was also identified as one of four dc-sign binding ligands in this study. running at~ kda on sds-page (fig. ) , gapdh was identified with three peptide matches, covering . % of the protein sequence. the calculated mass of the protein is , da and two potential n-linked glycosylation sites are present in the sequence, n st and n as (table , fig. ). these two potential nlinked glycosylation sites may be occupied by carbohydrate structures required for dc-sign binding via its crd. this protein has significant homology to the gapdh enzymes indentified in group a streptococcus, enteropathogenic e. coli, and candida albicans (parker and bermudez, ) . gapdh is an important enzyme in both prokaryotic and eukaryotic metabolism that catalyzes a step of glycolysis, converting glyceraldehyde- -phosphate to glycerate , -bisphosphate. gapdh is more commonly recognized as a cytosolic enzyme found on the inner surface of the cell membrane. even though there is no apparent signal sequence or stretch of hydrophobic residues to indicate a transmembrane region (fig. ) , studies have reported that a kda protein homologous to gapdh is expressed on the outer cell membrane of hematopoietic cells (allen et al., ) and also on many microorganisms such as group a streptococcus, enteropathogenic e. coli, candida albicans, mycobacterium avium and schistosoma mansoni (goudot-crozel et al., ; pancholi and fischetti, ; kenny and finlay, ; gil-navarro et al., ; parker and bermudez, ) . m. avium expresses gapdh on its cell surface, whereupon gapdh can bind to human epidermal growth factor. in the presence of recombinant human epidermal growth factor the rate of growth of m. tuberculosis and m. avium is rapidly increased (parker and bermudez, ) . another dc-sign ligand purified by affinity chromatography was identified as lprg, a kda lipoprotein. lprg actually runs with an apparent molecular weight of kda on sds-page (fig. ) and was identified with only one peptide hit with a protein score of . , covering . % of the protein sequence. the calculated mass of the protein is , da (fig. ) . the identification of lprg was supported by western blot analysis. as shown in fig. , in eluted fractions de - , de - and de - from dc-sign affinity chromatography, a strong band can be seen representing lprg. lprg has two potential n-linked glycosylation sites, one of which (n pt) is unoccupied or only partially occupied since it lies in one of the peptides identified by mass spectrometry. the other site, n at may be occupied. ligand blot analysis (fig. ) of whole m. bovis bcg lysate incubated with either i-dc-sign or i-dc-signr revealed that dc-sign and dc-signr both bind the same protein at around kda, which corresponds to lprg in our sds-page system, and is the only ligand detected by this method. dc-sign and dc-signr binding to lprg can therefore still occur when the mycobacterial protein has been denatured by sds-page. this strongly suggests that lprg binds to dcsign predominantly or entirely via protein-carbohydrate interactions. in other studies looking at the importance of lprg in m. tuberculosis, knockout of the lprg operon was shown to attenuate m. tuberculosis, indicating that it has a prominent role in the pathogenic behavior of the bacterium (bigi et al., ) . furthermore, lprg has been identified as a ligand for tlr- on macrophages, and lprg-tlr- interactions lead to reduced mhc class ii presentation (gehring et al., ) . there is also growing evidence indicating that intracellular signaling via dc-sign modifies transduction pathways downstream from tlrs, driving immunosuppressive responses (gringhuis et al., (gringhuis et al., , . several other m. tuberculosis lipoproteins that are either glycosylated or presumed to be glycosylated also have been identified as key antigens with immunomodulatory functions (herrmann et al., ) . lpqh ( kda) was confirmed to have seven o-linked glycosylation sites (herrmann et al., ) . it has the same protein sequence in m. tuberculosis as in m. bovis bcg and was previously identified as a ligand for dc-sign (pitarque et al., ) possibly binding via glycans. we were unable accurately to detect lipoproteins below bovis bcg lysate in mm hepes, mm nacl, mm cacl ph . . the sepharose was placed in a column and washed and bound proteins were eluted with mm hepes, mm nacl, mm edta ph . . eluted fractions were concentrated with strataclean beads and prepared in reducing conditions for analysis by sds-page. concentrated eluates were run on %- % gradient gel. as a negative control, underivatised sepharose was incubated with the lysate in the same way (results not shown). ls, µl of m. bovis bcg lysate; rt, µl lysate proteins not bound to the column ("run-through"); de - , concentrated eluted fractions - from the dc-sign column. bands marked by black arrows were used for mass spectrometry analysis. results are representative of three independent experiments. kda in the affinity chromatography experiment shown in fig. due to limitations in the sds-page system used, but in fig. (ligand blotting) no band in the position of lpqh is seen. this suggests either that lprg is a much better ligand (more abundant or higher affinity) or that lpqh does not bind via glycans. lprg binds to both dc-sign and dc-signr. dc-signr is expressed in the liver, lymph nodes but has also been described in the lung (pöhlmann et al., ; jeffers et al., ) . in humans, both dcs and alveolar macrophages express dc-sign in the lungs. although dc-signr has a different expression pattern from dc-sign, it has similar binding properties to dc-sign (bashirova et al., ; mitchell et al., ; pöhlmann et al., ) . while dc-sign has been shown to mediate endocytosis and protein trafficking as a recycling receptor and the release of bound ligand at reduced ph, dc-signr does not endocytose nor demonstrate ph-sensitive ligand binding (guo et al., ) . dc-sign has been implicated as an important receptor in the establishment of m. tuberculosis infection. although many dc-sign ligands have been identified at the cell-surface of the mycobacterium, studies suggested that there were more ligands present that had not yet been identified. here, we have shown dc-sign binds to whole m. bovis bcg in both ca + -dependent and ca + -independent modes. we have identified four novel ligands for dc-sign. of these only one, lprg appears to bind predominantly via the glycan binding site. lprg is also a ligand for dc-signr. dendritic cells present in the lung migrate in order to prime t lymphocytes in the lymph nodes. it is believed that m. tuberculosis resides within the phagosome of the dc and exploits the migration thereby circulating within the host undetected (fenton and vermeulen, ; henderson et al., ; banchereau and steinman, ) . the discovery of new dc-sign binding ligands: dnak, cpn . , gapdh and lprg, may help further research into designing inhibitors to prevent interactions between dc-sign and m. tuberculosis with the aim of blocking uptake and intracellular survival of mycobacterial cells. liquid cultures of mycobacterium bovis bcg (pasteur strain) were grown as described previously (carroll et al., ) in middlebrook h liquid medium containing . % (v/v) glycerol, . % (v/v) tween- , and % (v/v) albumin-dextrose-catalase (adc, bd bbl prepared culture medium: becton dickinson, oxford, uk). fresh cultures were inoculated from ml glycerol stock of m. bovis bcg to generate a ml culture. the 'first passage' was grown for four to five days at °c in roller bottles at rpm until the bacteria had reached the exponential growth phase (od nm = . − . ). only the first passages of the strains were used for experimental work. m. bovis bcg cell cultures ( ml) were harvested at exponential phase and cells were washed three times in mm nacl, . mm kcl, . mm na hpo and . mm kh po , ph . (pbs). cells were resuspended in ml mm tris, mm nacl, . % triton x- , ph . in the presence of protease inhibitors (protease inhibitor cocktail, roche diagnostics, mannheim germany) and kept on ice for min. the cells were then ribolysed in ribolysing tubes containing lysing matrix b (mpbiomedicals, illkirch, france) for s at speed setting . in a ribolyser (fastprep fp ). lysate was placed on ice for min before being spun down. to reduce viscosity, mycobacterial lysate was incubated with μg/ml of rnase a (r sigma aldrich,poole uk) for min at °c. lysate buffer was adjusted to . mm cacl , . mm mgcl and incubated with μg/ml dnase ii (d , sigma aldrich) for min at °c. the lysate was then stored at − °c until needed. recombinant, tetrameric dc-sign and dc-signr (complete extracellular domains, lacking the transmembrane segment) were made and purified as described previously . these were used in either unmodified, biotinylated or radioiodinated form. biotinylation was performed using n-hydroxysuccinimide biotin (sigma-aldrich, poole, uk) at a molar ratio of : reagent : protein at ph . , °c for min. radioiodination was done as a standard iodogen-catalyzed reaction (krarup et al., ) with µg of protein in pbs and uci of na i (ge healthcare, uk, product ims- ). sp-a was purified from human alveolar proteinosis broncho-alveolar lavage fluid as described by jäkel et al. ( a) . m. bovis bcg ( × cells) were fixed in . % paraformaldehyde in pbs, mm cacl . cells were washed in µl mm hepes, mm nacl, mm cacl , ph . (assay buffer) and resuspended in µl of the same buffer. cells were incubated with , , , µg of biotinylated-dc-sign or biotinylated-dc-signr for h at room temperature in assay buffer. incubations were also carried out in the presence of mm mannose and mm edta as potential inhibitors figure . western blot confirmation of lprg binding to dc-sign-sepharose. sds-page of concentrated eluted fractions were transferred to a pvdf membrane and blocked. the membrane was incubated with rabbit anti-lprg antiserum, then washed and incubated with goat anti-rabbit-horseradish peroxidase (hrp)-conjugated antibody. the membrane was washed and exposed to enhanced chemiluminescence western blot detection reagents. the bands were visualized by exposing the membrane to xray film for a few seconds. results are representative of independent experiments. ls, lysate; rt, run-through; de - , eluted fractions from the dc-sign column; ge - , eluted fractions from the guard (underivatised sepharose) column. of binding to m. bovis bcg. cells were washed and incubated with : dilution of streptavidin-pe solution ( bd pharmingen, oxford, uk) for min in µl assay buffer and fixed in µl of . % paraformaldehyde in pbs, mm cacl . binding to the cells was measured by flow cytometry using a facscan instrument (becton dickinson immunocytometry systems, san jose, ca, usa). aquisition and processing of data from , cells per sample were carried out with the cellquest software (becton dickinson). surfactant protein-a (sp-a) was used as positive control (downing et al., ; pasula et al., ; weikert et al., ) and was detected using a biotinylated anti-sp-a monoclonal antibody (antibodyshop, gentofte, denmark); biotinylated bsa was used as a negative control for binding to m. bovis bcg. soluble recombinant dc-sign extracellular domain protein ( ml, mg/ml) in mm hepes, mm nacl, mm cacl , ph . was incubated with ml hydrated cnbr-activated sepharose (ge healthcare, chalfont st. giles, uk) for h at room temperature with rotation. the resin was washed twice in m nacl and then incubated in ml mm ethanolamine, ph . for h at room temperature with rotation. the resin was washed twice in m nacl and stored in mm hepes, mm nacl, mm edta, ph . . fifteen percent of the dc-sign supplied remained unbound, as assessed by measuring protein od in the supernatant after binding. capacity of the dc-sign-sepharose for capturing glycoprotein ligand was confirmed using a test solution containing µg of yeast invertase ( % oligomannose by mass) loaded onto the column in ml of mm hepes, mm nacl, mm cacl ph . (equilibration buffer) and eluted with mm hepes, mm nacl, mm edta ph . (eluting buffer). successful capture and elution of ligand was visualized by sds-page. the dc-sign-sepharose column was regenerated with mm hepes, m nacl, mm edta ph . (regeneration buffer). the column was then equilibrated with equilibration buffer. lysate treated with rnase and dnase was diluted with one volume of mm hepes, mm nacl, . mm cacl , ph . to obtain ml with a protein concentration of about mg/ml. as a control, a second column ( ml) was made from underivatised sepharose (guard column) and prepared in equilibration buffer. lysate ( ml) was added to the guard column and the beads were stirred at intervals during an incubation period of h at °c. the lysate was then run off and loaded onto the dc-sign column. beads were resuspended and incubated with the lysate as above. both columns were washed exhaustively with equilibration buffer. bound ligands were eluted with eluting buffer and . ml fractions collected. eluted proteins were detected by reading od , and positive fractions were pooled and the protein concentrated by binding to µl strataclean beads (stratagene, cedar creek, tx, usa) per ml of eluted fraction. beads were incubated with eluates on a rotary stirrer for h. beads were spun down and prepared for analysis by sds-page. sds-page was performed using the invitrogen nupage® system (invitrogen, cambridge, uk). samples were prepared as described by fairbanks et al. ( ) . a total of µl strataclean beads per concentrated fraction were prepared in reducing conditions for sds-page and loaded per well. sds-page was run with seeblue ® plus prestained standard (invitrogen) to facilitate band size estimation. protein bands were transferred to a polyvinylidene fluoride (pvdf) microporous membrane (millipore, billerica, massachusetts, usa) in mm tris-hcl, mm glycine, % (v/v) methanol, ph . (transfer buffer) for h using a semi-dry blotter (whatman international ltd. banbury, uk). the membrane was blocked with pbs, . % tween- , mg/ml bsa for h. the membrane was washed with pbs, . % tween- , . mm edta (washing buffer) and incubated with : dilution of rabbit anti-lprg antiserum (bigi et al., ) in pbs, mg/ml bsa for h at room temperature. the membrane was washed in washing buffer and incubated with : , dilution goat anti-rabbit-horseradish peroxidase-conjugated antibody (sigma aldrich, a ) in pbs, mg/ml bsa for h. the membrane was washed in washing buffer and exposed to enhanced chemiluminescence western blot detection reagents (ge healthcare) for detection. bands were detected by exposing the membrane to x-ray film. sds-page of reduced m. bovis bcg lysate was run and protein bands were transferred to a pvdf microporous membrane and blocked as above. the membrane was washed with mm hepes, figure . radiolabelled dc-sign and dc-signr binding to m. bovis bcg blot. sds-page of m. bovis bcg lysate was run and protein bandswere transferred to a pvdf membrane, blocked and incubated with ml of , dpm/ml of either i-dc-sign or i-dc-signr. the bands were then visualized by exposing the membrane to x-ray film for week. mm nacl, mm cacl , . % tween- ph . and incubated with ml of , dpm/ml of either i-dc-sign or i-dc-signr for h at room temperature. the membrane was washed with mm hepes, mm nacl, mm cacl , . % tween- ph . and bands were visualized by exposing the membrane to x-ray film in a lightproof cassette for week. protein bands from sds-page gels were stained with either safestain (invitrogen) or coomassie blue r- stain (fairbanks et al., ) and destained in % (v/v) acetic acid, % (v/v) ethanol. individual bands were excised and subjected to ms-ms analysis. mass spectrometric analysis was carried out using a q-tof (micromass, manchester, uk) coupled to a caplc (waters, milford, usa). in-gel trypsin digestion was carried out as described by shevchenko et al. ( ) . tryptic peptides were concentrated and desalted on a µm id/ mm c pre-column and resolved on a µm id/ cm c pepmap analytical column (lc packings, san francisco, ca, usa). peptides were eluted to the mass spectrometer using a min %- % (v/v) acetonitrile gradient containing . % (v/v) formic acid at a flow rate of nl/min. spectra were acquired in positive mode with a cone voltage of v and a capillary voltage of v. the ms to ms/ms switching was controlled in an automatic data-dependent fashion with a s survey scan followed by three s ms/ms scans of the most intense ions. precursor ions selected for ms/ms were excluded from further fragmentation for min. spectra were processed using proteinlynx global server . . and searched against the swissprot_ . and ncbinr_ databases using the mascot search engine (matrix science, london, uk). database searches were performed with the taxonomy restricted to mycobacteria. carbamidomethyl cysteine was set as a fixed modification and oxidised methionine as a potential variable modification. data was searched allowing . da error on all spectra and up to one missed tryptic cleavage site. identification of the -kda protein displaying a variable interaction with the erythroid cell membrane as glyceraldehyde- -phosphate dehydrogenase cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific icam- -grabbing nonintegrin on dendritic cells the mannose cap of mycobacterial lipoarabinomannan does not dominate the mycobacterium-host interaction phagosome-lysosome interactions in cultured macrophages infected with virulent tubercle bacilli. reversal of the usual nonfusion pattern and observations on bacterial survival dendritic cells and the control of immunity a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection a novel kda lipoprotein antigen from mycobacterium bovis the knockout of the lprg-rv operon produces strong attenuation of mycobacterium tuberculosis multiple routes of complement activation by mycobacterium bovis bcg characterization of the mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited surfactant protein a promotes attachment of mycobacterium tuberculosis to alveolar macrophages during infection with human immunodeficiency virus electrophoretic analysis of the major polypeptides of the human erythrocyte membrane structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr immunopathology of tuberculosis: roles of macrophages and monocytes mycobacterial -kd heat shock protein induces release of proinflammatory cytokines from human monocytic cells groel heat shock protein of haemophilus ducreyi: association with cell surface and capacity to bind to eukaryotic cells expasy: the proteomics server for in-depth protein knowledge and analysis mycobacterium tuberculosis lprg (rv c): a novel tlr- ligand that inhibits human macrophage class ii mhc antigen processing identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-sign, a dendritic cell-specific hiv- -binding protein that enhances transinfection of t cells mycobacteria target dc-sign to suppress dendritic cell function the glycolytic enzyme glyceraldehyde- -phosphate dehydrogenase of candida albicans is a surface antigen the major parasite surface antigen associated with human resistance to schistosomiasis is a -kd glyceraldehyde- p-dehydrogenase c-type lectin dc-sign modulates toll-like receptor signaling via raf- kinase-dependent acetylation of transcription factor nf-kappab carbohydrate-specific signaling through the dc-sign signalosome tailors immunity to mycobacterium tuberculosis, hiv- and helicobacter pylori structural basis for distinct ligand-binding and targeting properties of the receptors dc-sign and dc-signr the listeria monocytogenes dnak chaperone is required for stress tolerance and efficient phagocytosis with macrophages activation of human dendritic cells following infection with mycobacterium tuberculosis groel (hsp ) of clostridium difficile is involved in cell adherence analysis of post-translational modification of mycobacterial proteins using a cassette expression system mycobacterium tuberculosis cpn . and dnak are located on the bacterial surface, where cpn . facilitates efficient bacterial association with macrophages a mycobacterium tuberculosis mutant lacking the groel homologue cpn . is viable but fails to induce an inflammatory response in animal models of infection the human lung surfactant proteins a (sp-a) and d (sp-d) interact with apoptotic target cells by different binding mechanisms surfactant protein a (sp-a) binds to phosphatidylserine and competes with annexin v binding on late apoptotic cells surfacebound myeloperoxidase is a ligand for recognition of late apoptotic neutrophils by human lung surfactant proteins a and d cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus protein secretion by enteropathogenic escherichia coli is essential for transducing signals to epithelial cells mycobacterium tuberculosis expresses two chaperonin- homologs simultaneous activation of complement and coagulation by mbl-associated serine protease cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd and a coreceptor mycobacterium tuberculosis chaperonin . is a more potent cytokine stimulator than chaperonin . (hsp ) and contains a cd -binding domain the cell surface receptor dc-sign discriminates between mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr. subunit organization and binding to multivalent ligands a major surface protein on group a streptococci is a glyceraldehyde- -phosphate-dehydrogenase with multiple binding activity sequence and characterization of the glyceraldehyde- -phosphate dehydrogenase of mycobacterium avium: correlation with an epidermal growth factor binding protein surfactant protein a (sp-a) mediates attachment of mycobacterium tuberculosis to murine alveolar macrophages deciphering the molecular bases of mycobacterium tuberculosis binding to the lectin dc-sign reveals an underestimated complexity dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans in-gel digestion for mass spectrometric characterization of proteins and proteomes dc-sign; a related gene, dc-signr; and cd form a cluster on p mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells the dnak/dnaj chaperone machinery of salmonella enterica serovar typhimurium is essential for invasion of epithelial cells and survival within macrophages, leading to systemic infection dc-sign: escape mechanism for pathogens cd genetic polymorphism and tuberculosis disease sp-a enhances uptake of bacillus calmette-guérin by macrophages through a specific sp-a receptor flow cytometric analysis of the heat shock protein expressed on the cell surface of helicobacter pylori cpn . , kda chaperonin- ; dc, dendritic cell; dc-sign/cd , dendritic-cell-specific intercellular adhesion molecule- -grabbing non-integrin; gapdh, glyceraldehyde- -phosphate dehydrogenase key: cord- - ny j ny authors: cuddon, paul a title: the weak and ataxic or paralyzed cat date: - - journal: problem-based feline medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ny j ny nan ataxia and paresis occur when there is physical or functional disruption to both the motor and sensory pathways of the nervous system. paresis is a deficit of voluntary movement, leading to weakness in one limb (monoparesis), both pelvic limbs (paraparesis), the limbs on one side of the body (hemiparesis), or all four limbs (tetraparesis). it is caused by disruption of the voluntary motor pathways anywhere from the cerebral cortex through the brainstem and spinal cord to the spinal cord segments and the peripheral nerves supplying muscles. neurological testing to determine whether a cat is paretic includes gait analysis and postural reaction testing, which includes hopping, wheel-barrowing, hemi-standing/hemi-walking, and the extensor postural thrust. the term paralysis is reserved for the patient who has a complete loss of any voluntary movements. ataxia is a lack of coordination of the limbs or trunk produced by disruption of the sensory proprioceptive pathways of the spinal cord and brainstem. lesions of the cerebellum and the vestibular system also can produce ataxia. sensory ataxia, the disruption of sensory proprioceptive pathways, can be assessed via such tests as proprioception and tactile placing. most cats with spinal cord disease have a combination of both ataxia and paresis, since most myelopathies cause disruption of both the motor and sensory systems. in many circumstances, therefore, separation of ataxia and paresis becomes almost impossible on routine neurological examination. cats presenting solely with ataxia and paresis/paralysis most commonly have spinal cord disease. weakness and ataxia can also occur with cerebral and brainstem disease. • however, cats with cerebral disease will usually show seizures, behavior change, aimless wandering, and pacing along with the ataxia and contralateral hemiparesis or tetraparesis. • cats with brainstem disease will demonstrate cranial nerve abnormalities as well as an ipsilateral hemiparesis or tetraparesis. most polyneuropathies manifest as generalized weakness without ataxia. in many cases of peripheral nerve disease, sensory tests such as proprioception and tactile placing will be normal despite obvious muscle weakness. localization of myelopathies is dependent on determining whether the thoracic and pelvic limbs have upper (umn) or lower motor neuron (lmn) signs. cats with umn paresis will demonstrate normal to increased segmental spinal reflexes (myotatic or tendon reflexes, muscle tone and withdrawal reflexes) whereas cats with lmn paresis will show decreased to absent segmental spinal reflexes. an umn bladder, likewise, will have increased tone and be difficult to express due to external (+/− internal) urethral sphincter hypertonia, whereas a lmn bladder will be flaccid (or atonic) and easily expressed due to external urethral sphincter hypo-to atonia. the classic spinal cord divisions when localizing a myelopathy are as follows: • cervical spinal cord (c -c ) -umn tetraparesis to tetraplegia. • cervical intumescence (c -t ) -lmn signs to thoracic limbs and umn signs to pelvic limbs. the most common causes of spinal cord ataxia and paresis in cats are infectious (including feline infectious peritonitis virus (coronavirus)), neoplasia (lymphosarcoma) and trauma. primary spinal neoplasia (meningioma), inflammation (feline polioencephalomyelitis), and ischemia (aortic thromboembolism and fibrocartilaginous embolism) are less common. intervertebral disc disease is very rare. spinal lymphosarcoma*** • chronic progressive ataxia. • asymmetric paraparesis. • focal areas of spinal pain. lymphosarcoma is the most common feline neoplasia. - % of cats with lymphoreticular malignancies develop neurologic involvement. % of cats with cns involvement show thoracic and lumbar myelopathy, although the brain may also be affected. epidural lymph channels and extramedullary hematopoietic tissue are possible sites for development of primary spinal lymphosarcoma. tumor growth commonly occurs longitudinally along the spinal canal (the epidural space is a low-resistance channel). % of cats with spinal lymphosarcoma have solitary epidural lesions. multifocal lesions are also possible, as is tumor involvement of multiple nerve roots. spinal lymphosarcoma is most commonly seen in young cats (≤ years). males are more commonly affected. most cats present with an initial insidious course of neurologic dysfunction followed by acute deterioration. tumors occur predominantly between t -l . involvement of the brachial intumescence and plexus also can occur. cervical spinal cord involvement is rare. the most common clinical signs include progressive ataxia, asymmetric paraparesis and focal spinal pain. if there is involvement of the brachial or lumbosacral intumescences, lmn signs will occur. nerve root involvement may result in lameness and limb pain. acute exacerbation of signs is commonly associated with hemorrhage. weight loss is the most common extraneural sign. the most common extraneural tumor site associated with spinal lymphosarcoma is the kidney. complete blood count, biochemistry parameters and urinalysis are often normal, although non-regenerative anemia or leukemia may occasionally be seen. many cats are seropositive for felv or positive on indirect fluorescent antibody testing of bone marrow. a monomorphous population of neoplastic lymphocytes will be seen in some cases. survey spinal radiographs are usually normal. mri scan or myelography shows single or multiple asymmetric extradural spinal cord compression(s). infectious diseases, such as feline infectious peritonitis, toxoplasmosis and cryptococcosis also may produce signs of progressive spinal cord dysfunction. however, unlike spinal lymphosarcoma, most infectious conditions usually result in multifocal neurologic dysfunction, with involvement of the cerebrum, brainstem and/or cerebellum. spinal lymphosarcoma is radiosensitive. • rapid reduction in tumor volume can occur within hours of delivering a single large palliative dose of radiation ( - gy). chemotherapy is important for systemic control of lymphosarcoma although most chemotherapeutic agents do not cross the blood-brain barrier. cytosine arabinoside may also help control spinal lymphosarcoma. • this anti-metabolite drug crosses the blood-brain barrier and reaches appropriate csf levels. • the recommended dosage is mg/m /day as a constant rate infusion intravenously for days. • an alternative regimen is mg/m subcutaneously twice daily for days. • potential toxicity includes myelosuppression (at - days); vomiting; and anorexia. • tumor cells rapidly become resistant to this drug's action. surgical decompression of the spinal cord and/or nerve roots, and tumor resection or debulking is a potential treatment option, especially if the tumor is localized. follow-up radiation and chemotherapy is recommended. prognosis is poor for long-term survival, especially when most cats are infected with the felv virus. the only effective means of prevention is test and removal programs directed at controlling the spread of felv. feline infectious peritonitis (fip) may produce a nonsuppurative meningoencephalitis in % of infected cats. the cns signs are usually associated with the noneffusive ("dry") form of fip (cell-mediated immunity is defective but not absent) the neurological form of fip has no breed or sex predilection, although most cases are < - years of age. in most cases, the neurological signs are multifocal with a predominance of caudal fossa signs (brainstem, vestibular nuclei and cerebellum). • signs can include nystagmus (positional, nonconstant, rotatory, horizontal or vertical in nature), head tilt, body lean or rolling, facial paralysis, an ipsilateral hemiparesis or tetraparesis, intention tremor, and hypermetria or dysmetria. the spinal cord and the cerebrum also can be involved. • signs of cerebral disease can include seizures, behavior change, decreased mentation, decreased menace response, compulsive walking, ipsiversive wide circling and head pressing. • spinal cord involvement will manifest as umn and/or lmn tetraparesis or paraparesis with ataxia. tetraplegia or paraplegia are the most severe consequences of spinal cord involvement. myelopathy is seen either as the sole abnormality or as part of a multifocal distribution in % of cases. the onset of fip can be acute, although most cats have a chronic progressive course over week to months. extraneural signs may include fever, cachexia, poor body condition, dehydration, lethargy, muscle atrophy, chorioretinitis, dyspnea and gastrointestinal/hepatic signs. many cats present with the neurological form of fip without extraneural signs. the diagnosis is best established via csf analysismarked elevation in protein (usually greater than the csf fip viral titer is usually positive if cns signs are present. it is probable that csf anti-fip viral immunoglobulin g is produced within the cns. serum biochemical changes often consist of an elevation in total protein and hypergammaglobulinemia. serum fip antibody titers are not reliable, and are elevated in only % of cats with the neurological form of fip and are even negative in some cats. numerous other infectious and immunological diseases can produce meningitis and encephalomyelitis in cats. felv-associated cns lymphosarcoma can usually be differentiated by csf analysis, and a positive felv serum titer. cryptococcal organisms are usually visible in csf with confirmation made by a latex agglutination assay or culture. cns toxoplasmosis is rare in the cat, usually producing signs of intracranial disease rather than myelopathy. recent or active infection is diagnosed by demonstrating a positive igm titer, a four-fold increase or decrease in igg serum titer, a csf igm antibody titer or a csf igg titer present at a higher csf:serum ratio than another antibody present in serum that is not produced in csf, e.g. calicivirus. feline polioencephalomyelitis usually produces only a mild to moderate increase in protein (mean of . g/l [ mg/dl] with a range of . - . g/l [ - mg/dl]) and a mild mononuclear pleocytosis (mean of cells/mm [ cells/μl] with a range of - cells/mm [ - cells/μl]) on csf. this is not typical of fip. no effective therapy is known against fip. immunosuppressive corticosteroid therapy may slow the disease course ( - mg/kg prednisone or prednisolone orally twice daily). all cats with the neurological form of fip will eventually die from their disease. • acute variably located spinal cord dysfunction (umn or lmn signs). • focal spinal hyperesthesia. • spinal crepitus. • external abrasions and bruising. external spinal trauma can result from automobile accidents, gunshot injuries, falls from heights and blunt trauma. sacrocaudal fractures most often occur when the cat's tail is forcefully pulled away from the body. • this most commonly occurs when the cat's tail is trapped under the tire of an automobile. the rotating tire luxates the tail at the sacrococcygeal junction or between two coccygeal vertebrae, resulting in damage to the coccygeal, sacral and lumbar nerve roots (cauda equina). most spinal fractures occur at the junction of mobile and immobile segments of the spine, such as the thoracolumbar, lumbosacral and sacrocaudal junctions. traction on the cauda equina in sacrocaudal luxations can also damage the nerve cell bodies in the caudal lumbar and sacral spinal cord due to cranial transmission of the forces through the dura mater and filum terminale of the cord. the degree of spinal cord compression; the length of time of compression; and the velocity of the initial impact injury are important contributors to the resultant spinal cord injury. there are two forms of spinal cord injury -mechanical (primary) injury, consisting of physical disruption of vessels and axons and ischemic (secondary) injury. the latter leads to energy compromise within the spinal cord, cellular accumulation of calcium and resultant intracellular stimulation of enzymes. this leads to protein and lipid breakdown (phospholipid hydrolysis), release of arachidonic acid, free radical and eicosanoid formation, and cell death. gray matter hemorrhages and white matter edema occur within minutes following acute spinal cord injury, progressing rapidly over the - -hour posttraumatic period. signs depend on the injury level and the degree of physical or functional disruption of the cord. signs in cats with sacrocaudal injuries range from hyperesthesia over the tail base to flaccid analgesic tails with varying degrees of urinary reflex dyssynergia or lmn urinary/fecal incontinence. • reflex dyssynergia involves a failure of urethral relaxation when the cat attempts to urinate and the bladder undergoes contraction. this leads to the production of only very small quantities of urine. the bladder will still be large and the cat will often dribble urine in between attempts to urinate. many cats with sacrococcygeal trauma also show signs of lmn paraparesis (sciatic nerve injury), consisting of dragging of the hind paws on their dorsum and a failure to flex the pelvic limb(s) when walking or when the withdrawal reflex is performed. there will also be a hypo-to areflexia of the cranial tibial and gastrocnemius reflexes. palpation of the sacrocaudal area of the spine will result in crepitus and pain. the tail will often be malaligned with the sacrum and the rest of the vertebral column. cats with spinal trauma commonly have concurrent thoracic, abdominal or pelvic trauma. neurological and physical examination determine the localization and severity of the cord injury. non-contrast spinal radiographs (lateral and across the table ventrodorsal views of the entire spine) establish the type of vertebral disruption (fracture vs. subluxation) and the location(s) of the vertebral trauma. myelography, mri or ct scans will determine the degree of spinal cord compression. any other cause of acute myelopathy must be considered as a differential diagnosis in cats with spinal trauma. most other causes can be eliminated based on history and physical examination -external abrasions, bruising, splintering of claws and spinal pain are seen with trauma. sacrocaudal fractures must be differentiated from aortic thromboembolism, which also can produce an acute onset of lmn paraparesis to paraplegia, with pain. • however, cats with this disease do not have dysfunction associated with the perineum, bladder, rectum and tail. • the lack of femoral pulses and the presence of cold, cyanotic pelvic limbs would strongly support aortic thromboembolism. pathologic vertebral fracture secondary to neoplasia will present as an acute, painful myelopathy, and may appear superficially similar to a traumatic fracture on non-contrast spinal radiographs. however, closer examination should reveal areas of lysis within the involved vertebral body. ischemic myelopathy secondary to fibrocartilaginous embolism most commonly involves the lumbosacral intumescence as is the case with sacrococcygeal trauma. • a differentiating feature of ischemic myelopathy is the absence of spinal pain. initial medical emergency treatment should include intravenous methylprednisolone sodium succinate ( mg/kg), administered within hours (and preferably within hours) of the spinal trauma. continued treatment consists of a constant rate intravenous infusion of methylprednisolone at . mg/kg/hour or, if not possible, a second bolus intravenous injection ( mg/kg) at hours after the initial treatment, followed by mg/kg at hours and then times daily for - hours. most cases of spinal trauma will require emergency surgical intervention (spinal cord decompression [laminectomy and/or hemilaminectomy] and spinal fixation). the type of decompression and stabilization technique is determined by non-contrast and contrast spinal radiographs or ct/mri scans. surgical spinal stabilization is rarely necessary in sacrocaudal fractures/luxations and, due to the oftenmarked separation of the involved vertebrae, is difficult to achieve. surgical decompression of the cauda equina in sacrocaudal trauma is also unnecessary since it is a traction, not compressive, injury. if the cat is incontinent, attention should be paid to cleanliness, bladder and bowel management, and prevention of decubital ulcers, urine scalding, and cystitis. if there is umn or lmn urinary incontinence, manual expression or indwelling catheterization of the bladder is required. pharmacological management of umn urinary incontinence or reflex dyssynergia, related to sacrocaudal trauma, consists of phenoxybenzamine, an alpha-adrenergic receptor antagonist, to relax the internal sphincter ( mg orally three times daily) together with diazepam ( . - mg/cat orally two to three times daily) to relax the external sphincter. bladder detrusor dysfunction can then be treated with bethanechol, a parasympathomimetic agent ( - mg orally two to three times daily). bethanechol may help lmn urinary incontinence (encourages detrusor muscle contraction). fecal retention can be treated by increasing the bulk of the feces with bran, psyllium or canned mashed pumpkin. in cats with sacrocaudal injury with only tail paresis, conservative medical management will often result in return of tail function. • if this does not occur after - months, amputation of the tail is recommended. prognosis is based on the severity of the spinal cord injury. the milder the neurological deficits, the better the prognosis for recovery. cats with severe myelopathy or cauda equina injury with analgesia have a very poor to hopeless prognosis since they commonly have physical or functional spinal cord or cauda equina transection. • only % or less of cats with flaccid tail, perineal analgesia and lmn urinary and fecal incontinence from a sacrocaudal injury will regain neurologic function. • % of cats with only tail flaccidity can regain tail function. prognosis should never be determined based on the degree of radiographic displacement of the involved vertebra(e). the only preventative measure is for cats to avoid situations where trauma is a distinct possibility. • chronic, progressive umn or lmn tetraparesis or paraparesis dependent on tumor location. • focal areas of spinal pain. although most meningiomas are found intracranially, they may also develop along the spinal cord with an intradural, extramedullary predilection site. meningiomas arise from any cell of the meningesblood vessels, fibroblasts or arachnoid cells. meningiomas produce signs by compressing the adjacent spinal cord causing vasogenic edema occasionally, other tumors such as an astrocytoma occur. most cats are > years of age. the nature of the ataxia and paresis (umn versus lmn signs) depends on the tumor's location. a focal area of spinal pain or more diffuse spinal discomfort may occur weeks prior to the development of neurological dysfunction. signs are slowly progressive. non-contrast spinal radiographs may reveal thinning or deformation of the vertebral lamina secondary to pressure necrosis from the expanding tumor. lumbar csf most commonly reveals increased protein (> . g/l [ mg/dl]) without an accompanying pleocytosis. tumor cells are rarely seen. any cause of chronic, progressive myelopathy should be considered. the focal nature of the myelopathy with meningioma eliminates most infectious and inflammatory myelopathies, with the exception of felv-associated spinal lymphosarcoma. ischemia and trauma produce an acute myelopathy and can usually be eliminated. age of disease onset for meningiomas would eliminate congenital and inherited myelopathies. differentiation from nerve root tumors often requires histopathology following surgical removal. surgical removal of the tumor is the only definitive treatment. post-operative radiation therapy is recommended. corticosteroids only transiently improve neurological function. the long-term prognosis is guarded to fair if surgical removal of the tumor appears complete. follow-up radiation probably further improves this prognosis. without surgery, the long-term prognosis is poor. there is no known prevention of spinal meningiomas in cats. • acute lmn paraplegia. • severe pain associated with the pelvic limb musculature. • vocalization and anxiety. • weak or absent femoral pulses. • cyanotic and cold nail beds and foot pads. aortic thromboembolism results from a thrombus that is dislodged from within the left heart or aorta, leading to obstruction of the aortic trifurcation and severe ischemia to pelvic limb muscles and nerves. in cats it is most commonly associated with hypertrophic, dilated and restrictive cardiomyopathy, with thrombus formation in the left atrium. it also occurs with thyrotoxic cardiac disease. thrombus formation requires either damage to the endocardium as occurs in cardiomyopathy, especially the dilated left atrium; blood stasis which also occurs in the dilated left atrium; or altered blood coagulability. disseminated intravascular coagulation (dic) was found in % of cats with cardiomyopathy and was associated with consumptive or liver-mediated coagulopathy or thromboembolism. embolism occurs when the thrombus lodges in a blood vessel. the thrombus most often lodges in the distal aorta at the trifurcation, leading to a saddle thrombus and occlusion of blood supply to the hindlimbs. ischemic neuromyopathy occurs and is most severe distal to the stifle. signs are usually asymmetrical. occasionally the right brachial artery is occluded, resulting in lameness or paresis of the right forelimb. occasionally cats with thromboembolic disease do not have underlying heart disease. thrombosis may also occur with infectious or neoplastic disease. there is no breed or age predilection (average years, range - years) although males are twice as likely to be affected. aortic thromboembolism usually produces an acute onset of lmn paraplegia and severe pain. the hindlimbs drag behind the cat, as the hocks cannot be flexed. hip extension and flexion is present. less severe ischemia leads to mild to moderate paraparesis or pelvic limb lameness. the gastrocnemius muscles usually are firm, but soften - days after embolization. femoral pulses are weak or absent and the nail beds and footpads are cyanotic and cold. the distal limbs are often swollen. extraneural signs include vocalization, tachypnea/ dyspnea, a cardiac murmur and arrhythmias. heart failure is present in about % of cats with aortic thromboembolism. dehydration and hypothermia are often present. tail movement, perineal reflexes and urinary function remain intact. increases in serum creatine kinase (often - iu/l [normal: - iu/l]) occur secondary to severe muscle damage. creatinine and bun are increased in more than % of cats and may be prerenal or renal. in some cats, concurrent embolization of the renal artery occurs. thoracic radiography reveals cardiomegaly ( - % of cats) and pulmonary edema and/or pleural effusion ( %). electrocardiography most commonly reveals sinus tachycardia. atrial fibrillation, and supra-/ventricular arrhythmias can also be seen. doppler (color flow) will often demonstrate the decrease in blood flow through the site of thromboembolism. mri angiography will reveal a complete or partial blood flow obstruction at the aortic trifurcation. a major feature that differentiates aortic thromboembolism from traumatic, infectious, neoplastic and ischemic myelopathies is the presence of paraparesis to paraplegia without involvement of the perineum, tail and bladder. cold, cyanotic pelvic limbs without femoral pulses and firm gastrocnemius muscles also are unique. with current treatment modalities, the results of pallative therapy are as good as aggressive thrombolytic therapies or physical removal of the thrombus, and considerably less expensive. • physical removal is via surgery or balloon embolectomy. • thrombolytic agents include tissue plasminogen activator, urokinase and streptokinase. -current information should be consulted on administration and monitoring of thrombolytic and intensive anti-thrombotic therapies if they are to be used. • reperfusion effects are a major problem with aggressive thrombolytic therapies and physical removal. rapid onset of severe hyperkalemia associated with reperfusion is common and often fatal. renal hemorrhage may also occur during thrombolysis, adversely affecting survival. clinical hemorrhage may require a blood transfusion to control. pallative therapy consists of relieving pain, heparin to help prevent another clot forming in the left atrium, fluid, electrolyte and nutritional support, warmth, physiotherapy (passive massage and flexing/extending legs) and treatment of underlying heart disease. potassium concentrations should be monitored carefully. injectable analgesics and a fentenyl patch should be used to provide adequate levels of pain relief. excessive licking or chewing of the affected limb may occur resulting in self-mutilation. loose bandaging of the limb or an elizabethan collar are usually effective. dehydration and electrolyte imbalances, especially hyper-and hypokalaemia, need correcting. nutritional needs should be met in anorexic cats by the placement of a nasoesophageal tube for feeding. acutely, heparin ( u/kg subcutaneously followed by u/kg subcutaneously three times daily) can be given to prevent further activation of the coagulation cascade, although it will not have any effect on the formed thrombus. continue palliative therapy for - days and look for rewarming of the toes and returning pulses as an initial sign of improvement. doppler is useful for detecting blood flow. if there is no sign of reperfusion after days, the prognosis is hopeless and the cat should be euthanized. various other treatments have been advocated but there are no studies showing increased survival. these include aspirin, periactin, acepromazine and vasodilator agents. aspirin may be beneficial during and after an episode of thromboembolism due to its antiplatelet effects and decreased production of the vasoconstrictor thromboxane a , as well as analgesic effects. the dosage is / × mg adult aspirin ( mg) every second to third day. blockade of prostacyclin production by the endothelium with aspirin is of concern, and some advocate lower doses of aspirin more frequently ( - mg/cat q h). prostacyclin inhibits platelet aggregation and vasoconstriction. acepromazine ( . - . mg subcutaneously three times daily) can be used for sedation and vasodilation. vasodilation with alpha-blockers is advocated by some but unproven. long-term warfarin therapy may decrease the frequency of rethrombosis, but needs careful monitoring using the international normalization ratio (inr) for pt to achieve a inr between and . doses of . - . mg/kg po q h have been advocated. a lower dose of . mg/cat po q h has also been used. only - % of cats survive the initial episodes and go home. most will re-embolize -long-term prognosis is poor (average survival is - months with therapy) with less than % surviving year. • % of cats have rethrombosis even when treated with warfarin. most cats that survive an initial episode will show varying degrees of neurological recovery to their pelvic limbs, although this often takes weeks to months. rarely does the neuromuscular function fully recover. hypothermia and azotemia prior to therapy and hyperkalemia during thrombolysis are negatively associated with survival. echocardiographic evidence of another thrombus in the left atrium also decreases the long-term prognosis. little can be done to prevent aortic thromboembolism if the underlying cardiac disease has not been previously recognized. if cardiac disease is diagnosed, the risk of thromboembolism may be reduced by appropriate treatment of the cardiomyopathy (see chapter ). efficacy of longterm aspirin (low dose - mg/cat q h or a regular dose mg/cat, q - h po) or warfarin therapy in preventing thromboembolism has not been demonstrated, and most cats rethrombose. • stiff staggering gait. • inability to jump. • chronic pelvic limb ataxia and paraparesis. • inability to retract their claws. • thoracolumbar hyperesthesia. • thoracic limb paresis and ataxia. • decreased mentation and seizures. • fever (in % of cases). feline polioencephalomyelitis is a chronic, progressive disease affecting the spinal cord and brain of cats. the cause is unknown, although neuropathology is suggestive of a neurotrophic virus. recently, specific antibodies to the borna disease virus have been found in % of cats with feline polioencephalomyelitis in sweden. feline polioencephalomyelitis is a sporadic worldwide disease. affected cats range from months to years of age. both male and female cats are affected. all domestic cat breeds as well as large non-domesticated cat species can contract the disease. neurologic signs usually develop over - months with a subacute to chronic progressive course. the most striking neurologic signs include a stiff staggering gait, inability to jump, pelvic limb ataxia and paraparesis. • some cats have decreased spinal reflexes and some are unable to retract their claws. other signs may include thoracic limb ataxia and paresis, hyperesthesia over the thoracolumbar and lumbosacral regions, decreased mentation, behavior change, decreased pupillary light reflexes, hypersensitivity to external stimuli, impaired vision, seizures, increased salivation, intention tremors and pruritis. with disease progression, paraplegia eventually occurs. a fever is present in approximately % of cats. serologic testing for fiv and felv antibodies is negative. definitive diagnosis can only be made on necropsy. histopathology reveals a disseminated meningoencephalomyelitis with lymphocytic perivascular cuffs that is most severe in the spinal cord (gray matter) and brainstem. infectious diseases (fip and toxoplasmosis) also may produce signs of progressive spinal cord dysfunction, making differentiation based on neurological signs difficult. uveitis, chorioretinitis and optic neuritis are often present with cns infections, but not with polioencephalomyelitis. cats with spinal cns lymphosarcoma commonly are felv positive and may have other systemic signs of tumor involvement. csf analysis may help differentiate between the infectious diseases, lymphosarcoma and feline polioencephalomyelitis (see sections on lymphosarcoma and fip), although the range of values overlap between the diseases. immunosuppressive steroid therapy (prednisone - mg/kg bid) is the recommended treatment against this disease, although data are lacking. this may be combined with other immunosuppressive agents such as cytosine arabinoside. aggressive and persistent therapy may result in remission and possible cure after several months. prognosis is guarded. the disease tends to be progressive if untreated. since this disease is of unknown etiology and very sporadic, there are no recommendations for prevention. • umn or lmn paraparesis or tetraparesis dependent on the anomaly and its location. • scoliosis (lateral curvature of the spine), lordosis (downward arching spine), or kyphosis (upward arching spine) resulting from a hemivertebra. • cervical pain and umn tetraparesis to paralysis (atlantoaxial subluxation). • lmn paraparesis to paraplegia (spina bifida and sacrococcygeal dysgenesis). • lmn urinary and fecal incontinence (spina bifida and sacrococcygeal dysgenesis). spinal cord and vertebral anomalies are classified into abnormalities originating in the tissues of mesodermal origin (vertebral body and intervertebral disc) and those arising from tissues of ectodermal origin (spinal cord and meninges). • block vertebrae. -caused by improper segmentation of somites, resulting in stable vertebral fusion. • butterfly vertebrae. -abnormal persistence of the notochord (the embryological precursor of intervertebral discs), producing a midline cranial to caudal cleft in the vertebral body (when viewing the vertebra from the dorsoventral direction). • hemivertebrae. -produced by fusion of one lateral somite to one on the contralateral side that is not directly opposed or by a lack of vascularization, leading to a failure of ossification in part of the vertebral body. this produces a wedge-shaped vertebra, and scoliosis, lordosis or kyphosis. • transitional vertebrae. -sacralization of the last lumbar vertebra is the most common in cats. in this condition, the last lumbar vertebra has characteristics of both lumbar vertebrae and the sacrum. clinical signs are dependent on the type of congenital malformation and its location. block vertebrae are usually stable. • occasionally they can be stenotic or angulated, causing extradural spinal cord compression. • disc extrusion can occur at disc spaces immediately cranial and caudal to the block vertebrae. butterfly vertebrae are generally incidental findings. hemivertebrae commonly lead to scoliosis, lordosis or kyphosis. • signs are due to spinal cord compression or repeated trauma associated with vertebral instability. instability produces osseous changes that secondarily compress the spinal cord. • signs may be acute, chronic, progressive or intermittent, but are usually first noted within the first - years of life. • conformation may be visibly or palpably abnormal. breed association (manx cat). signs are most commonly observed in immature animals (< months of age). the cat's conformation may be visibly or palpably abnormal (scoliosis, skin dimple, etc.). • block vertebra -joining of two adjacent vertebral bodies. • butterfly vertebra -sagittal vertebral body cleft. • hemivertebra -abnormal vertebral wedging and shortening. • atlantoaxial subluxation -abnormally shaped or absent dens. • spina bifida -vertebral arch defect. mri scan or ct myelography reveals soft tissue abnormalities associated with a spinal cord that is tethered (fixed) by either meningocutaneous attachments (spina bifida) or by a failure of the terminal cord-like attachment from the spinal cord to the dura to stretch with growth. they will also reveal cord compression. major differentials for hemivertebra include traumatic or pathologic fracture with secondary collapse of the vertebral body. • hemivertebrae, however, have smooth end plates and well-formed adjacent disc spaces. • difficulty in differentiation arises when vertebral osteophytes are present. differential diagnoses for block vertebrae include vertebral fusion secondary to discospondylitis, vertebral fracture/luxation, collapsed disc space secondary to disc extrusion, or previous disc surgery. however, all have associated reactive bone, which is not present with block vertebrae. atlanto-axial subluxation and spina bifida have few rule-outs, except trauma. spinal cord compression from a hemivertebra can be treated via surgical decompression and stabilization (if necessary). treatment for atlantoaxial subluxation involves stabilization of the atlantoaxial joint via ventral crosspinning or screws (preferred) or dorsal wiring. specific treatment for spina bifida and sacrococcygeal dysgenesis is rarely attempted. • if signs are associated with spinal cord tethering or there is a meningocutaneous fistula without primary spinal cord anomalies, reconstructive surgery may be possible. prognosis depends on the type of congenital anomaly and the degree of dysfunction. cats with mild signs due to a surgically treatable hemivertebra or block vertebra, have a guarded to good prognosis for improvement. morbidity and mortality is high with surgical stabilization of atlantoaxial subluxation. • with successful stabilization, however, cats with initially mild to moderate neurological dysfunction have a fair to good prognosis. prognosis for cats with spina bifida or sacrococcygeal dysgenesis with anomalies associated with the spinal cord or cauda equina, is grave. these cats should be euthanized if the neurological dysfunction is incompatible with a good quality of life. cats with spinal cord tethering (spina bifida) have a potentially fair to good prognosis with surgery. prevention of further congenital malformations is best achieved by a spay/neuter program. • cervical and thoracic limb hyperesthesia and rigidity. • thoracic limb lameness. • chronic progressive ataxia. • reluctance to move. • chronic tetraparesis to paralysis. hypervitaminosis a is a skeletal disease, secondary to excessive intake of vitamin a (liver or vitamin a supplementation (cod liver oil) (> iu/ml). affected cats are usually - years of age. signs include cervical and thoracic limb hyperesthesia and rigidity due to extensive confluent exostosis in the cervical and thoracic spine, thoracic limb lameness, ataxia, reluctance to move, and tetraparesis to paralysis. other signs include lethargy, anorexia, constipation, weight loss and an unkempt haircoat due to an inability to groom. exostoses develop insidiously with the above signs occurring only after the disease is advanced. non-contrast spinal radiographs show new bone formation involving the cervical vertebrae. • the sternum, costal cartilages, and long bone metaphyses also show new bone formation. • joints may show arthrodesis. analgesics may symptomatically treat the clinical signs. • aspirin - mg/kg po q h. • narcotic analgesics such as morphine ( mg/ml) - . mg/kg po three times to four times daily. • non-steroidal anti-inflammatory drugs. • fentanyl patch. • acute, non-progressive, non-painful, asymmetrical lmn paraparesis to paraplegia. • lmn urinary and fecal incontinence. spinal cord infarction secondary to fibrocartilaginous embolism is uncommon. the embolus is histochemically identical to the fibrocartilage of the nucleus pulposus. it is unknown how the embolus reaches the spinal vasculature from its origin. embolization of arteries, veins or a combination of the two may occur. sudden increases in intra-abdominal pressure (hard exercise) may facilitate retrograde passage of disc material through the venous sinuses and spinal veins. embolism results in segmental hemorrhagic necrosis and malacia of the spinal cord. clinical signs reflect the location of the lesion along the spinal cord. in cats, the lumbosacral intumescence is the most common site of myelopathy, resulting in an acute lmn paraparesis to paraplegia, and lmn urinary and fecal incontinence. other findings include a lack of spinal pain, lack of disease progression and marked asymmetry of neurological dysfunction. diagnosis is based on exclusion of other etiologies. history and neurological exam are important. survey radiographs are normal. csf analysis is variable, ranging from normal, hemorrhagic, or showing a mild mixed pleocytosis ( - cells/mm [ - cells/μl]). myelography will either be normal or occasionally show cord edema. mri diffusion studies may reveal the ischemic area of spinal cord, if the affected area is large enough. definitive diagnosis can only be made at necropsy. differential diagnosis includes any cause of acute paraparesis to paraplegia. the unique clinical signs and usual absence of abnormalities on work-up separate fibrocartilaginous embolism from other acute myelopathies. immediate treatment consists of methylprednisolone sodium succinate (see spinal trauma). embolic myelopathy is non-surgical. long-term corticosteroid therapy is not recommended. prognosis depends on density of neurological dysfunction and degree of irreversible cord damage. cats with severe lmn paraplegia, absent pain sensation and lmn urinary incontinence associated with involvement of the lumbosacral intumescence have a poor to hopeless prognosis. cats with less severe signs have a guarded to favorable prognosis for partial to full recovery. there are no preventive measures that can be undertaken. a review of the clinical diagnosis of feline infectious peritonitis viral meningoencephalomyelitis jr (ed) consultations in feline internal medicine ed) textbook of veterinary internal medicine congenital abnormalities of the spinal cord and vertebrae, chapter jr (ed) consultations in feline internal medicine feline non-suppurative meningoencephalomyelitis. a clinical and pathological study inflammatory disorders of the central nervous system encephalitis and meningitis. in: bagley r (ed) veterinary clinics of north america small animal practice -intracranial disease feline thrombotic disease veterinary information network vascular disorders, chapter based on the clinical and neurological examinations, other differentials include infectious meningitis and myelitis, vertebral neoplasia, mucopolysaccharidosis and discospondylitis (rare).non-contrast spinal radiographs eliminate all differentials with the possible exception of mucopolysaccharidosis vi, a rare autosomal recessive storage disease produced by deficiency of the lysosomal enzyme, arylsulfatase b. • mucopolysaccharidosis vi occurs in - -monthold siamese cats, producing spinal cord compression secondary to fusion of the cervical and thoracolumbar vertebrae with bony proliferation. • these cats also have a flat, broad face, widely spaced eyes, corneal clouding and enlarged feet. remove excess vitamin a from the diet to prevent further development of exostoses.use a balanced commercial cat diet.skeletal improvement is monitored by radiography and neurological examinations. key: cord- -npug c p authors: liu, yang; liu, jianying; pang, xiaojing; liu, tao; ning, zhijie; cheng, gong title: the roles of direct recognition by animal lectins in antiviral immunity and viral pathogenesis date: - - journal: molecules doi: . /molecules sha: doc_id: cord_uid: npug c p lectins are a group of proteins with carbohydrate recognition activity. lectins are categorized into many families based on their different cellular locations as well as their specificities for a variety of carbohydrate structures due to the features of their carbohydrate recognition domain (crd) modules. many studies have indicated that the direct recognition of particular oligosaccharides on viral components by lectins is important for interactions between hosts and viruses. herein, we aim to globally review the roles of this recognition by animal lectins in antiviral immune responses and viral pathogenesis. the different classes of mammalian lectins can either recognize carbohydrates to activate host immunity for viral elimination or can exploit those carbohydrates as susceptibility factors to facilitate viral entry, replication or assembly. additionally, some arthropod c-type lectins were recently identified as key susceptibility factors that directly interact with multiple viruses and then facilitate infection. summarization of the pleiotropic roles of direct viral recognition by animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. animal lectins will benefit our understanding of host-virus interactions and could provide insight into the role of lectins in antiviral drug and vaccine development. keywords: lectin; virus; direct interaction; antiviral immunity; viral pathogenesis lectins, a highly diverse group of proteins that recognize carbohydrates, have been demonstrated to play a vital role in numerous life processes and to be critical for several viral infections and pathogeneses in a variety of organisms [ , ] . based on their conserved structure of sequence motifs for sugar binding and carbohydrate specificities, lectins have been categorized into many families conventionally designated as calnexin, c-type, l-type, p-type/mannose- -phosphate receptors (mprs), i-type/siglecs, m-type, f-type (absent in mammals), r-type, f-box, chitinase-like lectins, galectins and intelectins (table ) [ ] . the features of carbohydrate recognition domains (crds), such as structure peculiarity, carbohydrate binding selectivity and geometrical arrangement of multiple crds, determine the different properties of lectins [ ] [ ] [ ] . furthermore, the diversity of locations and functions indicates the importance of lectins in the basic life processes of organisms. lectins are exploited as susceptibility factors to facilitate viral entry, replication or assembly ( figure ). insight into direct lectin-based viral recognition will provide a deep understanding of host-virus interactions. mammalian lectins have been categorized into multiple classes according to the features of their crds, as well as their sugar recognition specificity. some lectins generally play a role outside the cell, whereas others are predominantly intracellular and located on cytoplasmic organelles. extracellular lectins, including c-type, r-type, i-type/siglecs lectins and galectins, are secreted into the extracellular milieu or are localized to the plasma membrane and are capable of mediating cell adhesion, immune signaling and pattern recognition activities for host-pathogen interactions. however, intracellular lectins, such as the calnexin family, m-type, l-type and p-type lectins, are located in luminal compartments of the secretory pathway and function in the trafficking, sorting and maturation of glycoproteins [ ] [ ] [ ] . as lectins play diverse roles in mammalian physiological processes, we only focus herein on a portion of lectins that directly interact with viral components and describe their functions in viral propagation and pathogen-host immune responses. c-type lectins (ctls) are a large group of proteins in metazoans that were originally named according to their property of ca + (c-type)-dependent carbohydrate binding. sequence and structural comparisons of c-type lectins have suggested that their carbohydrate-binding activity is mediated by a specific crd that is conserved in a variety of organisms. although some c-type lectins do not possess carbohydrate-binding activity, all of them show distinct sequence similarity and are believed to descend from a common ancestor during evolution [ ] [ ] [ ] . to date, c-type lectins have been divided into subgroups according to their domain architecture and the phylogenetic relationship between their crd sequences [ ] . in general, c-type lectins can be separated into two groups, mannose-binding and galactose-binding c-type lectins, based on the specificity of their carbohydrate-binding activity. the binding specificity of these two groups is mediated by diverse residues flanking the conserved cis-proline in the long loop region, in which the sequence of the core motif is e-p-n for mannose-binding and q-p-d for galactose-binding specificity [ , ] . previous studies have demonstrated that interchange of the e-p-n and q-p-d sequences is sufficient to switch the mannose-and galactose-binding specificity ( figure ) [ ] . however, several lectins are exceptions to this rule. for example, surfactant protein a, possessing an e-p-k but not an e-p-n motif, binds to mannose sugars [ , ] . although human tetranectin contains a galactose-type q-p-d motif, it is not responsible to the lectin-carbohydrate interaction [ ] . therefore, other determinants, including modifications around the binding sites and stereochemical factors, should be taken into consideration when examining binding specificity [ ] [ ] [ ] . robust investigations have shown that multiple mannose-/galactose-binding c-type lectins play important roles in viral infections in mammals. mbl, one of the most intensively studied lectins, is a member of the collectin family, a subgroup of c-type lectins ( figure a ). the mbl molecule contains four domains that are standard for collectin family proteins: a cysteine-rich region, a collagen-like domain, a neck region and a carbohydrate recognition domain. the native functional form of mbl is a hexamer; however, although mbl can form several oligomeric forms, the dimers and trimers do not have biological activity, and at least a tetramer form is needed to activate the complement cascade [ ] . mbl functions as a soluble pattern recognition receptor in the host complement system and is involved in resistance to many viral infections [ ] . the crds of mbl multimers recognize carbohydrate patterns on the virus surface, and consequently, the binding of mbl and viral particles results in activation of the lectin pathway of the complement system. masps (mannose-binding lectin-associated serine proteases), which are protease zymogens (an inactive form of an enzyme) similar to the c r and c s molecules of the classical complement pathway, are activated to cleave complement components c and c into c a/c b and c a/c b, respectively. interactions between c b and c b produce the c convertase, which continuously activates c further downstream in the cascade to eliminate viruses ( figure a ) [ ] . current investigations have reported a resistance role of mbl in infections of multiple human viruses, including human immunodeficiency virus (hiv) [ ] , hepatitis b virus (hbv) [ , ] , hepatitis c virus (hcv) [ ] , west nile virus (wnv) [ ] and dengue virus (denv) [ ] . specific recognition of these viral particles by mbl is a central event for activation of the lectin-based complement cascade. the mechanism of viral elimination by the mbl-based complement cascade is still unclear. unlike bacterial elimination by the complement system, mbl-based complement components do not appear to form a membrane-attacking complex on the viral surface; therefore, viral eradication may not be mediated by known complement mechanisms. three possible mechanisms have been proposed for mbl-mediated viral elimination. ( ) mbl-mediated complement c /c deposition onto the viral surface. viral neutralization can be processed in cell-free serum and is completely dependent on c and c activation, but not on c q and c , suggesting that neither opsonization nor the classical/alternative complement pathway is sufficient for viral neutralization [ ] . ( ) mbl can directly neutralize viruses. pre-incubation of serial concentrations of recombinant mbl with hiv cell-derived living particles was found to dramatically neutralize hiv infection [ , ] . however, other independent investigations have suggested that some primary hiv isolates resist direct neutralization by mbl [ ] , indicating that the possible neutralizing activity depends highly on the different carbohydrate structures on the surfaces of various viral strains. ( ) mbl can block the recognition of viruses and receptors. dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign), which is present on the surface of dendritic cells, functions as a key attachment factor used for the recognition and uptake of multiple viruses [ ] [ ] [ ] [ ] [ ] . a study reported that mbl can prevent interaction between hiv and dc-sign, thereby inhibiting the hiv infection of t cells, which is mediated by dc-sign [ ] . furthermore, mbl interacts with the viral envelope glycoproteins of ebola and marburg viruses (marv), resulting in the impairment of viral internalization by blocking virus-dc-sign interaction ( figure b ) [ ] . two soluble collectins, designated sp-a and sp-d ( figure b ,c), have been found to be involved in the recognition of viral particles for limiting infection in humans. sp-a is produced within the respiratory tract, gastrointestinal tract, and possibly other sites; conversely, sp-d is primarily synthesized in the respiratory tract [ ] . these factors are constitutively secreted into the lungs by alveolar type ii cells, unciliated bronchial epithelial cells and other mucosal tissue cells [ , ] . the specific location of surfactant proteins suggests a defensive role against viral invasion of the respiratory system. both sp-a and sp-d interact with different strains of influenza a virus (iav) via glycosylated hemagglutinin (ha) and neuraminidase (na) on the viral surfaces. the binding of iav to sp-a leads to agglutination of the virions, inhibition of iav infectivity and dissemination and also facilitates clearance by macrophages and neutrophils ( figure a ,b) [ ] . sp-d binds to iavs and thereby inhibits virus attachment and entry by viral aggregation ( figure b ) [ ] [ ] [ ] [ ] [ ] and also controls iav infection in human by activating neutrophil chemoattraction ( figure a ) [ , ] . respiratory syncytial virus (rsv) infects humans via the respiratory tract. sp-a has been reported to bind fusion (f) and adherence (g) glycoproteins on the surfaces of rsv virions, resulting in opsonization to reduce infection by enhancing viral uptake by peripheral blood mononuclear cells (pbmcs) and alveolar macrophages ( figure a ) [ , ] . sp-d also directly interacts with rsv surface g protein to modulate host immune responses to control rsv infection [ ] . dc-sign (cd ) and its homolog l-sign (also called dc-signr, cd l) are one of the most investigated c-type lectins involved in viral infection ( figure f ). unlike the main role of collectins in host defense, dc-sign and l-sign have been widely reported to be susceptibility factors that facilitate viral entry into host immune cells [ , , , ] . both dc-sign and l-sign are trans-membrane proteins that are composed of a short cytoplasmic tail, which is responsible for signaling and internalization, a transmembrane region, a neck domain, which consists of eight repeat regions of amino acids, and a carbohydrate recognition domain [ ] . the roles of dc-sign/l-sign in viral infections have been summarized and reviewed previously [ , ] . studies have shown that dc-sign/l-sign are capable of binding to the surface proteins of hiv [ ] , cytomegalovirus (cmv) [ ] , denv [ ] , wnv [ , ] , severe acute respiratory syndrome coronavirus (sars-cov) [ ] [ ] [ ] , hcv [ , ] , ebola virus [ ] and marv [ ] and consequently facilitating viral entry ( figure c ). differential glycosylation patterns of viral surface proteins strongly influence the efficiency of viral recognition by dc-sign/l-sign [ , ] . for example, only mannosylated envelope (e) glycoproteins on denv, but not e proteins with complex glycosylation, have been shown to interact with dc-sign-expressing cells [ ] . the mannose receptor (mr, cd ) is another c-type lectin that functions as a viral recognition receptor on the cell membrane ( figure g ). mr is mainly expressed in multiple immune cells, including macrophages and dendritic cells, and is a key susceptibility factor for denv infection of human macrophages. binding of mr to denv e glycoproteins enhances viral attachment, thus facilitating denv internalization into macrophages, and deglycosylation of the denv e glycoprotein enables the abrogation of this binding, and denv infection of primary human macrophages can be blocked by anti-mr antibodies [ ] . moreover, the interaction between mr and hbv surface antigen (hbsag) enhances viral uptake by dendritic cells (dcs), resulting in the impairment of dc function and the ineffective antiviral response of chronic hbv [ ] . the recognition of viral surface glycoproteins by mr is also beneficial to influenza virus [ , ] and hiv [ ] invasion into host cells ( figure c ). overall, dc-sign/l-sign and mr function as receptors/attachment factors for viral entry into particular cell types. clec a/mdl- (myeloid dap -associating lectin) is a c-type lectin associated with dap ( -kda dnax-activating protein) on myeloid cells such as monocytes, macrophages and neutrophils ( figure h ) [ ] . a recent study has found that clec a binds to dengue glycoproteins. however, in contrast to other c-type lectin receptors, the association between clec a and denv does not result in viral entry, but rather induces dap -mediated immune signaling to stimulate the release of pro-inflammatory cytokines that potentially contribute to the pathogenesis of dengue hemorrhagic fever [ , ] . clec a also directly interacts with japanese encephalitis virus (jev) to induce dap phosphorylation in macrophages and therefore plays a role in jev-induced neuro-inflammation and lethality ( figure c ) [ ] . the blocking of clec a in mice can significantly reduce the infiltration of jev-harboring leukocytes into the central nervous system, thus attenuating neuro-inflammation and protecting the animals from jev-induced lethality [ ] . the discovery of a role of clec a in flaviviral pathogenesis suggests that the extracellular crd modules are generally responsible for the recognition of viral glycoproteins; nonetheless, the intracellular modules determine the role of c-type lectins in viral infection. langerin (also known as cd ), containing a single ca + -dependent crd domain, is a type ii transmembrane c-type lectin that is specifically expressed on langerhans cells ( figure i ). the physiological function of langerin is to trigger the cellular membrane superimposition and zippering that benefit birbeck granule (bg) formation [ ] . langerin is capable of directly binding to hiv- envelope protein gp and thus serves as a potential receptor for hiv- infection in langerhans cells ( figure c ) [ , ] . however, a recent study reported that langerin is a natural barrier for hiv- transmission among langerhans cells. langerin is capable of directly capturing hiv- and sequentially degrading it in bgs to promote t cell elimination of hiv- infection [ ] , suggesting that langerin plays a pleiotropic role in hiv infection. furthermore, langerin functions as an attachment factor to facilitate measles virus (mv) infection in langerhans cells [ ] . a large number of host proteins are abundantly glycosylated. therefore, microbial recognition by c-type lectins relies on the mechanism for distinguishing carbohydrate structures between self and non-self, and the c-type lectin structure largely influences binding avidity and selectivity in the recognition of self and non-self carbohydrate structures [ , ] . based on the x-ray crystal structures of mannose-binding lectin (mbl), the mbl crd sites in the trimer form are too far apart to spatially interact efficiently with common mammalian high-mannose oligosaccharides. however, the dense and repeated arrays of carbohydrates present on the microbial surface can span the distance between the binding sites in mbl, resulting in highly avid multivalent interaction [ , ] . moreover, the number of crds is another determinant for the avidity and strength of differential binding by c-type lectins. the eight different c-type crds of mr contribute to its high binding affinity for single sugars, even though each individual crd motif only displays weak affinity. the crd domain organization also confers mr with the ability to recognize the wide range of different carbohydrates found on the pathogen surface and to distinguish between self and foreign glycoproteins [ ] . galectins are a group of secreted proteins that associate with specific cell surface glycans containing beta-galactosides ( figure d ,e) [ ] . although mammalian galectins lack conventional signal sequences, they reach the cell surface via a particular mechanism. galectins accumulate directly beneath the plasma membrane and are subsequently involved in the establishment of membrane-bound vesicles that pinch off before release outside the cell; galectins then bind to glycoconjugates on the plasma membrane or remain in the extracellular matrix [ ] [ ] [ ] . fifteen galectins have been identified in mammals and are categorized into three structural forms: dimeric, tandem or chimeric. dimeric galectins, also called prototypical galectins, are homodimers and include galectin- , - , - , - , - , - , - , - and - . tandem galectins contain at least two distinct crds within one polypeptide and include galectin subtype- , - , - , - and - . galectin- is specific to mammals, has one crd and a long non-lectin domain, and exists in either a monomeric form or a multivalent complex associated via the non-lectin domains of monomers [ ] ; this property allows galectin- to effectively bridge different ligands to form adhesive networks. current investigations have indicated that direct recognition between galectin- /- and viral-surface glycoproteins is important for host-virus interaction. [ ] . because of its particular binding specificity for galactosides, galectin- recognizes the surface envelope proteins of many human viruses and therefore is involved in viral infection. galectin- binds to niv-f, a viral envelope glycoprotein of nipah virus (niv), to reduce the niv-f-mediated fusion of endothelial cells and thereby inhibit niv-induced syncytium formation [ ] . galectin- directly interacts with the envelope glycoproteins of influenza a/wsn/ virus and inhibits its hemagglutination activity, resulting in the reduction of influenza virus infectivity ( figure b) [ ] . however, galectin- has also been reported to be a susceptibility factor for viral entry. hiv- exploits galectin- to enhance gp -cd interaction, leading to faster viral entry and more robust viral replication ( figure c ) [ ] [ ] [ ] [ ] . in addition to galectin- , the role of galectin- in viral infection has been elucidated by several studies. galectin- has been shown to interact with herpes simplex virus- (hsv- ). rnai-mediated knockdown of galectin- in human corneal keratinocytes significantly impaired hsv- infection, suggesting that hsv- exploits galectin- to enhance its attachment to host cells [ ] . recently, proteomic-based studies have identified galectin- as a host-binding partner of parvovirus minute virus of mice (mvm). the authors proposed that galectin- binding facilitates the access of mvm to its receptor(s) at the plasma membrane and thus promotes mvm endocytosis ( figure c ) [ ] . the above-mentioned evidence indicates a pleiotropic role of galectins during viral infections. the endoplasmic reticulum (er) of mammalian cells contains molecular chaperones and foldases, which are required for forming the active structures of newly synthesized peptides and thus serve as components of the er quality control system. the er-resident chaperones include bip, calnexin ( figure l ) and calreticulin (a calnexin-like soluble form without the transmembrane region) [ , ] . both calnexin and calreticulin are lectin-like, membrane-bound molecular chaperones that associate with newly synthesized proteins in the er. in addition, several studies have indicated that calnexin and calreticulin preferentially interact with glycoproteins that carry monoglucosylated n-linked oligosaccharides [ ] [ ] [ ] [ ] . the maturation of virus-encoded proteins occurs in the er, and calnexin family proteins have been shown to transiently interact with multiple viral proteins that consequently undergo rapid maturation ( figures d and a) . both calnexin and calreticulin can transiently interface with envelope glycoproteins f and hn of sendai virus (sev) [ ] and glycoproteins g /g of uukuniemi virus (uukv) (bunyaviridae family) [ ] to facilitate the rapid maturation of these proteins. during sars-cov infection, maturation of the viral s protein due to its interaction with calnexin is essential for the formation of infective virions [ ] . calnexin/calreticulin also plays a role in the assembly and secretion of hbv middle (m) envelope protein [ ] , hiv- envelope protein gp [ ] and gp [ ] . furthermore, during the rotavirus life cycle, calnexin binds to the er-associated viral transmembrane protein nsp , a nonstructural glycoprotein that acts as a toxin capable of inducing diarrhea in animals [ ] [ ] [ ] . calnexin/calreticulin is also associated with the glycosylation of hantaan virus (htnv, also known as hantavirus) envelope proteins gn and gc and plays a crucial role in the folding of htnv glycoproteins with a high content of high-mannose oligosaccharides [ ] . accumulated evidence suggests that the lectin-like calnexin proteins interact with viral components to largely facilitate viral assembly and protein maturation. the p-type lectin/mannose -phosphate receptors (mprs) are transmembrane glycoproteins that target lysosomal enzymes located in either intracellular organelles or the plasma membrane ( figure j ,k). mprs can bind newly synthesized lysosomal hydrolases in the trans-golgi network (tgn) and deliver them to pre-lysosomal compartments. the mpr crd was originally identified in two types of proteins, cation-independent and cation-dependent mannose -phosphate receptors (ci-mpr and cd-mpr, respectively), both of which recognize mannose- -phosphate (m- -p) to identify and route lysosomal enzymes to the lysosomal compartment [ ] . a previous study demonstrated that human herpes simplex virus (hsv) glycoprotein d (gd) binds to both ci-mpr and cd-mpr. these mprs sort glycoproteins modified with m- -p to lysosomes in the trans-golgi compartment and divert them to the endosomal pathway ( figures e and b) [ , ] . mprs were also found on the surfaces of mammalian cells as serving as putative cellular receptors for hsv entry and cell-cell viral spread; furthermore, chemo-or immuno-blocking mprs was shown to inhibit hsv entry and the production of hsv plaques in monkey cells ( figure c ). mouse cells lacking both ci-mpr and cd-mpr remain sensitive to hsv infection [ ] , suggesting that the expression of mprs is not essential for hsv invasion. varicella zoster virus (vzv) is known as a highly infectious human pathogen, and multiple vzv envelope glycoproteins are modified by m- -p; therefore, ci-mprs appear to be important for vzv infection. intracellular ci-mpr contributes to the transport of enveloped vzv to late endosomes, and the plasmalemmal form is necessary for cellular entry through cell-free vzv particles [ ] . l-type lectins are widely distributed in plants and animals. animal l-type lectins are intracellular luminal proteins that are involved in protein sorting in the luminal er-golgi compartments of animal cells. there are four l-type lectins in mammals: ergic- ( figure m) , ergl, vip , and vipl [ , ] . a recent study has shown that intracellular cargo receptor ergic- interacts with the glycoproteins of arenavirus, hantavirus, coronavirus, orthomyxovirus, and filovirus particles. ergic- is also essential for the propagation of arenavirus, coronavirus, and filovirus; in the absence of ergic- , viral particles can be formed but are noninfectious (figures e and c ) [ ] . in addition to the above-mentioned mammalian lectins, there are some other lectin classes in animals, e.g., m-type, r-type, i-type, chitinase-like, f-box lectins and intelectins. as we have not found reports on direct interactions between these lectins and viral components, we cannot determine the role of these lectins in viral infection based on the current knowledge. lectin crds are conserved throughout evolution, and many lectin homologues have been identified and reported in invertebrates. a homolog of galectin plays a role in opsonization for bacterial clearance in marsupenaeus japonicus [ ] . similarly, a galectin-like factor is expressed on the surface of oyster hemocytes and plays a role in oyster physiology through the recognition of oligosaccharides [ , ] . several proteins identified in crassostrea hongkongensis [ ] , venerupis philippinarum [ ] and trypanosoma cruzi [ ] have been categorized as homologs of mammalian i-type lectins/siglecs with high sialic acid-binding activity. in arthropods, multiple lectins identified in shrimp, such as l-type, p-type/mprs, m-type, and calnexin family factors, have been proposed to be important in shrimp innate immunity [ ] . many c-type lectin homologues in aedes and anopheles mosquitoes have been found to be involved in insect immune responses and pathogenesis [ ] [ ] [ ] . the current investigations of the immune roles of arthropod lectins mainly focus on their anti-bacterial or anti-parasite functions, including microorganism-induced lectin up-regulation, lectin-mediated microorganism recognition and opsonization [ , ] . however, little is known about the molecular details of lectins in arthropod immunity and pathogenesis, especially with regard to the function in arthropod-virus interactions. a recent study on c-type lectins in aedes aegypti initially assessed lectin functions in viral infections of arthropods. tens of c-type lectins were identified in aedes [ , ] and anopheles [ ] mosquitoes, and most are soluble forms [ ] . previous studies have shown that an aedes aegypti c-type lectin, mosquito galactose-specific c-type lectin- (mosgctl- ), interacts with wnv in a calcium-dependent manner to form a mosgctl-virus complex. this complex consequently interacts with mosquito protein tyrosine phosphatase- (mosptp- ), a mosquito homolog of human cd in a. aegypti, to enable viral attachment to the plasma membrane and enhance viral entry. in vivo experiments showed that mosgctl- and mosptp- function as part of the same pathway and are critical for wnv infection of mosquitoes [ ] . further investigations identified that another mosgctl paralogs facilitate dengue infection of mosquitoes. these mosgctls interact with denv- surface e protein and virions, functioning as susceptibility factors for dengue viral entry into mosquito cells. however, mosptp- did not influence dengue infection in mosquitoes, suggesting that other membrane receptors may recruit the denv-mosgctl complex onto the cell membrane for viral entry [ ] . in agreement with the findings in mosquitoes, a recent study has identified a c-type lectin in the shrimp marsupenaeus japonicus that interacts with an envelope protein of white spot syndrome virus (wssv) and consequently associates with a cell-surface calreticulin, which serves as a membrane receptor that facilitates viral entry in a cholesterol-dependent manner [ ] . the study therefore suggested that c-type lectins might play a broad role in expediting many viral infections of arthropods. the role might not be limited to wnv/denv in mosquito and wssv in shrimp but might extend to other virus infections in arthropods. lectins are potential targets for the development of antiviral drugs and vaccines. such lectin-based antiviral strategies are divided into two parts: ( ) lectin-based immune activation and ( ) blockade of lectin receptors against viral entry [ ] . many envelope viruses are protected by their dense carbohydrate shield against efficient recognition and persistent neutralization by the host immune system. various natural and synthetic carbohydrate-binding agents have been screened to refine candidates that can reinforce the recognition of specific pathogens, enhance the cascade amplification of the innate immune response and interrupt virus attachment to receptors. in fact, lectins have been considered as drug targets for many years. several heterologous lectins derived from various organisms have been already selected and introduced into pre-clinic trials for hiv therapy, including svn (scytovirin), a . -kd lectin isolated from aqueous extracts of the cyanobacterium scytonema varium [ ] , and uda (stinging nettle lectin), a . -kd plant lectin isolated from urtica dioica [ , ] . furthermore, the combined usage of uda with hha (amaryllis lectin, from a hippeastrum hybrid) and gna (snowdrop lectin from galanthus nivalis), another two carbohydrate-binding agents, showed broad anti-viral activity against four serotypes of denv in monocyte-derived dendritic cells by preventing virus attachment [ ] . additionally, an interesting monoclonal antibody, g , which interacts with specific, highly conserved glycosylation sites on hiv envelop protein gp shows a broad anti-hiv neutralizing activity. the mechanism of this antibody specifically targeting n-linked glycans is very similar to that of lectins [ ] [ ] [ ] . with regard to arthropod-borne viruses, vector ligands that interact with pathogens are ideal targets for interfering with the successful acquisition of the virus from the vertebrate host. due to the importance of c-type lectins in dengue infection of mosquitoes, these lectin factors may be proposed as targets for the development of vaccines or antiviral drugs. studies show that treatment with mosgctls antisera dramatically interrupted denv- infection of mosquitoes through blood feeding. therefore, the humoral response against mosgctls in mammals could feasibly impair dengue infection of mosquitoes. the approach to blocking mosquito c-type lectins may direct a future avenue for the development of a transmission-blocking vaccine that interrupts the mosquito-borne viral life cycle and reduces disease burden [ ] . lectins comprise highly diverse proteins with different carbohydrate recognition activities and play pleiotropic roles in the immune responses and pathogenesis of many viral infections (summarized in table ). the interaction between lectins and viral glycoproteins may lead to the three following consequences: ( ) lectins, such as mbl and sps, function as pattern recognition molecules that bind a repertoire of viruses and activate antiviral immune responses; ( ) lectins are employed as attachment factors that recruit viral particles to the cell membrane to enhance viral entry, e.g., some mammalian lectins (dc-sign, l-sign, mr and mprs) or their homologs in arthropods (mosgctls); and ( ) some intracellular lectins, such as calnexin and ergic- , function as susceptibility factors associated with virus-encoded proteins to facilitate viral replication or assembly (please refer to figures and ) . interestingly, the same lectin may show opposing roles in different virus infections. for example, galectin- binds to niv to inhibit syncytium formation and recognizes iav to reduce its infectivity [ , ] ; however, galectin- was also reported to be a susceptibility factor that enhanced gp -cd interactions, thus facilitating hiv entry [ ] [ ] [ ] [ ] . the current accumulated knowledge indicates that lectins are crucial host factors with complex and profound roles in the process of viral infection. lectins for investigation of proteins and carbohydrates molecular structure animal lectins animal lectins: a historical introduction and overview demonstration of carbohydrate recognition activity in diverse proteins which share a common primary structure motif two distinct classes of carbohydrate-recognition domains in animal lectins mannose-binding proteins isolated from rat liver contain carbohydrate-recognition domains linked to collagenous tails. complete primary structures and homology with pulmonary surfactant apoprotein the c-type lectin-like domain superfamily engineering galactose-binding activity into a c-type mannose-binding protein binding of sugar ligands to ca( +)-dependent animal lectins. ii. generation of high-affinity galactose binding by site-directed mutagenesis studies on the carbohydrate-binding characteristics of human pulmonary surfactant-associated protein a and comparison with two other collectins: mannan-binding protein and conglutinin the major lung surfactant protein, sp - , is a calciumdependent, carbohydrate-binding protein interaction of tetranectin with sulphated polysaccharides and trypan blue. scand lectin-carbohydrate interactions: different folds, common recognition principles mechanism of n-acetylgalactosamine binding to a c-type animal lectin carbohydrate-recognition domain mechanism of ph-dependent n-acetylgalactosamine binding by a functional mimic of the hepatocyte asialoglycoprotein receptor human mannose-binding protein carbohydrate recognition domain trimerizes through a triple alpha-helical coiled-coil collections and ficolins: humoral lectins of the innate immune defense two mechanisms for mannose-binding protein modulation of the activity of its associated serine proteases interaction of mannose-binding lectin with hiv- is sufficient for virus opsonization but not neutralization high frequency of variant alleles of the mannose-binding lectin (mbl ) gene are associated with patients infected by hepatitis b virus mannose-binding lectin in chronic hepatitis b virus infection mannose-binding lectin mbl gene polymorphisms and outcome of hepatitis c virus-infected patients direct complement restriction of flavivirus infection requires glycan recognition by mannose-binding lectin a human serum mannose-binding protein inhibits in vitro infection by the human immunodeficiency virus glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type binding and neutralization by mannose-binding lectin dc-sign, a dendritic cell-specific hiv- -binding protein that enhances transinfection of t cells c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses dc-sign (cd ) mediates dengue virus infection of human dendritic cells inhibition of dc-sign-mediated trans infection of t cells by mannose-binding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization localization of lung surfactant protein d on mucosal surfaces in human tissues immunocytochemical localization of surfactant protein d (sp-d) in type ii cells, clara cells, and alveolar macrophages of rat lung expression sites of the collectin sp-d suggest its importance in first line host defence: power of combining in situ hybridisation, rt-pcr and immunohistochemistry mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins pulmonary surfactant protein d in first-line innate defence against influenza a virusinfections site-directed mutagenesis of cys- and cys- of pulmonary surfactant protein d. expression of a trimeric protein with altered anti-viral properties mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro inhibition of influenza viral neuraminidase activity by collectins evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses recombinant sp-d carbohydrate recognition domain is a chemoattractant for human neutrophils surfactant protein a binds to the fusion glycoprotein of respiratory syncytial virus and neutralizes virion infectivity surfactant protein a enhances uptake of respiratory syncytial virus by monocytes and u macrophages respiratory syncytial virus and pulmonary surfactant c-type lectin receptors on dendritic cells and langerhans cells dc-sign: escape mechanism for pathogens the c type lectins dc-sign and l-sign: receptors for viral glycoproteins nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection dc-sign enhances infection of cells with glycosylated west nile virus in vitro and virus replication in human dendritic cells induces production of ifn-alpha and tnf-alpha dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign -sign) is a receptor for severe acute respiratory syndrome coronavirus l-sign (cd l) is a liver-specific capture receptor for hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dc-signr dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals the mannose receptor mediates dengue virus infection of macrophages the mannose receptor acts as hepatitis b virus surface antigen receptor mediating interaction with intrahepaticdendritic cells involvement of themannose receptor in infection of macrophages by influenza virus reading pc macrophage receptors for influenza a virus: role of the macrophage galactose-type lectin and mannose receptorin viral entry oligomerization of the macrophage mannose receptor enhances gp -mediated binding of hiv- immunoreceptor dap bearing a tyrosine-based activation motif is involved in activating nk cells clec a is critical for dengue-virus-induced lethal disease clec a is critical for dengue virus-induced inflammasome activation in human macrophages clec a regulates japanese encephalitis virus-induced neuroinflammation and lethality langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules the role of dendritic cell c-type lectin receptors in hiv pathogenesis diversity of receptors binding hiv on dendritic cell subsets langerin is a natural barrier to hiv- transmission by langerhans cells human langerhans cells capture measles virus through langerin and present viral antigens to cd + t cells but are incapable of cross-presentation the c-type lectin superfamily in the immune system self-and nonself-recognition by c-type lectins on dendritic cells trimeric structure of a c-type mannose-binding protein galectins: a family of animal beta-galactosidebinding lectins secretion of the galectin family of mammalian carbohydrate-binding proteins secretion of the baby hamster kidney -kda galactose-binding lectin from polarized and nonpolarized cells: a pathway independent of the endoplasmic reticulum-golgi complex plasma membrane targetting, vesicular budding and release of galectin from the cytoplasm of mammalian cells during secretion galectins: regulators of acute and chronic inflammation galectins: structure, function and therapeutic potential endothelial galectin- binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation galectin- binds to influenza virus and ameliorates influenza virus pathogenesis galectin- promotes hiv- infectivity in macrophages through stabilization of viral adsorption galectin- acts as a soluble host factor that promotes hiv- infectivity through stabilization of virus attachment to host cells galectin- and hiv- infection host-soluble galectin- promotes hiv- replication through a direct interaction with glycans of viral gp andhost cd binding of transmembrane mucins to galectin- limits herpesvirus infection of human corneal keratinocytes proteomic analysis identifies a novel function for galectin- in the cell entry of parvovirus role of n-oligosaccharides endoplasmic reticulum processing reactions in glycoprotein folding and degradation lectins as chaperones in glycoprotein folding association of folding intermediates of glycoproteins with calnexin during protein maturation role of n-linked oligosaccharide recognition, glucose trimming, and calnexin in glycoprotein folding and quality control transient, lectin-like association of calreticulin with folding intermediates of cellular and viral glycoproteins glycan-dependent and -independent association of vesicular stomatitis virus g protein with calnexin kinetics of interactions of sendai virus envelope glycoproteins, f and hn, with endoplasmic reticulum-residentmolecular chaperones, bip, calnexin, and calreticulin transient association of calnexin and calreticulin with newly synthesized g and g glycoproteins of uukuniemivirus (family bunyaviridae) monitoring of s protein maturation in the endoplasmic reticulum by calnexin is important for the infectivity of severe acute respiratory syndrome coronavirus role for calnexin and n-linked glycosylation in the assembly and secretion of hepatitis b virus middle envelopeprotein particles calreticulin interacts with newly synthesized human immunodeficiency virus type envelope glycoprotein, suggesting a chaperone function similar to that of calnexin effects of inefficient cleavage of the signal sequence of hiv- gp on its association with calnexin, folding, and intracellular transport the rotavirus nonstructural glycoprotein nsp possesses membrane destabilization activity rotavirus nonstructural glycoprotein nsp alters plasma membrane permeability in mammalian cells the molecular chaperone calnexin interacts with the nsp enterotoxin of rotavirus in vivo and in vitro analysis of n-linked glycosylation of hantaan virus glycoproteins and the role of oligosaccharide side chains in protein folding and intracellular trafficking lysosomal enzyme binding to mouse p d macrophage membranes lacking the -kda mannose -phosphate receptor: evidence for the existence of a second mannose -phosphate receptor herpes simplex virus glycoprotein d acquires mannose -phosphate residues and binds to mannose -phosphate receptors role of mannose- -phosphate receptors in herpes simplex virus entry into cells and cell-to-cell transmission mannose -phosphate receptor dependence of varicella zoster virus infection in vitro and in the epidermis during varicella and zoster a putative novel class of animal lectins in the secretory pathway homologous to leguminous lectins structures of the carbohydrate recognition domain of ca + -independent cargo receptors emp p and emp p the intracellular cargo receptor ergic- is required for the production of infectious arenavirus, coronavirus, and filovirus particles a galectin from the kuruma shrimp (marsupenaeus japonicus) functions as an opsonin and promotes bacterial clearance from hemolymph hemocytes and plasma of the eastern oyster (crassostrea virginica) display a diverse repertoire of sulfated and blood group a-modified n-glycans the galectin cvgal from the eastern oyster (crassostrea virginica) binds to blood group a oligosaccharides on the hemocyte surface a novel sialic acid binding lectin with anti-bacterial activity from the hong kong oyster (crassostrea hongkongensis) cloning and characterization of a sialic acid binding lectins (sabl) from manila clam molecular interaction of siglecs (sialic acid-binding ig-like lectins) with sialylated ligands on trypanosoma cruzi diversity and multiple functions of lectins in shrimp immunity transmission-blocking antibodies against mosquito c-type lectins for dengue prevention cloning and characterization of a mannose binding c-type lectin gene from salivary gland of aedes albopictus the genome sequence of the malaria mosquito anopheles gambiae purification, characterization and cdna cloning of a novel lipopolysaccharide-binding lectin from the shrimp penaeus monodon a novel c-type lectin (fclec ) facilitates the clearance of vibrio anguillarumin in vivo in chinese white shrimp a c-type lectin collaborates with a cd phosphatase homolog to facilitate west nile virus infection of mosquitoes collaboration between a soluble c-type lectin and calreticulin facilitates white spot syndrome virus infection in shrimp targeting the c-type lectins-mediated host-pathogen interactions with dextran overexpression and purification of scytovirin, a potent, novel anti-hiv protein from the cultured cyanobacterium scytonema varium the mannose-specific plant lectins from cymbidium hybrid and epipactis helleborineand the (n-acetylglucosamine)n-specific plant lectin from urtica dioicaare potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp a new therapeutic concept to hit the achilles heel of hiv broad antiviral activity of carbohydrate-binding agents against the four serotypes of dengue virus in monocyte-derived dendritic cells the broadly neutralizing anti-human immunodeficiency virus type antibody g recognizes a cluster of alpha → mannose residues on the outer face of gp antibody domain exchange is an immunological solution to carbohydrate cluster recognition dissection of the carbohydrate specificity of the broadly neutralizing anti-hiv- antibody g key: cord- -lnik k authors: celerino da silva, ronaldo; segat, ludovica; crovella, sergio title: role of dc-sign and l-sign receptors in hiv- vertical transmission date: - - journal: hum immunol doi: . /j.humimm. . . sha: doc_id: cord_uid: lnik k the innate immune system acts in the first line of host defense against pathogens. one of the mechanisms used involves the early recognition and uptake of microbes by host professional phagocytes, through pattern recognition receptors (prrs). these prrs bind to conserved microbial ligands expressed by pathogens and initiate both innate and adaptative immune responses. some prrs located on the surface of dendritic cells (dcs) and other cells seem to play an important role in human immunodeficiency virus type (hiv- ) transmission. dendritic cell–specific intercellular adhesion molecule– grabbing non-integrin, cd (dc-sign) and its homolog, dc-sign-related (dc-signr or l-sign) receptors are pprs able to bind the hiv- gp envelope protein and, because alterations in their expression patterns also occur, they might play a role in both horizontal and vertical transmission as well as in disseminating the virus within the host. this review aims to explore the involvement of the dc-sign and l-sign receptors in hiv- transmission from mother to child. the joint united nations program on hiv- /aids estimates that . million children worldwide are infected with human immunodeficiency virus type (hiv- ) [ ] and that more than , children were newly infected in . mother-to-child transmission, also known as vertical transmission, accounts for more than % of all hiv- infections in children [ ] and can occur in three ways [ ] [ ] [ ] : during pregnancy through the placenta (transplacental or intrauterine transmission); during delivery (intrapartum transmission) through amniotic fluids, infected blood and cervical secretions; and during the process of breastfeeding. in addition, some independent factors have been associated with vertical transmission of hiv- , such as high maternal viral load, low amount of cd ϩ t cells, vaginal delivery and lower gestational age [ , ] . global estimates show that, without specific medication, the rate of transmission of hiv- from mother to child is around - % [ ] ; using antiretroviral therapies, this percentage has sharply dropped to %. however, although prophylactic antiretroviral therapy can reduce mother-to-child transmission to %, the limited access to timely diagnosis and drugs in many developing countries reduces the potential impact of this strategy [ ] . despite the sharp fall in the rate of viral transmission, a significant percentage of children are still being infected with hiv- ; the mechanisms used by the virus to escape the immune response and infect targets cells of children born to infected mothers treated with antiretroviral therapies still remain to be clarified. a better understanding of the immunologic mechanisms acting at the maternal-fetal interface and of host-pathogen interaction is essential for the development of alternative interventions aimed to prevent viral transmission. this review aims to explore the involvement of the dendritic cell-specific intercellular adhesion molecule- grabbing non-integrin, cd (dc-sign) and dc-sign-related c-type lectin domain family , member m (l-sign) receptors in hiv- transmission from mother to child. the innate immune system is the first line of host defense against pathogens; it involves the early recognition and uptake of microbes by host professional phagocytes, such as dendritic cells (dcs) and macrophages, through germline-encoded receptors, known as pattern recognition receptors (prrs) [ ] . these proteins bind to conserved microbial ligands expressed by the pathogens, and initiate both innate and adaptative immune responses. prrs are involved in phagocytosis, and antigen presentation could activate intracellular signaling and cytokine secretion. the efficiency of this initial pathogen recognition may have important consequences in the pathogenesis of infectious diseases [ ] . some prrs located on the surface of dcs and other cells seem to play an important role in hiv- transmission. of particular interest are the dc-sign and its close relative, l-sign (also known as dc-sign related [dc-signr], cd l or clec m) receptors [ , ] . dc-sign and l-sign receptors, two c-type lectins, are long type ii integral membrane proteins [ , ] that are involved in both innate and adaptive immunity [ ] [ ] [ ] . they present strong dependence on calcium and act as cellular adhesion's receptors and are involved in pathogen recognition [ , ] . as pathogen-recognition receptors, both these lectins recognize a wide range of microorganisms, some of which have a major impact on public health. for example, dc-sign captures viruses, such as ebola virus [ ] , hepatitis c virus [ , ] , dengue virus [ ] , cytomegalovirus [ ] , and sars coronavirus (sars-cov) [ , ] , bacteria such as mycobacterium tuberculosis [ ] and helicobacter pylori [ ] , and parasites such as leishmania pifanoi [ ] . l-sign is able to capture viruses such as ebola virus [ ] , hepatitis c virus [ , , ] , and more recently sars-cov [ , ] , as well as bacteria such as m tuberculosis [ ] , and leishmania infantum [ ] . both dc-sign and l-sign can recognize and capture human immunodeficiency virus by binding to the gp glycoprotein [ ] [ ] [ ] [ ] [ ] . dc-sign and l-sign receptors are organized into three structurally distinct regions ( fig. ) : an intracytoplasmatic tail domain responsible for internalization and signal transduction [ ] that consists of n-terminal, ll, and yksl motifs and a triacidic group (eea), then a transmembrane domain and finally an extracellular domain, which is further divided into two structures, the neck repeat region and the carbohydrate recognition domain (crd) [ ] . the neck repeat region usually consists of full and incomplete tandem repeats of a sequence of highly conserved amino acids, but the number of repeats can vary in the population: the neck repeat region of the l-sign receptor is highly polymorphic ( - repetitions are often found), while the one of dc-sign is less polymorphic (mainly seven repetitions) [ , , ] . the neck repeats region plays a crucial role in tetramerization and supports carbohydrates' recognition, thus directly influencing the receptor's binding affinity to pathogens [ , , ] . the number of tandem-neck repeats determines the multimerization status (fig. ). feinberg et al. [ ] showed that the lack of two repeats (five-repeat allele) results in partial dissociation of the final tetramer, whereas the lack of even more than five repeats causes a reduction in the overall stability of the molecule. by using a series of recombinant soluble receptors with different number of repeats, synder et al. [ ] showed that the binding affinity to hiv- gp glycoprotein was affected by the length of the repeats and multimerization status, with tetrameric forms presenting a higher affinity than shorter monomeric forms. the crd, both in dc-sign and l-sign, is flexibly connected to the neck repeat region, allowing a departure from the membrane, which enables the binding of pathogens in a calcium-dependent manner [ ] . dc-sign and l-sign belong to the cd gene family and probably originated following a gene duplication event [ ] . the human genes encoding dc-sign and l-sign map on p . - and extend approximately for kb [ , ] . in addition, they share the same introns and exons organization (consisting in seven exons and five introns) and the encoded proteins present a high similarity at the amino acid level ( % identity) [ ] . the two receptors are characterized by different expression patterns. dc-sign is highly expressed in monocytes and cd ϩ monocyte-derived dcs and in subsets of immature and mature dcs in various tissues such as dermis, mucosa, spleen, placenta (special- ized macrophages of decidua and hofbauer cells in the chorionic villi), and lung (specialized macrophages in the alveoli) [ , , ] . in contrast, l-sign is not expressed by dcs or monocyte-derived dcs in vitro, and its expression is limited to some tissues, such as lymph nodes (endothelial cells in the subcapsular sinus), liver (sinusoidal endothelial cells), placenta (capillary endothelial cells), lung (alveolar cells and endothelial cells), and intestine (villi capillaries in the lamina propria of the terminal ileum, peyer's patches) [ , ] . as a specific adhesion receptor, dc-sign mediates the interaction between dcs and t cells by binding with high affinity to icam- [ , ] . the interaction between dc-sign and icam- expressed on t cells contributes to a close interaction between dcs and t cells required for an efficient antigen presentation. dc-sign can also facilitate the capture of viral antigens by class i and class ii mhc, leading to activation of specific cd ϩ and cd ϩ t cells [ ] . the interaction between dc-sign and l-sign and the hiv- has been already well studied [ , , [ ] [ ] [ ] , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . as mentioned before, dc-sign can bind to the gp protein of hiv- [ , , ] capturing the virus and possibly increasing hiv- transmission [ ] . similar to dc-sign, l-sign also captures the hiv- virus by binding to the gp and promoting the enhancement of t cell infection by hiv- in trans [ , ] ; however, l-sign can also internalize the virus and promote virus degradation in a proteasomedependent manner, possibly directly affecting the outcome of the infection by hiv- [ ] . the presence of l-sign on the surface of endothelial cells in lymph node sinuses represents an obvious mechanism by which the virus can be transmitted to cd ϩ cells that traffic into lymph nodes via the afferent lymphatics. in addition, since l-sign binds to icam- and may bind to other cell surface receptors, interactions between t-cells and the endothelial cell surface may occur more frequently, increasing the likelihood of virus transmission [ ] . although dc-sign and l-sign are not direct receptors for hiv- infection, they work efficiently in the capture of hiv- from the periphery, thus facilitating viral transmission to secondary lymphoid organs rich in t cells and increasing the infection of target cd ϩ cells [ , , ] . the hiv- virus can infect cells through two different paths: the trans-and the cis-infection (fig. a, b) . although infection in trans occurs when dc-sign is expressed on a separate cell from the one that becomes infected, the infection in cis may occur when dc-sign is co-expressed with cd and chemokine receptor (e.g., ccr ) on a permissive cell type, such as macrophages [ , , ] . co-expression of cd , ccr , and cxcr receptors with dc-sign receptor has been blamed for the increase in the efficiency of hiv- infection, suggesting the involvement of the dc-sign receptor in viral transmission, besides increasing the efficiency of the infection in cis [ ] . both dc-sign and l-sign receptors are involved in hiv- infection in trans [ ] . however, the hiv- infection in cis, has only been observed so far for dc-sign receptor. the interaction of dc-sign and l-sign molecules with the hiv- occurs through the connection between their crd with the gp viral envelope glycoprotein [ , ] . binding of the gp to dc-sign and l-sign molecules may induce a conformational change in the gp itself that enables a more efficient interaction with cd and/or the chemokine receptor and subsequent membrane fusion with t cells. alternatively, the binding of viral particles to the dcs may increase the probability that entry will occur after binding to the cd and co-receptor complex on target cells [ ] . the ability of dc-sign and l-sign receptors to capture and transmit the hiv- to t cells may largely depend on their membrane organization in rafts or their capability to multimerize [ ] . in addition, alternative splicing events can occur in dc-sign e l-sign, leading to the production of a vast repertoire of membrane-bound and soluble isoforms, which may also differently affect the process of hiv- transmission [ , , ] . the human placenta is responsible for a close juxtaposition between fetal and maternal blood; however, this apparent barrier is permeable enough to display the hiv- from the mother, a fact that leads to fetal exposure to virus [ ] . thus, the placenta can play an important role in the transmission of hiv- infection. most cases of vertical transmission via uterus occur through of the placenta (fig. ) , especially during the third trimester of pregnancy [ ] . it is estimated that the trans-placental or intrauterine hiv- transfer comprise - % of cases of vertical transmission, but little is known about the mechanisms involved in the transmission of the virus [ ] . some cell types are pointed out as likely targets for the viral spread, such as macrophages present in the decidua and spe- cialized macrophages present in the placenta (fig. ) , also known as hofbauer cells [ ] . both decidual macrophages and hofbauer cells play important roles in the placental physiology, the firsts promoting the development and control of blood flow and the latter acting in the defense against infectious agents (hofbauer cells) [ ] . hofbauer cells, specialized macrophages from human placenta, support infection by hiv- both in vitro and in vivo [ ] . this ability to support hiv- infection is probably associated with the expression of some cellular receptors related to the infection of t lymphocytes, such as cd , ccr , cxcr , and dc-sign, also expressed in these cells [ , ] . during pregnancy, there is an increased expression of dc-sign by hofbauer cells in the chorionic villi, and this expression has been correlated with increased rates of hiv- vertical transmission [ ] . thus, dc-sign can enhance the binding of hiv- on the surface of hofbauer cells, providing an efficient mechanism by which the virus can be transmitted to other receivers permissible to hiv- in trans. thus, one can wonder: how is the contact between native virus-bound infected cells with the fetal cells expressing receptors for hiv- ? the main physical barrier between fetal hofbauer cells and maternal fluids is the wall of the trophoblast cells; however trophoblast cells can express receptors for hiv- entry, so they can be infected by the virus [ ] . moreover, breaches in the wall of the trophoblasts can originate from spontaneous processes or in consequence of infectious diseases (such as corioamniotite) and be-havioral habits such as smoking and drug use [ , ] , allowing a direct contact between fetal hofbauer cells dc-sign ϩ cd ϩ ccr ϩ cxcr ϩ and viral particles adsorbed to the maternal decidual macrophages or dcs expressing dc-sign, present in maternal blood [ , , ] . therefore, the contact between maternal and fetal cells through the wall of the trophoblasts, allows the efficient spreading of hiv- to fetal cells expressing receptors for viral binding and entry, allowing establishment of the infection [ ] . a study has suggested that the mechanism of hiv- association with the cells, such as hiv- adsorbed to the dc-sign receptor, operates more efficiently in pregnancies where the viremia remains low because of the administration of antiretroviral therapies [ ] . why does this happen? some authors explain that binding of viral particles to dc-sign may focus or concentrate the virus particles at the surface of the dc and may thus increase the probability that entry will occur after binding to the cd and co-receptor complex on target cells [ ] . in addition, the hiv- virus can remain viable for several days on the dc-sign-expressing cell, and then can be more efficiently transferred to t cells than to the transfer executed by free cells [ , ] . for the l-sign receptor, this fact is not observed. in addition, some authors propose three different mechanisms to explain how the hiv- virus is transmitted from mother to child, via the placenta. the first mechanism suggests that hofbauer cells infected with hiv- or with the virus adsorbed to their cell membrane through receptors, such as dc-sign, may enter the fetus through the umbilical vein [ , ] . the second mechanism is that hofbauer cell infected by hiv- or carrying the virus adsorbed on the surface, remains in situ in the chorionic villi, promoting hiv- antigen presentation and subsequent t lymphocytes infection. however, this mechanism seems unlikely, since t lymphocytes are inconspicuous in chorionic villi [ , ] . the third mechanism argues that hofbauer cells may become infected by hiv- and may release infectious viral particles, which may become adsorbed to l-sign on the immediately adjacent placental capillary endothelium. the endothelium may, in turn, mediate infection of hiv- receptorpositive t-lymphocytes circulating in the blood. infected t lymphocytes or hofbauer cells either productively infected with hiv- or simply with the virus adsorbed to their surface may then travel between the placenta and the fetus in umbilical cord blood [ , , ] . in the intra-and post-partum vertical transmission of hiv- , the virus is delivered and transmitted because of the contact with amniotic fluid, maternal blood, and cervical secretion (intrapartum) or with breast milk (post-partum) [ , ] . it has been reported that, in the absence of a prophylactic antiretroviral therapy, the breast milk of infected mothers is responsible for more than % of cases of children infected with hiv- via vertical transmission [ ] [ ] [ ] ] . some factors, such as the viral load in the plasma and breast milk may be relevant for vertical transmission of hiv- [ ] . transmission of the hiv- virus from mother to child via breast milk can occur by free virus particles and/or viral particles associated with cells [ ] ; in this case, the expression of cellular receptors for recognition and adhesion of pathogens is required. among the cell types involved in the transmission of hiv- via breastfeeding, macrophages and mammary epithelial cells should be mentioned. breast milk is the only bodily fluid that contains a large number of macrophages, comprising more than % of all cells present in colostrum [ ] . expressing ccr , macrophages are prime targets of the hiv- virus, which uses the co-receptors ccr for viral entry [ ] . in addition, macrophages, derived from peripheral blood monocytes (pbmo), are present in different concentrations throughout lactation, acting as immunoprotective in situ [ ] . moreover, it has been reported that macrophages also express dc-sign receptors [ ] . in certain situations, the expression levels of dc-sign on macrophages can be quite high, especially when stimulated with interleukin (il)- , which also promotes reduction in the expression of ccr and cxcr , suggesting the need for changes in local inflammatory th dominance for an acceleration of hiv- transmission via breastfeeding [ - ] . local production of il- during infectious processes, as in mastitis, can over-regulate the expression of dc-sign on macrophages, suggesting the association of mastitis with high viral load in breast milk and high risk of vertical transmission of the virus [ ] . along with the macrophages, mammary epithelial cells may also be infected by hiv- , through the co-receptor cxcr [ ] . some studies suggest the possibility of a hiv- compartmentalization between blood and milk, suggesting that the virus could be produced in and transmitted by the milk, through the mammary epithelial cells. in this sense, the viruses that derived from mammary epithelial cells can determine the tropism of hiv- transmitted to cells located in the gastrointestinal tract [ ] . after being introduced in the organism through infected breast milk, the virus reach the mucosa of the upper intestine, where, in the lamina propria, a large pool of lymphocytes expressing ccr and cxcr facilitate viral replication. the presence of dcs expressing a series of receptors, such as cd /ccr , dc-sign, and dc , has been reported in the human gut [ ] . from the mucosa, the virus is systemically spread and produces a profound depletion of cd ϩ t cells, with monocytes and macrophages also acting as cellular reservoirs for hiv- [ ] . the viral entry through the mucosa of the gastrointestinal tract can be mediated by the binding of the dc-sign receptor, expressed on dcs, to the viral gp protein [ ] . this interaction appears to be more pronounced in the tonsils at the top of the esophagus and intestinal tract [ ] . the infectivity of viruses associated with cells and captured by dc-sign is stable even in presence of the acidification process occurring in the gastrointestinal tract, suggesting that the virus bound to dcs through dc-sign is protected from the action of the gastric juice. the fact that the free viral particles loose their infectivity when exposed to acidic environments, suggest that the transmission of the virus to free cells in milk is hampered by gastric juice [ ] . however, it is possible that free cells become infected in the oral mucosa and esophagus, where the acidity is not high. studies with raji cells expressing dc-sign, preincubated with pbs and with the hiv- virus, showed an efficient viral transfer. however, raji cells expressing dc-sign, incubated with hiv- virus and uninfected human milk, showed a significant reduction of the binding of hiv- gp to dc-sign receptor. this suggests that in human milk some factors that could prevent the interaction between the gp and dc-sign receptor exist [ ] . similar tests were conducted for l-sign receptor, however, breast milk did not inhibit the interaction between the viral proteins and the receptor, suggesting that l-sign receptor can be used by the virus to increase its infectivity [ ] . some studies also report that exclusive feeding with uninfected mothers' milk during the first months of life protects against a variety of infections, including hiv- , and help fight morbidity and mortality, suggesting that there should be certain components in the human milk that may protect against the transmission of the virus [ ] . so, what are these likely factors in breast milk, and how do they act in protection against the infection caused by hiv- ? breast milk is provided with a series of antimicrobial compounds, such as lactoferrin, lysozyme, secretory leukocyte protease inhibitor, lactodifucotetrase, lacto-n-fucopentose i, ii, and iii, and monofucosilacto-n-hexose iii, among others, which are associated with a reduced rate of hiv- transmission [ , ] . some studies attribute this reduction in viral transmission to certain antigens, such as lewis structures, which compete with the gp for a binding site in the dc-sign receptor, inhibiting the viral transfer to cd ϩ t cells [ ] . inhibition of the binding between gp and dc-sign receptor is probably due to the size of the compound, which contains many lewis structures that mask the interaction sites [ ] . compounds containing lewis structures and present in breast milk were shown to interact with dc-sign, blocking the response of th cells and resulting in an increased responsiveness of th cells, suggesting that these compounds may influence the immune response by acting as immunomodulatory factors [ ] . a constituent of human milk, bile salt-stimulated lipase, a lewis x (le x )-containing glycoprotein secreted by the pancreas as well as by mammary gland, has been shown to inhibit dc-sign binding to hiv and dc-sign-mediated transfer of hiv- to cd ϩ lymphocytes, by competing with the virus for the binding to dc-sign [ ] . the binding of bile salt-stimulated lipase to dc-sign can be prevented using an antibody against le x , thus demonstrating the importance of the le [ ] epitope. others constituents of human milk, such as human milk oligosaccharides and muc (epithelial mucin), have shown promising results and could be used to develop drugs that inhibit the hiv- binding to dc-sign [ , ] . hong et al. [ ] found a reduction of more than % in the interaction between gp and the receptor protein dc-sign when using human milk oligosaccharides at a concentration of . g/l, the one usually present in breast milk. additionally, saeland et al. [ ] also described the blocking of the interaction between the gp and dc-sign receptor in the presence of muc factor, present in human milk. because of the blockade, there was the prevention of the virus transmission to cd ϩ t cells. blocking dc-sign may be a double-edged sword. it may reduce the entrance of certain viruses, such as hiv- , but at the same time it may also reduce the ability of the infant's immune system to detect and fight other pathogens [ ] . for some genes, susceptibility and/or resistance to certain (infectious but not only) diseases has been associated with gene expression levels and with the presence of gene variations/mutations. can this happen also for dc-sign and l-sign? can variations in the gene encoding dc-sign and l-sign be associated with vertical transmission of hiv- ? to date, except one study regarding the l-sign gene, no other genetic studies trying to associate mutations in the genes encoding for dc-sign and l-sign with the vertical transmission of the hiv- have been performed. boily-larouche et al. [ ] performed an association study in a well-characterized cohort of hiv- -infected mothers and their children from zimbabwe, and found that children with two copies of h and/or h haplotype of l-sign were about . times more at risk for intrauterine transmission of hiv- and . times at risk for intrapartum transmission. the h and h haplotypes are characterized by two single nucleotide polymorphisms in the promoter region (p- a) and the intron (int - a) that associate with a reduction of the transcriptional activity. the same study also showed that infants homozygous for the h haplotype showed a more than fourfold decrease in the level of placental l-sign transcripts, and in particular of the membrane linked isoforms [ ] . a reduced expression of l-sign (especially of the membrane isoforms) in the endothelial cells of capillaries in the placenta may facilitate the binding of hiv- to viral entry receptors of endothelial cells, such as ccr , which can facilitate the migration of maternal hiv- across the placental barrier, resulting in intrauterine transmission of hiv- [ ] . the membrane bound l-sign receptors are responsible for catching the virus. after capture, the virus adhered to l-sign may undergo degradation processes or be presented as antigens. thus, these receptors act to protect the infant against infection by hiv- [ ] . boily-larouche et al. [ ] explain these discoveries with the hypothesis that when the levels of placental l-sign-bound membrane are reduced, virus fails to bind to l-sign and binds preferentially to ccr receptors on endothelial cells of capillaries, resulting in loss of integrity of the placental barrier and increase the passage of cells infected by hiv- in fetal circulation, leading to vertical transmission. in view of what has been discussed, much evidence exist that the dc-sign and l-sign receptors are involved in the transmission of hiv- from mother to child. therefore, the dc-sign and l-sign receptors should be likely targets for the development of new drugs and antiretroviral therapies, to challenge the spread of viral transmission. in addition to this, given that only a few genetic studies have been performed to investigate the possible involvement of dc-sign and l-sign receptors in the genetic mechanisms correlated with vertical transmission of the hiv- virus, we believe that more detailed studies aiming to elucidate the role of genetic variants from different worldwide populations in susceptibility and/or resistance to hiv- infection are needed. human milk oligosaccharides reduce hiv-gp binding to dendritic cell-specific icam -grabbing nonintegrin dcsigns) macrophage hiv infection and the gastrointestinal tract reservoir component in human milk binds dc-sign and inhibits hiv transfer to cd ϩ t lymphocytes functional genetic variants in dc-signr are associated with motherto-child transmission of hiv mother-to-child transmission of hiv: a global perspective the evolution and genetics of innate immunity promoter and neck region length variation of dc-sign is not associated with susceptibility to tuberculosis in tunisian patientes the role of dc-sign and dc-signr in hiv and siv attachment, infection, and transmission dc-sign, a c-type lectin on dendritic cells that unveils many aspects of dendritic cell biology dc-sign: a guide to some mysteries of dendritic cells polymorphic varieants in dc-sign, dc-signr and sdf- in high risk seronegative and hiv patients in northern asian indians molecular characterization of dendritic cells operating at the interface of innate of acquired immunity dc-sign and dc-signr genetic diversity among different ethnic populations: potential implications for pathogen recognition and disease susceptibility c-type lectins on dendritic cells: key modulators for the induction of immune responses dc-sign (dendritic cell-specific icam- grabbing non-integrin) and dc-sign-related dcsignr): friend or foe? the signs for infection c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans cd l) and dcsign(cd ) mediate transinfection of liver cells by hepatitis c virus hepatitis c virus glycoproteins interact with dc-sign and dcsignr cd ) mediates dengue virus infection of human dendritic cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells helicobacter pylori modulates the t helper cell /t helper cell balance through phase-variable interaction between lipopolysaccharide and dcsign role of the c-type lectins dc-sign and l-sign in leishmania interaction with host phagocytes dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein cd l lsigns) is a receptor for severeacute respiratory syndrome coronavirus identification of the mycobacterial carbohydratestructure that binds the ctype lectins dc-sign, l-sign and signr structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr dc-sign, a dendritic cell-specific hiv-binding protein that enhances trans-infection of t cells identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin dcsigns)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv infection dendritic-cell interactions with hiv: infection and viral dissemination impact of polymorphisms in the dc-signr neck domain on the interaction with pathogens extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr characterization of dc-sign/r interaction with human immunodeficiency virus type gp and icam molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor influence of polymorphism in dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin-related. dcsignr) gene on hiv trans-infection most dc-signr transcripts at mucosal hiv transmission sites are alternatively spliced isoforms association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection dc-sign, a dentritic cell-specific hiv receptor present in placenta that infects t cells in trans-a review dendritic cells and transmission of hiv binding of human immunodeficiency vÎrus type to immature dendritic cells can occur independently of dc-sign and mannose binding c-type lectin receptors via a cholesteroldependent pathway dc-sign on b lymphocytes is required for transmission of hiv to t lymphocytes the polymorphisms in dc-signr affect susceptibility to hiv type infection evolution of dc-sign use revealed by fitness studies of r hiv variants emerging during aids progression abundant and superficial expression of c-type lectin receptors in ectocervix of women at risk of hiv infection cd gene polymorphisms in south indian hiv and hiv-tb patients cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd and a co-receptor extensive repertoire of membrane-bound and soluble dendritic cell-specific icam- -grabbing nonintegrin . dcsigns; and dc-sign isoforms effect of intrauterine hiv exposure on the frequency and function of uninfected newborns' dendritic cells placental expression of dc-sign may mediate intrauterine vertical transmission of hiv transplacental transmission of hiv: a potential role for hiv binding lectins mechanisms and timing of mother-to-child transmission of hiv vertical transmission of hiv: parameters which might affect infection on the study of in utero transmission of hiv dendritic cells transmit hiv through human small intestinal mucosa muc in human milk blocks transmission of human immunodeficiency virus from dendritic cells to t cells transmission of macrophage-tropic hiv by breast-milk macrophages via dc-sign breast milk macrophages spontaneously produce granulocyte-macrophages colonystimulating factor and differentiate into dendritic cells in the presence of exogenous interleukin- alone bile salt-stimulated lipase from human milk binds dc-sign and inhibits human immunodeficiency virus type transfer to cd ϩ t cells we thank the laboratory of immunopathology keizo asami, the department of genetics, federal university of pernambuco, the graduate program in genetics and molecular biology for supporting physical and scientific, as well as facepe and cnpq, for financial support. s.l. is recipient of a fellowship grant (apq- - . / ) from facepe key: cord- -ybj lwdb authors: platt, simon r. title: vestibular disorders date: - - journal: consultations in feline internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: ybj lwdb nan cats have the ability to control posture and movements of the body and eyes relative to their external environment. the vestibular system mediates these activities through a network of receptors and neural elements. this system integrates peripheral sensory information from vestibular, somatosensory, and visual receptors, in addition to motor information from the cerebellum and cerebral cortex. central processing of these inputs occurs rapidly and provides coordinated relevant muscle movements. although the vestibular system is considered as a special sense, most vestibular activity is conducted at a subconscious level. disease leading to dysfunction of the vestibular system can lead to dramatic signs of dysequilibrium. treatment and prognosis for causes of dysequilibrium differ, depending on whether the peripheral or central components of the system are affected. this chapter outlines relevant anatomy of the vestibular system with emphasis on the clinical signs of vestibular dysfunction. additionally, an overview of the diseases responsible for vestibular dysfunction in cats is provided. the vestibular system can be divided into peripheral components located in the inner ear, and central nervous system (cns) components. three major cns areas receive projections from the peripheral sensory receptors of the vestibular system: the cerebral cortex, the spinal cord, and the cerebellum. projection pathways to the cerebral cortex incorporate extensions to the extraocular muscles. three neurons make up the pathway responsible for the sensory input of head and body position and movement to the cerebral cortex ( figure - ). the location for the first-order neuron is within the vestibular ganglion of cranial nerve viii or the vestibulocochlear nerve. the axon projects to the ipsilateral vestibular nuclei. these neurons receive input from the vestibular receptors contained within the membranous labyrinth that is surrounded by a bony labyrinth located in the petrous temporal bone. the membranous labyrinth consists of four fluid-filled, communicating compartments; these include the saccule and utriculus, three semicircular ducts, and a cochlear duct (figure - ). [ ] [ ] [ ] [ ] specifically, the vestibular portion of the eighth cranial nerve innervates five sites: the crista of the ampulla of each of the three semicircular ducts and the maculae of the utricle and saccule. each semicircular duct is orientated at right angles to the others and connects at both ends with the utriculus, which in turn communicates with the saccule. movement of endolymph contained within the membranous labyrinth is responsible for stimulation of the receptors; the endolymph is thought to be derived from blood. the crista detects head movement. neuroepithelial cells are stimulated by the movement of the crista's gelatinous cupula secondary to the flow of endolymph as the head turns; any movement deflects the cupula and cilia, which leads to their depolarization and propagation of nerve impulses to the vestibular neurons. primary function of the crista involves dynamic equilibrium with regard to acceleration and deceleration. [ ] [ ] [ ] [ ] , the maculae detect head orientation; the macula of the utricle is parallel to the ground with its "hairs" pointing dorsally, and the macula of the saccule is vertical with its "hairs" pointing laterally. constant input from gravity acts upon the neuroepithelial cells of each macula, subsequently causing them to fire. these slow-adapting receptors are responsible for sensing static position of the head and linear acceleration and deceleration. [ ] [ ] [ ] , the sensory neurons of the vestibulocochlear nerve consist of cell bodies in the spiral ganglion located within the modiolus of the cochlea. the vestibulocochlear nerve and the facial nerve exit the petrous temporal bone through the internal acoustic meatus. the nerve enters the medulla of the brainstem at the ventrolateral margin of the trapezoid body. a branch of the vestibulocochlear nerve enters the medulla directly, and a branch travels within the acoustic stria on the dorsal surface of the medulla and caudal cerebellar peduncle before entering the brainstem. the course of the vestibulocochlear nerve stays within the cranium. , , , the location of the cell bodies for the second-order neuron is the vestibular nuclei in the medulla oblongata. four paired vestibular nuclei exist: the caudal vestibular nucleus located medial to the caudal cerebellar peduncle, the medial vestibular nucleus that lies medial to the caudal nucleus, the lateral vestibular nucleus positioned dorsal to the caudal nucleus, and the rostral vestibular nucleus (figure - ) . the lateral and the rostral nuclei are juxtapositioned dorsally to the cerebellar peduncles. , , , similar to the auditory pathway, axons from the vestibular nuclei have ipsilateral and contralateral projections. some axons course within the medial longitudinal fasciculus and project to the contralateral medial geniculate nucleus of the thalamus. ascending fibers within the fasciculus give off numerous branches to the motor nuclei of cranial nerves iii, iv, and vi before synapsing in the medial geniculate nucleus. this pathway coordinates conjugate eye movements associated with changes in position of the head. the medial longitudinal fasciculus also contains fibers that descend to the spinal cord. some axons have afferent projections from the vestibular nuclei to the vomiting center located within the reticular formation. , , , neuron cell bodies for the third-order neuron are located in the medial geniculate nucleus, within the medial geniculate body. these axons project to the cerebral cortex via the internal capsule and via a poorly defined pathway to the temporal lobe. , , two vestibulospinal pathways, termed lateral and medial vestibulospinal tracts, correspond with their origin from the vestibular nuclei. fibers from the lateral vestibular nucleus descend ipsilaterally the entire length of the spinal cord in the ventral funiculus to synapse with alpha and gamma motor neurons of the extensor muscles. this pathway is facilitatory to ipsilateral extensor muscles and inhibitory to ipsilateral flexor muscles and contralateral extensor muscles. fibers from the medial vestibular nucleus descend the spinal cord in the medial longitudinal fasciculus located in a dorsal area of the ventral funiculus. these fibers synapse in the cranial area of the thoracic spinal cord with cervical motoneurons that control head position and maintain equilibrium. projection pathways between the vestibular nuclei and the cerebellum course through the caudal cerebellar peduncle. fibers from the vestibular nuclei synapse in the ipsilateral flocculonodular lobe (the flocculus of the hemisphere and the nodulus of the caudal vermis) and the fastigial nucleus of the cerebellum. fibers from the fastigial nucleus of the cerebellum synapse in the vestibular nuclei. projections from the cerebellum have a strong influence over the activity of the vestibular nuclei. the vestibular system maintains equilibrium through ipsilateral tonic input to the muscles of the head, neck, and torso. an asymmetrical lesion causes loss of the ipsilateral extensor system and causes the extensor system on the contralateral side to become functionally "dominant." clinical signs are recognized as ipsilateral hypotonicity and contralateral hypertonicity. unilateral vestibular disease produces ipsilateral dysfunction. common clinical signs of vestibular disease are head tilt, nystagmus, and ataxia; these may be present as single entities or as a combination of signs ( figure - ) . the primary aim of the neurological examination is to determine if these vestibular signs are due to a peripheral vestibular system (inner ear) disease or a central vestibular system (brainstem and/or cere-bellum) disease. neuroanatomical localization determines the most appropriate diagnostic tests, the differential diagnoses, and the prognosis. essential determination of whether these signs are due to a peripheral or central disease may be possible by the identification of associated neurological signs that are associated only with central disease. , signs of central vestibular syndrome suggest brainstem involvement and are not present in patients with inner ear disease except in cases of direct extension of the disease process, such as can be seen with otitis media/interna and neoplasia. loss of equilibrium most commonly is represented clinically as a head tilt (figure - and table - ) . a head tilt may be present with either central or peripheral vestibular disease. the head tilt is always toward the side of the lesion with peripheral disease but may be to either side with central disease. a head tilt that is opposite to the side of the lesion is paradoxical. this can be seen with lesions of the flocculonodular lobe of the cerebellum or the supramedullary part of the caudal cerebellar peduncle, with sparing of the vestibular nuclei in the rostral medulla. the head tilt often is accompanied by ipsilateral cerebellar signs, paresis, and postural reaction deficits. , , , the mechanism by which the paradox of vestibular signs is contralateral to the lesion is not well understood. a loss of cerebellar inhibition over intact vestibular nuclei could result in hyperactivity of the latter, which simulates a relative loss of function on the other side. cats with bilateral peripheral vestibular disease do not have asymmetrical lesions such as a head tilt, but have a characteristic "side-to-side" head movement (figure - ) . pathological or spontaneous nystagmus is an involuntary rhythmic oscillation of both eyes, which can occur when the head is still or can be induced with a change in head position. this is a sign of altered vestibular input to neurons of cranial nerves that innervate the extraocular eye muscles. this is in contrast to physiological nystagmus, which can be induced in normal cats by moving the head from side to side, best achieved by holding the cat in the air and moving the whole cat's body the fast phase is toward the direction of the head movement and represents the corrective repositioning of the eye as the extraocular muscles reach their stretch threshold after the slow phase. delayed physiological nystagmus can be seen with peripheral or central vestibular disease. pathological nystagmus may be horizontal, rotatory, or vertical in direction (figure - ) . vertical nystagmus implies a central vestibular lesion. if nystagmus of any direction is induced only when the head is placed in an unusual position, it is known as positional nystagmus ( figure - ) , which may be more common with, but not specific for, central disease; this term also may refer to nystagmus that changes its predominant direction with altered head positions. a reliable way to induce positional nystagmus is to decompensate the cat by quickly positioning the cat on its back. eye movements typically are described to have a slow and fast phase. damage to the vestibular system on one side impedes the resting baseline activity on this side, with the normal side continuing to emit baseline activity, now interpreted as head rotation to the normal side. , , , therefore, the nystagmus occurs with the fast phase away from the damaged side and with the slow phase directed commonly toward the affected side; the exception is in the case of paradoxical disease (see section on head tilt above). the direction also can depend on whether the lesion is irritative or destructive to the vestibular system. with acute onset nystagmus, the eyelids may be seen to contract at a rate corresponding to that of the nystagmus. nystagmus may disappear in chronic lesions as a result of adaptation, particularly with peripheral disease; however, its presence usually indicates an active disease process within the vestibular apparatus. cats with bilateral vestibular disease do not have pathological or physiological nystagmus. caloric nystagmus is a type of physiological nystagmus that can be induced by irrigating the ear canal with ice-cold water ( ∞ c) or warm water ( ∞ c) for to minutes. the water causes the flow of endolymph within the ducts. absence of response or asymmetry between sides may indicate vestibular dysfunction, but this often is too unreliable to use in the clinical case. , , ataxia is a loss of muscular coordination or an irregularity of muscle action. it generally is associated with an abnormality of the cerebellar, vestibular, or proprioceptive pathways. cats with vestibular dysfunction assume a wide-based stance and may lean or drift toward the side of a lesion.* with disease of the flocculonodular lobe of the cerebellum or the supramedullary part of the caudal cerebellar peduncle, the ataxia may be directed to the side opposite the lesion as part of the paradoxical central vestibular syndrome. cats with bilateral vestibular disease usually have a symmetrical ataxia and may fall to either side. strabismus is an abnormal position of the eye and often is present in cats with vestibular disease. strabismus can be induced when the head is moved dorsally and is thus termed positional; normally, when the head and neck are extended, the eye should remain centered within the palpebral fissure. the deviation often is ventral and lateral on the ipsilateral side but is not due to paralysis of any of the cranial nerves innervating the extraocular muscles of the eye.* the eyeball occasionally can be noted to deviate without extension of the head and neck, which appears as a lower motor neuron strabismus, corrected by inducing the patient to move its eyeballs to gaze in different directions. the presence of positional strabismus does not help with the determination of a peripheral or central vestibular disease. dysconjugate strabismus implies deviation of both eyes in different directions and is an uncommon finding, which may be more common with central disease. rarely, the opposite eyeball exhibits a dorsal strabismus. cranial nerve vii, the facial nerve, enters the internal acoustic meatus of the petrosal bone, and courses through the facial canal to exit the stylomastoid foramen located dorsal to the tympanic bulla. its course is near the components of the peripheral vestibular system and is affected commonly with destructive lesions to the peripheral vestibular system. the resulting signs are those of facial paresis, paralysis, or more rarely spasm. the owners may report that the patient drools excessively or drops food from the mouth on the affected side. the menace response and palpebral and corneal reflexes often are reduced or absent because of an inability to close the eyelid. , , , because the facial nerve also supplies preganglionic parasympathetic fibers to the lacrimal gland and salivary glands, neurogenic keratoconjunctivitis sicca may accompany facial nerve paralysis associated with middle ear disease, in addition to the presence of xeromycteria. , hemifacial spasm may be seen early in the course of middle ear diseases. inflammation of the facial nerve may cause the facial muscles on the affected side to become hypertonic, causing the face and nose to be pulled caudally. a narrowed palpebral fissure may exist, which is caused by partial closure of the eyelids, elevation of the ear, and wrinkling of the face. these signs may precede those of facial paresis and paralysis. horner's syndrome (miosis, ptosis, enophthalmos, and protrusion of the third eyelid) of the ipsilateral eye may be present with middle or inner ear disease, causing peripheral vestibular dysfunction ( figure - ). , , , this association is seen because the vagosympathetic trunk synapses in the cranial cervical ganglion deep to the tympanic bulla. the postganglionic fibers pass with the internal carotid artery into the middle ear cavity through the tympano-occipital fissure, which is in close proximity to the vestibulocochlear nerve ( figure - ) . , horner's syndrome is associated rarely with central vestibular syndrome. , , sympathetic hyperirritability has been reported in early otitis media, because of disease of the post-ganglionic sympathetic fibers resulting in dilation of the pupil , , and exophthalmos. this has been likened in human beings to pourfour du petit syndrome. paresis suggests abnormal neurological function (weakness) without complete paralysis, which implies that some voluntary motion remains. locomotion is thought to be initiated in the brainstem of animals, and so paresis usually is seen with any lesion within the neuraxis caudal to the level of the red nucleus in the midbrain. with unilateral focal central vestibular diseases, paresis of the ipsilateral limbs (hemiparesis) may be seen if the motor pathways in the medulla oblongata also are affected. large or multifocal lesions can cause an asymmetric tetraparesis. strength always is maintained with peripheral vestibular dysfunction, which is a key finding on neurological examination. a tremor is an involuntary, rhythmic, oscillatory movement of all or part of the body. it results from alternating contraction of antagonistic muscles of variable frequencies. localized tremor usually involves the head and in most cases this is an intention tremor. intention tremors occur commonly with goaloriented tasks such as when an animal "intends" to perform a task such as eating or drinking. these tremors indicate underlying cerebellar dysfunction. cerebellar dysfunction in conjunction with vestibular dysfunction implies central vestibular disease. disorders causing central vestibular dysfunction may be accompanied by altered mentation. the reticular activating system of the brainstem facilitates the alert-awake state in animals. damage to this area may cause disorientation, stupor, or coma. , , peripheral vestibular disease often causes disorientation, which makes the assessment of mentation more difficult. central vestibular syndrome may be accompanied by other cranial nerve dysfunction. cranial nerves v, vi, vii, ix, x, and xii may be affected. clinical signs suggesting involvement of these cranial nerves include ipsilateral facial hypalgesia, atrophy of the masticatory muscles, reduced jaw tone, facial paralysis, tongue weakness, and loss of the swallow or gag reflex. an ipsilateral loss of menace response accompanying vestibular dysfunction usually implies cranial nerve vii dysfunction, or multifocal disease affecting the forebrain or optic nerve. a loss of menace response also can be associated with cerebellar dysfunction. possible causes include an alteration of the menace response pathway from the visual cortex to the facial nucleus through the cerebellum or a loss of cerebellar influence on the cerebrocortical neurons. falling or leaning toward the side of the lesion indicates asymmetrical vestibular disease. cats with unilateral vestibular dysfunction show reduced extensor tone ipsilaterally and increased extensor tone contralaterally. this is manifested clinically as leaning, falling, and a tendency for tight circling toward the side of the lesion.* shaking the head induces falling or leaning. decerebellate posturing can be observed with severe and acute central vestibular dysfunction. this posture is characterized by opisthotonus with thoracic limb extension, normal mentation, and flexion of the pelvic limbs. decerebellate posturing can be intermittent and misinterpreted as seizure activity. dorsiflexion of the neck sometimes elicits this posture in cats with cerebellar dysfunction. the vomiting center is located within the reticular substance of the medulla, with direct connections to and from the vestibular nuclei. [ ] [ ] [ ] [ ] [ ] , , vomiting can occur in cats with acute vestibular dysfunction. middle and/or inner ear disease also may cause hearing loss through conductive or sensorineural impairment, respectively. conductive deafness occurs with impedance of sound wave transmission through the middle ear caused by structural defects such as ceruminoliths, a ruptured tympanum, bony ossicle damage, fluid accumulation, or aural neoplasms. , , external ear canal lavage can affect hearing thresholds in dogs and the same is assumed for cats. sensorineural deafness results from abnormalities of the inner ear structures, cochlear nerve, or central auditory pathway. , , deafness associated with central disease is considered rare. the diagnostic approach for a cat with vestibular dysfunction depends upon whether the neuroanatomical localization is peripheral or central (figure - ) . signalment, assessment of the clinical history, and thorough physical and neurological examinations are essential. peripheral vestibular dysfunction results from disease of the middle and inner ear affecting the receptors in the labyrinth and the vestibular portion of cranial nerve viii. central vestibular dysfunction results from disease affecting the brainstem and or the cerebellum. testing procedures are performed in a logical sequence, which depends on the cost expenditure and amount of invasiveness. diagnosis of a central vestibular disorder may require performance of most of the testing procedures (see figure - ). hematology, serum biochemistry, thyroid hormone testing, and urinalysis are useful to screen for other underlying metabolic disorders. thoracic radiography and abdominal ultrasound are recommended in older cats or in cats with central vestibular dysfunction to evaluate for multisystemic disease or metastatic neoplasia. an ophthalmological examination may reveal evidence of inflammatory cns disease. serology can assist with the diagnosis of some infectious diseases. cats with peripheral vestibular disease require examination of the ears and pharynx under general anesthesia. both ears should be examined with an otoscope. the tympanum is examined for color, texture, and integrity. otitis media is suspected when the tympanum is dark gray or brown. an intact tympanum does not rule out otitis media; visualization of a ruptured tympanum without other associated abnormalities also is unreliable for diagnosis of otitis media. bulging (convex appearance) of the tympanum can indicate fluid accumulation within the middle ear (see figure - ), whereas retraction (and a concave appearance) suggests a partially filled middle ear with obstruction of the auditory tube. examination of the pharynx may reveal evidence of inflammation, polyp formation from the eustachian tube, or other masses associated with the choanae. radiography is useful for evaluation of the osseous tympanic bulla. skull radiographs are performed under general anesthesia to achieve adequate positioning. lateral, dorsoventral, ventrodorsal, and oblique views are advised for tympanic bullae *references , , , , , . assessments. positioning for radiography of the bullae has been described. the normal tympanic bulla is a thin-walled gas-filled structure with well-defined, smooth borders. bilateral sclerosis of the bullae can be normal in older animals or a residual finding of previous ear disease. the external acoustic meatus is rounded with distinct smooth margins. myringotomy is the deliberate puncture or incision through the tympanic membrane. a -gauge spinal needle is used to puncture the ventrocaudal part of the tympanic membrane. the needle is connected to a -ml or -ml syringe, and fluid is aspirated for cytological analysis and culture. , , purulent or particulate matter within the middle ear may prevent needle aspiration and a larger hole may be needed for adequate drainage. a myringotomy knife can be used to make a curvi- linear or radial incision. care must be taken not to incise the tympanum too deeply and damage contents within the middle ear. similarly, forceful flushing of the middle ear should be avoided. a normal tympanum heals within to days. brainstem auditory evoked potential (baep) testing is used to assess the integrity and function of the peripheral and central auditory pathways, and to evaluate the closely associated vestibular pathways indirectly. baep are recordings of sound-evoked electrical activity in the auditory pathway between the cochlea and the auditory cortex. because of the level of patient "cooperation" with cats, sedation or a light plane of general anesthesia often is needed for this test to be performed and interpreted properly. small ( -gauge) needle electrodes are placed subcutaneously in the scalp and connected to sensitive amplifiers that can record signals in the microvolt range. the electrodes are arranged with the positive electrode over the bregma on the dorsum of the skull, the negative electrode just rostral to the base of the pinna of the ear to be tested, and the reference electrode in the same position relative to the untested ear (figure - ) . the brain activity, resulting from broad-spectrum sounds, such as clicks delivered at to hz through earphones inserted into the external ear canal, usually is averaged for milliseconds (ms) for the early latency or brainstem potentials averaging for ms includes a record of middle latency responses, but these are not as well documented in cats. the baep recording consists of six to seven positive time-locked peaks (i through vii) beginning at approximately ms after the stimulation (figure - ). wave i represents acoustic nerve activity, and subsequent waves mark peak activities as sound is being processed through ascending portions of the auditory pathway ( figure generally, cerebrospinal fluid (csf) analysis is a useful adjunctive test for determination of the cause of central vestibular disease but rarely is specific. risk of iatrogenic cns trauma or cerebellar herniation after cisterna magna puncture in cats with space-occupying lesions should not be underestimated. obtaining advanced imaging studies of the brain (see below) before csf tapping is recommended, especially if a caudal fossa lesion is suspected. i frequently use a hypodermic needle for csf acquisition in cats rather than a spinal needle and stilet to lessen risks of iatrogenic cns damage. serology is useful for determining titers for presence of antigens but nonspecific for evaluation of antibody. polymerase chain reaction analysis of csf is now performed in specialized laboratories to evaluate for the presence of some infectious agents. computed tomography (ct) and magnetic resonance imaging (mri) have revolutionized the diagnosis of vestibular diseases. the physics and interpretation details of both of these modalities have been described in detail. ct evaluation of the peripheral vestibular system is particularly useful if radiographs have not determined an underlying cause, if nasopharyngeal polyps or neoplasia are suspected, or if the patient is a potential surgical candidate. the same interpretive principles used for the radiographic diagnosis of peripheral vestibular diseases apply to ct. findings are more apparent on transverse ct images, however, because of reduced superimposition of structures in comparison to radiographs. ct can allow for an earlier diagnosis of subtle lesions. on a well-positioned study, both bullae should appear symmetrical, although subtle variations occur. lumina of the tympanic bulla and the external ear canals are gas filled (see figure - ). the tympanic bulla has a thin well-defined wall. optimal resolution of the inner ear is achieved with high-resolution ct, but it still may be inferior to high-field mri. ct evaluation for central vestibular diseases is less helpful because of beam hardening artifacts. the density of the petrous temporal bones obliterates the visualization of the medulla. mri is used less than radiography and ct for the diagnosis of peripheral vestibular disease because of its comparative limited availability and high cost. mri allows for multiplanar views when compared to ct. improved soft tissue resolution allows for better assessment of neoplastic and inflammatory processes that affect the vestibular system. a typical mri study consists of t -weighted (t w), t -weighted (t w), and proton density-weighted sequences. transverse, sagittal, and dorsal planes are used to evaluate the brain and cranium. a t w sequence is obtained after intravenous administration of a gadolinium-based contrast agent. transverse and dorsal planes with t w and t w sequences are suggested for mri of the middle ear in cats. post-contrast sequences are recommended if a mass is present in the tympanic bulla or external ear canal. congenital vestibular disorders have been reported in siamese and burmese kittens (tables - and - ). signs of peripheral vestibular dysfunction and concurrent deafness may be detected by to weeks of age and show clinical improvement within to months. a hereditary abnormality has not been proven. diagnosis is based on history, excluding other causes, and baep results. neoplasms that involve the peripheral vestibular system include squamous cell carcinoma, fibrosarcoma, osteosarcoma, chondrosarcoma, and ceruminous gland and sebaceous gland adenocarcinoma. squamous cell carcinoma is the most common malignant tumor of the middle and inner ear in cats. nonkeratinizing squamous epithelial cells are found normally in the eustachian tube and the middle and inner ear. clinical signs of peripheral vestibular dysfunction have been documented but vary depending upon lesion extension. , neoplasms of the middle/inner ear also can cause oropharyngeal signs that present with pain on palpation of the bulla or when manipulating the jaw. in addition to an examination of the external ear cavity and the tympanum for masses, the oropharynx should be examined for swelling or deviations of the soft palate. suspicious lesions should be aspirated for cytological analysis. radiography of the skull can reveal soft tissue opacity in the tympanic bulla, osteolysis, and periosteal reaction (figure - ) . , ct is a more accurate method for determining lesion extent. opacity within the tympanic bullae can indicate fluid or a soft tissue mass effect. lesion extent within the horizonal and vertical ear canals is identified. bony lysis involving osseous bulla, petrous temporal bone, and adjacent calvarium may be visualized with aggressive neoplasms. some neoplasms contrast enhance. mri characteristics described for neoplasms of the middle ear include lysis of the osseous tympanic bulla and petrous temporal bone that can extend to adjacent structures. however, a malignant melanoma involving the external ear canal and dorsalateral compartment of the tympanic bulla has been described in the cat, in which destruction of the bulla was not present and contrast enhancement of the mass did not occur. radical surgical resection and adjunctive radiotherapy often is recommended as a treatment for neoplasms involving the middle ear. median disease-free interval of months has been reported for cats with ceruminous gland adenocarcinoma after surgery alone. bacterial otitis interna or labyrinthitis can cause clinical signs of peripheral vestibular dysfunction. often otitis interna and media occur concurrently. organisms isolated commonly from the bullae include staphylococcus spp., streptococcus spp., pasteurella spp., proteus spp., escherichia coli, enterococcus spp., pseudomonas spp., and obligate anaerobes. yeast infections are an uncommon cause of otitis media. diagnosis is based on otoscopic examination, myringotomy, and imaging. otitis externa may be evident but is not necessarily the origin of the bacterial infection. bulging and discoloration of the tympanum may be identified if the bulla contains fluid or an exudate. fluid within the middle ear can be collected by myringotomy for cytological examination and anaerobic and aerobic culture/sensitivity. the external ear canal also is cultured. skull radiography is performed with the cat under general anesthesia. the latero- -degree ventrolaterodorsal oblique and rostral- -degree ventral-caudodorsal open-mouth oblique views are best for evaluation of the tympanic bullae. common radiographic findings associated with otitis media/interna include soft tissue opacity in the bulla and/or petrous temporal bone and bony proliferation of the petrous temporal bone (figure - ) . if the infection is severe enough, lysis of the tympanic bullae also can be visible. ct findings with otitis media/interna include thickening and irregularity of the tympanic bulla wall, lysis of the bulla, and radiopacity within the bulla, which suggests fluid or a soft tissue mass (see figure - , b) . a study that compared ct with radiography for diagnosis of otitis media/interna found ct to have per cent false-positives and per cent false-negatives for diagnosis confirmed by surgical findings. ct was a more sensitive but less specific technique than skull radiography. , neither radiography nor ct was able to detect early lesions associated with otitis media/interna when no osseous involvement occurred. otitis interna is difficult to assess with ct except in cases of severe destruction of the inner ear. mri findings that are compatible with otitis media include mediumsignal intensity material in the tympanic bulla on a t w sequence and hyperintense on a t w sequence. the inner margin of the tympanic bulla also may enhance after gadolinium administration. osseous lesions of the tympanic bulla are more difficult to assess with mri. an mri finding of otitis interna is a lack of signal intensity of the labyrinthine fluid on t w sequences. this may represent replacement of the fluid with fibrous tissue; however, similar findings are seen in normal ears. meningeal enhancement on post-contrast t w sequences also has been described secondary to otitis interna. treatment consists of long-term ( to weeks) antibiotic therapy and prognosis usually is good. improvement often occurs within to weeks of therapy. refractory cases may require surgical drainage of the tympanic bulla. , cryptococcosis more often causes central vestibular dysfunction. however, three cats have been reported with peripheral vestibular disease referable to otitis media/interna because of cryptococcosis. the infection was isolated from the tympanic bulla in two cats and the eustachian tube in one cat. all cats responded well to surgical drainage and medical therapy. nasopharyngeal polyps are pedunculated masses that can arise from the epithelial lining of the tympanic cavity, eustachian tube, or nasopharynx. , , nonseptic otitis media/ interna may occur secondary to occlusion of the eustachian tube because of a nasopharyngeal polyp, and polyps may occur as a result of chronic middle ear infection or from ascending infection from the nasopharynx. , polyps are especially common in young adult to middle-age cats, with no apparent gender or breed predisposition. clinical signs include peripheral vestibular dysfunction, head-shaking, aural discharge, facial nerve paralysis, and horner's syndrome. clinical signs of nasopharyngeal involvement include dysphagia, stertorous respiration, respiratory distress, and change in phonation. a secondary suppurative meningoencephalitis has been documented in a young cat with lesion extension of an inflammatory polyp within the tympanic bulla. inflammatory polyps of the middle ear can be visualized using otoscopy or by inspection of the oropharynx with the cat under general anesthesia. a lateral skull radiograph can reveal a soft tissue mass in the nasopharyngeal area and assist with the identification of nasopharyngeal polyps (figure - ) . other radiographic findings associated with polyps include unilateral or bilateral soft tissue opacity within the tympanic bulla and sclerosis of the osseous bulla. , transverse, sagittal, and parasagittal ct images of nasopharyngeal polyps in cats have been described. , ct can lateralize the lesion and assess the lesion extent. mri of polyps is recommended because of the superior soft tissue resolution of this modality. two cases of inflammatory polyps have been described in which signal intensity on post-contrast t w sequences was increased. in one cat, a nonuniform increase occurred in signal intensity on t w sequences. treatment involves traction and avulsion of the mass through the external acoustic meatus or from the nasopharyngeal cavity. bulla osteotomy can facilitate polyp removal from the tympanic bulla. prognosis usually is good, although a residual head tilt is not uncommon. the recurrence rate after polyp removal is approximately per cent. recurrence is more likely in cats with aural polyps and more severe signs of otitis externa and less likely if treated with steroids after surgery. idiopathic feline vestibular syndrome (ifvs) is a disease of peracute peripheral vestibular dysfunction (less than hours). the incidence is highest during the months of july and august in the united states. no sex predilection exists, and the median age of affected cats in one study was years. no confirmed cause exists; however, as in meniere's disease in human beings, abnormal endolymphatic flow or electrolyte aberrations in the perilymph have been hypothesized. with lack of a structural lesion, other associated neurological deficits such as horner's syndrome or facial nerve paralysis would not be expected. bilateral disease can occur but this is uncommon (less than per cent). clinical signs of ifvs often are preceded by upper respiratory tract disease ; additionally, excessive vocalization can be seen, which probably is due to the generalized feeling of disorientation. diagnosis of ifvs is made through exclusion of other causes of peripheral vestibular disease (see table - ). no specific treatment exists for ifvs besides managing the clinical signs such as anorexia, which may accompany this condition. prognosis for spontaneous recovery is good although this may take to weeks, and per cent of affected cats may have residual deficits such as a head tilt. i have seen recurrence of signs with ifvs to be more common in cats than dogs with idiopathic vestibular disease. cats with cns cuterebriasis have been documented to present most commonly during the months that coincide with the occurrence of ifvs. , this similarity has led to the hypothesis that cuterebra larval migration may account for some idiopathic vestibular cases in cats in the united states. however, clinical signs of cns cuterebriasis and idiopathic vestibular disease are dissimilar, and most cats with idiopathic vestibular disease recover in a few weeks, which makes this hypothesis less plausible. migration of a cuterebra larva through the ear canal to the peripheral vestibular apparatus still remains as a potential cause of peripheral vestibular disease. peripheral vestibular disease can be caused by ototoxic agents. an ototoxic agent is a substance that can produce cochlear or vestibular damage by causing unilateral or bilateral damage to structures of the inner ear. parenteral or oral administration of ototoxic drugs reaches the structures of the inner ear by the hematogenous route. topical drugs applied into the external ear canal reach the middle ear through a ruptured tympanic membrane and subsequent penetration into the inner ear via the round or oval window. the membrane of the round window is more permeable to macromolecules when otitis media is present. the ototoxic substance passes into the perilymph, which is contiguous within the osseous labyrinths of the cochlea and vestibule. many agents are listed in the literature as "potentially" ototoxic, but much of the information is based on anecdotal reports. studies also are extrapolated from species other than cats, and use dose formulations that far exceed the concentrations in proprietary medication. as an example, chlorhexidine and gentamycin often are quoted as ototoxic drugs when administered topically; however, no vestibular abnormalities were seen when these drugs solutions were administered at . per cent and . per cent concentrations, respectively. a list of potential ototoxic agents for cats is shown in table - . aminoglycosides can damage the neuroepithelium of the macule and crista of the vestibular apparatus, in addition to the hearing apparatus. the severity of vestibular toxicity may be directly proportional to the duration and concentration of aminoglycoside given. , other antibiotics, such as erythromycin, minocycline, chloramphenicol, vancomycin, and topical polymyxin b, have been reported to cause vestibular damage in human beings, but this has not been observed in cats. loop diuretics (e.g., furosemide) cause ototoxicity in human beings, but this has not been reported in cats when standard clinical doses have been prescribed. regarding antiseptics, many studies have been performed to document the ototoxic effect of intratympanic application of chlorhexidine. at per cent concentration, chlorhexidine is obviously ototoxic to the cochlea and vestibular system, but the damage is much more subtle at . per cent, and no clinical effects are seen. peripheral vestibular disease has been reported after the offlabel use of intraaural per cent fipronil solution for otoacariosis in two cats. the cats developed vestibular dysfunction and signs of horner's syndrome within hours after two drops of the solution were administered in each ear. both cats showed signs of improvement within days, but one of the cats had a residual head tilt. diagnosis of toxicity in peripheral vestibular disease is based on history and results of otoscopic examination and baep testing. treatment consists of cessation of the ototoxic agent and initiation of supportive care. prognosis for recovery from the vestibular signs is good in most instances. cranial trauma. peripheral vestibular signs may follow any trauma to the head, secondary to a fracture of the petrosal part of the temporal bone or tympanic bulla. this often is accompanied by facial paresis/paralysis. skull radiography or advanced imaging will be necessary for an accurate diagnosis (figure - ) . treatment is supportive and should be focused on any concurrent injuries sustained during the trauma. iatrogenic trauma. peripheral vestibular disease can be seen immediately after a bulla osteotomy, especially in cases of vigorous curettage of the petrous temporal bone. supportive care and appropriate antibiosis are necessary, but resolution usually occurs because of compensation by the animal. three cats with signs of unilateral ocular sympathetic hyperactivity (mydriasis and exophthalmos) have been reported after middle ear flushing procedures; however, the cats had signs of peripheral vestibular dysfunction because of otitis media/interna before the procedure. cerebellar cortical abiotrophy. in contrast to dogs, this condition in cats is exceedingly rare. sporadic anecdotal cases have been mentioned in the literature. late-onset cerebellar abiotrophy has been documented in adult cats, but it would be expected primarily in kittens. , lysosomal storage diseases. specific lysosomal storage disorders have been reviewed (see consultations in feline internal medicine, volume , chapter ). lysosomal storage diseases documented to cause central vestibular disease include gm -gangliosidosis, niemann-pick disease type c (sphingomyelinosis), and alpha-mannosidosis. hydrocephalus. this disease is not common in cats but may be the result of obstructive processes such as neoplasia or inflammation elsewhere in the neuraxis. enlargement of the fourth ventricle may cause central vestibular dysfunction because of the anatomical location of the vestibular nuclei. diagnosis requires advanced imaging ( figure - ) , but a csf tap also would be warranted to rule out an underlying inflammatory disease. treatment is possible with either the use of prednisone ( . mg/kg po q h) or surgical placement of a ventriculoperitoneal shunt. thiamine deficiency. this is the most common nutritional deficiency affecting the cns, usually resulting in lesions of the oculomotor and vestibular nuclei, the caudal colliculus, and lateral geniculate nucleus. the earliest neurological sign is vestibular ataxia, progressing to seizures, dilated nonresponsive pupils, and ultimately coma. treatment is administration of thiamine, parenterally ( to mg q h) or intravenously. , neoplasms neoplasms can affect the medulla of the brainstem or vestibular pathways associated with the cerebellum directly (parenchymal compression or invasion) or indirectly to cause central vestibular dysfunction. neoplasms can affect these regions indirectly by ( ) causing an obstructive hydrocephalus affecting the fourth ventricle and/or ( ) increasing intracranial pressure, causing a rostrocaudal shift of the forebrain and/or hindbrain with subsequent cerebellar herniation through the foramen magnum. space-occupying lesions in the region of the cerebellomedullary pontine angle often can be responsible for paradoxical vestibular syndrome. , rarely, middle ear tumors in cats may extend medially to involve the brainstem. the most common neoplasms in cats that affect this region are meningioma and lymphoma, but a cerebellar oligodendroglioma causing paradoxical signs also has been described in the cat. , [ ] [ ] [ ] in a study of intracranial tumors in cats, five meningiomas, lymphomas, and three glial cell tumors were documented to occur in the region of the cerebellomedullary angle and the region of the fourth ventricle. although meningiomas have been observed in cats from to years of age, the majority of cats are older than years. , the imaging characteristics of feline meningiomas have been well described ( figure - ) . , surgical resection of tumors in this area is challenging but can be achieved with improvement of the clinical signs, although recurrence is common (see chapter ) . a -year-old cat has been diagnosed with a medulloblastoma, a type of primitive neuroectodermal tumor. the cat presented with a -month history of an ipsilateral ataxia, which progressed to develop nystagmus, ipsilateral paresis, and dysmetria. magnetic resonance imaging using a t w sequence demonstrated an irregularly shaped hypointense mass within the cerebellar parenchyma that contrast-enhanced and was irregularly hyperintense on t w images. surgical resection was possible but no follow-up was documented. the same cat seems to have been described in another report, which documented a -day postsurgery survival. any inflammatory disease that affects the cns has the potential to cause central vestibular signs, usually as part of a multifocal syndrome. these diseases have been discussed in detail and are documented in table - . the more common infectious agents are discussed briefly. bacterial meningoencephalitis/abscessation from otitis media and otitis interna can extend into the intracranial cavity and result in bacterial meningoencephalitis (see chapter ) . seven such cats with otitis media/interna have been documented, in one study, with cns dysfunction that included central vestibular signs. mri was extremely effective in demonstrating the location, extent, and relationship to normal structures of inflammation of the middle ear and brainstem in all cases. a mild to severe neutrophilic pleocytosis was present in the csf of four of five cats tested. marked neurological improvement was seen in all the cats, which underwent surgical drainage in addition to prolonged antibiotic therapy. extension of bacterial infection into the cns also has been documented in a -month-old male maine coon cat with an inflammatory polyp of the middle ear. the cat required a ventral bulla osteotomy to remove the polyp in addition to broad-spectrum antibiotic therapy for the secondary suppurative meningoencephalitis but made a good recovery with a residual head tilt. mri is useful in detecting brain abscessation secondary to otitis media/interna. abscessation with extension of an inner ear infection can affect the brainstem and has a heterogenous signal intensity on t w and t w images (figure - ) . a ring-enhancing lesion with extension into the tympanic bulla can be seen after intravenous contrast administration. feline infectious peritonitis. feline infectious peritonitis (fip) results from infection with a mutated form of feline enteric coronavirus and represents the most common cause of inflammatory brain disease in cats. neurological disease usually is seen with the noneffusive form of fip, and up to a third of cats with this form of disease develop neurological disease. some affected cats have evidence of disease only localized to the cns. insidious multifocal or diffuse cns clinical signs are seen, which commonly include vestibular dysfunction. analysis of csf is the most useful antemortem diagnostic test, which often reveals a neutrophilic pleocytosis with a marked protein elevation (more than mg/dl). however, this test cannot be relied upon to be either sensitive or specific for fip. positive coronavirus antibody titers in the csf are the most reliable indicator of the disease, but only if an albumin quotient and igg index rule out serum protein translocation across a disrupted blood-brain barrier. polymerase chain reaction (pcr) testing of the csf recently has become available; unfortunately, only a third of cats with neurological fip have positive csf pcr results, and only two thirds of brain tissue specimens actually are pcr-positive. advanced imaging reveals the presence of hydrocephalus in the majority of affected cats. no documented effective treatment exists, and the prognosis is poor. toxoplasmosis. cats are the definitive hosts of toxoplasma gondii. occasionally, cats develop central neurological disease because of this organism. after the initial enteroepithelial life cycle, tachyzoites are disseminated through the blood and lymph. the immune system generally can suppress proliferation of tachyzoites with subsequent development of cysts. these cysts remain dormant for long periods and have a predilection for sites such as the brain. diseases associated with toxoplasmosis can be due to recrudescence of local infection. multifocal neurological signs are a common clinical manifestation. a definitive diagnosis is difficult. csf analysis reveals a mixed pleocytosis and elevated protein levels. comparison of csf and serum antibody titers may aid in the diagnosis of the disease. pcr analysis for protozoal disease on the csf is now available but may not be highly sensitive. advanced imaging can reveal multifocal areas of irregular contrast-enhancing lesions within the brain parenchyma. clindamycin ( . mg/kg po q h for to weeks) is advocated for treatment of this disease; however, the prognosis is guarded and residual signs and recrudescence may be common. cryptococcosis. cryptococcosis is the most common systemic mycosis of cats. feline cryptococcosis has been reviewed extensively. more than per cent of cats present with signs of nasal cavity disease, including sneezing, nasal discharge, respiratory stridor, and subcutaneous masses at the nostrils (see figure - ). the cns occasionally is involved, manifesting with multifocal neurological signs, including central vestibular dysfunction. csf analysis is the most helpful diagnostic test in cats with cns cryptococcosis. the organism may be identified cytologically or cultured from the csf. a positive capsular antigen test can provide a definitive diagnosis. treatment consists of triazole drugs (fluconazole, itraconazole) for at least months beyond resolution of the clinical signs. fluconazole crosses the blood-brain-barrier readily and is the preferred antifungal agent. the decision to discontinue therapy is based upon repeat csf analysis results, serology, and resolution of clinical signs. often patients require long-term antifungal therapy. prognosis is considered guarded. although not common, central vestibular signs have been reported in cats after chronic high-dose therapy with metronidazole. clinical signs reversed in two of the cats reported within a few days of drug withdrawal and with appropriate supportive care. diazepam administration has improved the recovery time in dogs with metronidazole toxicity; this remains to be determined for cats. unlike metronidazole toxicosis in dogs, nystagmus is an uncommon clinical finding. lead. the most common clinical signs of lead toxicosis in cats are anorexia, vomiting, and seizures. central vestibular abnormalities, including vertical nystagmus and ataxia, have been reported. old paint is the most common source of exposure for cats. recovery can be complete after standard treatment. central vestibular signs subsequent to head trauma often imply brainstem involvement; occasionally, the signs may be due to elevated intracranial pressure, causing a rostrocaudal transtentorial herniation or a cerebellar herniation through the foramen magnum. diagnosis is supported by history and skull fractures on radiographs; however, it is not necessary for the skull to be fractured for central vestibular damage to occur. advanced imaging studies can be used to assess for intracranial hemorrhage and edema. principles for management of head trauma address the pathophysiologic sequelae to traumatic brain injury such as edema. feline ischemic encephalopathy (fie). fie is a poorly understood syndrome of brain infarction in cats. onset of clinical signs is peracute. fie affects cats of all ages and most commonly in the months of july and august. the main clinical signs are acute in onset, focal, and lateralizing; these include depression, blindness, circling, and central vestibular dysfunction. this has been associated with cuterebra spp. migration. although central vestibular signs have been reported with this abnormality, other neurological signs supportive of forebrain disease are more common. diagnosis is based on focal lesions identified by advanced imaging and csf analysis. treatment is supportive care. gradual improvement of clinical signs can occur over several months, but residual signs are likely. severe cases can be fatal. the damaged vestibular system can compensate over time with central reprogramming of eye movements and postural responses in addition to reliance on visual and other sensory input that replaces lost vestibular input. , , histamine is thought to be involved in the recovery of vestibular function, although the mechanism is unclear. if the underlying disease process can be targeted, the prognosis for a functional recovery can be good. residual signs, such as a head tilt, are not uncommon. recurrence of vestibular dysfunction can occur at times of stress, recurrent disease, or after an anesthetic episode. supportive care often is essential in cats with vestibular dysfunction, because anorexia is a frequent complication. vomiting, salivation, and possible nausea associated with vestibular disease can be treated medically. drugs used commonly include the phenothiazine derivative chlorpromazine ( . to . mg/kg sq q h); and the antihistamines diphenhydramine ( to mg/ kg po or im q h), dimenhydrinate ( to mg/kg po q h), and meclizine ( . mg q h). betahistine dihydrochloride is a histamine-like substance that is used in human beings with meniere's syndrome and also has been shown to accelerate the recovery process from a central vestibular syndrome in experimental cats when used at daily doses of mg/kg. clinical use has not been documented. vestibular system-special proprioception cortical and brainstem control of motor function vestibular dysfunction disorders of the vestibular system king as: special senses otitis media and otitis interna recognition and localization of intracranial disease neurologic evaluation of the ear meningoencephalitis secondary to bacterial otitis media/interna in a dog feline vestibular diseases-new developments cerebellar meningioma with paradoxical vestibular signs vestibular diseases strain gm: aetiology, prevalence and diagnosis of deafness in dogs and cats facial neuropathy in dogs and cats: cases ( - ) cranial nerves and cutaneous innervation of the head essentials of veterinary ophthalmology horner's syndrome in dogs and cats: cases ( - ) iatrogenic pupillary dilation resembling pourfour du petit syndrome in three cats paradoxical vestibular syndrome in a cat with a cerebellar meningioma effects of otitis on hearing in dogs characterized by brainstem auditory evoked response testing healing of experimentally perforated tympanic membranes demonstrated by electrodiagnostic testing and histopathology congenital deafness and its recognition deafness in the dog and cat the normal ear diagnostic imaging of the canine and feline ear imaging techniques in the diagnosis of middle ear disease microbial flora and antimicrobial susceptibility patterns of isolated pathogens from the horizontal ear canal and middle ear with otitis media otitis externa and otitis media: diagnostic and medical aspects insertion of a transtympanic ventilation tube for the treatment of otitis media with effusion electrodiagnostic evaluation of hearing and vision middle and long latency auditory evoked potentials in cats. ii. component distributions and dependence on stimulus factors use of a multiplex polymerase chain reaction assay in the antemortem diagnosis of toxoplasmosis and neosporosis in the central nervous system of cats and dogs advanced imaging concepts: a pictorial glossary of ct and mri technology intracranial neoplasia magnetic resonance imaging of the normal and diseased feline middle ear feline vestibular disorders. part ii: diagnostic approach and differential diagnosis adenocarcinoma of the middle ear with osteolysis of the tympanic bulla in a cat vestibular dysfunction caused by squamous cell carcinoma involving the middle and inner ear in two cats peripheral vestibular disease in a cat with middle and inner ear squamous cell carcinoma oral and peripheral vestibular signs in a cat with squamous cell carcinoma the cranial and nasal cavities-canine and feline results of magnetic resonance imaging in dogs with vestibular disorders: cases ( - ) results of surgery in cats with ceruminous gland adenocarcinoma clinical neurology in small animals: localization, diagnosis and treatment. international veterinary information service a comparison of radiographic versus surgical diagnosis of otitis media radiographic and computed tomographic evaluation of otitis media meningeal enhancement on magnetic resonance imaging in dogs and cats feline vestibular disorders. part i: anatomy and clinical signs peripheral vestibular disease associated with cryptococcosis in three cats tumors of the ear canal inflammatory polypoid growths in the ear canal of cats nasopharyngeal diseases in cats: a retrospective study of cases ( - ) inflammatory polyp in the middle ear with secondary suppurative meningoencephalitis in a cat computed tomographic appearance of inflammatory polyps in three cats feline nasopharyngeal polyps results of surgery and long-term follow-up in cats with nasopharyngeal polyps feline inflammatory polyps: historical, clinical, and pcr findings for feline calicivirus and feline herpesvirus- in cases review of idiopathic feline vestibular syndrome in cats clinical and clinicopathological features in cats with cuterebra larvae myiasis of the central nervous system gentamicin tympanoclysis: effects on the labyrinthine cells off-label treatment for otoacariosis current trends in the treatment of sarcoptes, cheyletiella and otodectes mite infestations in dogs and cats tympanic bulla osteotomy for treatment of middle-ear disease in cats: cases ( - ) adult-onset cerebellar cortical abiotrophy and retinal degeneration in a domestic shorthair cat late onset cerebellar abiotrophy in a siamese cat cerebellar cortical atrophy in a kitten study of hereditary cerebellar degeneration in cats degenerative brain disease meningiomas in cats: a retrospective clinical study of cases clinical and pathological features of a cerebellar oligodendroglioma in a cat cerebral meningiomas: diagnostic and therapeutic considerations magnetic resonance imaging features of feline intracranial neoplasia: retrospective analysis of cats feline intracranial neoplasia: retrospective review of cases early diagnosis of feline medulloblastoma in the vermis medulloblastoma in a cat: clinical and mri findings inflammatory disorders of the central nervous system otitis media/interna with central extension in dogs and cats: clinical signs, magnetic resonance imaging features, and outcome after surgical intervention magnetic resonance imaging features of brainstem abscessation in two cats diagnostic features of clinical neurologic feline infectious peritonitis cryptococcosis: new perspectives on etiology, pathogenesis, diagnosis, and clinical management metronidazole neurotoxicosis in two cats succimer for treatment of lead toxicosis in two cats feline ischemic encephalopathy in a cat cerebrospinal cuterebriasis in cats and its association with feline ischemic encephalopathy vestibular compensation in the cat: the role of the histaminergic system betahistine dihydrochloride treatment facilitates vestibular compensation in the cat key: cord- -c r n o authors: pöhlmann, stefan; tremblay, michel j. title: attachment of human immunodeficiency virus to cells and its inhibition date: journal: entry inhibitors in hiv therapy doi: . / - - - - _ sha: doc_id: cord_uid: c r n o the entry of enveloped viruses involves virus adsorption followed by close apposition of the viral and plasma membranes. this multistep process is initiated by specific binding interactions between glycoproteins in the viral envelope and appropriate receptors on the cell surface. in the case of hiv- , attachment of virions to the cell surface is attributed to a high affinity interaction between envelope spike glycoproteins (env, composed of the surface protein gp and the transmembrane protein gp ) and a complex made of the primary cd receptor and a seven-transmembrane co-receptor (e.g., cxcr or ccr ) (reviewed in [ ]). then a chain of dynamic events take place that enable the viral nucleocapsid to penetrate within the target cell following the destabilization of membrane microenvironment and the formation of a fusion pore. the entry of enveloped viruses involves virus adsorption followed by close apposition of the viral and plasma membranes. this multistep process is initiated by specific binding interactions between glycoproteins in the viral envelope and appropriate receptors on the cell surface. in the case of hiv- , attachment of virions to the cell surface is attributed to a high affinity interaction between envelope spike glycoproteins (env, composed of the surface protein gp and the transmembrane protein gp ) and a complex made of the primary cd receptor and a seven-transmembrane co-receptor (e.g., cxcr or ccr ) (reviewed in [ ] ). then a chain of dynamic events take place that enable the viral nucleocapsid to penetrate within the target cell following the destabilization of membrane microenvironment and the formation of a fusion pore. although it is generally accepted that hiv- attachment to its major cellular reservoirs (i.e., t helper cells and macrophages) occurs through the twostage receptor-interaction pathway, there is accumulating evidence indicating that the initial attachment step is a more complex phenomenon than initially thought. indeed it seems that adsorption of hiv- to the cell surface is modulated by a large variety of interactions between the viral entity and the target cell surface (reviewed in [ ] ). this retrovirus may also attach to some cell types by cd -independent interactions involving highly glycosylated groups or basic residues found on gp and polyanionic sulfated chains or lectin-like domains on some specific cell surface receptors (reviewed in [ ] ). for example, heparan sulfate proteoglycans, which are expressed at high levels on different cell types, such as epithelial and endothelial cells, can interact with the envelope spike glycoprotein and serve as docking structures for hiv- [ ] . heparan sulfate proteoglycans such as syndecans serve as the main class of attachment receptors for hiv- on different cell types, e.g., macrophages and endothelial cells, and are thought to play a cardinal role in virus transmission [ , ] . gp can bind also to galactosyl ceramide and its sulfated derivative (i.e., sulfatide) [ , ] that are found on macrophages and neural, glial and colon epithelial cells [ ] [ ] [ ] . it can also associate with the mannose-specific macrophage endocytosis receptor (mr) [ ] and other cellular lectins. in fact, the determinant role played by dendritic cells (dcs) in hiv- transmission might rely on specific interactions between gp and c-type lectins, of which the dc-specific intercellular adhesion molecule- (icam- ) grabbing nonintegrin (dc-sign) and dc-signr (for dc-sign-related) are the best studied [ , ]. these two lectins are expressed on certain dc populations and endothelial cells, respectively, and are described in more detail in this article. hiv- , as an enveloped virus, is released by budding through the plasma membrane of the productively infected cell. in addition to its own virus-encoded envelope glycoproteins, the virus incorporates many different cellular proteins normally found on the cell surface (reviewed in [ ] [ ] [ ] [ ] the process of incorporation of host cell membrane proteins was found to be conserved among all tested hiv- subtypes and strains that were expanded in natural cellular reservoirs, such as mitogen-activated peripheral blood lymphocytes and human lymphoid tissue cultured ex vivo [ ] [ ] [ ] [ ] [ ] [ ] . the physiological significance of this phenomenon is provided by two previous reports showing that host-encoded cell surface constituents were incorporated in plasma-derived clinical hiv- isolates [ , ] . although different host cell constituents can be found embedded within hiv- , the incorporation process seems to be selective. for example, cd is the most abundant leukocyte cell surface glycoprotein [ ] , but is not acquired by hiv- [ , ] . the cxcr , ccr , and ccr co-receptors are also excluded from hiv- [ ] . this ability to incorporate discriminatory host antigens into mature virions has allowed two groups to demonstrate that cell-type-specific antigens can serve as markers of the cellular origin of hiv- replication [ , ] . it has been estimated that between and hla-dr molecules are found associated with hiv- iiib emerging from h cells [ ] . this observation suggests that virally embedded host hla-dr outnumbered virus envelope (env) glycoprotein gp by a factor of . to . considering that hiv- possesses an average of between and gp molecules per virion [ ] . the molecular basis governing the selective incorporation of cell surface proteins within emerging hiv- particles is only beginning to be exposed. it was established that the virus envelope spike glycoproteins (i.e., gp and gp ) are not essential to achieve insertion of icam- into hiv- [ ] . interestingly, icam- incorporation is governed by an intimate association between the cytoplasmic domain of icam- and the viral gag precursor polyprotein pr gag [ ] . it can be proposed that besides interactions between gp and multiple attachment receptors, interactions can also occur between host-derived cell surface components incorporated within emerging virions and their natural counter-ligands. this scenario has been confirmed in numerous studies where such host cell membrane molecules were found to retain their biological activity when located on the virus. for example, hla-dr can increase hiv- infectivity for cd -expressing t cells by about twofold [ ] , whereas icam- alone augments virus infectivity for lfa- + target cells by up to -fold depending of the lfa- conformational state [ , , ] . activation of primary human cd + t lymphocytes was found to result in lfa- clustering, an event that promotes the early events of hiv- replication cycle through an interaction between virus-embedded host icam- and lfa- clusters [ ] . confocal analyses showed that hiv- is concentrated in microdomains rich in lfa- clusters [ ] . virus entry studies including subcellular fractionation experiments with primary human cd + t cells illustrated that the acquisition of icam- by nascent hiv- modified the entry route of the virus within such target cells [ ] . it was established that the icam- mediated increase in virus infectivity was linked with a more productive entry process into primary cd + t lymphocytes (i.e., cytosolic delivery of viral material) [ ] . it has been reported that the higher susceptibility of memory cd + t cells (cd ro + subset) to hiv- infection is due to secondary interactions between virus-associated icam- and cell surface lfa- [ ] . the presence of host-encoded cd in newly formed hiv- particles resulted in a close to -fold augmentation in virus infectivity when using target cells that express high levels of cd and cd , two natural ligands of cd [ ] . in addition, an increase in virus infectivity was also seen following insertion of host-encoded costimulatory molecules cd and cd within progeny viruses [ ] . given that attachment of hiv- to host cells can be modulated by the additional interactions provided by virus-anchored host cell membrane proteins, it is thus not surprising to discover that virus susceptibility to blocking agents is affected. for example, icam- -bearing virions are more resistant to antibodymediated neutralization and this decreased sensitivity is even more dramatic when target cells expressed on their surface the activated form of lfa- [ , ] . additionally, it was reported that virions carrying host icam- on their surface are more resistant to the fusion inhibitor t- than are isogenic viruses lacking host icam- [ ] . although the physical presence of such host constituents on the exterior of virions might be detrimental for the infected individual, the propensity of hiv- to acquire numerous host cell surface components could be exploited to control viral load. indeed, it has been shown in numerous reports that hiv- infectivity can be efficiently neutralized, both in vitro and in vivo, with antibodies specific for such host membrane proteins [ , , , , , , , ] . interestingly, it was demonstrated that hiv- replication is diminished upon treatment with statin compounds (e.g., lovastatin) [ ] , the primary drugs used in the treatment of hypercholesterolemia. the antiviral potency of lovastatin seems to be linked with its capacity to inhibit interactions between virusassociated host icam- and cell surface lfa- . this in vitro work was confirmed by a proof-of-concept small-scale clinical study [ ] . in this provocative study, six a stage hiv- patients not receiving combined therapy were given lovastatin for a month as their only medication. this short-term statin treatment clearly reduced serum viral rna loads in all patients and in general increased their cd + t cell counts. discontinuation of treatment was followed by a rebound in viral load. the prevention of hiv- infection by microbicides, topically applied inhibitors that block access of sexually transmitted hiv- to the host system, is an attractive strategy [ ] . understanding which cell types are first targeted by sexually transmitted hiv- and how these cells interact with hiv- is key to the generation of effective microbicides. several studies suggest that dcs, professional antigen-presenting cells, might be intimately involved in the early local and subsequent systemic spread of sexually transmitted hiv- [ ] . langerhans dcs in the top layer of the anogenital mucosa are probably the first cells exposed to sexually transmitted hiv- . mucosal macrophages and submucosal dcs might subsequently get into contact with virus crossing the mucosal barrier via local breaches or with progeny virions generated by infected langerhans cells. dcs and macrophages express cd and chemokine receptors, and are thus permissive to hiv- infection, albeit infection of dcs is often relatively inefficient and depends on the subpopulation analyzed [ ] . it has been proposed, however, that mere attachment of hiv- to mucosa asso-ciated dcs might be sufficient to promote hiv- spread, since these motile cells might ferry bound virus into lymphoid tissue, the major compartment of hiv- replication, as part of their migratory and antigen-presenting functions within the immune system [ ] . several cellular lectins have been implicated in virus attachment to dcs, macrophages and other cell types relevant to hiv- spread. here, we discuss the role of the lectins dc-sign, dc-signr, mr and langerin in hiv- infection and introduce strategies to inhibit hiv- interactions with these molecules. dc-sign has initially been identified as a gp -binding calcium-dependent lectin expressed in placental tissue [ ] . the lectin has been "rediscovered" in when geijtenbeek and colleagues [ ] showed that dc-sign is expressed on dcs and is involved in hiv- binding and subsequent transfer of the virus to t cells, the latter process presumably involving dc-sign-dependent endocytosis and conservation of infectious hiv- in a low ph compartment [ ] . dc-sign seemed to mainly account for the ability of dcs to promote hiv- infection of cocultured t cells, and it was proposed that dcs might function as trojan horses, which take up hiv- via dc-sign and transport the virus into lymphoid tissue [ , ]. geijtenbeek and coworkers also provided evidence that dc-sign interacts with icam- on endothelial cells [ ] and icam- on t cells [ ] , and proposed that these interactions contribute to extravasation of dcs from blood vessels into tissues and to the close contact between dcs and t cells required for efficient antigen presentation, respectively. thus, a scenario emerged in which dc-sign was involved in dc functions critical for the establishment of an effective immune response and simultaneously allowed hiv- to misuse dcs to ensure its spread in the host. a critical contribution of dc-sign to dc interactions with t cells/endothelial cells or hiv- has subsequently been challenged. it was reported that dc-sign or the related protein dc-signr bind to icams with submicromolar affinities similar to that observed for nonspecific cellular proteins [ ] , suggesting that icam recognition might not account for a potential role of dc-sign in cell-cell interactions. it was also documented that hiv- capture by dcs does either not dependent on dc-sign [ , ] or that the contribution of dc-sign is relatively modest with other factors playing an important role [ ] [ ] [ ] [ ] . in fact, truville and colleagues [ ] provided evidence that different dcs bind to hiv- gp via different lectins or via cd , as discussed below. moreover, it has been demonstrated that transformed cells frequently used to assess dc-sign function were not thp- monocytes, as reported [ ], but most likely raji b-cells [ ] , and that these cells as well as monocyte-derived dcs were permissive to infection by hiv- [ ] [ ] [ ] . the latter observation suggests that the ability of dc-sign-expressing cells to maintain hiv- infectious over prolonged time is most likely due to productive infection of these cells [ ] [ ] [ ] . indeed, dc-sign-dependent hiv- transmission is probably a short-lived process (fig. ) , which is only observed a few hours after the dc-sign-positive, hiv- -exposed cells make contact with target cells. mainly, hiv- might be endocytosed and processed for mhc presentation ( [ , ] , fig. ). finally, two reports indicate that dc-sign might not be a good marker for dcs in vivo [ , ] , with dc-sign-positive cells in lymphoid tissue being of macrophage origin [ ] . how these results relate to a series of previous studies demonstrating dc-sign expression on tissue dcs [ , , ] is currently unclear. s. pöhlmann and m.j. tremblay can a significant contribution of dc-sign to dc interactions with hiv- , and thus to sexual transmission of hiv- , be disregarded in the light of these results? probably not, since several studies also provide evidence for a role of dc-sign in hiv- capture and transmission by dcs. for example, arrighi and colleagues [ ] demonstrated that sirna-mediated down-modulation of dc-sign diminishes hiv- capture by dcs. the contribution of dc-sign to this process might be due to an involvement of this lectin in the formation of an infectious synapse [ ] , a microenvironment established between hiv- bearing cells and target cells, which promotes efficient transfer of infectious virions [ ] . interestingly, dc-sign did not contribute to hiv- infection of target cells in cervical explants but, together with cd , was mainly responsible for hiv- uptake by migratory cells present in these explants [ ] , suggesting that in hiv- -infected individuals dc-sign might indeed contribute to hiv- dissemination by motile cells expressing this lectin. in this regard, it is noteworthy that platelets have been shown to express dc-sign and to capture hiv- in a largely dc-sign-dependent manner [ , ] . these cells might bind hiv- via dc-sign once the virus has reached the blood stream and might promote its dissemination in the host system. similarly, a recent report suggests that a subset of b cells expresses dc-sign and facilitates hiv- transmission to t cells in a dc-sign-dependent manner [ ] . finally, two groups found that certain polymorphisms in the dc-sign gene are associated with decreased risk of hiv- infection [ , ] , highlighting that dc-sign might modulate important events leading to the establishment of hiv- infection. thus, further research is needed to clarify the role of dc-sign in hiv- infection and to evaluate whether this protein is a potential target for microbicides. , also termed l-sign (for liver sign) [ ] , shares % sequence identity with dc-sign and is expressed by sinusoidal endothelial cells in liver (lsecs) and in lymph nodes, alveolar macrophages [ ] and enterocytes of the small intestine [ ] . moreover, dc-signr transcripts have been detected at sites of mucosal hiv- transmission [ ] . dc-signr, like dc-sign, binds to high-mannose carbohydrates and captures hiv- , hiv- and simian immunodeficiency virus [ , ] . binding to icam proteins has also been demonstrated [ ] . however, the natural function of dc-signr is currently unclear. expression of dc-signr in lymph node sinusoids might concentrate hiv- in this compartment, while dc-signr on lsecs might promote infection of this cell type, which was shown to be permissive in vitro [ ] and in vivo [ , ] . lsecs might therefore constantly release progeny virus into the blood stream, thereby promoting hiv- spread. dc-sign and dc-signr are both organized into a n-terminal intracellular domain, a transmembrane domain, a neck region containing . repeats of a -amino acid-comprising sequence and a c-terminal lectin domain. in contrast to the neck domain of dc-sign, which is highly conserved among individuals, the neck domain of dc-signr is polymorphic. while . repeats are most often found and are considered wild type (wt), alleles with . and . repeats are also frequent ( . % and . %, respectively, in the caucasian population [ ] ). the impact of polymorphisms in the dc-signr neck region on susceptibility to hiv- infection has been analyzed by two studies. lichterfeld and colleagues [ ] found no significant differences in dc-signr allele distribution between hiv- -infected individuals and healthy controls. also, no correlation between dc-signr allele frequency and course of hiv- disease was observed [ ] . in contrast, liu and colleagues [ ] found that the / genotype was significantly less frequent in high-risk hiv- -seronegative individuals compared to hiv- -seropositive individuals, while the / genotype was associated with some protection against hiv- infection. it is currently unclear, however, how such a protective effect can be explained on the molecular level. thus, dc-signr variants with and repeats were found to form stable homo-oligomers [ ] and to augment hiv- infection [ ] with similar efficiency as the wt protein. also, coexpression of dc-signr alleles with and repeats allowed formation of stable hetero-oligomers and did not result in decreased hiv- interactions when compared to controls expressing the / allele combination [ ] . a linkage between dc-signr polymorphisms and alterations in unrelated genes determining susceptibility to hiv- infection can therefore at present not be excluded. the observation that dcs can bind to hiv- independently of dc-sign raised the question whether related lectins might be involved. a detailed analysis of gp interactions with different dc subsets revealed that mr on dermal dcs might contribute to gp capture by these cells [ ] . mr is an endocytic receptor that harbors multiple lectin domains and recognizes ligands bearing mannose, fucose or n-acetylglucosamine (glcnac) [ ] . the lectin is expressed on dcs, macrophages and some endothelial cells [ ] and might contribute to capture of hiv- virions by these cells. in fact, it has been demonstrated that an mr-specific antibody can reduce hiv- attachment to macrophages [ ] . langerin contains a single carbohydrate recognition domain specific for mannose, fucose and glcnac and is expressed exclusively by langerhans cells [ , ] . expression of langerin triggers formation of birbeck granules, which are part of the endosomal recycling machinery of langerhans cells [ , ] . the lectin might function as an antigen uptake receptor that releases ligands upon exposure to low ph in endosomal compartments [ ] . while langerin recognizes hiv- gp , it needs to be determined whether it contributes to infection of langerhans cells, which are sus-ceptible to hiv- in culture and in patients [ , ] , or to transmission of hiv- from langerhans cells to adjacent target cells. lectin-dependent hiv- attachment to cells can be prevented by interfering with lectin expression or by targeting domains in the lectin required for efficient ligand recognition. alternatively, carbohydrate structures in hiv- -gp , which are recognized by relevant lectins, are targets for intervention. down-modulation of lectin expression can be achieved by specific sirna [ , ] and by sanglifehrin a [ ] , an immunosuppressant that diminishes ctype lectin expression on dcs. however, issues with delivery (sirna) and possible unwanted side effects (sanglifehrin a) need to be addressed. several inhibitors that impede the interaction of dc-sign with hiv- or other viruses have been described. a synthetic, branched molecule that presents mannose residues on its surface has been shown to inhibit hiv- -gp binding to dc-sign [ ] and to block dc-sign interactions with reporter viruses bearing the ebola virus glycoprotein [ ] , a well-established dc-sign ligand [ , ] . the antiviral activity of comparable molecules bearing sialic acid, the structure recognized by influenza hemagglutinin, has also been demonstrated in a mouse model for influenza infection [ ] , underlining the feasibility of this approach. the inhibitory substances used to target lectinmediated hiv- attachment must not necessarily be of synthetic origin, since bovine lactoferrin [ ] and a substance in human milk which harbors lewis x carbohydrates [ ] were shown to bind to dc-sign and to inhibit hiv- transmission by dcs. similarly, a dc-sign inhibitory activity was identified in human cervicovaginal lavage fluid [ ] . these natural substances might modulate the risk of hiv- transmission and merit further investigation. finally, inhibition of ligand binding to lectins can be achieved by monoclonal antibodies, and a variety of dc-sign-or dc-signr-specific monoclonal antibodies that inhibit pathogen interactions with these lectins have been generated [ , , , ] . while several lectins expressed at the cell surface can mediate hiv- attachment, soluble human-, plant-and bacteria-derived lectins can be employed to inhibit this process. thus, mannose-binding lectin (mbl), a soluble lectin that is involved in innate immunity and is known to bind to hiv- -gp [ ] , inhibits dc-sign-dependent hiv- transmission to target cells, probably by competing with dc-sign for binding sites in hiv- -gp [ ] . a similar observation was reported for ebola virus [ ] , validating that lectins with overlapping carbohydrate specificity can compete for binding sites in gp , which can result in reduction of viral attachment. in fact, soluble lectins were shown to be effective against hiv- transfer by dcs and direct infection of dcs [ ] , highlighting that lectins applied within a microbicide formulation might help to block hiv- infection upon sexual transmission. a promising candidate microbicide is cyanovirin n, a mannose-specific lectin obtained from the cyanobacterium nostoc ellipsosporum [ ] . cv-n binds to the hiv- -gp protein and inhibits hiv- interactions with dcs in vitro [ ] and, when applied topically, infection of macaques with a simian/human immunodeficiency hybrid virus upon vaginal and rectal challenge [ , ] . a more complete understanding of the possible contribution of virus-associated host proteins to the hiv- life cycle is crucial because it might lead to the development of alternative approaches for the treatment of hiv- infection and/or the design of an efficient vaccine strategy. interestingly, a therapeutic or vaccine strategy targeted at virus-associated host cell surface proteins might circumvent problems due to the great genetic variability displayed by hiv- . elucidation of the molecular mechanisms underlying hiv- capture by cellular lectins and assessment of the contribution of this process to hiv- dissemination in and between individuals might help to define novel strategies for preventive or therapeutic intervention. moreover, lectins on dcs can be used as tools to target hiv- antigens to these important antigen-presenting cells [ ] [ ] [ ] [ ] , which might facilitate the generation of an effective hiv- vaccine. cell surface receptors, virus entry and tropism of primate lentiviruses hiv- attachment: another look human immunodeficiency virus type attachment to hela cd cells is cd independent and gp dependent and requires cell surface heparans syndecans serve as attachment receptors for human immunodeficiency virus type on macrophages syndecan captures, protects, and transmits hiv to t lymphocytes infection of colonic epithelial cell lines by type human immunodeficiency virus is associated with cell surface expression of galactosylceramide, a potential alternative gp receptor inhibition of entry of hiv- in neural cell lines by antibodies against galactosyl ceramide acquisition of host cell-surface-derived molecules by hiv- host protein incorporation is conserved among diverse hiv- subtypes presence of host icam- in laboratory and clinical strains of human immunodeficiency virus type increases virus infectivity and cd (+)-t-cell depletion in human lymphoid tissue, a major site of replication in vivo host cell-dependent alterations in envelope components of human immunodeficiency virus type virions cellular compartments of human immunodeficiency virus type replication in vivo: determination by presence of virion-associated host proteins and impact of opportunistic infection detection of hla-dr associated with monocytotropic, primary, and plasma isolates of human immunodeficiency virus type cd : a prototype for transmembrane protein tyrosine phosphatases cd (b - ), and major histocompatibility complex class i and ii molecules into human immunodeficiency virus type virions and microvesicles: implications for viral pathogenesis and immune regulation exclusion of hiv coreceptors cxcr , ccr , and ccr from the hiv envelope human immunodeficiency virus type derived from cocultures of immature dendritic cells with autologous t cells carries t-cell-specific molecules on its surface and is highly infectious cellular proteins bound to immunodeficiency viruses: implication for pathogenesis and vaccines envelope glycoprotein incorporation, not shedding of surface envelope glycoprotein (gp /su), is the primary determinant of su content of purified human immunodeficiency virus type and simian immunodeficiency virus envelope glycoproteins are not required for insertion of host icam- into human immunodeficiency virus type and icam- -bearing viruses are still infectious despite a suboptimal level of trimeric envelope proteins interaction between the cytoplasmic domain of icam- and pr gag leads to acquisition of host icam- by human immunodeficiency virus type the presence of host-derived hla-dr on human immunodeficiency virus type increases viral infectivity host-derived icam- glycoproteins incorporated on human immunodeficiency virus type are biologically active and enhances viral infectivity role of the leukocyte function antigen- conformational state in the process of human immunodeficiency virus type -mediated syncytium formation and virus infection regulation of lfa- activity through cytoskeleton remodeling and signaling components modulates the efficiency of hiv type- entry in activated cd + t lymphocytes presence of host icam- in human immunodeficiency virus type virions increases productive infection of cd + t lymphocytes by favoring cytosolic delivery of viral material lfa- is a key determinant for preferential infection of memory cd + t cells by human immunodeficiency virus type new insights into the functionality of a virion-anchored host cell membrane protein: cd vs hiv type insertion of host-derived costimulatory molecules cd (b . ) and cd (b . ) into human immunodeficiency virus type affects the virus life cycle interaction between virion-bound host icam- and the high affinity state of lfa- on target cells renders r and x isolates of hiv- more refractory to neutralization virion-bound icam- and activated lfa- : a combination of factors conferring resistance to neutralization by sera from human immunodeficiency virus type -infected individuals independently of the disease status and phase susceptibility of hiv type to the fusion inhibitor t- is reduced on insertion of host intercellular adhesion molecule in the virus membrane macaques immunized with hla-dr are protected from challenge with simian immunodeficiency virus immunization with class i histocompatibility leukocyte antigen can protect macaques against challenge infection with sivmac- h statin compounds reduce human immunodeficiency virus type replication by preventing the interaction between virion-associated host intercellular adhesion molecule and its natural cell surface ligand lfa- statins inhibit hiv- infection by downregulating rho activity inhibiting sexual transmission of hiv- infection the interaction of immunodeficiency viruses with dendritic cells sequence and expression of a membrane-associated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection dc-sign, a c-type lectin on dendritic cells that unveils many aspects of dendritic cell biology dc-sign-icam- interaction mediates dendritic cell trafficking identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses characterization of dc-sign/r interaction with human immunodeficiency virus type gp and icam molecules favors the receptor's role as an antigen-capturing rather than an adhesion receptor rhesus macaque dendritic cells efficiently transmit primate lentiviruses independently of dc-sign binding of human immunodeficiency virus type to immature dendritic cells can occur independently of dc-sign and mannose binding c-type lectin receptors via a cholesterol-dependent pathway dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin/cd is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells cell type-dependent retention and transmission of hiv- by dc-sign functional evaluation of dc-sign monoclonal antibodies reveals dc-sign interactions with icam- do not promote human immunodeficiency virus type transmission diversity of receptors binding hiv on dendritic cell subsets raji b cells, misidentified as thp- cells, stimulate dc-sign-mediated hiv transmission immunodeficiency virus uptake, turnover, and -phase transfer in human dendritic cells covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells dc-sign promotes exogenous mhc-i-restricted hiv- antigen presentation dendritic cells and hiv-specific cd + t cells: hiv antigen presentation, t cell activation, viral transfer tlr activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells expression of dc-sign by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells dc-sign-mediated infectious synapse formation enhances x hiv- transmission from dendritic cells to t cells recruitment of hiv and its receptors to dendritic cell-t cell junctions blockade of attachment and fusion receptors inhibits hiv- infection of human cervical tissue lentivirus degradation and dc-sign expression by human platelets and megakaryocytes dc-sign and clec- mediate human immunodeficiency virus type capture by platelets dc-sign on b lymphocytes is required for transmission of hiv- to t lymphocytes association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection analysis of genetic polymorphisms in ccr , ccr , stromal cell-derived factor- , rantes, and dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin in seronegative individuals repeatedly exposed to hiv- a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus homozygous l-sign (clec m) plays a protective role in sars coronavirus infection most dc-signr transcripts at mucosal hiv transmission sites are alternatively spliced isoforms primary cultures of endothelial cells from the human liver sinusoid are permissive for human immunodeficiency virus type detection of hiv rna and p antigen in hiv -infected human liver presence of hiv- in human parenchymal and non-parenchymal liver cells in vivo the tandem-repeat polymorphism of the dc-signr gene does not affect the susceptibility to hiv infection and the progression to aids repeat-region polymorphisms in the gene for the dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin-related molecule: effects on hiv- susceptibility all but the shortest polymorphic forms of the viral receptor dc-signr assemble into stable homo-and heterotetramers impact of polymorphisms in the dc-signr neck domain on the interaction with pathogens pattern recognition receptors: doubling up for the innate immune response involvement of macrophage mannose receptor in the binding and transmission of hiv by macrophages the monoclonal antibody dcgm recognizes langerin, a protein specific of langerhans cells, and is rapidly internalized from the cell surface characterization of carbohydrate recognition by langerin, a ctype lectin of langerhans cells reproduction of langerin/cd traffic and birbeck granule formation in a human cell line model langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules epidermal langerhans cells -a target for htlv-iii/lav infection langerhans' cells are an actual site of hiv- replication rnai-directed inhibition of dc-sign by dendritic cells: prospects for hiv- therapy the novel cyclophilinbinding drug sanglifehrin a specifically affects antigen uptake receptor expression and endocytic capacity of human dendritic cells mannose hyperbranched dendritic polymers interact with clustered organization of dc-sign and inhibit gp binding mannosyl glycodendritic structure inhibits dc-sign-mediated ebola virus infection in cis and in trans dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers lactoferrin prevents dendritic cell-mediated human immunodeficiency virus type transmission by blocking the dc-sign-gp interaction lewis x component in human milk binds dc-sign and inhibits hiv- transfer to cd + t lymphocytes human cervicovaginal lavage fluid contains an inhibitor of hiv binding to dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin internalizing antibodies to the c-type lectins, l-sign and dc-sign, inhibit viral glycoprotein binding and deliver antigen to human dendritic cells for the induction of t cell responses mannose binding lectin (mbl) and hiv inhibition of dc-sign-mediated trans infection of t cells by mannose-binding lectin mannose-binding lectin binds to ebola and marburg envelope glycoproteins, resulting in blocking of virus interaction with dc-sign and complement-mediated virus neutralization sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type (hiv- ) infection and dendritic-cell-directed hiv- transfer the highly specific carbohydrate-binding protein cyanovirin-n: structure, anti-hiv/ebola activity and possibilities for therapy cyanovirin-n inhibits aids virus infections in vaginal transmission models cyanovirin-n gel as a topical microbicide prevents rectal transmission of shiv . p in macaques dc-sign-specific liposomal targeting and selective intracellular compound delivery to human myeloid dendritic cells: implications for hiv disease high efficiency transduction of dendritic cells by adenoviral vectors targeted to dc-sign effective induction of naive and recall t-cell responses by targeting antigen to human dendritic cells via a humanized anti-dc-sign antibody recombinant adenovirus type vectors that target dc-sign, chemr and alpha(v)beta integrin efficiently transduce human dendritic cells and enhance presentation of vectored antigens we acknowledge numerous contributions from various laboratories whose references were not cited in this review due to space limitations. the work presented in this review is supported by grants to key: cord- -vf xbaug authors: dysko, robert c.; nemzek, jean a.; levin, stephen i.; demarco, george j.; moalli, maria r. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - / - sha: doc_id: cord_uid: vf xbaug nan status as a cooperative companion animal of reasonable size. dogs were used in the mid- s by william harvey to study cardiac movement, by marcello malpighi to understand basic lung anatomy and function, and by sir christopher wren to demonstrate the feasibility of intravenous delivery of medications (gay, ) . the use of dogs continued as biomedical research advanced, and they were featured in many noteworthy studies, including those by pavlov to observe and document the conditioned reflex response and by banting and best to identify the role of insulin in diabetes mellitus. for a comprehensive but concise review of the use of the dog as a research subject, the readers are directed to the manuscript by gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger-breed dogs for use in surgical research studies. some specific breeds with congenital or spontaneous disorders are also maintained by research institutions (see specific examples below). random-source dogs used in research are most frequently mongrels or larger-breed dogs (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for beagle for the years - , approximately % of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. the next most common areas of research using beagles were dental and periodontal disease and surgery ( % of publications), orthopedic surgery and skeletal physiology ( %), and radiation oncology ( %). other research areas that utilized beagles included canine infectious disease, surgery, imaging, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established, and the organs of larger-breed dogs are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies is maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of blood, most notably the neutrophil population. these dogs are used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease), and the development of spontaneous diabetes mellitus and hypothyroidism in a variety of dogs has also been studied for comparisons with the human conditions. although historically the dog has been a common laboratory animal, the use of dogs in research has been waning over the past few years. according to the u.s. department of agriculture ( ) , the number of dogs used in research has declined from a high use of , in , in to only , in . this decrease was caused by a variety of factors, including (but not limited to) increased cost, decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purpose-bred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola and icterohemorrhagiae, and bordetella bronchiseptica. rabies virus vaccination may also be included. purpose-bred dogs are also usually treated prophylactically for helminths and ectoparasites, intestinal coccidia, and bacterial ear infections (r. scipioni and j. ball, personal communication, ) . random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting), retired racing dogs, or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. randomsource dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations. options for procurement of dogs for biomedical research typically include purchase from a u.s. department of agriculturedesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . , definitions, and . , requirements and application. briefly, class a licensees are breeders who raise all animals on their premises from a closed colony (suppliers of purpose-bred dogs are typically class a dealers). class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and then resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and recordkeeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. regulations regarding the sale of pound dogs to research facilities or class b dealers vary from state to state and include some bans on this practice. the best resource for identification of possible vendors is the "buyer's guide" issue of the periodical lab animal. typically the last issue of each year, the "buyer's guide" lists sources for both purpose-bred and random-source dogs and denotes such features as pathogen-free status, documentation of health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within that issue of the journal. welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare. regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats, of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures--additional requirements for dogs), because the space required for housing dogs is calculated using the length of the dog rather than the body weight (which is used for other species and also for dogs, according to national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute of laboratory animal research (ilar) has written the "guide for the care and use of laboratory animals" (seventh edition, ) . the "guide" is the primary document used by institutional animal research programs to develop and design their programs, as well as by the association for assessment and accreditation of laboratory animal care international (aaalac international) and other animal care evaluation groups to facilitate site visits and inspections. the ilar committee on dogs has also written "dogs: laboratory animal management " ( ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. growth data for beagles from a purpose-bred dog breeding facility are provided in table i . table ii features hematology data from beagles from the same commercial facility. table iii lists serum and urine chemical data for beagles. normal physiologic data for dogs (no breed specified) are provided in table iv . the information presented in the tables represents a range of normal values that can vary, depending on the analytical method and equipment used as well as the age, breed, gender, and reproductive status of the animal. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal good nutrition and a sound, balanced diet are essential to the health, performance, and well-being of the animal. the basic nutrient requirements for dogs have been compiled by the nrc and represent the average amounts of nutrients that a group of animals should consume over time to maintain growth and prevent deficiencies (national research council, ) . the reader is referred to these guidelines for useful reference points for management of an animal's diet during various physiologic states (e.g., gestation, lactation, maturational age). most commercially available balanced dog diets are "closedformula" diets, in which the labeled specific minimum requirements for protein and fat, and the maximum values for ash and fiber, are met. these diets do not necessarily provide the identical composition of ingredients from batch to batch. ingredient composition varies, depending on the cost relationships of the various ingredients as the manufacturer attempts to achieve the label requirements at the lowest ingredient cost. an "openformula" (or "fixed-formula") diet provides more precise dietary control. in these diets the ingredients are specified, and the percentage of each ingredient is kept constant from batch to batch. "semipurified" diets provide for the strictest control of ingredients and are formulated from the purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are generally safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf life, but the best strategy is to use each lot based on the date of manufacture in order to prevent food from expiring and to ensure that only fresh diets are fed. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the basal metabolic rate, or basal energy requirement (ber), refers to the amount of energy expended following sleep, - hours after food consumption, and during thermoneutral conditions (kleiber, ; lewis et al., ) . the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment, which in the dog is approximately twice the ber (lewis et al., ) . for dogs weighing greater than kg, the mer may be calculated using this simplified linear equation: mer (metabolizable kcal/day) = ( weightkg + ) (national research council, ; lewis et al., ) . the quantity of a correctly balanced diet to be fed to each dog can then be determined by dividing the mer by the energy density of the diet. fat provides three major dietary functions, including absorption of fat-soluble vitamins (a, d, e, and k), enhancement of palatability, and provision of essential (unsaturated) fatty acids. dietary fat is an excellent, highly digestible energy source, providing . times more energy on a per weight basis than either soluble carbohydrates or proteins (lewis et al., ) . however, fats are not needed for this purpose when adequate carbohydrate and protein are present. consumption of fat in excess of an animal's ability to metabolize it results in steatorrhea and has been related to the development of acute pancreatitis, whereas lack of dietary fat may lead to a fatty acid/energy deficiency. fatty acid deficiency is associated with poor growth, poor physical performance, reduced reproductive performance, and weight loss. dogs are considered to be "easy keepers," because they do not have as many absolute nutritional requirements as their domestic counterpart, the cat. however, they do possess a unique requirement for certain polyunsaturated fatty acids, a deficiency of which may predispose them to decreased growth rates and dermatologic abnormalities, such as "hot spots." dogs require linoleic (f - ) acid, an essential fatty acid (national research council, ) , and more recently it has been demonstrated that the f - fatty acids may play a role in maintaining healthy skin (logas and kunkle, ) . supplementation with a balanced essential fatty acid product (e.g., derm caps) may alleviate allergy-related dermatoses such as flea-bite dermatitis and pyoderma (logas and kunkle, ; miller, ) . essential fatty acid deficiency can occur in dogs receiving low-fat dry dog food that has been stored too long, particularly under warm, humid conditions (lewis et al., ) . there are a-amino acids, of which cannot be synthesized in sufficient quantity to meet a dog's normal metabolic demands for growth and maintenance. hence, as their name implies, these essential amino acids are required by all dogs and must be provided in the diet. the essential amino acids and the minimal requirements for growth are listed elsewhere (lewis et al., ) . chronic excessive protein intake may be detrimental to the kidney by contributing to accelerated renal aging and subsequent glomerulosclerosis (lewis et al., ) . conversely, inadequate protein intake results in retardation of growth and adata graciously provided by r. scipioni and j. ball of marshall farms usa, inc., north rose, new york. beagles tested for period / / - / / . b s.d., standard deviation; wbc, white blood cells; rbc, red blood cells; hgb, hemoglobin; hct, hematocrit; mcv, mean corpuscular volume; mch, mean corpuscular hemoglobin; mchc, mean corpuscular hemoglobin concentration; rdw, red cell distribution width; hdw, hemoglobin distribution width; plt, platelets; mpv, mean platelet volume; neut, neutrophils; lymp, lymphocytes; mono, monocytes; eos, eosinophils; baso, basophils; luc, large unstained cells; li, lobularity index; mpxi, mean peroxidase activity index reduction in production and/or performance. protein deficiency, a potential consequence of decreased food intake, results in decreased energy intake. as a compensatory mechanism for a lack of fat or carbohydrate, body protein catabolism ensues in order to meet energy demands, thus exacerbating the negative protein balance and contributing to the clinical signs of edema/ascites, unkempt appearance, lethargy, and weight loss. thus, caloric needs must be met before protein needs (lewis et al., ) , an important concept to bear in mind in the event of research experiments that may predispose to anorexia. in general, providing a good quality commercial diet that supplies the required amount of amino acids and caloric requirements of the animal, while avoiding excess protein, will ensure nutritional stability and promote longevity. appropriate mineral balance in the diet is very important. the best approach in the laboratory setting is to feed a commercial diet that has been formulated with the proper amount and balance of minerals for normal growth. the recommended amount of dietary minerals and the major causes and clinical signs of deficiencies are published elsewhere (lewis, ) . determining the specific mineral involved in an imbalance can be a diagnostic challenge, because the clinical signs for several excesses/ . (basal) . (anestrus) . c < . c - c - r (continues) deficiencies are similar and nonspecific. a definitive diagnosis is often made only after the diet has undergone analysis of the mineral components. once the imbalance has been identified, the safest resolution to the problem is to discard the entire lot of misformulated diet. attempting to correct the imbalance through oral supplementation is likely to be more harmful than beneficial, and it risks intensifying the problem by creating additional mineral imbalances. vitamins function as enzymes that regulate a wide variety of physiologic processes. they are divided into two groups based on their solubility. the fat-soluble vitamins include a, d, e, and k, whereas the rest are water-soluble. a list of the vitamins, their requirements, and clinical signs associated with deficiencies and toxicities is published elsewhere (lewis et al., ) . cases of dietary deficiency are rarely encountered in the research setting, because laboratory dog chows are fortified with vitamins. additional vitamin supplementation may occasionally be required during prolonged clinical illnesses, such as polyuria or diarrhea, which predispose to loss of water-soluble vitamins (b complex and c) (lewis et al., ) . however, as with minerals, routine supplementation of vitamins may induce inadvertent toxicity and exacerbation of an imbalance. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. this section is largely based on information assimilated from texts such as "miller's anatomy of the dog" (evans and christensen, ) , "veterinary reproduction and obstetrics" (arthur et al., ) , and an issue of veterinary clinics of north america: small animal practice devoted to pediatrics of puppies and kittens (hoskins, ) . the ovaries of the bitch are attached to the dorsolateral walls of the abdominal cavity caudal to the kidneys by the broad ligaments and are not palpable abdominally. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the birchard and sherding ( ) . ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. catheterization of the cervix is usually not possible in the normal bitch at any stage of the reproductive cycle, except during or immediately following parturition. thus, semen is deposited at the external cervical os during natural or artificial insemination. the vagina is a long musculomembranous canal that extends from the uterus to the vulva. when the vagina is examined, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva so as to avoid the deep ventral clitoral fossa. examination should proceed at an angle of approximately ~ until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the bitch has a monoestrous cycle, with clinical estrus occurring predominantly in january or february and again in july or august (although it can occur at any time of year). the estrous cycle consists of four stages: proestrus, estrus, diestrus, and anestrus. the average duration of proestrus is days. during this stage the vulva is enlarged, turgid, and firm, and a sanguinous vaginal discharge is present. endocrinologically, proestrus is the follicular stage of the cycle, and estrogen levels peak at this time. estrus generally lasts days, and the vulva is softer and smaller than in proestrus. a vaginal discharge persists during estrus and may remain serosanguinous or become straw-colored. the endocrine feature of estrus is the luteinizing hormone (lh) surge, followed by ovulation within - hours. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. serum progesterone levels peak during diestrus. the duration of anestrus is approximately months. anestrus is the stage of reproductive quiescence, characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. components of the canine spermatic cord include the ductus deferens, the testicular artery and vein, the lymphatics and nerves, and the cremaster muscle. the cremaster muscle and pampiniform plexus aid in thermoregulation of the testicles, which are maintained at ~ lower than basal body temperature. sweat glands in the scrotum assist in lowering the scrotal temperature through evaporation. the penis is a continuation of the muscular pelvic urethra and is attached to the ischiatic arch by two fibrous crura. it is composed of fibrous tissue and three cavernous sinuses: corpus cavernosum, corpus spongiosum penis, and corpus spongiosum glandis. the accessory sex glands of the dog consist of only a well-encapsulated prostate gland that surrounds the pelvic urethra, and ampullary glands at the termination of the vas deferens in the urethra. the dog does not have seminal vesicles or bulbourethral glands. the onset of puberty ranges from to months of age and is affected by breed, season, and nutritional and disease status. testicular growth is rapid at this time, and the seminiferous tubules begin to differentiate. the sertoli cells form the bloodtestis barrier, the tubules become hollow, and spermatogenesis commences. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial, or leydig's, cells. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, while inhibin and estrogen play a role in a feedback loop on the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from initiation of spermatogonial mitosis to delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality and number of sperm produced. problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities that suppress sexual behavior. animals with poor hindlimb conformation or with trauma to the back or hindlimbs may be unable to properly mount the female. there is a positive correlation with the size of the testicles as measured by scrotal circumference and the number of sperm produced. finally, parameters used to assess the quality of sperm include motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities (such as a kinked tail) is a good indicator of normal male fertility. in general, erection, which involves muscular contractions and increased arterial blood flow to the penis, is controlled by the parasympathetic nervous system, whereas ejaculation is under sympathetic control. on mounting, the initial thrusting and ejaculation of semen last about minute. the bulbus glandis becomes enlarged, which lodges the penis in the female reproductive tract. the male then dismounts and brings one hindleg over the female, and the two continue to be joined "rear to rear," a position classically termed "the tie." ejaculation of the accessory gland fluid continues for - minutes. the continued expulsion of prostatic fluid during the "tie" may serve to propel the semen from the vagina through the cervix into the uterus. fertilization occurs in the oviduct and may occur as late as days after coitus, because of the long life span of sperm in the dog. however, once ovulated, oocytes generally remain viable for only - hours. therefore, the bitch should be bred prior to ovulation to ensure the presence of sperm for fertilization of live oocytes. cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation, nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear-cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear "anuclear" and are classified as "cornified" or "anuclear squames." cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. the percentage of cornified cells (of the total number of epithelial cells) decreases gradually to zero after the onset of diestrus. the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear-cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, it is not a substitute for observation of behavioral estrus, which is the best criterion to use in breeding management. during proestrus the male is attracted to the bitch and will investigate her hindquarters, but she will not accept breeding. the behavioral hallmark of estrus is standing receptivity toward the male. during this stage the bitch will exhibit "flagging," or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate, and the number of pups whelped per egg ovulated, it is recommended to breed the bitch on days , , and of the standing heat. fertilization is completed in the mid-to distal oviduct. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary and deciduate, indicating that the placental villi are arranged in a belt and that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy, and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility. pregnancy detection can be performed by abdominal palpation of the uterus days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings in the early gravid uterus. they are approximately inches in length at - days, the time at which pregnancy is most easily and accurately diagnosed. by day the uterus begins to enlarge diffusely, so that the vesicles (and, therefore, pregnancy) are difficult to identify by palpation. fetal skeletons become calcified and are radiographically evident by day . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. real-time ultrasound can be utilized for pregnancy detection of vesicles as early as - days. an abrupt drop in body temperature to less than ~ indicates impending parturition within - hours. the process of parturition has been divided into three stages, stage of labor lasts - hours and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include hard panting and increased pulse and respiration rates. fetal expulsion occurs during stage , which lasts approximately - hours. as the fetus engages the cervix, the neuroendocrine system induces the release of oxytocin; this is referred to as the ferguson reflex. oxytocin strengthens the uterine contractions and may elicit voluntary abdominal contractions as well. the bitch is usually recumbent during stage but is able to inhibit this stage if labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between delivery of each pup is irregular, but the average time lapse is less than hour between pups until parturition is complete. veterinary assistance is necessary if the bitch remains in stage for more than hours without delivering the first pup, or for more than hours before delivering subsequent pups. the placentas are expelled during stage of labor, immediately following delivery of a pup, or up to minutes thereafter. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats, and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged as they have the potential to coat the umbilical cord, which may predispose to ascending infections. heat lamps may be placed hours prior to parturition and remain until all neonates dem-onstrate vigorous and successful suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. thus, monitoring of parturition is important, but human intervention should be minimal in order to prevent stress-induced cannibalism. weak or debilitated puppies may be cannibalized by the bitch before the research staff recognizes the need for veterinary attention. the postpartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. in these cases, - units of oxytocin may be administered intramuscularly. uterine involution occurs during anestrus within - weeks of parturition. during this time a greenish to red-brown vaginal discharge, or lochia, may be noted. although lochia is normal, the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the sixth postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as strictures, or when the bitch refuses to stand for breeding. semen for ai is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the bitch's scent and manually stimulated. after collection of the first two fractions, a sufficient amount of the third fraction, which consists predominantly of prostatic fluid, is collected to bring the total semen volume to - ml. the semen is then drawn into a sterile or ml syringe attached to a sterile disposable insemination pipette. the bitch is inseminated either standing or with raised hindquarters. a gloved index finger is inserted into the dorsal commissure of the vulva and directed craniodorsally until it is over the ischial arch. the tip of the insemination pipette is introduced and guided by the gloved finger toward the external cervical os. the semen is injected, and - ml of air are then flushed through the syringe and pipette. the pipette is withdrawn, and the gloved finger is used to feather the ceiling of the vagina until contractions of the vaginal musculature are palpable. the bitch's hindquarters are subsequently elevated to promote pooling of semen around the external cervical os. as with natural breeding, ai should be performed on days , , and of standing heat, or on the days of maximal vaginal cornification. the bitch should be palpated for pregnancy approximately weeks after the first insemination. false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. however, in the event of extreme discomfort due to mammary gland enlargement, bitches may be treated with mibolerone (cheque drops) at an oral dose of ~tg/kg q hr for - days (brown, ) . reproductive performance in the bitch is optimal prior to years of age. although normal cycle lengths are reported to occur up to the ages of - years, the interestrous interval tends to increase by years of age. cycling does not completely cease; however, after years of age, bitches demonstrate significant decreases in conception rate and number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia are extremely common. beagles have been a popular animal model because of their docile nature. they are easily handled and for the most part respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiogram (ecg) recordings, oral gavage, and venipuncture. dogs are sexually mature by - months of age, but they are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from - weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. the extent to which breed affects behavior has been the subject of popular speculation but is difficult to prove. in general, breed-specific patterns do tend to emerge. for example, it appears that beagle pups are very motivated by food reward (overall, ). this is not surprising, because the breed was selected to work with its nose, and this may be a useful attribute for laboratory investigations that are predicated on food restriction. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the third chapter of "clinical behavioral medicine for small animals" (overall, ). by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of the disorders that can affect this species. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting. especially noted in this chapter are infectious diseases associated with the use of random-source dogs that have unknown vaccination history and have had intensive contact with other similar animals at pounds and/or shelters, or conditions seen frequently in the beagle, the most common breed used in biomedical research. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks, such as the "current veterinary therapy" series (j. d. bonagura and r. w. kirk, eds.), "veterinary internal medicine" (s. j. ettinger and e. c. feldman, eds.), and "infectious diseases of the dog and cat" (c. e. greene, ed.) . full citations of some chapters from these texts are listed in the references (w. b. saunders co. of philadelphia publishes all three texts.) canine infectious tracheobronchitis (kennel cough complex) etiology. infectious tracheobronchitis (itb) is a highly contagious illness of the canine respiratory tract that usually manifests as an acute but self-limiting disease. several organisms have been incriminated as causative for this condition: bordetella bronchiseptica; canine parainfluenza virus (cpiv); canine adenovirus types and (cav- , cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasms and ureaplasms. clinical signs. clinical infectious tracheobronchitis can be subdivided into mild or severe forms. the mild form is the more common presentation and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough is easily elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise the dog is typically asymptomatic, with normal body temperature, attitude, and appetite. mild tracheobronchitis usually lasts - days, even if left untreated. the severe form of tracheobronchitis generally results from mixed infections complicated by poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and may be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. these cases require more aggressive treatment and may be fatal. bordetella bronchiseptica is considered to be the respiratory tract of infected animals (bemis, ) . this bacterium is very easily spread by aerosol and direct contact, and fomite transmission is also possible (bemis, ) . transmission is favored by confined housing of multiple animals. in experimental studies, b. bronchiseptica transmission to susceptible individuals was % (thompson et al., ; mccandlish et al., ) . the incubation period is - days. cpiv and cav- are also spread by aerosols. of these two viruses, cav- is the most persistent, lasting for up to several months in the environment, whereas cpiv is fairly labile (hoskins, a) . both viruses can be destroyed by quaternary ammonium disenfectants. pathogenesis. the most common clinical isolates are cpiv and bordetella bronchiseptica. however, b. bronchiseptica may be a commensal organism, and it is often recovered from asymptomatic animals. in cases of clinical infection, b. bronchiseptica attaches to the cilia on the mucosal surface of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv or cav- alone are usually subclinical; coinfections with b. bronchiseptica or other microbes may result in clinical itb (keil and fenwick, ; wagener et al., ) . the characteristic lesion from cpiv or cav- infection is necrotizing tracheobronchiolitis (dungworth, ) . pathogenic infection of the upper airways typically results in inflammation and ciliary dysfunction. diagnosis and differential diagnosis. diagnosis of infectious tracheobronchitis is often based on clinical signs. isolation of bordetella bronchiseptica or mycoplasma by nasal swabs allows only a presumptive diagnosis. viral isolation or paired serology can be done but is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. canine distemper virus infection, pneumonia, heartworm disease, tracheal collapse, and mycotic infections are differential diagnoses for dogs with similar signs. bronchial compression as a result of left atrial enlargement, hilar lymphadenopathy, or neoplasia may also elicit a nonproductive cough (johnson, ) and should be considered as a differential for itb. prevention. prevention is best achieved by avoiding exposure to infected animals, but this is oftentimes not practical. dogs should be vaccinated prior to, or at the time of, admission to the animal research facility. intranasal vaccine combinations for bordetella bronchiseptica and cpiv are preferred. intranasal vaccines protect against both infection and disease, can be given to dogs as young as weeks of age, and can produce immunity within days. control. sanitation and ventilation are critical for control. the animal care staff must practice proper hygiene to prevent fomite transmission. symptomatic animals should be isolated, and animal-to-animal contact avoided. kennels should be disinfected with agents such as bleach, chlorhexidine (nolvasan) or quaternary ammonium chloride (roccal-d). proper ventilation and humidity are important in controlling spread of these infectious agents; - air changes per hour at % relative humidity are recommended (sherding, ) . no specific treatment is available for viral infections. bordetella bronchiseptica is typically sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur, and it should be continued for days. use of empirical antibiotic treatment in mild cases may hasten the resolution of clinical signs. for severe or unresponsive infection, treatment should be based on bacterial culture sensitivity patterns; nebulized gentamicin may be helpful. cough suppressants (e.g., dextromethorphan) should be avoided if the cough is bringing up mucus (productive); however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction and minimizing discomfort. tis results in altered respiratory tract histology and impaired mucociliary clearance, infected animals should not be used for pulmonary studies. animals with clinical disease would also be poor surgical candidates. etiology. [ -hemolytic lancefield's group c streptococcus (streptococcus zooepidemicus) is a gram-positive non-spore-forming coccus and an etiologic agent for pneumonia and septicemia in dogs. clinical signs. clinical signs vary based on the organ system affected. pneumonic disease is typically associated with coughing, weakness, fever, dyspnea, and hematemesis. peracute death without clinical signs has been reported in a previously healthy research dog (bergdall et al., ) , and conjunctivitis can also be caused by this organism (murphy et al., ) . epizootiology and transmission. lancefield's group c streptococci have been isolated as commensal flora in the upper respiratory tract and the vagina of clinically normal dogs (olson et al., ) . epizootics have been reported in both racing greyhounds and research colonies (sundberg et al., ; garnett et al., ) . in these epizootics, and in the reported case of peracute death (bergdall et al., ) , recent transportation (within days) was associated with the disease. as such, lancefield's group c streptococcus may be an opportunistic pathogen in dogs. pathologic findings. in the peracute case reported (bergdall et al., ) , hemorrhage from the mouth and nose and within the pleural cavity was the most striking lesion. ecchymotic and petechial hemorrhages were seen on other organ surfaces. the lungs were heavy and wet, and blood oozed from cut surfaces. "bull's-eye" lesions were observed on the pleural surface of affected lung lobes, similar to ischemic lesions seen with fungal infections (fig. ) . histologically, the lungs were characterized by areas of hemorrhage surrounding foci of degenerative neutrophils, blood, and necrotic debris. gram-positive cocci were seen in both the lung and the tonsils. pathogenesis. the pathogenesis for disease caused by lancefield's group c streptococcus is unclear. strain variation with respect to virulence and host immune factors is probably significant. diagnosis and differential diagnosis. definitive diagnosis is made based on bacterial culture and identification. any cause of pneumonia and/or peracute death in dogs needs to be considered as a differential diagnosis. bacterial pneumonias or septicemias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and bordetella bronchiseptica. nonbacterial causes include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control. too little is known about the pathogenesis of lancefield's group c streptococcus to make any recommendations about prevention and control. treatment. antibiotic therapy should be provided, based on culture and sensitivity. intravenous fluids are indicated for febrile or systemically ill patients. for dyspneic patients, oxygen therapy and strict activity restriction are required. research complications. clearly, dogs with severe hemorrhagic pneumonia or septicemia are not appropriate for any research study. the association between epizootics of this disease and transportation shipment supports the philosophy of providing acclimation periods to animals upon arrival at research facilities to evaluate health status and enable the animals to normalize physiologically. etiology. serovars of the spirochete leptospira interrogans sensu lato cause canine leptospirosis. disease in dogs is primarily due to serovars canicola, icterohemorrhagiae, grippotyphosa, pomona, and bratislava. clinical signs. leptospirosis may present as either an acute or a chronic problem. clinical signs are nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, and vomiting. animals may be febrile and may be reluctant to move, because of muscle or renal pain or meningitis. icterus, congested mucous membranes, or signs referable to disseminated intravascular coagulation (petechial/ecchymotic hemorrhages, melena, epistaxis, or hematemesis) are also possible. animals with peracute leptospirosis are characterized by septicemia, shock, vascular collapse, andrapid death. uveitis, abortions, and stillbirths have also been associated with leptospirosis. epizootiology and transmission. vaccination and reduced exposure to reservoir hosts have markedly decreased the prevalence of leptospirosis over the past years. wild animals, cattle, and rodents are reservoirs for leptospira. the epidemiology of the disease is not static, and recent changes have been observed. serovars pomona, grippotyphosa, and bratislava are becoming more common causes of canine disease, with canicola and icterohemorrhagiae becoming less common. this may be due to vaccination practices and increased movement of wildlife reservoirs (raccoons, skunks, and opossums) into urban/suburban areas. rats have been implicated as important in the transmission of serovars canicola and icterohemorrhagiae (rentko et al., ; brown et al., ; kalin et al., ) . transmission occurs primarily by environmental contact, and not directly from animal to animal. infected hosts shed leptospires in urine, thereby contaminating the environment; naive animals are infected when the organisms contact mucous membranes or abraded skin. recovered animals may shed organisms in their urine for months to years. the organisms are actually labile in the environment; moisture, moderate temperatures, and alkaline soil favor survival and subsequent transmission. close contact, bites, ingestion of infected meat, and transplacental and venereal transmission are also possible. leptospirosis is a zoonotic disease. pathologic findings. the kidneys consistently have gross and microscopic lesions. in the acute phase of the infection, kidneys are swollen and have subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs and liver may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and focal areas of necrosis (searcy, ) . in chronic stages of leptospirosis the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly. several days postinfection the renal tubular epithelium (and, to a variable extent, the liver) is colonized. the hematogenous phase lasts - days. acute renal failure or progressive renal failure leading to oliguria or anuria may occur. the most common clinical syndrome is chronic or subclinical infections after recovery from the acute phase (greene, ) . the nephritis may or may not be accompanied by hepatitis, uveitis, and meningitis. icterus, if it develops, is most common in the acute phase. the combination of azotemia and icterus should alert the clinician to the possibility of leptospirosis. disseminated intravascular coagulation is often a secondary complication. the severity and course of leptospirosis depend on the causative serovar and the age and immune status of the patient. diagnosis and differential diagnosis. zinc toxicity in dogs most closely mimics the clinical syndrome of leptospirosis. other causes of acute and chronic renal failure, icterus, and acute hepatic failure must also be considered. paired serology is the most reliable means of definitive diagnosis; however, seroconversion may not occur until after the first week of infection. prevention and control. vaccination for leptospirosis is standard veterinary practice. bivalent inactivated bacterins for serovars of l. interrogans canicola and serovars of l. interrogans icterohemorrhagiae are commercially available. however, immunization does not prevent development of the carrier state or protect against other serovars. for outdoor-housed dogs, an effective program to prevent contact with wildlife reservoirs is important. control requires identification and either treatment or elimination of carrier animals. treatment. penicillins are the drugs of choice for treating leptospiremia, and prompt use reduces fatal complications. aggressive fluid therapy and supportive care may also be needed. elimination of renal colonization and the carrier state can be accomplished with dihydrostreptomycin or doxycycline administration. should not be used in research studies because of the effects of the disease on renal and hepatic function. etiology. campylobacteriosis in dogs is caused by campylobacter jejuni, a thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rod. clinical signs. most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, small volumes of mucoid or watery diarrhea, with or without frank blood, are most commonly noted. these signs are usually mild, may be intermittent, and typically last - days. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea. epizootiology and transmission. the role of c. jejuni as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . clinical signs of disease most often occur in dogs less than months of age, although any age may be affected. stress or immunosuppression may make animals more susceptible to clinical disease. pound and shelter populations have the highest rates of fecal excretion of c. jejuni (sherding and johnson, ) . transmission is via the fecal-oral route, mostly through fecally contaminated food or water. unpasteurized milk, poultry, and meat are other sources of infection. campylobacter jejuni can be zoonotic; children and immunocompromised individuals are at the greatest risk. pathologic findings. the actual lesions observed depend upon the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated fluid-filled bowel loops, with little or no histopathologic alteration. in cytotoxin-mediated disease, hyperemia and a friable, hemorrhagic mucosal surface are noted. on histopathology the mucosal surface is irregular and ulcerated, and a lymphocytic-plasmacytic ileitis or colitis may be seen. when translocation occurs, the lamina propria becomes edematous and congested, with focal accumulation of granulocytes in the crypts and lamina propria. focal areas of epithelial hyperplasia and decreased numbers of goblet cells are also noted. with warthin-starry silver staining, c. jejuni may be seen between enterocytes but only rarely inside them. pathogenesis. clinical disease may be produced by several different mechanisms after the campylobacter has populated the intestinal tract (van kruiningen, ) . after colonization of the enterocyte surface, c. jejuni can produce an enterotoxin that causes a secretory diarrhea. campylobacterjejuni can also cause an erosive enterocolitis by invasion of the ileal and colonic epithelium along with production of a cytotoxic agent; this may be the mechanism that causes hematochezia. in addition, c. jejuni can produce illness by translocation, i.e., multiplication in the lamina propria and transportation to regional lymph nodes by macrophages. this causes mesenteric lymphadenitis. diagnosis and differential diagnosis. fresh feces (per rectum) are best for ensuring an adequate diagnostic sample. presumptive diagnosis may be made by demonstration of highly motile curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of ~ any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis, including canine parvovirus, coronavirus, distemper virus, giardia, and salmonella infections; helminth infestations; and hemorrhagic gastroenteritis. clinical signs. based on experimental infections in dogs, three phases to the disease have been described: acute, subclinical, and chronic. clinical signs observed vary with the phase of the disease, and the acute and subclinical phases are often missed or misdiagnosed (c. g. couto, personal communication, ; waddle and littman, ; woody and mcdonald, ) . a history of tick exposure may be noted prior to onset of signs. in the acute phase, clinical signs range from mild to severe and may last - weeks. they include inappetance, lethargy, fever, generalized lymphadenopathy, hepatosplenomegaly, exercise intolerance or dyspnea, petechial or ecchymotic hemorrhages, and peripheral edema. central nervous system (cns) signs may also be present such as hyperaesthesia, myoclonus, and cranial nerve deficits. clinical laboratory abnormalities noted during the acute phase include thrombocytopenia, anemia, neutropenia or neutrophilia, and bicytopenia or pancytopenia. hyperplastic bone marrow, mild hyperglobulinemia, and elevated hepatic enzymes may be noted during this phase (kuehn and gaunt, ) . clinical signs are generally absent during the subclinical phase. mild thrombocytopenia, anemia, or leukopenia may be seen. the chronic phase develops - months after the initial infection, and signs may be subclinical to severe. an extremely varied clinical picture can emerge during this time and can mimic several other clinical syndromes. the following constellation of clinical signs may be observed: chronic lethargy, weight loss, inappetance or anorexia, fever, generalized lymphadenopathy, hepatosplenomegaly, petechial or ecchymotic hemorrhages, epistaxis, hematuria, melena, pallor, anterior or posterior uveitis, chorioretinitis, peripheral edema, ataxia, upper and lower motor neuron deficits, altered mentation, cranial nerve deficits, and seizures. persistent thrombocytopenia is the most consistent laboratory abnormality noted for all three stages. many other hematologic abnormalites may be found, such as regenerative or nonregenerative anemia (more frequently the latter), positive coombs' test, bicytopenia or pancytopenia, and splenic plasmacytosis or lymphocytosis. on bone marrow evaluation, plasmacytosis along with hypoplasia of erythroid, myeloid, and/or megakaryocyte lines may be seen. hyperglobulinemia as a result of polyclonal or occasionally monoclonal gammopathy has been noted in - % of e. canis seropositive or infected dogs (kuehn and gaunt, ; breitschwerdt et al., ; shimon et al., ) . proteinuria and/or hypoalbuminemia have also been seen. epizootiology and transmission. ehrlichia canis is an obligate intracellular parasite that infects mononuclear cells. the definitive hosts are arthropods; domestic and wild canids are parasitized secondarily. the primary vector and reservoir is the brown dog tick, rhipicephalus sanguineus. ehrlichia canis is found worldwide and follows the distribution of the vector. infection in dogs is most prevalent in tropical and subtropical areas (greene, ) . in the united states, cases are concentrated in the southeastern and southwestern states but have been reported in almost every state (breitschwerdt, ) . transmission is primarily by tick bites, but it can also occur via blood transfusions from dogs infected for as long as years. ticks become infected by feeding on an infected dog that is in the first - days of an acute infection (lewis et al., ) , and ticks can shed the organisms for up to months. within the tick population, e. canis is transmitted transstadially (within developmental stages) but not transovarially (from female to offspring) (groves et al., ) . pathogenesis. in experimental infections, the incubation period prior to the onset of the acute phase is - days. during the acute phase, which can last from - weeks, the bacteria replicate within circulating and tissue monocytes, resulting in lymphoreticular hyperplasia in affected tissues. infected monocytes then spread hematogenously to other organs in the body, in particular the lungs, kidney, and meninges. infected cells adhere to the vascular endothelium and induce vasculitis, which is the primary mechanism whereby the organism causes disease. the thrombocytopenia during the acute phase is due to both sequestration and destruction, and the development of anemia is a result of red blood cell destruction and suppression of erythrocyte production. the subclinical phase of the disease occurs - weeks after initial infection. during this stage, dogs that can mount an effective immune response clear the infection. those that cannot mount such a response progress to the chronic stage. infection does not confer protective immunity in dogs that recover. german shepherds and doberman pinschers seem to be more severely affected than other breeds. pathologic findings. gross lesions are varied and change, depending on the phase of the disease. the most common findings are petechial and ecchymotic hemorrhages and edema of dependent tissues (woody and hoskins, ) . the most common histologic abnormality noted is lymphocytic-plasmacytic inflammation of numerous organs. mononuclear phagocytic system hyperplasia, extramedullary hematopoiesis, and splenic erythrophagocytosis may also be seen. diagnosis and differential diagnosis. the most sensitive, specific, and commonly employed method for diagnosing e. canis infections is the indirect fluorescent antibody (ifa) test. antibodies can be detected as early as days postinfection, although some dogs may not seroconvert until days postinfection (buhles et al., ) . cross-reaction may occur between e. canis, e. chaffeensis, and e. ewingii. titers greater than : are considered positive and indicative of infection and may persist for up to year. effective treatment typically produces seronegative results in - months. in some cases, asymptomatic dogs may remain seropositive for years after treatment or may be seropositive with a persistent hematologic abnormality (bartsch and greene, ) . the exact mechanism for this finding has not been elucidated. ehrlichia canis morulae can be demonstrated in circulating monocytes of giemsa-stained blood smears. however, this method is labor-intensive and has low sensitivity, as morulae are present transiently and in low numbers. using buffy coat smears from capillary blood may increase the diagnostic yield. polymerase chain reaction (pcr) assays are also available to identify e. canis. differential diagnoses include immune-mediated hemolytic anemia/thrombocytopenia, multiple myeloma, chronic lymphocytic leukemia, and lymphoma. prevention. preventing laboratory animals from contacting ticks is the primary means to avoid monocytic ehrlichiosis in research dogs. avoid exercising dogs in areas infested with ticks. use topical acaricides to prevent tick infestations. keep kennel areas tick-free. dogs used as blood donors and dogs from unproven sources should be tested for e. canis. treatment. doxycycline is the drug of choice for treating monocytic ehrlichiosis. oral doses of either . - mg/kg q hr or mg/kg q hr for days are very effective at eliminating the organism. tetracycline, chloramphenicol, and enrofloxacin are also effective antibiotics; however, chloramphenicol should not be used in animals with cytopenias. in chronic cases, antibiotic treatment should be extended for an additional - weeks. research complications. the most significant research complication is the thrombocytopenia that persists for all stages of the disease. additionally, there is probable alteration in immune function and increased susceptibility to infectious agents. for these reasons, dogs positive for antibodies to e. canis should not be used in research. etiology. this disease, caused by ehrlichia platys, was first described as cyclic thrombocytopenia by harvey et al. in . clinical signs. in most cases, infection with e. platys results in subclinical disease. a generalized lymphadenopathy may be noted. epizootiology and transmission. the vector for e. platys is assumed to be a tick; however, this mode of transmission has not been established. experimental studies by simpson et al. ( ) failed to demonstrate rhipicephalus sanguineus as a vector for e. platys. coinfection with e. canis has been reported, which suggests a common vector for both organisms (french and harvey, ; kordick et al., ) . dogs have been experimentally infected by inoculation with infected blood or infected platelets from other dogs (harvey et al., ; gaunt et al., ) . the geographic distribution of thrombocytic ehrlichiosis is assumed to follow that of other ehrlichia organisms. the highest concentration of cases seems to be in southeastern states, but isolated cases have been reported as far north as michigan and as far west as oklahoma (wilson, ; mathew et al, ) . the prevalence of seropositive dogs can be high in some parts of the country. a study by bradfield et al. ( ) reported that % of the dogs entering a research institute's quarantine facility from sources in eastern north carolina were seropositive for e. platys. hoskins et al. ( ) reported a . % seropositive prevalence in healthy dogs from kennels in louisiana. pathologic findings. gross and histopathologic findings during experimental e. platys infection in dogs have been described by baker et al. ( ) . generalized lymphadenopathy was the only gross lesion noted. follicular hyperplasia and plasmacytosis were the predominate findings in lymphoreticular tissues. all dogs also had extramedullary hematopoiesis, erythrophagocytosis, and crescent-shaped hemorrhages in the spleen. multifocal kupffer's cell hyperplasia was noted in the liver, and mild multifocal lymphocytic-plasmacytic interstitial inflammation was seen in the kidneys. pathogenesis. the pathogenesis of e. platys in dogs has primarily been determined through experimental infection (harvey et al., ) . after inoculation the organism directly infects platelets. thrombocytopenia occurs by day - and fluctu-ates, along with parasitemia, at to day intervals. in some cases the rebound may be within the normal range for thrombocyte counts. the nadir can be lower than , platelets/~d. concurrent with low platelet counts is the development of megakaryocytic hyperplasia in the bone marrow. interestingly, despite extremely low platelet counts, spontaneous bleeding has not been reported in cases of e. platys infection. the mechanism responsible for the cyclic nature of the infection has not been elucidated. diagnosis and differential diagnosis. ehrlichia platys infection may be diagnosed on stained blood smears by visualization of the organisms within platelets. however, this method is very unreliable due to the cyclic nature of the parasitemia and the low numbers of infected thrombocytes. available ifa assays are much more sensitive and specific, and there is reportedly no serologic cross-reaction with other ehrlichia species. dogs usually develop detectable titers - weeks postinfection. pcr assays for e. platys have now been developed as well (chang and pan, ; mathew et al., ) . differential diagnoses for thrombocytic ehrlichiosis include e. canis infection, immunemediated thrombocytopenia, and disseminated intravascular coagulation (dic). platys is the same as described for e. canis, above. research complications. ehrlichia platys infection may increase the risk of bleeding during surgical or traumatic procedures. coinfection with e. platys may potentiate the pathogenicity of other infectious agents, in particular e. canis (breitschwerdt, ) . etiology. lyme disease is caused by borrelia burgdorferi sensu lato, a microaerophilic spirochete that is primarily an extracellular pathogen. clinical signs. clinical signs may be highly variable; lameness due to polyarthritis has been reported as the most common sign. the onset of lameness may be acute or chronic, shift from limb to limb, and be accompanied by swelling and joint pain. synovial fluid analysis from affected joints is consistent with a diagnosis of suppurative arthritis. other clinical signs include fever, anorexia, lethargy, lymphadenopathy, and weight loss. over the course of the disease, signs may wax and wane over a period of weeks to months. dogs rarely develop erythema chronicum migrans (the characteristic rash seen in infected people) and do not exhibit the severe arthritis and neurologic sequelae seen in human beings (greene, ; manley, ) . hematologic and biochemical profiles are generally unremarkable. lyme disease is thought to be the most common arthropod-borne disease of human beings (and possibly of dogs) in the united states. it affects humans and dogs worldwide. the geographic distribution of canine borreliosis is assumed to follow that of the human disease and is related to the range of the arthropod vectors. three major endemic foci that have been identified in the united states account for % of reported human cases (appel and jacobson, ). the distribution of these cases is as follows: northeast/mid-atlantic focus, %; midwestern focus (michigan, wisconsin, minnesota, iowa, illinois, and missouri), %; and california and oregon, %. for the most part, dogs in the remainder of the country are not at risk for contracting lyme disease. borrelia burgdorferi is transmitted exclusively by ixodes ticks. other arthropod hosts may carry the organism but have not as yet been implicated in the transmission of disease. ixodes scapularis, a three-host tick with a to year life cycle, is the prototypical vector for north america. the spirochetes are spread by tick bites from both nymphs and adults. ticks become infected by feeding on an infected mammal and by transstadial transmission (transovarial passage is rare). in endemic areas, - % of adult ticks may be infected (appel and jacobson, ) . the primary reservoir for the organism is the whitefooted deer mouse, peromyscus ieucopus, which can carry spirochetes for its life span without becoming ill. evidence also indicates that the eastern chipmunk, tamias striatus, is an important reservoir (slajchert et al., ) , and birds may also be a significant reservoir. deer, however, serve only as hosts for the tick vectors and not as a reservoir for the spirochete. pathogenesis. the pathogenesis of lyme disease is poorly understood, primarily because of a lack of good animal models and the chronic nature of the disease. infection can be induced experimentally by the bite of a single infected tick. clinical signs develop - days postinfection. some evidence points to the host's inflammatory response to the organism as etiologic for disease (pershing et al, ; greene, ) . seroconversion in dogs occurs - weeks after infection with b. burgdorferi. antibody titers may remain extremely elevated for at least months. igm titers also remain elevated for several months and are indicative of neither acute nor active infection (appel and jacobson, ) . because antibiotic treatment may not eliminate the organism, persistent infections in dogs (treated for days with antibiotics) can be reactivated by steroid treatment up to days postinfection (straubinger et al., ) . diagnosis and differential diagnosis. appel and jacobson ( ) recommend that three of the following four criteria be met to establish a diagnosis of lyme disease in dogs: ( ) history of exposure to ixodes ticks in an endemic area, ( ) characteristic clinical signs, ( ) positive serology, and ( ) rapid resolution of clinical signs with antibiotic therapy. ifa or elisa tests for borrelia antibodies are the assays of choice. it should be re-membered, however, that a positive titer in an endemic area indicates exposure and not necessarily disease and that vaccinated dogs will also have a positive titer. responses to vaccine versus infection may be distinguished by western blot. culture or identification of the organism provides a definitive diagnosis but is very difficult to perform. differential diagnoses include immune-mediated polyarthritis and septic arthritis from other etiologic agents. prevention and control. prevention and control are the same as for the other tick-borne diseases (see discussion of monocytic ehrlichiosis, section iii,a,l,e above). a vaccine against b. burgdorferi is available but should not be necessary in a research setting. treatment. doxycycline is the drug of choice for treating lyme borelliosis. a typical dosing regimen is mg/kg q hr for - weeks. amoxicillin, tetracycline, and the quinolones are also effective. of significant note is that antibiotic treatment results in resolution of clinical signs but may not result in elimination of the organism. (fox and lee, ) . "helicobacter heilmannii" and h. bizzozeronii are thought be the same species, with the latter being the updated nomenclature. this species, as well as h. rappini and h. canis, is considered to be zoonotic (fox and lee, ) . clinical infections may present with vomiting, diarrhea, fever, and anorexia, pica, or polyphagia. epizootiology and transmission. the epizootiology and transmission of helicobacter spp. in the dog remains to be elucidated. the prevalence of canine helicobacter infections in colony or shelter situations has been reported to range from % to almost % (fox, ; hermanns et al., ) . both oral-oral and fecal-oral routes for transmission have been suggested. pathologic findings. no gross lesions are noted; the primary lesion is that of histologic gastritis. this is typically characterized by reduced mucus content of the surface epithelium; vacu-olation, swelling, karyolysis, and karyorrhexis of parietal cells; and multifocal infiltrates of plasma cells and neutrophils into the subepithelium, primarily around blood vessels and between the gastric pits (hermanns et al., ) . focal areas of lymphocytic inflammation and lymphoid follicles may also be seen. pathogenesis. some helicobacter spp. colonize the gastric epithelium exclusively and other species colonize lower parts of the gastrointestinal tract. helicobacter felis and "h. heilmannii" infections have been linked to gastric lesions in laboratoryraised beagles (fox and lee, ) . the mechanism by which these organisms cause disease may be related to the host's inflammatory response to colonization and the helicobacter's ability to produce urease. urease splits urea into ammonia and bicarbonate; ammonia is toxic for the epithelial cells, and bicarbonate may help the organism survive the acidic environment (marshall et al., ; shimoyama and crabtree, ). diagnosis and differential diagnosis. any of the numerous causes of acute or chronic vomiting and diarrhea in the dog (including canine distemper, viral or bacterial gastroenteritis, and ingested toxicants) should be considered as differential diagnoses. definitive diagnosis for dogs requires either endoscopic or surgical biopsy. confirmation of infection with helicobacter spp. requires demonstration of the organism in biopsy samples by histopathology, culture, or recognition by pcr. a positive urease test on a biopsy sample may give a presumptive diagnosis, but only for those species that produce urease. the use of warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. prevention and control. until more is known about the epizootiology and transmission of helicobacter spp. in the dog, specific recommendations cannot be made about prevention and control in this species. treatment. combination therapy has proven to be the most effective method for treating helicobacter spp. infections in dogs. combination therapy of amoxicillin ( mg/kg q hr), metronidazole ( mg/kg q hr), and sucralfate ( . - . mg/kg q hr) for days has been suggested for dogs (hall and simpson, ) . replacing the sucralfate with famotidine ( . mg/kg q hr), omeprazole ( . mg/kg q hr), or bismuth subsalicylate ( . ml/kg q - hr) may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . the benefits of antimicrobial therapy in dogs still need to be established by controlled therapeutic studies. research complications. helicobacter spp. infections could result in altered gastrointestinal responses to drugs and toxic or carcinogenic compounds. therefore, dogs used in gastric physiology or oral pharmacology studies should be free from helicobacteriosis. clinical signs. clinical signs of canine parvovirus usually appear days after inoculation by the fecal-oral route and are characterized by anorexia, fever, depression, and vomiting. profuse, intractable diarrhea ensues, which may become hemorrhagic. approximately % of affected dogs develop severe leukopenia, with a total granulocyte/lymphocyte count ranging from - wbc/~d or less. repeated hemograms may provide prognostic value, because rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. parvovirus can infect dogs of any age, but puppies between and weeks of age appear to be particularly susceptible. puppies less than weeks of age are generally protected from infection by passive maternal antibody. adult dogs probably incur mild or inapparent infections that result in seroconversion. pathogenesis. canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes an acute, highly contagious enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis. parvovirus can be detected in fecal samples with a commercially available elisa from cite. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. other lesions include myeloid degeneration and widespread lymphoid depletion. parvovirus can also be demonstrated in frozen sections by fluorescent antibody techniques. differential diagnoses should include other viral enteritides, salmonellosis, and small intestinal obstruction. prevention and control. prevention of transmission begins with isolation of affected animals and quarantine for week after full recovery. disinfection of potentially infected kennel and diagnostic areas with diluted bleach ( : ) or commercially prepared disinfectant (such as kennesol, available from alphatech, lexington, massachusetts) is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a commercially available modified live vaccine until - weeks of age. young rottweilers and doberman pinschers appear to be predisposed to parvoviral enteritis and should be vaccinated every weeks ( times) from - weeks of age. treatment. treatment is largely supportive and is aimed primarily at restoring fluid and electrolyte balance. research complications. infection with parvovirus obviously precludes the use of a particular dog in an experimental protocol. given the potential for significant discomfort of the affected animal, and the cost of therapy, humane euthanasia is usually the option chosen in a research setting. canine coronavirus infection is usually inapparent or causes minimal illness. this epitheliotropic virus preferentially invades the enterocytes of the villous tips, resulting in destruction, atrophy, and fusion and subsequent diarrhea of varying severity. subclinical infections are most common, but abrupt gastrointestinal upset accompanied by soft to watery, yelloworange feces is possible. definitive diagnosis by virus isolation or paired sera is usually not made, because supportive therapy generally results in rapid resolution of the diarrhea. inactivated coronavirus is present in commercially available combination vaccines, which are administered immunoprophylactically at - , - , and - weeks of age and then annually thereafter. the role of these vaccines in protection from coronaviral infection is unknown, because the virus typically causes inapparent or mild illness (hoskins, ) . etiology. canine distemper virus (cdv) belongs to the family paramyxoviridae, within the genus morbillivirus, which includes human measles virus and rinderpest virus of ruminants. although there is only one serotype of cdv, there is a wide difference in strain virulence and tissue tropism. some strains produce mild clinical signs that are similar to tracheobronchitis, whereas other strains cause generalized infections of the gastrointestinal tract, integument, and central nervous system, resulting in enteritis, digital hyperkeratosis, and encephalitis, respectively. other factors contributing to the severity and progression of clinical signs include environmental conditions, immune status, and age of the host. a transient subclinical fever and leukopenia occur - days after exposure, with a subsequent fever spike - days later, accompanied by conjunctivitis and rhinitis. other clinical signs associated with acute distemper include coughing, diarrhea, vomiting, anorexia, dehydration, and weight loss. secondary bacterial infections can cause progression to mucopurulent oculonasal discharge and pneumonia. an immune-mediated pustular dermatitis may develop on the abdomen; this is usually a favorable prognostic sign (greene and appel, ) , because dogs that develop skin lesions often recover. neurologic complications of distemper infection may occur weeks to months after recovery from an acute infection. dogs that develop late-onset disease are usually immunocompetent hosts, suggesting that the virus may have escaped complete elimination by the immune system, possibly because of protective effects by the blood-brain barrier. classic neurologic signs that may occur in acute or chronic cdv infection include ataxia, incoordination, vocalization, "chewing gum" seizures, and myoclonus with or without paresis of the affected limb. canine distemper is the most common cause of seizures in dogs less than months of age. dogs with extensive neurologic involvement often have residual clinical deficits, including flexor spasm and olfactory dysfunction. cdv has also been associated with two forms of chronic encephalitis in mature dogs: multifocal encephalitis and "old dog encephalitis." epizootiology and transmission. the virus is highly prevalent and contagious to dogs and other carnivores, especially at the age of - months, coincident with the waning of maternal antibody. transmission is primarily by aerosolization of infective droplets from body secretions of infected animals. pathologic findings. the predominant histopathologic lesion in neurologic forms of distemper is demyelination, which may .. be accompanied by gliosis, necrosis, edema, and macrophage infiltration. acidophilic cytoplasmic inclusions can be found in epithelial cells of mucous membranes, reticulum cells, leukocytes, glia, and neurons, while intranuclear inclusions are often present in lining or glandular epithelium and ganglion cells. diagnosis and differential diagnosis. diagnosis of cdv is based on history of exposure and clinical signs. young dogs who have not received routine immunoprophylaxis (or similarly, mature dogs with a questionable vaccination history) and present with rhinitis, mucopurulent oculonasal discharge, plus or minus hyperkeratosis of the footpads and neurologic signs, are highly likely to have cdv. ophthalmologic examination may reveal chorioretinitis with acute disease or retinal atrophy in chronic cases. definitive diagnosis of acute infection can be made by fluorescent antibody testing of intact epithelial cells from conjunctival and mucous membranes. attenuated strains of cdv, found in modified live vaccines, are not disseminated from lymphoid tissue to epithelial cells and thus are not detected by the fluorescent antibody. serologic testing is usually not useful, because dogs frequently fail to mount a measurable immunologic response. because of the variety of clinical signs, there are many differential diagnoses for canine distemper. an important differential diagnosis for respiratory illness is infectious tracheobronchitis (kennel cough). bacterial, viral, and protozoal causes of gastroenteritis must be considered for cases presenting with vomiting and diarrhea, and rabies, pseudorabies, bacterial meningitis, and poisonings are differential diagnoses for dogs with central nervous system disorder. prevention and treatment. a series of three immunizations from to weeks of age, followed by yearly boosters, is a recommended preventative. treatment is largely supportive, but because of the profound immunologic effects and significant morbidity of cdv, humane euthanasia is usually undertaken in the research setting. etiology. canine herpesvirus (chv) infection causes a generalized hemorrhagic disease with a high mortality rate in newborn puppies less than weeks of age. in adult dogs, chv causes a persistent, latent infection of the reproductive tract with recrudescence and shedding during periods of physiologic stress. clinical signs. clinically affected puppies do not suckle, cry persistently, become depressed and weak, and fail to thrive. petechial hemorrhages of the mucous membranes and erythema of sparsely haired regions such as the caudal abdomen and inguinal area are evident. older puppies, aged - weeks, develop less severe clinical signs and are likely to survive with neurologic sequelae such as ataxia and blindness resulting from reactivation of latent infection. infection in adult dogs may result in stillbirths, abortions, and infertility. lesions in adult bitches include raised vesicular foci in the vaginal mucosa, accompanied by mild vaginitis. adult males have preputial discharge due to vesicular lesions at the base of the penis and on the preputial mucosa. passage of puppies through the birth canal or venereally in adult dogs. puppies can also be horizontally infected by littermates. entire primiparous litters may be lost, with subsequent litters protected by colostral antibody. pathologic findings. pathologic findings include multifocal ecchymotic hemorrhages of the kidneys, liver, lungs, and gastrointestinal tract. basophilic intranuclear inclusions in necrotic areas of parenchymal organs are characteristic findings. diagnosis and differential diagnosis. diagnosis of canine herpesvirus infection in adult dogs is based on a history of reproductive infertility and the presence of genital vesicular lesions. differential diagnoses for stillbirths, abortions, and infertility include canine brucellosis, canine distemper virus and parvovirus infections, and pyometra. the diagnosis in infected puppies is usually made based on clinical history and characteristic lesions (multifocal systemic hemorrhages) (carmichael and greene, ) . differential diagnoses for the disease in neonates would include canine ehrlichiosis and causes of disseminated intravascular coagulation, including bacterial endotoxemia. there is no effective curative treatment. supportive therapy is unrewarding, and death usually ensues within hours in in-fected neonates. in general, adult bitches that have multiple abortions, stillbirths, or persistent infertility should be culled from the breeding colony. examination of these animals may reveal raised vesicular lesions on the vaginal mucosa. adult male dogs that have vesicular lesions on the base of the penis and preputial mucosa should be similarly culled. adult dogs would obviously interfere with production operations, and affected animals should be culled based on the criteria noted above in the discussion of prevention and treatment. because of the severity of clinical illness in puppies, such animals should be humanely euthanatized. etiology. rabies virus is a member of the rhabdovirus family and is essentially contagious to all species of warm-blooded animals. clinical signs. clinical progression of neurologic disease occurs in three stages. the first, or prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, or furious, stage animals are easily excited or hyperreactive to external stimuli and will readily snap at inanimate objects. the third, or paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death usually occurs within - days of the onset of clinical signs, due to respiratory failure. epizootiology and transmission. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact of infected saliva from a rabid to a naive animal (or human), usually via bite wounds. pathogenesis. the incubation period for rabies is generally - weeks from the time of exposure to the onset of clinical signs but can range from week to year. bites of the head and neck typically result in shorter incubation periods because of the proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to the central nervous system and eventually to neurons within the brain, resuiting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis. diagnosis of rabies is based on clinical signs; differential diagnoses include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. definitive diagnosis is based on fluorescent antibody demonstration of the virus in negri bodies of hippocampal cells. prevention and treatment. puppies should be vaccinated at - months of age, "boostered" in year, then vaccinated annually or triennially, depending on state and local laws and which vaccine product is used. treatment of rabies is not recommended, because of the risk of human exposure. research complications. in a research setting, dogs are often not vaccinated for rabies, because of the low incidence of exposure to wild-animal reservoirs. a healthy, purpose-bred dog that bites a human in a research facility should be quarantined for days and observed for signs of rabies. this quarantine interval is based on the knowledge that dogs do not shed rabies in the saliva for more than a few days before the onset of neurologic disease. a random-source dog with an unknown vaccination history that bites a human should be immediately euthanized. the brain should be examined for rabies virus to determine if the dog was infected, and if the test is positive, postexposure immunization should be initiated for the human patient. a rabies vaccine licensed for use in humans is available, and immunoprophylaxis is recommended for animal care and research personnel who may have high work-related risks of exposure. a. protozoa i. giardiasis etiology. giardiasis is a small-intestinal disease of the dog caused by giardia duodenalis (lamblia), a binucleate flagellate protozoan. clinical signs. most giardia infections are subclinical. when dogs are clinically affected, diarrhea is the most prominent sign. the diarrhea is a result of intestinal malabsorption and is often characterized as voluminous, light-colored, foul-smelling, and soft to watery. weight loss has also been associated with clinical infection. clinical illness is more often seen in young animals. epizootiology and transmission. giardia has a direct life cycle. dogs (and people) typically become infected when they consume water (or food) contaminated with giardia cysts. the ph change from the stomach (acid) to duodenum (neutral) causes excystation. trophozoites migrate to the distal duodenum and proximal jejunum and attach to the villus surface. eventually the trophozoites encyst and pass in the feces to perpetuate the life cycle. pathologic findings. giardiasis is rarely fatal. on histopathology of duodenal or jejunal specimens, giardia trophozoites can be seen attached to enterocytes. mucosal inflammation and ulceration, and villous atrophy, have been observed. pathogenesis. the exact pathogenesis of giardia-induced illness is unknown. it is thought that tissue invasion, although occasionally observed, is unimportant for pathogenesis. it is suspected that illness is caused by physical obstruction of enteric absorption, enterotoxicity, competition for nutrients, excess mucus production, and/or secondary bacterial overgrowth. diagnosis and differential diagnosis. definitive diagnosis requires observation of the organism in fecal or intestinal samples. direct fecal smears are considered best for observing trophozoites, and zinc sulfate flotation is preferred for detection of cysts. commercial elisa kits and direct immunofluorescent tests are available to detect fecal giardia antigens, but the diagnostic specificity and/or sensitivity of these tests may not be sufficient to warrant substitution for the less expensive direct fecal examination or zinc sulfate preparation (barr, ) . differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. prevention. high-quality water sources will eliminate the possibility of infection developing within an animal research facility. use of dogs with a known husbandry and medical background will minimize the chances of giardiasis developing in a research colony. control. once giardiasis has been diagnosed in a canine population, segregation of infected animals will help to reduce further infection (provided other dogs were not preinfected at the same source location as the signal case). disinfection with quaternary ammonium compounds, bleach, or steam is usually successful in eradication of giardia cysts. treatment. the most common treatment for giardiasis is metronidazole (flagyl) at - mg/kg per os twice per day for - days. quinacrine hydrochloride (atabrine) at mg/kg per os once per day for days, furazolidone (furoxone) at mg/kg per os twice per day for - days, and the anthelmintics albendazole and fenbendazole have been proposed for use against metronidazole-resistant strains of giardia. a bendazole is recommended at mg/kg per os q hr for days, and fenbendazole at mg/kg per os q hr for days. fenbendazole was thought to be safer for both puppies and pregnant females (nonteratogenic) (barr, ) . research complications. typical asymptomatic infections probably have no consequence on research protocols, with the exception of intestinal physiology or immunology studies. clinical diarrhea would clearly need to be treated before a dog could be used as a research subject. ii. coccidiosis etiology. intestinal coccidia that have been associated with enteropathy in dogs include cystoisospora canis, c. ohioensis, c. burrowsi, and c. neorivolta. clinical signs. dogs are typically asymptomatic when infected with intestinal coccidia, and oocysts are an incidental finding on fecal flotation or direct smear. dogs that are clinically infected usually develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. epizootiology and transmission. cystoisospora oocysts are typically spread by fecal-oral transmission, usually by ingestion of fecal-contaminated food or other objects in the environment. an indirect form of transmission is also possible, whereby the dog consumes a rodent or other animal that is serving as a transport host. once inside the small intestine, the cyst releases sporozoites that infect enteric epithelium. several generations of asexual reproduction can occur in the enterocyte before sexual reproduction produces gamonts. the gamonts fuse to become a zygote, which encysts, ruptures the enterocyte, and passes in the feces. once in the environment the cyst sporulates and is now an infective stage for ingestion by another host. pathologic findings. dogs with coccidiosis may have hyperemia or fluid retention at affected intestinal segments. the mucosa may appear normal, raised, or ulcerated. histologically, there may be necrosis of enterocytes, hyperemia, and submucosal inflammation. the oocysts are usually readily apparent within the epithelial cells (van kruiningen, ) . pathogenesis. intestinal coccidia are opportunistic organisms; they do not typically cause illness unless other predisposing factors are present. such factors include immunodeficiency, malnutrition, and/or concurrent disease. overcrowding and unsanitary conditions can also promote clinical coccidiosis by providing a high population of infective oocysts to stressed animals. diagnosis and differential diagnosis. diagnosis is somewhat difficult, as coccidian oocysts (of both cystoisospora and non-cystoisospora spp.) can be seen on fecal examinations of clinically healthy dogs, as well as animals with diarrhea. other causes for diarrhea (e.g., parvovirus, roundworms, giardia spp., campylobacter jejuni, and inflammatory bowel disease) should be excluded before a coccidial etiology is implicated. prevention. clinical coccidiosis can be readily prevented by adhering to proper sanitation guidelines, reducing any over-crowding, and providing as stress-free an environment as possible. treatment. treatment for the presence of coccidial oocysts may often not be necessary, because cystoisospora infections are typically self-limiting and clinically insignificant. treatment may, however, help to limit the number of oocysts shed in a kennel housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/lb per os for days), trimethoprim sulfa ( mg/lb per os for days), or quinacrine ( mg/lb per os for days). amprolium, which is not labeled for dogs, can also be used as a coccidiostat. it can be given in gelatin capsules for - days at a daily dose of mg for small-breed pups and mg for larger breeds. research complications. as with any enteric disease, the presence of clinical coccidiosis can cause aberrations in gastrointestinal physiological parameters. dogs used in intestinal pharmacokinetic studies should be confirmed to be free of cystoisospora infections. b. nematodes i. ascarids etiology. the most common ascarid of dogs is toxocara canis. toxascaris leonina can also infect both dogs and cats. clinical signs. ascarid infestations are most commonly subclinical. however, large worm burdens can cause diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization. puppies may have a classical "potbellied" appearance and dull hair coat. heavy infestations can cause intussusception and/or intestinal obstruction, in which case the young dogs may be found dead. visceral larval migrans caused by toxocara canis can cause pneumonia. epizootiology and transmission. toxocara canis typically infects puppies. in fact, a unique characteristic of t. canis is its ability to infect prenatal puppies by transplacental migration, and neonatal puppies by transmammary migration. ingestion of infective eggs that have been shed in the feces is another common route of transmission, and infection by ingestion of a transport or intermediate host is also possible. pathologic findings. puppies that die from ascarid infestations typically have large worm populations in the lumen of the small intestine. such populations can cause intestinal obstruction and may also result in intussusception or intestinal perforation. puppies that experience lung migrations of large larval worm populations can have severe pulmonary parenchymal damage and develop fatal pneumonia. pathogenesis. the infective stage of t. canis is the third-stage larva (l ). infections initiated by ingestion of infective eggs have three possibilities for larval migration: liver-lung migration (which leads to intestinal infection), somatic tissue migration, and intestinal wall migration. older dogs that become infected typically have an age-related resistance to liver-lung migration and instead experience the other two migratory patterns. these larval migrations are often asymptomatic, and progression of the l larvae is arrested in the tissues. it is these larvae that become reactivated in a pregnant bitch, thus establishing the transplacental and transmammary routes of transmission. if the source of infection is transplacental, puppies may be born with l larvae in their lungs, because larval migration is already in progress (sherding, ). diagnosis and differential diagnosis. the characteristic large ( - ~tm in diameter) and relatively round ascarid eggs can be readily diagnosed by standard fecal flotation methods. prevention and control. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. most anthelmintics are effective for treatment of ascariasis. pyrantel pamoate (nemex) and fenbendazole (panacur) are commonly used. treatment should be started early in puppies ( , , , and weeks) because of the possibility of prenatal or neonatal infection. pyrantel pamoate, dosed at mg/kg per os, is safe for puppies and is also effective in treatment of hookworms (see section iii,a, ,b,ii). in breeding colonies in which ascarid infestation is a known problem, treatment of the pregnant and nursing bitch may be advantageous. extended fenbendazole therapy ( mg/kg per os twice per day for days or once per day from day of gestation through day of lactation) has been shown to be experimentally safe and effective in decreasing ascarid burdens in puppies. research complications. puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. ii. hookworms etiology. the most common and most pathogenic hookworm of dogs is ancylostoma caninum. other, less pathogenic canine hookworms found in north america are a. braziliense, which can be found in the american tropics and southern united states, and uncinaria stenocephala, which is distributed in the northern united states and canada. clinical signs. only a. caninum infestation typically results in clinical illness, because of the amount of blood that it con-sumes. puppies with a. caninum infestations are typically pale and weak (from anemia), with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. epizootiology and transmission. infective larvae (l ) are typically ingested by puppies and develop directly in the intestinal tract. ingestion can be from the bitch's milk (transmammary migration occurs with a. caninum), from food or objects contaminated with infective larvae, or from ingestion of a paratenic host. transplacental migration does occur with a. caninum, but to a much lesser extent than is seen with toxocara canis. larvae can also penetrate intact skin, migrate to the lung via somatic or circulatory routes, and be coughed and swallowed to reach the intestine. the prepatent period is weeks. pathologic findings. infected puppies often have severe anemia and eosinophilia. the anemia can be from acute blood loss or can also be an iron-deficiency anemia caused by chronic blood loss coupled with limited iron reserves. on gross necropsy, the small-intestinal tract contains worms admixed with intestinal contents containing fresh or digested blood (fig. a) . ulcerative enteritis caused by hookworm attachment is evident on histopathologic examination, and worms with mouthparts embedded in the mucosa can be identified in some sections (fig. b) . pathogenesis. the severe pathogenicity of a. caninum is a direct result of its voracious consumption of blood and body fluids. each adult hookworm can consume . - . ml of blood; thus an extensive infection could deplete a puppy of ml of blood per day, which is approximately % of the blood volume of a . kg animal. in contrast, a. braziliense and u. stenocephala consume . and . ml per worm, respectively. diagnosis and differential diagnosis. diagnosis of ancylostomiasis is made by identification of eggs or larvae from fecal samples by either flotation or direct smear. parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in the diagnosis of a young dog with anemia. prevention and control. purchase of purpose-bred animals will limit the exposure to hookworm larvae, and effective sanitation programs will easily eradicate the infective larvae. unlike ascarid eggs, hookworm eggs are readily killed by drying, sunlight, or cold; however, they do survive readily in warm, moist environments. monthly administration of milbemycin or ivermectin plus pyrantel pamoate (heartgard plus) is recommended for prevention and control of canine ascarid infestation (hall and simpson, ) . treatment. pyrantel pamoate (nemex) is the anthelmintic of choice because it is safest in young ill animals and is also effective against ascarids and other enteric helminths. because of the possibility of transplacental or milk-borne infection, puppies should be treated every weeks from weeks - . a follow-up treatment at weeks is recommended to kill any larvae that have migrated and matured since the initial therapy. severely ill puppies may require supportive fluid therapy and possibly whole blood transfusions and iron supplementation. research complications. anemic puppies with large worm burdens make poor research subjects and should be treated aggressively before placement on an experimental study. iii. strongyloides etiology. strongyloides stercoralis is a small strongyle that can cause hemorrhagic enteritis in puppies. it is found in warm, humid climates such as the southeastern united states. fects dogs and other animals by third-stage larval penetration of the skin or mucous membranes. larvae migrate via the circulatory system to the lung and then are coughed and swallowed to initiate the intestinal parasitism. the eggs of s. stercoralis hatch within the gut lumen, and so it is the first-stage larvae that pass in the feces and need to be identified by diagnostic examination. once passed, the larvae can either develop into the infectious third-stage larvae or mature into free-living, nonparasitic adults. diagnosis and differential diagnosis. the baermann procedure is usually performed on fresh feces in order to detect the motile first-stage larva ( - ~tm x - ~tm). the larvae must be distinguished from larva of filaroides hirthi and hatched ancylostoma caninum. treatment. the usual treatment for s. stercoralis is fenbendazole (panacur) at mg/kg per day for days. iv. whipworms etiology. trichuris vulpis, the canine whipworm, can cause acute or chronic large-intestinal diarrhea. the adult whipworm typically resides in the cecum or ascending colon. clinical signs. most whipworm infections are subclinical. in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss are also seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. severe dehydration with electrolyte imbalance has occurred occasionally as an acute crisis episode. life cycle. adult worms residing in the canine large intestine intermittently release eggs that pass in the feces. the eggs are very hardy and can persist for years. in optimal conditions, the eggs develop into an infective embryo within days. after ingestion by a dog, the larvae hatch in the small intestine, burrow into the small-intestinal mucosa, and then reemerge several days later to travel and burrow into the cecal and colonic mucosa. the prepatent period is typically - months long. pathologic findings. dogs do not typically die from whipworm infestations. lesions seen as incidental findings feature adult worms embedded into the colonic and cecal mucosae, causing local granulomatous inflammatory reactions and mucosal hyperplasia. pathogenesis. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to the clinical development of diarrhea. factors that influence the possible.development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. diagnosis and differential diagnosis. whipworm infestation is diagnosed by the presence of characteristic trichurid eggs on fecal flotation. these eggs are barrel-shaped, with thick walls and bipolar plugs. because of the intermittent release of eggs by the adult female worms, negative fecal flotation does not exclude the possibility of clinical whipworm infection. adult worms can be seen on colonoscopy (jergens and willard, ) . differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. prevention and control. trichuris eggs are resistant to disinfection, making control difficult. dessication or incineration is the only completely effective means to eradicate whipworm eggs from the environment. treatment. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . treatment is also suggested in cases wherein whipworm infestation is suspected but not confirmed by multiple fecal flotation. rapid response to treatment would be indicative of a correct diagnosis; lack of response should prompt further diagnostic efforts. research complications. whipworm infestation has not been documented to interfere with research protocols, although one would anticpate that aberrations in local enteric immune function and absorptive functions of the large intestine could result from trichuriasis. etiology. heartworm disease of dogs is caused by the filarial worm, dirofilaria immitis. adult heartworms reside in the pulmonary artery; severe infestations can result in the presence of worms in the right ventricle and atrium. microfilariae, the immature worms produced by the adults, circulate in the bloodstream until a mosquito (intermediate host) ingests them. clinical signs. most heartworm infestations are asymptomatic. the most common clinical signs observed are coughing and dyspnea. clinical signs of exercise intolerance and rightsided heart failure can be seen in severe infestations. epizootiology and transmission. successful heartworm transmission requires the presence of mosquitoes. for this reason, random-source dogs or dogs housed in outdoor kennels are much more likely to have heartworm infestations than indoor, purpose-bred dogs. mosquitoes become infested with heartworm microfilariae when they take a blood meal from the dog. the microfilaria progress through several larval stages within the mosquito, eventually terminating at the third stage. this stage is then returned to the canine bloodstream during feeding. this stage matures within the dog's circulatory system, and the adults reside in the pulmonary artery. male and female heartworms will then sexually reproduce to create more microfilariae and propagate the parasitic life cycle. in the united states, transmission of heartworm by mosquitoes occurs over a month or shorter period, except for the southeastern and gulf coast states. here, climatic conditions enable longer survival of the mosquitoes (possibly year-round), thus resulting in the highest prevalence of heartworm infestation (knight, ) . pathologic findings. on necropsy, the small, slender worms can be seen in the pulmonary artery, right ventricle, and/or right atrium (fig. a ). there may be no histologic abnormalities associated with a minor worm burden, although typically the arterial endothelium in these areas is hyperplastic (fig. b) . endothelial cell hyperplasia, vascular smooth muscle hyperplasia, inflammation, and thrombosis of the pulmonary arteries and arterioles characterize more significant infestations. severe infestations can lead to right-sided heart failure and its pathologic sequelae of ascites, pleural effusion, hepatomegaly, and right heart and pulmonary artery enlargement. verminous pulmonary embolism can result from treatment of dogs with anthelmintics when a worm burden is present. immune responses to circulating microfilariae can cause pathologic lesions, most commonly glomerulonephritis. pathogenesis. the physical presence of the worms in the pulmonary artery is partially responsible for clinical signs observed in severe cases. however, the host immunologic response to this infestation, coupled with secretion by the heart-worms of physiomodulative factors, contributes significantly to the complications seen with this disease. endothelial cell proliferation, damage, and sloughing stimulates periarteritis and proliferation of the vascular media of pulmonary arteries and arterioles. these changes lead to thrombosis of these vessels and the arterial truncation that can be seen radiographically in severe infestations. the heartworms also release circulating factors that affect vascular tone and can promote bronchoconstriction (dillon, ) . these factors are discussed in more detail below, under "research complications." diagnosis and differential diagnosis. for dogs used in biomedical research, diagnosis of asymptomatic heartworm disease is important, especially if the dogs are used in cardiovascular, pulmonary, or long-term studies. a diagnosis of dirofilariasis is typically made by detection of adult heartworm antigens in a blood sample. use of adult heartworm antigen tests has virtually eliminated the historical status of "occult" heartworm disease, which was caused by infestation of adult worms without corresponding microfilarial circulation. commercial test kits that assay for the presence of adult heartworm antigens, and designed for use by veterinary practitioners, are readily available. false-negative results can occur during the prepatent period after initial infection (first - months), and when the adult worm burden is light or predominantly male. infections consisting of more than three mature female worms are usually detected by antigenic serology (knight, ) . a significant feature of these tests for circulating antigen is that they have a very high specificity (low rate of false-positive resuits). if a dog were negative on initial testing because of prepatency or small worm burden, it will more than likely be detected on a follow-up test months later. examination for circulating microfilariae could be used to confirm an antigenic diagnosis of dirofilariasis or to establish that microfilarial production had occurred. microfilarial detection can be done by microscopic examination of the buffy coat of a microhematocrit tube or by concentration techniques, such as the modified knott test and filter tests. tests that examine for microfilariae have the inherent problem of false positives caused by microfilariae of dipetalonema reconditum, a nonpathogenic filarial worm. other serologic diagnostic tests that were more common historically, and that may still be useful, include detection of antibodies to either adult heartworm antigens or microfilarial antigens. these same techniques can be used to diagnose clinical heartworm disease. additional diagnostic tests that can augment a diagnosis of clinical heartworm disease include thoracic radiography (pulmonary artery and right-heart enlargement), electrocardiography (right-heart enlargement), and hematology (eosinophilia). differential diagnoses for symptomatic heartworm disease (coughing, dyspnea, and exercise intolerance) include canine distemper, canine infectious tracheobronchitis (complicated), streptococcal or other bacterial pneumonia, nocardiosis, and congestive heart failure. prevention and control. for dogs used in biomedical research, prevention is primarily via insect control and housing of the dogs in a controlled, indoor environment. purpose-bred dogs reared in such an environment are usually free from dirofilariasis. however, any dog (random-source or purposebred) exposed to mosquitoes could become inoculated with infective larvae and, if untreated, could develop adult heartworm disease. there are many commercial anthelmintic preparations used to prevent heartworm infestation by killing the larval stages in the canine bloodstream before they become adult worms (e.g., ivermectin, milbemycin, and diethylcarbamazine). these could be used in a research setting in which heartwormnegative dogs are housed outdoors and thus could potentially be infected through mosquito bites. if a research facility is conditioning random-source dogs for long-term use, the presence of circulating adult heartworm antigen should disqualify an animal from the conditioning program. treatment. treatment for eradication of heartworms (adults, juveniles, and microfilaria) is a long process that can pose a significant risk to the patient with regard to both drug side effects (hoskins, ) and immunologic reactions to dead worms lodged in the pulmonary vasculature. for this reason, medical treatment of heartworm disease is not usually attempted in research dogs. in a rare instance when such treatment was in the best interest of a long-term canine experiment, thiacetarsamide (caparsolate) and ivermectin (ivomec) were used to eradicate adults and microfilariae, respectively (authors' personal experience). alternative choices include melarsomine (immiticide) as an adulticide and milbemycin (interceptor), levamisole (levasol), or fenthion (spotton) as microfilaricidal agents. dosing regimens for these agents are detailed in dillon ( ) . research complications. the physiomodulative properties of heartworm infection have been studied. such studies have looked at factors released by adult heartworms, as well as changes in the function of host tissues in response to the worm presence. probably the most consistent finding is that endothelial cell-dependent relaxation of pulmonary arterial smooth muscle is depressed in heartworm-infected dogs as compared with control dogs, indicative of alterations in local endothelial cell behavior (maksimowich et al., ; matsukura et al., ; mupanomunda et al., ) . the extension of this effect on peripheral arteries (in vivo and in vitro) has been supported in some studies (kaiser et al., ) but refuted in others (tithof et al., ) . it is thought that the endothelium is perturbed by a factor released from the adult dirofilaria, possibly a cyclooxygenase product such as prostaglandin d (kaiser et al., (kaiser et al., , . these products have also been demonstrated to cause constriction in in vitro rat tracheal ring preparations (collins et al., ) , suggesting that bronchoconstriction could be an aspect of the pathogenesis of the infestation. platelet reactivity was also been found to be enhanced in dogs naturally infected with dirofilaria, when compared with uninfected controis (boudreaux and dillon, ) . based on these data, dogs that are positive for adult heartworm antigen should be considered inappropriate for use as research subjects and, if used, should be restricted to nonsurvival preparations that do not require physiological measurements. etiology. several species of cestodes (tapeworms) parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. clinical signs. most cestode infestations are subclinical. severe infestations with dipylidium can be associated with diarrhea, weight loss, and poor growth. epizootiology and transmission. the cestode life cycle requires an intermediate host. for dipylidium caninum, the intermediate hosts are fleas and lice. thus this species of tapeworm can be readily transmitted by ingestion of arthropods that are canine parasites in and of themselves. taenia pisiformis requires small ruminants, rabbits, or rodents for intermediate hosts, so spread is less likely, especially in a research setting. echinococcus granulosus uses not only sheep as an intermediate host but also human beings, and thus the zoonotic potential of this cestode must be considered. pathologic findings. adult cestodes in the small intestine are usually an incidental finding at necropsy. diagnosis and differential diagnosis. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. dipylidium egg packets are large ( x bm) and contain - eggs per packet (hall and simpson, ) . prevention and control. the most significant means to limit cestode infestation is to control the population of fleas and/or lice infesting the colony. see the sections on these ectoparasites for effective means to treat infested dogs and kennels. treatment. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against dipylidium caninum (hall and simpson, ) . clinical signs. most lung fluke infestations are inapparent, but coughing can develop in cases that prompt a strong inflammatory response. pneumothorax has been a sequela of cyst rupture, in which case dyspnea with reduced lung sounds would be the typical presentation. epizootiology and transmission. the lung fluke life cycle requires two intermediate hosts: a snail and then a crayfish. dogs become infested after eating crayfish, which essentially limits this disease to random-source dogs. on ingestion, the immature flukes (metacercariae) migrate to the lungs and encyst in the pulmonary parenchyma. eggs produced by adult flukes are passed into the bronchioles, coughed up, swallowed, and passed in the feces to complete the life cycle. pathologic findings. grossly, the trematode cysts containing adult flukes can be seen in the lung parenchyma. areas of eosinophilic inflammation surround the cysts, and eosinophilic granulomas can also be seen encircling released eggs. pleural hemorrhages may also be caused by the migrating metacercariae (lopez, ) . pathogenesis. clinical illness is usually a result of a severe eosinophilic inflammatory response, pneumothorax caused by cyst rupture, or secondary bacterial pneumonia. diagnosis and differential diagnosis. definitive diagnosis of paragonimus infestation requires identification of the characteristic ovoid eggs ( - ~tm long) with a single operculum in either the feces or a transtracheal wash. identification from fecal samples requires sedimentation techniques. other causes of coughing in dogs (e.g., infectious tracheobronchitis, dirofilariasis, congestive heart failure) need to be considered. radiographically, the appearance of (multi)focal densities within the air-filled lung field needs to be differentiated from pulmonary neoplasia (primary or metastatic) or systemic fungal pneumonias. prevention. use of purpose-bred dogs virtually eliminates the chance of pulmonary trematodiasis in a research animal. treatment. praziquantel (at mg/kg q hr x days) or fenbendazole ( - mg/kg q hr x - days) are recommended for treatment of canine paragonimus infestation (hawkins, ) . effectiveness is monitored by fecal sedimentation tests for eggs and resolution of radiographic lesions (which may never resolve entirely). early diagnosis of pulmonary trematodiasis should warrant discontinuation of a dog from a long-term study because of the possibility of more serious clinical sequelae, such as pneumothorax. research complications. experimental studies involving the immune system, especially eosinophilic or local pulmonary responses, would be significantly affected by even minor infestations. clinical illness would complicate almost any research project and makes dogs poor anesthetic risks. radiographic lesions may confound diagnostic evaluation for pulmonary metastasis of tumors. e. mites i. demodicosis etiology. canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles. it is considered to be normal fauna of dog skin, but certain conditions (i.e., immunosuppression) cause development of clinical illness. clinical signs. demodex canis infestation is typically asymptomatic. clinical demodicosis presents with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and the face and around the ears (demanuelle, a). secondary bacterial pyoderma is a common complication. epizootiology and transmission. demodex canis mites pass to nursing pups from the dam. they live their entire lives on one dog and are not considered contagious to other dogs or humans. certain breeds are predisposed to the generalized form of demodex dermatitis (see "pathogenesis," below). beagles are among the predisposed breeds, as are german shepherds, doberman pinschers, old english sheepdogs, collies, boxers, and shorthair brachycephalic breeds (muller et al., ) . pathologic findings. histologically, demodex infections are characterized by perifolliculitis and folliculitis with mites and keratin debris visible in the hair follicles. cases with generalized demodicosis (see "pathogenesis," below) may have a minimal cellular response with no eosinophils, indicative of severe immunosuppression . pathogenesis. when clinical demodicosis develops, it is classified into "localized" or "generalized" (e.g., more than one foot affected, or five or more small areas, or one large body area). localized demodicosis is typically seen in juvenile dogs (< months) and usually resolves without treatment as natural immunological control develops. generalized demodicosis can develop in juvenile or adult populations. juvenile-onset generalized demodicosis occurs in dogs with a genetic predisposition, thought to be an inherited t-lymphocyte dysfunction. adult-onset generalized demodicosis is usually indicative of an underlying endocrine (hyperadrenocorticism, diabetes mellitus, hypothyroidism) or neoplastic disorder or can develop as a result of immunosuppressive therapy (such as corticosteroid administration). diagnosis and differential diagnosis. demodex is readily identified from deep skin scrapings of lesioned areas (campbell, ; noli, ) . demodex canis has a characteristic "cigar shape," with short, stubby legs on a body - ~tm long. differential diagnoses for local demodicosis include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma; remember, however, that bacterial pyoderma is a common secondary complication of the generalized form of this parasitism. prevention and control. dogs with generalized demodicosis should not be maintained in a breeding colony. treatment: ivermectin (ivomec) at - ~tg/kg and oral milbemycin (interceptor) at - mg/kg/day have been found to be effective treatments. these parasiticides are probably the most practical to use in a research setting, although they are not labeled for treatment of demodex canis. amitraz (mitaban) dips ( ppm every days) can be used for more problematic cases. treatment duration can be extensive and must be accompanied by repeated skin scrapings. research complications. dogs with generalized demodicosis should not be used in research studies, because this disease is indicative of another underlying disorder (endocrine or immunological). dogs that receive immunosuppressive agents or paradigms could develop generalized demodicosis as an unexpected consequence of the experimentation. ii. sarcoptic mange etiology. canine sarcoptic mange is caused by sarcoptes scabiei var. canis. clinical signs. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas such as the ear pinnae, elbows, and ventral thorax and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. epizootiology and transmission. sarcoptes mites live their entire lives in the stratum corneum of the host animal; however, they can survive for - weeks away from the host, and it is this ability that enables them to spread from dog to dog. sarcoptes scabiei var. canis can also infect cats and humans. pathologic findings. histologic examination can be unrewarding because mites are rarely seen on tissue sections, and the associated dermatitis is nondiagnostic: perivascular and interstitial dermatitis with hyperkeratosis, with or without eosinophilic infiltration. suggestive histopathologic lesions are epidermal "nibbles," small foci of edema, exocytosis, degeneration, and necrosis . pathogenesis. lesions and illness are a result of the female mites burrowing through the epidermal layers to deposit eggs, and the larvae migrating back to the surface. the typical locations of mange lesions are a result of the mite's preference for relatively hairless areas. diagnosis and differential diagnosis. sarcoptic mange can be difficult to diagnose because multiple skin scrapings can yield negative results with this parasitic disorder. hopefully, adult mites, mite eggs, or mite feces can be observed on superficial skin scrapings. even if scrapings are negative, however, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige in either the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ). an important differential diagnosis is flea allergy dermatitis; in contrast, mange is nonseasonal and contagious. prevention and control. use of purpose-bred dogs limits the possibility of having research animals with sarcoptic mange. for random-source dogs, an ectoparasite control program should be in place to limit possible infestations. many institutions use ivermectin as a means to control both endoparasites and ectoparasites. treatment. unless treatment would interfere with research objectives, all dogs with sarcoptic mange (no matter how minor the lesions) and their kennel mates should be treated because of the contagious nature of the disease and its zoonotic potential. in research colonies, the usual means of treatment is either ivermectin (ivomec) at - ~tg/kg q days or milbemycin (interceptor) at oral doses of mg/kg q days . neither of these agents is approved for treatment of sarcoptic mange, but they are considered to be effective. acaricidal dips (e.g., lime sulfur, organophosphates, amitraz) can also be used. research complications. the local skin inflammation and systemic immune response to sarcoptic mange probably make infected dogs poor subjects for dermatologic and immunologic studies. f lice and ticks i. lice etiology. dogs can be infested by one species of sucking louse (linognathus setosus) and two species of biting lice (trichodectes canis and heterodoxus spiniger). clinical signs. mild cases of pediculosis may be asymptomatic or may cause pruritic areas of dry skin. more severe infestations can cause significant pruritus and produce alopecia, papules, and crusts. these lesions lead to excoriation and secondary bacterial dermatitis. severe linognathus infestations could cause anemia, because this species feeds on blood. epizootiology and transmission. louse infestations are uncommon in both pet animal practice and the research setting. they would most likely be seen in random-source dogs that were obtained from a pound or shelter. transmission is usually by direct contact, for lice spend their entire lives on the host species. lice are host-specific and not zoonotic. pathogenesis. the biting lice usually cause more local irritation than the sucking louse and therefore are more apt to induce clinical dermatologic signs. trichodectes canis can serve as vector for the canine tapeworm dipylidium caninum. the most severe complication of infestations by the sucking louse is the potential anemia. diagnosis and differential diagnosis. pediculosis is diagnosed by direct observation of the lice or nits (eggs) on the dog's skin. cellophane tape can be used to pick up surface debris from skin lesions, which may include nits or immobilized lice (muller et al., ) . differential diagnoses include dermal acariasis, flea allergy dermatitis, and seborrhea. prevention. use of high-quality conditioned dogs for research should prevent pediculosis from ever being seen within a research facility. random-source dogs should be shampooed or treated prophylactically with topical insecticide before being permitted to enter the research colony. treatment. most commercially available insecticide shampoos and dips readily treat louse infestations. treatment should be repeated in - days, because any nits that were not killed would have hatched by that time (muller et al., ) . there is probably minimal interference with research, unless severe linognathus infestations cause anemia. ii. ticks etiology. ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. except for the brown dog tick (rhipicephalus sanguineus), ticks have a wide host range and are not especially host-specific; so any number of tick genera and species can be found on dogs. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, and ixodes. the primary significance of tick infestation is the tick's ability to be a vector for many other infectious diseases, including rocky mountain spotted fever (caused by rickettsia rickettsii), lyme disease (borrelia burgdorferi), and the canine forms of ehrlichiosis (ehrlichia canis and e. platys), babesiosis (babesia canis), haemobartonellosis (haemobartonella canis), and hepatozoonosis (hepatozoon canis). clinical signs. as an entity unto itself, tick infestation causes minimal clinical signs. most infestations are subclinical, although some dogs may lick and bite at the site, aggravating the local lesion. some dogs can develop a hypersensitivity reaction after several tick bites; these dogs develop a more granulomatous response at the location of the bite (merchant and taboada, ) . some species of ticks (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that can cause an ascending flaccid paralysis (malik and farrow, ) . the paralysis develops within - days of tick attachment and can result from a single tick. this paralysis is fatal once the respiratory musculature is affected. epizootiology and transmission. in dogs used for biomedical research, tick infestation may occasionally be seen in randomsource dogs, because these dogs are more likely to have been in tick habitats than purpose-bred dogs. ticks commonly reside in wooded areas until they contact a suitable host for a blood meal. the brown dog tick may reside within kennels (attics, bedding, wall insulation) (garris, ) . pathologic findings. under most circumstances, tick infestation will be an incidental finding on necropsy (unless tick paralysis was the cause of death). pathogenesis. tick-bite paralysis is caused by the presence of a salivary neurotoxin released by female ticks of certain genera (e.g., dermacentor) while consuming a blood meal (malik and farrow, ) . interestingly, dogs seem to be most affected by this condition, whereas cats appear to be resistant. the primary dysfunction appears to be at the neuromuscular junction, as stimulation of the motor nerves fails to elicit a response, but direct stimulation of the muscle tissue results in contractions. tick bites can also transmit pathogen microorganisms to the dog, because ticks serve as vectors for several infectious diseases, including lyme borreliosis, ehrlichiosis, babesiosis, and rocky mountain spotted fever. diagnosis and differential diagnosis. for uncomplicated tick bites and tick-bite paralysis, definitive diagnosis is made by identification of the offending arachnid (and improvement of paralysis after removal). differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . prevention. purpose-bred dogs should be free from all ectoparasites, but ticks can occasionally be seen on randomsource animals. research dogs should not be exercised in outdoor areas infested with ticks, and kennels must be cleaned properly and regularly so as to remain free of ticks and other parasites. treatment. removal of the offending tick is the primary treatment for both local inflammation as well as tick-bite paralysis. dogs with tick-bite paralysis usually show improvement within hr, with complete recovery within hr (malik and farrow, ) to remove an attached tick from a dog, forceps should be used to grasp the tick as close to the dog's skin as possible. the tick should not be grabbed by the body, as this may cause the parasite to either rupture or inject its body contents into the dog. the tick should be pulled away from the dog with steady pressure. many of the diseases transmitted by ticks are zoonotic so precautions, such as wearing gloves, should be taken. use of topical acaricide/insecticides on newly arrived random-source dogs should help to limit infestations. probably have minimal impact on research variables. the significant concern for tick infestation is the possible development of tick-bite paralysis or of any one of a number of systemic diseases spread by ticks (see sections iii,a,l,e-g). g. other i. flea infestation etiology. fleas are laterally flattened wingless insects that feed on animal blood. the most common flea to infest dogs is ctenocephalides felis, the cat flea. other fleas that can affect dogs are ctenocephalides canis, pulex irritans, and echidnophaga gallinacea. the fleas are speciated by the shape of their head and by the presence or absence of ctenidae (spiny combs on or behind the head) (campbell, ) . clinical signs. flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop the more severe "flea allergy dermatitis," which features papules and crusting. acute moist dermatitis ("hot spots") can also be seen in these cases, and secondary pyoderma or seborrhea can develop. lesions from flea allergy dermatitis generally appear in the dorsal lumbosacral region, as well as the flanks, thighs, and abdomen (muller et al., ) . the lesions are typically worse in the summer and autumn months and are progressively more severe as the dog ages. epizootiology and transmission. fleas are readily transmitted between animals and even between host species. they move readily between the host and the environment, making transmission easy and control difficult. because fleas require host blood for food, they can survive off of a host for only - months (muller et al., ) . pathologic findings. biopsy samples are usually nondiagnostic in cases of flea allergy dermatitis. lesions are typically characterized by perivascular eosinophilic inflammation and may feature pustules and folliculitis if secondary pyoderma develops (muller et al., ) . pathogenesis. fleas are parasites that require animal blood for their meals. when they bite host animals, they inject some saliva into the host's skin. if the host develops an allergic response to the flea saliva, it will develop the more pruritic flea allergy dermatitis. fleas can also transmit or serve as vectors for other pathogens (e.g., dipylidium tapeworms). flea allergy dermatitis are definitively diagnosed by observing the fleas on the host's skin. given that this may be difficult because of the mobility of the flea and the majority of the time it spends off of the host, diagnosis is often based on clinical signs, history, and lesion distribution. sometimes the presence of flea excrement ("flea dirt") on the dog's skin can support a presumptive diagnosis (demanuelle, b) . circulating eosinophilia is seen in some dogs with flea allergy dermatitis. differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs (e.g., food, drug, or contact hypersensitivity). prevention. most dogs obtained from high-quality purposebred facilities should be free from flea infestations. dogs received from pounds, shelters, or licensed dealers would be more likely to be affected by fleas (or any ectoparasitism). thorough knowledge of prevention, control, and treatment measures at these facilities should be obtained, and dogs from sources where proper prevention and/or therapy are not practiced should be evaluated and/or empirically treated upon arrival at the facility. control. thorough cleaning of the dog's housing environment should remove the risk of perpetuating or transmitting flea infestation in the colony. treatment. treatment for fleas needs to address treatment of both the dog and the environment. many insecticide formulations such as shampoos, sprays, dips, powders, and oral systemics can be used for initial treatment of the individual dog. the active ingredients include pyrethrins, pyrethroids, carbamates, and organophosphates. flea control in the kennel may need to include outdoor areas in warm climates. typically combinations of adult insecticides and juvenile growth regulators are used for environmental treatment. directed sprays are the most effective means of treating housing areas, because flea "bombs" or foggers do not penetrate adequately into tight areas where fleas might hide (demanuelle, b) . in addition to insecticide therapy, dogs with flea allergy dermatitis may also require anti-inflammatory medication to relieve clinical signs. oral prednisone or prednisolone at . mg/kg q hr for - days has been proposed as a starting therapy (muller et al., ) . the use of hyposensitization with flea-bite antigens is controversial and not practical for the research setting. research complications. mild flea infestation probably has minimal impact on most research protocols, and treatment measures may in fact be more detrimental to the experimental objective than the actual ectoparasitism. in a research setting, the residual effects of insecticides may preclude their use in experimental animals. such treatments should be used judiciously to ensure that experimental results are not more seriously affected by the therapy rather than the infestation. dogs with flea-allergy dermatitis are more severely affected by the flea infestation and should be treated apigropriately; however, systemic corticosteroids may also interfere with experimental objectives, especially in studies involving functions of the immune system. the ability of fleas to transmit other parasitic diseases must also be considered. etiology. dermatophytoses ("ringworm") are fungal skin infections, which in dogs in the united states are usually caused by either microsporum canis, m. gypseum, or trichophyton mentagrophytes (muller et al., ) . clinical signs. uncomplicated superficial dermatophytoses are characterized by circumscribed circular areas of alopecia, usually with minimal to no inflammation. these skin lesions are usually seen around the face, neck, and forelimbs but can be found anywhere on the body. secondary bacterial infections can develop; these lesions are called kerions and are selflimiting, for the fungus cannot survive in inflamed skin (muller et al., ) . ep&ootiology and transmission. the fungi that cause skin infections are very contagious and readily transmissible between dogs and other species (including human beings), but they can also be obtained from the soil. pathologic findings. on close inspection of skin samples, broken hair shafts (and not complete hair loss) would be seen with uncomplicated dermatophytosis. histologically, fungal elements can be seen within the stratum corneum or in and around the hair and hair follicles (muller et al., ) . stains that facilitate visualization of fungal elements include periodic acid-schiff (pas) or gomori methenamine-silver. the pattern of inflammation in the affected foci is very variable and can feature folliculitis, perivascular dermatitis, hyperkeratosis, and/or vesicular dermatitis. pathogenesis. the dermatophytes typically infect the hair shaft itself, the hair follicle, and possibly the skin around the affected hair. the hair follicle is not destroyed (unless by secondary bacterial infection), but the hair itself becomes brittle and breaks. this causes short stubbly hair to be seen within the lesion. as the lesion progresses, the hairs in the center recover from the infection, thus leading to the classic "ringworm" appearance of the alopecic areas. it is postulated that the inflammatory process produces an environment that is unfavorable for dermatophyte survival, whereas the periphery of the lesion still enables continued fungal growth (muller et al., ) . diagnosis and differential diagnosis. diagnosis of dermal fungal infection is typically made by scraping the affected area to obtain hair and superficial epidermal cells. these scrapings are then digested with potassium hydroxide to facilitate observation of fungal elements. fungal elements can also be seen on skin biopsy samples. for speciation of a fungus, skin scrapings can also be inoculated onto agars that promote fungal growth, such as sabouraud's medium or dermatophyte test medium (dtm). incubation should be at ~ with % humidity for - days. lesions caused by m. canis may fluoresce when inspected using a wood's ( . nm ultraviolet) light. unfortunately, some strains of m. canis do not fluoresce, and neither does m. gypseum or t. mentagrophytes. differential diagnoses for dermatomycosis include seborrhea, localized demodecosis, folliculitis, histiocytoma, and acral lick dermatitis (muller et al., ) . prevention. purpose-bred dogs are typically free of infectious dermatophytes, but ringworm may be diagnosed on randomsource animals. control. in cases of dermatophytosis, isolation of the affected animal(s) is prudent, because the fungi are easily spread to other dogs, as well as to people. treatment, if acceptable, should be started immediately. treatment. topical antifungal therapy is most commonly used. shampoos, rinses, and creams containing miconazole, ketoconazole, enilconazole, or chlorhexidine are commercially available to treat ringworm (stannard et al., ) . severe cases may require systemic therapy with griseofulvin, ketoconazole, itraconazole, or fluconazole. however, these systemic antifungal agents may have considerable side effects (such as vomiting and teratogenicity with griseofulvin). many of the newer agents are also expensive and not labeled for use in dogs. impact on most research applications for dogs. unfortunately, the zoonotic implications of dermatophytoses force the issue of aggressive treatment, and many antifungal agents may not be compatible with biomedical research studies. systemic fungal infections disseminate to multiple organ systems from a single mode of entry (usually through the respiratory tract). dogs are susceptible to several fungi that characteristically cause systemic mycosis, including blastomyces dermatitidis, histoplasma capsulatum, coccidioides immitis, and cryptococcus neoformans var. neoformans. these diseases are not typically seen in the research setting, because of the low overall incidence and noncontagious nature of these disorders and because of the use of purpose-bred animals. these conditions could, however, present in the rare random-source dog that was subclinical at its point of origin, especially if the animal becomes immunosuppressed (either naturally or by virtue of experimental manipulation). typical clinical signs include weight loss, fever, lymphadenopathy, and cough and dyspnea (if the lungs are affected). the reader is advised to read veterinary medical text chapters (e.g., taboada, ) for more complete information on these disorders and their possible treatments. although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology. the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. both of these causes result in a gradual loss of functional thyroid tissue (kemppainen and clark, ) . lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs. because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including alopecia, hyperpigmentation, seborrhea, and pyoderma (peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic normochromic nonregenerative anemia and increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bischel et al., ; panciera, ) , and so the relationship between hypothyroidism and these problems has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause bradycardia as a result of decreased myocardial conductivity. abnormalities that may be detected by ecg include a decrease in p and r wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these electrocardiographic abnormalities are caused by lowered activity of atpases and calcium channel function. several reports have suggested that hypothyroidism is associated with von willebrand's disease and bleeding abnormalities. however, the relationship is probably one of shared breed predilection and not a true correlation. it has been demonstrated that dogs with hypothyroidism are not deficient in von willebrand's factor when compared with other dogs. in addition, the replacement of thyroid hormone in dogs did not increase the levels of vwf:ag in naturally occurring (panciera and johnson, ) or experimentally induced (panciera and johnson, ) hypothyroidism. epizootiology. the prevalence of hypothyroidism in the general canine population has been reported to be less than % (panciera, ) . the disorder occurs most often in large-breed dogs but has been reported in several other breeds as well as mongrels. doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism when compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . in general, the problem is usually recognized in middle-aged animals, and some reports state that there is a higher incidence of hypothyroidism in spayed female dogs (panciera, ; peterson and ferguson, ). diagnosis and differential diagnosis. because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. the tests currently available and in popular use will be discussed further. however, a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t in the body and is heavily proteinbound. free t represents the unbound fraction that is available to the tissues (peterson and ferguson, ) . using the measurement of serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial is in order (peterson and ferguson, ) . however, it must be noted that nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present, and animals should not be treated solely on the basis of serum hormone levels if clinical signs are absent. if the clinical signs are equivocal or if only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone in the body, the measurement of serum t levels is an unreliable indicator of hypothy-roidism (peterson and ferguson, ; ferguson, ) . like t , serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released, and conversion of t to t may be enhanced in the hypothyroid dog (peterson and ferguson, ; ferguson, ) . t was within normal limits in % of the hypothyroid dogs in one study (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous tsh (thyroid-stimulating hormone) levels provide reliable information on thyroid status, and an assay for endogenous tsh is now available in dogs. however, tsh levels can be normal in some dogs with hypothyroidism, and high tsh levels have been noted in normal dogs. therefore, it is recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and hours after. a normal response to the administration of tsh should create an increase of t levels at least ktg/dl above the baseline levels or an absolute level that exceeds ~tg/dl (peterson and ferguson, ; wheeler et al., ) . treatment. the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . mg/kg once a day or . mg/m (body surface area)/day for very small or very large dogs. if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - hours after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are also frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must be aware of the development of obesity and the potential effect that it can have on research. etiology. obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy. excessive caloric intake resuits from overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household, because access to food is more restricted and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and thus the limitation to exercise reduces energy expenditure and predisposes dogs to weight gain. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy expenditure are followed (butterwick and hawthorne,. ). as in humans, genetics plays an important role in the development of obesity in dogs. it has been established that certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . in addition to genetics, several metabolic or hormonal changes are associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese when compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ) . in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . epizootiology. ewen, ) . obesity affects up to % of pet dogs (mac-diagnosis and differential diagnosis. the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see section iii,b,l,a), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment. restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to rebound and gain weight after restrictions are relaxed. there has been agreat deal of attention in humans as to the correct diet to be fed to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet in both humans and animals as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorierestricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick and thorne, ; butterwick and thorne, ) . it is important to control weight gain in research animals, because of the association of obesity and several metabolic changes. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . obesity in dogs over years of age appears to be related to an increase in cardiovascular problems (edney and smith, ) , and obesity has been linked to hypertension. joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity, and obesity induces hyperinsulinism in several experimental models (macewen, ) . in the laboratory setting, the majority of traumatic wounds will be small in size. in facilities with good husbandry practices and a diligent staff, traumatic wounds will generally be observed quickly and attended to promptly. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or. when the basic principles of wound management are not followed. to aid in the description of wounds and in decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a "golden period." it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the capability of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as either clean, clean-contaminated, contaminated, or dirty (see table v ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds, which are seen infrequently in the laboratory setting, require more aggressive therapy. dirty wounds can occur as postsurgical infections or complications of initial wound therapy. when one is in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of the wound's classification. when first recognized, the wound should be covered' with a sterile dressing until definitive treatment is rendered. bleeding should be controlled with direct waldron and trevor ( ) . pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . when the treatment of a wound begins, anesthesia or analgesia may be necessary, and the choice of anesthetic regimen will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed at this time. then a water-soluble lubricant gel may be applied directly to the wound. a wide margin of hair should then be clipped from around the wound, using a # blade. after the clipping, a surgical scrub is performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a,b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. two solutions, . % chlorhexidine diacetate in water (lozier et al., ) and % povidone-iodine in saline, are most often recommended for wound lavage (waldron and trevor, ) . the chlorhexidine solution may offer the advantage of greater bactericidal activity but does not significantly alter wound healing when compared with povidone-iodine (sanchez et al., ) . actually, the type of solution chosen may not be as important to wound care as the volume and pressure at which the solution is delivered. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a ml syringe and an -or gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure of the wound at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be place d. subcutaneous closure should be performed with absorbable suture such as polydioxanone (pds), polyglactin (vicryl), or polyglycolic acid (dexon). it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. initially, the wound can be covered by gauze sponges soaked in saline or chlorhexidine to create a wet-to-dry bandage. when the sponges are later pulled from the wound, dried exudates will also be removed. when the wound appears clean, the layer in contact with the wound may be changed to a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . secondintention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than surgical repair of a wound, and in the case of large wounds it will be more expensive because of the cost of bandaging materials. several factors must be weighed concerning the use of antibiotics in traumatic wounds, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated, and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. in skin wounds, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. cephalosporins, amoxicillin-clavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . etiology. pressure sores (decubital ulcers) can be a problem in long-term studies that require extended periods of recumbency. decubital ulcers usually develop due to continuous pressure from a hard surface contacting a bony prominence such as the elbow, the tuber ischii, tarsus, or carpus. the compression of the soft tissues between the hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores. these factors include poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting casts or bandages (swaim and angarano, ) . clinical signs. at first, the skin at the developing site will appear red and irritated. over time, constant trauma can result in full-thickness skin wounds and can progress to necrosis of underlying structures such as bone. the severity of the sores may be graded from i to iv, according to the depth of the wound and the tissues involved, from superficial skin irritation to bone necrosis. epizootiology. the problem usually occurs in large-breed dogs, but any type of dog can be affected. prevention and control. minimizing or eliminating those factors that can predispose to decubital ulcers is important to both the prevention and the control of this condition. if the dogs are going to experience long periods of recumbency, adequate bedding or padding must be provided. skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain good flesh and adequate healing is also important (swaim and angarano, ) . treatment. the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive therapy must be performed. the affected area should be radiographed to assess bone involvement, and the wound should be cultured. all of the damaged tissue should be debrided, and wound management guidelines should be followed (see section iii,c, ). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary. bandaging should be performed on all full-thickness wounds; however, it is important to remember that ill-fitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded, because this will increase the pressure over the wound. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound. this will displace the forces acting on the wound over a larger area and over healthier tissue. then the doughnut is incorporated into the bandage. if a cast has been applied to the area for treatment or for research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ). bandages should be removed at least once or twice a day to allow wound care. after wound care has been initiated the causative factors for the pressure sore must be addressed (see "prevention and control," above). recumbent animals should be moved frequently to prevent continuous compression on the wound. if the dog tends to favor a position that aggravates the problem, splinting the body part to reduce contact with hard surfaces may be necessary. etiology. acral lick granuloma is a psychodermatosis, a skin lesion caused by self-trauma. in a few cases, self-trauma begins because of identifiable neurologic or orthopedic causes (tarvin and prata, ) . however, the majority of the cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory setting is an environment that could promote this abnormal behavior and lead to acral lick granuloma. epizootiology. the lesions associated with acral lick granuloma are seen most often in large-breed dogs, but any type of dog can be affected (walton, ) . clinical signs. at first, lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ). the predilection for the limbs may be due to accessibility or possibly may be caused by a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated, and the wound has a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis. acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast-cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of these problems can be ruled out by the history of the animal. when in doubt, a biopsy should be taken. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control. behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. the environment of a dog with this problem can be enriched with exercise and the introduction of toys. in addition, the relief of boredom or anxiety can be attempted through the use of drugs such as phenobarbital, megestrol acetate, and progestins. these drugs may produce side effects, however (swaim and angarano, ) , and may interfere with experimental results. treatment. several treatments have been reported for acral lick granuloma, and none of them have been proven to be successful in ah cases. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been used in an attempt to treat acral lick granuloma by blocking endogenous opioids. in one study, either naltrexone ( mg/kg sq) or nalmefene ( - mg/kg sq) successfully reduced the excessive licking behavior in of dogs; however, lesions returned after the drug was discontinued (dodman et al., ) . the use of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide ( ml of banamine [schering] mixed with ml of synotic [diamond laboratories]) applied topically twice daily has also been shown to be effective (walton, ) . the prognosis for acral lick granuloma should be considered guarded, because the lesions often recur or new lesions develop when treatment is discontinued. etiology. hygromas are fluid-filled sacs that develop as a result of repeated trauma over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology. elbow hygromas are most frequently reported in large and giant breeds of dogs around - months of age (johnston, ; bellah, ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment for research dogs predisposes them to hygromas, because these animals spend a large amount of time on hard surfaces such as cage bottoms or cement runs. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs. a dog with an elbow hygroma presents with a unilateral or bilateral, painless, fluctuant swelling over the point of the elbow. the animals are not usually lame. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma is secondarily infected, the animal may exhibit pain and fever (johnston, ; bellah, ) . pathology. the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is yellow or red and is a serous transudate. this fluid is less viscous than joint fluid, and elbow hygromas do not communicate with the joint (johnston, ) . treatment. the treatment of elbow hygromas should be conservative whenever possible, and surgical options should be reserved for complicated or refractory cases. conservative management of the elbow hygroma is aimed at relieving pressure at the point of the elbow by providing a padded cage surface and/or bandaging the elbow in a manner similar to that used to treat pressure sores (see section iii,c, ). more aggressive therapy, including needle drainage and the injection of corticosteroid into the hygroma, has been described but is not recommended, because infection is a serious complication of this treatment (johnston, ) . likewise, simple surgical excision of elbow hygromas can be associated with complications such as wound dehiscence and ulceration (johnston, ) . a technique that has been used successfully involves placement of multiple penrose drains. the drains are kept in place for - weeks, and the limb remains bandaged for weeks with this technique (bellah, ) . another technique has been described that involves the removal of a crescent-shaped piece of the skin and capsule. the remaining dead space is closed with mattress sutures over stents, and then the wound is closed in a routine fashion. the stents are removed in - days, and the wound is bandaged until suture removal in - days (newton et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology. in the research environment, corneal ulcers are most often associated with either direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these cases would be rare in the laboratory setting. clinical signs. the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may not appear abnormal; however, in cases of deeper ulceration, the cornea may appear roughened or may have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers may be made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies or abnormal eyelids or cilia. treatment. the treatment of corneal ulcers will depend on the depth and size of the affected area. deep ulcers may require debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given times a day for - days usually provides adequate treatment. ointments are preferred over drops, because use of the former requires less frequent. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. indwelling intravascular catheters, including intracaths and vascular access ports, often play a vital role in research protocols. the catheters are most often placed in a central vein or artery where they may be used for repeated blood sampling, administration of anesthetics and experimental compounds, or measurement of hemodynamic parameters. although catheters vary in composition, number of ports, and port placement, the basic principles of their implantation and maintenance are similar. it is important that the laboratory animal veterinarian be familiar with these principles and the potential complications of catheter use. when appropriately maintained, indwelling catheters may remain functional for months without serious complication. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. this is due largely to the fact that many of the problems may be incidental findings or related to a particular research protocol. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters (exact vascular locations not specified). the lesions found were categorized as traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. the traumatic cardiac lesions consisted primarily of masses of fibrin and inflammatory cells on the heart valves. the visceral infarcts were noted in the spleen, kidney (fig. ) , and brain and resulted from fibrin embolization from either the valvular lesions or the catheter tip. fatal hemorrhages were most often found in animals with experimentally induced hypertension. these animals developed clinical signs of sepsis and later ruptured a major vessel associated with mycotic infection and aneurysm. etiology. the leading complication associated with the use of indwelling vascular catheters is infection, either systemic or local at the point of entry through the skin. septicemia may develop from bacterial colonization of either the tract around the catheter or the catheter lumen. clinical signs. the signs and treatment of systemic infection are covered in section iii,d, . problems with the skin defect associated with the catheter port vary from mild skin irritation to obvious infection. the signs may include redness and swelling of the skin around the external port, discharge from the skin wound, or even abscess formation. prevention. because indwelling catheters play an important role in many research protocols, it is highly desirable to prevent catheter complications that may result in loss of the device. the catheter should be made of nonthrombogenic material. in addition, it is recommended that catheters be as simple as possible. a catheter with extra ports or multiple lumens requires addi- tional management and supplies more routes for infection. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that a long extension of tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. the placement of the catheter should be verified by radiography. catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. after catheter placement, the animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide, as described below (see "treatment"). throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. treatment. the treatment of catheter infections almost invariably involves removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ) . superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. aerobic and anaerobic cultures of blood and locally infected sites should be performed (ringler and peter, ) . localized abscesses or sinus tracts may be managed by establishing drainage and flushing with chlorhexidine. again, the catheter should be removed. if retention of a catheter is important, the catheter lumen may be disinfected by filling with chlorine dioxide solution. it has been shown that there are no adverse effects from the use of chlorine dioxide in catheters (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used in the catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . when placed and managed correctly, these ports may remain in place for months without complications. the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., , kwei et al., . these infections lead to removal of the catheters despite treatment with local lavage and systemic antibiotics. there have also been reports of catheters dislodging from the intestinal tract and resulting in peritonitis. this complication has largely been eliminated with the improved security afforded by a synthetic cuff added to the end of the catheter (meunier et al., ) . the chapter authors have also seen migration of the catheter end within the lumen of the intestine (caused by peristaltic motion to egest the catheter), extensive intra-abdominal adhesions, and intestinal torsion (figs. a,b) as complications of intestinal access ports. the procedures for placement and maintenance of the catheters are similar to those outlined previously for indwelling vascular catheters. it is important that the catheters be firmly secured to the intestine to prevent migration or dislodgment. an omental patch placed over the site of entry may help form a firm adhesion. in addition, it is important to place the proper length of catheter within the peritoneal cavity; excess catheter length can promote adhesion formation, whereas insufficient catheter length to account for visceral organ motion can result in detachment. the placement and patency of the catheters can be verified periodically by contrast radiography using iodinated contrast material or by fecal occult blood testing after a small amount of blood has been injected through the catheter (meunier et al., ) . etiology. sepsis is defined as the systemic response to infection. most often, sepsis is a result of infection with gramnegative bacteria; however, sepsis may also be associated with gram-positive bacteria and fungi. in laboratory animals, sepsis is seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs. the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course of sepsis, dogs will present with signs of a hyperdynamic response, including an increased heart rate, increased respiratory rate, red mucous membranes, and a normal to increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals will show the classic signs of septic shock, including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis. the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such ~ as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis. in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of bands than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several systems should be evaluated for infection, including urinary tract, reproductive tract, abdominal cavity, respiratory tract, teeth, and heart valves (kirby, ) . treatment. the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is associated with the source of infection, the implant should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or third-generation cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no "magic bullet" for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology. in research animals, aspiration into the lungs may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. clinical signs. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on a history consistent with aspiration and the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. pathogenesis. aspiration of gastric contents or other compounds can create lung injury of variable severity, depending upon the ph, osmolality, and volume of the substance. the compounds aspirated can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - hr. treatment. the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. in cases in which a small amount of a relatively innocuous substance (e.g., barium) has been aspirated, treatment may not be necessary. when severe inflammation is present, systemic fluid therapy should be instituted. support of the cardiovascular system should be performed judiciously; fluid overload could lead to an increase in pulmonary edema. the use of colloids is controversial because of the increase in vascular permeability that occurs in the lungs. oxygen therapy is also controversial, because it may increase lung injury if administered at high concentrations for long periods of time (nader-djahal et al., ) . several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. in humans, antibiotics are reserved for use in cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be treated with antibiotics immediately when the aspirated material is either not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. etiology. in laboratory animals, accidental burns usually result from thermal injury (heating pads, water bottles) or harsh chemicals (strong alkalis, acids, disinfectants). the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs. the clinical signs vary with the type and degree of burn injury. initially, the injury may not be noticed. the first signs may be oozing from the skin and matting of the overlying hair. within a couple of days, progressive hair and skin loss may be observed (johnston, ) . the wounds may vary in severity from very superficial (involving only the epidermis) to those in which the epidermis and dermis are completely destroyed. superficial wounds appear as red, inflamed skin similar to sunburn in humans. the pain associated with these injuries usually subsides in - days, and the wound reepithelializes without complications in - days. deeper burns develop a thick covering, or eschar, composed of the coagulated proteins and desiccated tissue fluid. the wound heals by granulation under the eschar, which eventually sloughs or is removed to allow further healing by contraction and reepithelialization. within - days of injury, the burn wound will be colonized by grampositive bacteria that rapidly cover the entire wound. several days later, gram-negative organisms can appear in the burn wound (johnston, ) . at this point, signs of wound infection and sepsis may occur (see section iii,d, ). treatment. appropriate and timely treatment of a burn wound will reduce the extent of the injury. thermal injuries should be immediately cooled to reduce edema and pain (demling and lalonde, ) . chemical burns should be thoroughly lavaged for min after wounding. the damaged tissues may be unable to mount appropriate responses to changes in temperature; therefore, the lavage should be performed with warm water to prevent hypothermia. after the initial treatment, all burn wounds should be gently cleansed - times a day (demling and lalonde, ) . burns involving the epidermis and part of the dermis can be extremely painful, and analgesia should be addressed throughout the treatment period. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, topical wound dressings are recommended in the early stages of treatment. a thin film of a water-soluble broad-spectrum antibiotic ointment should be applied to the wound surface after each cleaning. silver sulfadiazine has a broad spectrum, penetrates eschar well, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it is associated with pain upon application (demling and lalonde, ) . when signs of wound or systemic infection are present, systemic antibiotics should be employed, and their ultimate selection should be based on culture and sensitivity results. after the topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds. when the eschar over a burn wound has formed and become fully defined, a small or moderately sized wound may be completely resected. prevention. obviously, prevention of burn wounds is preferable to a long course of treatment. care should be taken to prevent direct exposure to harsh chemicals. tables, floors, and other surfaces should be rinsed thoroughly after chemical use, prior to allowing any animal contact. electric heating pads should be avoided, and only heated water blankets or circulating warm-air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. etiology. research and/or anesthetic protocols may require the intravenous injection of various solutions. when these substances have a ph or osmolarity significantly different from that of the surrounding tissues, the accidental perivascular extravasation of the solutions may result in tissue damage. several drugs have been shown to cause problems when injected perivascularly, including pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin (swaim and angarano, ; waldron and trevor, ) . clinical signs. the immediate signs of perivascular injection are swelling at the injection site and withdrawal of the limb or other signs of discomfort. later, the area may appear red, swollen, and painful as inflammation progresses. often there will be eventual necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a to month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . prevention. because the degree of injury and extensive treatment associated with perivascular extravasation of a drug can be detrimental to research protocols and can cause severe discomfort to the dog, prevention of these injuries is preferred. prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. if a potentially caustic compound is to be used in a fractious subject, sedation of the dog is warranted if this will not interfere with the research protocol. whenever possible, insertion of an indwelling catheter is extremely important. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. prior to use, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. treatment. the treatment of perivascular injections will depend on the amount and type of substance injected. in most cases, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions (swaim and angarano, ). the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) , and local infiltration of hyaluronidase accompanied by warm compresses has been suggested for use in cases of vinblastine injection (waldron and trevor, ) . despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful, and analgesia should be addressed. etiology. hepatic encephalopathy is the result of the derangements in metabolism associated with abnormal liver function. this condition may be seen in young dogs with congenital portosystemic shunting of blood flow. however, in the research setting, encephalopathy occurs more often in canine models of hepatic disease that lead to liver failure. a well-developed knowledge of the pathophysiology of liver disease is necessary for the initial treatment and long-term management of hepatic encephalopathy. pathogenesis. when the liver function is severely impaired because of either portosystemic shunting of blood flow or loss of metabolically active hepatic tissue, the result is an accumulation of ammonia, toxic amines, aromatic amino acids, and short-chain fatty acids (hardy, ; center, ) . these compounds have several toxic effects that result in a decrease in cerebral energy metabolism and a decrease in excitatory neurotransmitter synthesis. concurrently, there is an increase in the concentration of false neurotransmitters and the inhibitory substance -aminobutyric acid (gaba). clinical signs. the signs of hepatic encephalopathy include lethargy, depression, muscle tremors, and convulsions. diagnosis and differential diagnosis. a presumptive diagnosis of hepatic encephalopathy may be based on the appearance of clinical signs following experimental manipulation of the liver. additional diagnostic tests to verify the loss of liver function can be performed to confirm the diagnosis. serum glucose and protein levels may be low if hepatic function is severely impaired. a low serum urea nitrogen level suggests that the normal hepatic metabolism of ammonia into urea has been impaired. elevated levels of serum bile acids and blood ammonia also verify the loss of liver function (hardy, ) . measurement of serum hepatic leakage enzymes are nondiagnostic, because they can be low, high, or normal. treatment. because of the severity of hepatic encephalopathy, treatment may be initiated based on a presumptive diagnosis. during initial treatment, supportive care with fluids and electrolytes should be instituted, based on the results of serum chemistry and blood gas analysis. the majority of animals with hepatic dysfunction will be hypokalemic, alkalotic, and hypernatremic; therefore, either . % sodium chloride or . % sodium chloride with . % dextrose, supplemented with potassium chloride, is recommended (hardy, ) . the type of drug to be used for seizure control is controversial. the short halflife of diazepam makes it an attractive choice compared with barbiturates, which have prolonged metabolism when hepatic function is impaired (maddison, ) . however, endogenous benzodiazepines mediate some of the cns signs seen with hepatic encephalopathy. therefore, the use of diazepam has been discouraged in favor of phenobarbital (johnson, ) . the drug selected for seizure control should be titrated carefully, given the altered liver metabolism. most importantly, the treatment of dogs with hepatic encephalopathy must be aimed at reducing the levels of toxic metabolites in the bloodstream. because protein metabolism is a major source of ammonia, all oral food intake should cease until the signs of hepatic encephalopathy have abated. because gastrointestinal bleeding may occur in individuals with liver failure and this is also a source of protein, the use of h blockers such as cimetidine or ranitidine is suggested (swalec, ) . in addition, lactulose retention enemas should be performed ( - ml/lb of a % solution in water, retained for - min) (hardy, ) . lactulose is an indigestible semisynthetic sugar that is metabolized in the gut to lactic and other acids. the decrease in colonic ph reduces ammonia levels in the bloodstream by converting intestinal ammonia into less diffusible ammonium ions. lactulose will also cause an osmotic diarrhea. antibiotics such as neomycin ( mg/lb, - times/ day) or metronidazole ( mg/lb, times/day) should also be used to reduce the intestinal load of urease-producing bacteria responsible for splitting urea into ammonia (hardy, ) . when the signs of hepatic encephalopathy have resolved, the dog may be fed a low-protein diet. diets suitable for dogs with renal insufficiency are recommended initially. this type of diet is not suitable for long-term use, however, because it appears that individuals with some types of hepatic disease actually have increased protein requirements. these requirements may be met by slowly increasing protein in the diet as long as signs of hepatic encephalopathy do not recur. to maintain the appropriate balance of aromatic and branched-chain amino acids, the diet should be based on vegetable and dairy protein instead of meat or fish protein (center, ) . in addition, the antibiotics suggested above should be continued to reduce the effects of increasing dietary protein levels. the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had either mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients but typically require local anesthesia. an instrument such as a tru-cut needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a mm x to . cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor, and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large soft-tissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology. lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid ( , -d) as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . clinical signs. multicentric and alimentary lymphomas account for most cases of canine lymphoma. in multicentric lymphoma, animals usually present with enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. alimentary lymphoma is associated with vomiting and diarrhea, in addition to previous clinical signs. less commonly, dogs develop mediastinal, cutaneous, and extranodal lymphomas. dogs with mediastinal lymphoma often present with respiratory signs secondary to pleural effusion. hypercalcemia is most frequently associated with this form of lymphoma and may result in weakness. cutaneous lymphoma varies in presentation from solitary to generalized and may mimic any of a number of other skin disorders. the tumors may occur as nodules, plaques, ulcers, or dermatitis. approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement. epizootiology. the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . pathologic findings. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. pathogenesis. all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. diagnosis and differential diagnosis. differential diagnoses for multicentric lymphoma include systemic mycosis; salmonpoisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections are frequently needed. treatment. therapy for lymphoma typically consists of one or a combination of several chemotherapeutic agents. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. macewen and young ( ) provide a thorough discussion of therapeutic options for the treatment of lymphomas in the dog. research complications. given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with signifcant clinical illness. etiology. the fibrosarcoma group of tumors encompasses not only malignant tumors of fibroblasts but also a number of indistinguishable tumors, all of which are capable of collagen production (pulley and stannard, ) . frequently classified in this group are undifferentiated leiomyosarcomas, liposarcomas, malignant melanomas, and malignant schwannomas. clinical signs. although these neoplasms can arise throughout the body, they are most commonly found in the skin, subcutaneous tissues, and oral cavity. fibrosarcomas are extremely variable in size and can grow to be quite large. in general, they are irregular and nodular, poorly demarcated, and nonencapsulated, and they frequently invade deeper tissues. epizootiology. most fibrosarcomas develop in adult and aged animals but can affect dogs as young as months or less. pathogenesis. fibrosarcomas exhibit rapid, invasive growth, recurring frequently after excision. metastasis occurs in only one-fourth of cases, usually by the bloodstream to the lungs. less frequently, spread to local lymph nodes is observed. diagnosis and differential diagnosis. differential diagnoses for fibrosarcomas vary with the location of the tumor. histopathologic exam should be used to distinguish these tumors from round cell tumors (mast cell tumors, histiocytomas, transmissible venereal tumors), papillomas, and other neoplasms. treatment. treatment of any soft-tissue sarcoma would begin with wide surgical excision. if the tissue margins indicate incomplete resection, radiotherapy could be used. for any highgrade tumors, adjuvant chemotherapy would be recommended (see macewen and withrow, a , for a complete discussion). research complications. because fibrosarcomas are locally invasive and often recur, dogs with these neoplasms should not be considered good subjects for long-term studies. etiology. neoplasms of lipocytes and lipoblasts are welldifferentiated tumors referred to as lipomas. clinical signs. these growths can be found as single or multiple round, ovoid, or discoid masses in the subcutaneous tissues of the lateral and ventral thorax, abdomen, and upper limbs. generally they are well circumscribed, encapsulated, and soft on palpation. further, the skin is freely movable over the tumor. epizootiology. lipomas occur principally in aged animals (average years), and the incidence increases with age (pulley and stannard, ) . the tumors are most commonly seen in overweight female dogs, but no breed predisposition is observed. pathologic findings. histologically, lipomas are indistinguishable from normal adipose tissue except when a fibrous capsule is present. pathogenesis: lipomas are typically slow-growing and do not recur after complete surgical excision. diagnosis and differential diagnosis. lipomas are not frequently confused with other tumors but can sometimes be difficult to distinguish from normal adipose tissue. generally, the distinction can be made from the clinical history. treatment. treatment for lipomas is not usually necessary unless the mass is causing problems with normal ambulation. in such cases, surgical excision is usually curative. research complications. lipomas usually do not complicate research studies unless they are interfering with other systemic functions or ambulation. etiology. histiocytomas are benign skin growths that arise from the monocyte-macrophage cells in the skin. some debate exists as to whether this growth is actually a neoplasm or a focal inflammatory lesion (pulley and stannard, ) . clinical signs. the most frequent sites for histiocytomas are the head (especially the pinna) and the skin of the distal forelegs and feet. the masses are usually domelike or buttonlike (often referred to as "button tumors") and usually measure - cm in diameter. epizootiology. histiocytomas are the most common tumors of young dogs, mostly occurring in dogs less than years of age. pathologic findings. histologically, these tumors contain round to ovoid cells with pale cytoplasm and large nuclei. the cells infiltrate the dermis and subcutis, displacing collagen fibers and skin adnexa. despite being benign lesions, histiocytomas characteristically have a high mitotic index. pathogenesis. this tumor typically exhibits rapid growth ( - weeks) but does not spread. most histiocytomas will spontaneously regress in less than months. diagnosis and differential diagnosis. histiocytomas must be distinguished from potentially metastatic mast cell tumors. this is accomplished by staining with toluidine blue, which would stain the cytoplasmic granules of mast cells red or purple. treatment. although most histiocytomas will spontaneously resolve, conservative surgery or cryosurgery will provide an expeditious resolution. research complications. histiocytomas should not interfere with most studies. etiology. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog (bostock, ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin. clinical signs. well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, to cm nodules in the skin. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema. mast cell tumors can be found on any portion of the dog's skin but frequently affect the hindquarters, especially the thigh and in-guinal and scrotal areas. mast cell tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. epizootiology. these tumors tend to affect middle-aged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ). pathologic findings. because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis, and grade iii the worst prognosis. grade i tumors are well differentiated, with round to ovoid uniform cells. the nuclei are regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have large, irregular nuclei with multiple prominent nucleoli. the cytoplasmic granules are few, but mitotic figures are much more frequent. in addition to skin lesions, mast cell tumors have been associated with gastric ulcers. these lesions are most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . the ulcers can be found in the fundus, pylorus, and/or proximal duodenum. although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis. mast cell tumors can be distinguished histologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue, which metachromatically stains the cytoplasmic granules of the mast cells red or purple. treatment. initial treatment for mast cell tumors is generally wide surgical excision ( to cm margins). even with wide surgical margins, approximately % of mast cell tumors may recur. if the site is not amenable to wide surgical excision, debulking surgery and radiation therapy may be used. other alternatives include amputation (if on a limb) or radiation therapy alone. as an adjunct to surgery, grier et al. ( grier et al. ( , found that deionized water injected into surgical margins reduced tumor recurrence by hypo-osmotically lysing any mast cells left behind. this technique has recently been refuted by jaffe et al. ( ) . for systemic mastocytosis, and nonresectable or incompletely excised mast cell tumors, chemotherapy can be used. treatment options would include oral prednisolone, intralesional triamcinalone, and the combination of cyclophosphamide, vincristine, and prednisolone (graham and o'keefe, ) . research complications. because of the possibility of systemic histamine release and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed on a regular basis (monthly). grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology. hemangiosarcomas are malignant tumors that originate from endothelial cells. clinical signs. these tumors may arise in the subcutis but are more commonly found in the spleen and the right atrium. clinical signs are associated with the site of involvement. vascular collapse is frequently observed secondary to rupture and hemorrhage from splenic masses. heart failure can be observed secondary to tumor burden or hemopericardium. when found in the skin, hemangiosarcomas are poorly circumscribed, reddish black masses that range in size from to cm in diameter. the most common cutaneous sites are the ventral abdomen, the prepuce, and the scrotum. epizootiology: hemangiosarcomas occur most frequently in -to -year-old dogs. the german shepherd dog is most commonly affected. pathologic findings. grossly, splenic hemangiosarcomas resemble nodular hyperplasia or hematomas (fig. ) . the masses are spherical and reddish black and can range in size up to - cm in diameter. on cut section the masses may appear reddish gray or black and have cavernous areas of clotted blood. when the masses are found in the heart, the endocardium may be covered by a thrombus, giving the to cm tumors a reddish gray or yellow appearance. histologically, hemangiosarcomas are composed of immature endothelial cells that form vascular channels or clefts. these spaces may be filled with blood or thrombi. the neoplastic cells are elongated with round to ovoid, hyperchromatic nuclei and frequent mitotic figures. pathogenesis. hemangiosarcomas can be found in one or many sites. in cases where multiple sites are involved, it may be impossible to identify the primary tumor. this neoplasia is highly malignant and spreads easily. metastasis occurs most frequently to the lungs but can be found in any tissue. diagnosis and differential diagnosis. splenic hemangiosarcoma may resemble nodular hyperplasia or some manifestations of lymphoma. when the heart is affected, other causes of heart failure must be ruled out. echocardiography is a valuable tool for identifying the primary lesion. histopathology should be used to differentiate dermal hemangiosarcoma from hemangiomas and other well-vascularized tumors. treatment. surgery is generally the first choice of treatment for hemangiosarcoma. dermal tumors are treated with radical resection, splenic tumors by total splenectomy, and heart tumors by debulking and pericardiectomy. because of the high likelihood of metastasis, adjunct chemotherapy should always be considered. research complications. dogs with dermal hemangiosarcoma may be cured after complete resection with margins, but monitoring should be done regularly for recurrence. the other forms of hemangiosarcoma have a much poorer long-term prognosis, and treatment is typically unwarranted in the research setting. etiology. also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the transmissible venereal tumor is transmitted to the genitals by coitus (nielsen and kennedy, ) . the origin of this tumor is still unknown but has been described as a tumor of lymphocytes, histiocytes, and reticuloendothelial cells. although this tumor has been reported in most parts of the world, it is most prevalent in temperate climates (macewen, ) . clinical signs. the tumors are usually cauliflower-like masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis (fig. ) . less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission. transmissible venereal tumors are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions are believed to be a result of trauma prior to exposure to the tumor. pathogenesis. tumor growth is rapid after implantation but later slows. metastasis is rare (< % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. diagnosis and differential diagnosis. transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. cytology may be of benefit in making a definitive diagnosis, so impression smears should be made prior to processing for histopathology. prevention. thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control. removing affected individuals from a breeding program should stop further spread through the colony. treatment. surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for - treatments will induce remission and cure in greater than % of the cases (macewen, ). research complications. experimental implantation of transmissible venereal tumors has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology. dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs. single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger and invasive and coalesce with adjacent tissues. epizootiology. mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. median age at diagnosis is - years. mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . extensive discussions of classification, staging, and histopathologic correlations can be found in macewen and withrow ( lb) and moulton ( ) . pathogenesis. mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. futher, schneider et al. ( ) showed that the risk of developing mammary tumors increased greatly after the first and second estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs. diagnosis and differential diagnosis. both benign and malignant mammary tumors must be distinguished from mammary hyperplasia and mastitis. prevention. mammary tumors can effectively be prevented by spaying bitches prior to the first estrus. this is commonly done in the general pet population at months of age. recently, the topic of spaying sexually immature dogs ( - weeks of age) has received much attention for the control of the pet population. kustritz ( ) reviewed the techniques for anesthesia and surgery, as well as possible pros and cons of spaying at this young age. treatment. surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. lumpectomy or nodulectomy should be elected in the case of small discrete masses, while mammectomy and regional or total mastectomies are reserved for more aggressive tumors. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. inguinal lymph nodes should be removed any time the fourth and fifth glands are excised (macewen and withrow, lb). research complications. because % of mammary tumors are benign, treatment may be rewarding, allowing dogs to con-tinue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. etiology. beagles are among the breeds with the highest prevalence of thyroid carcinomas. benjamin et al. ( ) reported a correlation between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in the beagle. clinical signs. thyroid carcinomas generally present as palpable cervical masses. affected animals may experience dysphagia, dyspnea, and vocalization changes. precaval syndrome resulting in facial edema is also observed in some cases. epizootiology and transmission. the mean age of dogs presented with thyroid carcinomas is years, with equal distribution of cases between the sexes. pathologic findings. grossly, thyroid carcinomas are multinodular masses, frequently with large areas of hemorrhage and necrosis. they tend to be poorly encapsulated and invade local structures such as the trachea, esophagus, larynx, nerves, and vessels. the masses are unilateral twice as often as bilateral (capen, ) . histologically, thyroid carcinomasare divided into follicular, papillary, and compact cellular (solid) types (see capen, , for complete discussion). pathogenesis. thyroid carcinomas tend to grow rapidly and invade local structures. early metastasis is common and occurs to the lungs by invasion of branches of the thyroid vein. diagnosis and differential diagnosis. nonpainful cervical swellings such as seen with thyroid tumors are also consistent with abscesses, granulomas, salivary mucoceles, and lymphomas. often a preliminary diagnosis can be made by fineneedle aspirate. treatment. surgery is the treatment of choice for thyroid carcinomas that have not metastasized. when the tumor is freely movable, surgery may be curative. surgical excision may be difficult for tumors that adhere to local structures, requiring excision of the jugular vein, carotid artery, and associated nerves. when bilateral tumors are observed, preservation of the parathyroid glands may not be possible. in such cases, treatment for hypoparathyroidism will be necessary. both chemotherapy and radiation therapy have been suggested for extensive bilateral tumors and/or after incomplete excision, but no controlled trials have been performed (ogilvie, ) . in the research setting, treatment of this tumor may not be rewarding. only freely movable tumors can be practically treated without seriously affecting research efforts. euthanasia is warranted in the more advanced cases when clinical illness is apparent. beagles are subject to many of the inherited and/or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, b) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). in addition, there are defects that affect so many breeds that the author simply lists "many breeds" for the breeds affected by those disorders. thus these defects could also affect beagles and include pectus excavatum, polydactyly, radial and ulnar dysplasia, hypoadrenocorticism, entropion, lens coloboma, factor viii deficiency (von willebrand's disease), renal agenesis or ectopia, and developmental defects of the reproductive and lower urinary tracts. at a commercial breeder of purpose-bred beagles, the most common birth defects were umbilical hernia ( . % of births) and open fontanelle ( . % of births) (r. scipioni and j. ball, personal communication, ) . other defects observed include cleft palate and cleft lip, cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology. cataract is an opacification of the lens or the lens capsule. it is the pathologic response of the lens to illness or injury, because the lens has no blood supply. cataracts can be caused by metabolic, inflammatory, infectious, or toxic causes and can be congenital, juvenile, or degenerative. nuclear sclerosis is an apparent opacification of the lens caused by the compression of older lens fibers in the center of the lens (nucleus) as a consequence of the production of new fibers. because the nucleus increases in size as the animal ages, the sclerosis is more apparent in older animals and may be mistaken as a senile cataract. the ability to see the fundus during ophthalmoscopy persists with nuclear sclerosis but is obstructed by a true cataract. clinical signs. the first clinical sign is typically the ability to visualize the opaque lens through the pupil of the dog's eye. dogs have an impressive ability to tolerate bilateral lens opacity (especially when development is gradual), and often visual impairment is detected late in the development of the condition (helper, ) . moderate vision loss may cause the dog to be hesitant in moving in new surroundings or unable to locate movable objects (such as a toy). rapid cataract development can result in a sudden vision loss, such as can occur with diabetic cataracts. epizootiology and transmission. certain dog breeds can be predisposed to the development of juvenile or senile cataracts or to metabolic disorders that result in cataract development, such as diabetes mellitus. dogs in studies for diabetes mellitus should be observed regularly for cataract development. toxicological studies may also induce formation of cataracts. pathogenesis. lens fibers respond to all biological or chemical insults by necrosis and liquefaction (render and carlton, ) , because they have no blood supply with which to recruit an inflammatory and repair process. disruption of these fibers by any means, therefore, leads to opacification. the exact processes by which the varieties of congenital and juvenile cataracts are produced have not been determined. in diabetic cataracts, the excess glucose is metabolized to sorbitol and fructose. as these alcohols and sugars accumulate in the lenticular cells, they produce an osmotic imbalance, which brings fluid into the cells, causing swelling and degeneration of lens fibers and resultant opacity (capen, ) . diagnosis and differential diagnosis. the ability to visualize the retina and fundus during ophthalmoscopy differentiates true cataracts from nuclear sclerosis. dogs with cataracts should be evaluated for possible causes, especially diabetes mellitus. diabetes mellitus will typically affect middle-aged dogs and feature rapid cataract formation, whereas juvenile and senile cataracts are slow to develop and affect younger and older dogs, respectively. progressive retinal atrophy can also cause secondary cataract formation; pupillary light response is maintained with primary cataracts (even if the lens is completely opaque), whereas this reflex is obtunded by retinopathy. prevention. most forms of cataracts cannot be prevented, for their exact etiologic pathogenesis is unknown. diabetic cataracts, however, can be prevented by proper regulation of blood glucose concentrations with insulin therapy and proper diet. treatment. because dogs do not need to focus visual images as accurately as human beings, proper lens clarity and function are not necessary for an adequate quality of life. many dogs adjust quite well to the visual impairment caused by persistent cataracts. lens removal can be performed for dogs seriously affected by cataracts, but this would not be anticipated for dogs in the research setting. information on surgical lens extraction procedures can be found in helper ( ) or other veterinary ophthalmology textbooks. research complications. research complications would be minimal with cataracts, unless the dogs were intended for use in ophthalmologic or visual acuity-based studies. etiology. hip dysplasia is a degenerative disease of the coxofemoral joint. a specific etiology is unknown, but the development of hip dysplasia has a strong genetic component (pedersen et al., ) , modified by age, weight, size, gender, conformation, rate of growth, muscle mass, and nutrition (smith et al., ) . clinical signs. the initial clinical abnormality caused by hip dysplasia is laxity of the coxofemoral joint. this may present as a gait abnormality without any indication of lameness or stiffness. eventually, affected dogs will have periods of lameness and, in protracted cases, will be rendered immobile by severe pain. epizootiology and transmission. hip dysplasia has been seen in most dog breeds, but it typically affects larger breeds of dogs. in the research setting, it is primarily a condition of randomsource large-breed dogs used for surgical research. diagnosis and differential diagnosis. hip dysplasia is classically diagnosed by radiography of the pelvis and hip joints. radiographic abnormalities consistent with hip dysplasia include shallow acetabula with remodeling of the acetabular rim, flattening of the femoral head, subchondral bone sclerosis (caused by erosion of articular cartilage and exposure of underlying bone), and osteophyte production around the joint (pedersen et al., ) . hip dysplasia needs to be differentiated from other musculoskeletal or neurological conditions that can cause unusual gaits and/or lameness. this may be somewhat difficult, because clinical signs of hip dysplasia may develop before radiographic abnormalities. radiographic calculation of the distraction index (di) to measure joint laxity has proven to be a good means to predict future hip dysplasia before other radiographic changes are evident (smith et al., ) . prevention. because of the genetic component, dogs with hip dysplasia should not be used in breeding colonies. dogs should be provided a good plane of nutrition but not be allowed to become overweight. dogs that were limit-fed at % of the food amount eaten by ad libitum-fed dogs had lower body weights and decreased severity of radiographic lesions of hip dysplasia (kealy et al, ) . treatment. in the stages when clinical signs are episodic, cage rest and analgesics for several days can be used to treat the symptoms. more advanced cases may require continuous analgesia. sectioning of the pectineus muscle or tendon may provide some pain relief but does not affect the progression of the disease (pedersen et al., ) . surgical treatments for hip dysplasia include femoral head ostectomy and total hip replacement. neither surgical treatment is likely in a research setting. research complications. long-term studies using large-breed dogs may be affected by the eventual development of hip dysplasia. in studies where hip dysplasia would be a serious complication or confounding variable (e.g., orthopedic research), dogs should be radiographed upon arrival to assess possibility of early coxofemoral joint degeneration and suitability for use in the study. etiology. benign prostatic hyperplasia (bph) is an agerelated condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs. bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum as it passes through the pelvic canal. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs for bph. epizootiology and transmission. bph typically affects older dogs (> years), although it has been seen as early as years of age. pathologic findings. in its early stages, canine b ph is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis. bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to a-dihydrotestosterone (kustritz and klausner, ) . diagnosis and differential diagnosis. bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis characterized by hemorrhage, at most. differential diagnoses include squamous metaplasia of the prostate, paraprostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve when castration is used for treatment of prostatic enlargement. prevention. castration is the primary means for prevention of benign prostatic hyperplasia. treatment. the first and foremost treatment for b ph is castration. in pure cases of b ph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases in which the dog is a valuable breeding male (e.g., genetic diseases), and semen collection is necessary. if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. newer drugs marketed for human males have also shown promise in treating canine bph. finasteride (proscar) is a a-reductase inhibitor that limits metabolism of testosterone to a-dihydrotestosterone. treatment at daily doses of - mg/kg has been effective in causing prostatic atrophy without affecting testicular spermatogenesis (kustritz and klausner, ) . dogs given . mg/kg were proven to still be fertile. there are also indications that lower doses may be effective in relieving b ph. androgen receptor antagonists (flutamide and hydroxyflutamide) have also been studied in the dog and found to be effective for treatment of bph while maintaining libido and fertility (kustritz and klausner, ) . unfortunately, both the areductase inhibitors and the androgen receptor antagonists are not presently labeled for use in male dogs in the united states. research complications. bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. it is presently unknown whether the use of the newer antihyperplastic agents systemically alters physiologic parameters outside of the prostate itself. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. etiology. juvenile polyarteritis syndrome (jps) is a painful disorder seen in young beagles (occasionally reported in other breeds). the lesion consistent with the syndrome is systemic necrotizing vasculitis. the cause of the vasculitis has not been established, but it appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs. clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to extend the neck ventrally. most dogs seem to be in pain when touched, especially in the neck region. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that a vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission. jps typically affects young beagles ( - months), with no sex predilection. pathologic findings. on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to medium-sized arteries are seen. these lesions are most noticeable in the three locations where gross lesions are observed, but they may be seen in other visceral locations. the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries (fig. a) . the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen (fig. b) . a subclinical vasculitis has also been diagnosed in beagles postmortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis. the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclical nature and respond to treatment with corticosteroids, and the affected dogs have elevated a -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis of some affected dogs has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resuited in / affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis. differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). are known at this time. no prevention and control measures brane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs. the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit (fig. ) . excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. treatment. clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q hr, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. pathologic findings. typically the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis. prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . research complications. because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology. "cherry eye" is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating mem-prevention. hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment. corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications. in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland, or its removal, might compromise ophthalmologic studies. etiology. interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes (fig. ). the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym "sterile pyogranuloma complex"). clinical signs. dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission. interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . pathologic findings. histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis. initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis. bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection, and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment. if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg ql h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require fig. . interdigital cyst between the third and fourth digits of the forelimb of a research beagle. surgical removal. excision includes removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weightbearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. post-therapy antibody titers in dogs with ehrlichiosis: follow-up study on patients treated primarily with tetracycline and/or doxycycline dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog saunders manual of small animal practice neurologic manifestations associated with hypothyroidism in four dogs neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats platelet function, antithrombin-iii activity, and fibrinogen concentration in heartworm-infected and heartworm-negative dogs treated with thiacetarsamide pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis ehrlichia platys infection in dogs the rickettsioses monoclonal gammopathy associated with naturally occurring canine ehrlichiosis variation in age at death of dogs of different sexes and breeds leptospira interrogans serovar grippotyphosa infection in dogs efficacy and dose titration study of mibolerone for treatment of pseudopregnancy in the bitch tropical canine pancytopenia: clinical, hematologic, and serologic response of dogs to ehrlichia canis infection, tetracycline therapy, and challenge inoculation comparison of campylobacter carriage rates in diarrheic and healthy pet animals. zentralbl advances in dietary management of obesity in dogs and cats effect of level and source of dietary fiber on food intake in the dog effect of amount and type of dietary fiber on food intake in energy-restricted dogs external parasites: identification and control tumors of the endocrine glands thomson's special veterinary pathology infectious diseases of the dog and cat nutritional support for dogs and cats with hepatobiliary disease specific amplification of ehrlichia platys dna from blood specimens by two step pcr detection of humoral antibody to the transmissible venereal tumor of the dog dirofilaria immitis: heartworm products contract rat trachea in vitro dogs: laboratory animal management management of septicemia in rhesus monkeys with chronic indwelling catheters client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis dirofilariasis in dogs and cats use of narcotic antagonists to modify stereotypic self-licking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma study of obesity in dogs visiting veterinary practices in the united kingdom miller's anatomy of the dog update on diagnosis of canine hypothyroidism helicobacter-associated gastric disease in ferrets, dogs, and cats the role of helicobacter species in newly recognized gastrointestinal tract disease of animals serologic diagnosis of infectious cyclic thrombocytopenia in dogs using an indirect fluorescent antibody test hemorrhagic streptococcal pneumonia in newly procured research dogs control of ticks platelet aggregation studies in dogs with acute ehrlichia platys infection health benefits of animal research: the dog as a research subject soft tissue sarcomas and mast cell tumors textbook of veterinary internal medicine infectious diseases of the dog and cat canine lyme borreliosis mast cell tumor destruction by deionized water mast cell tumour destruction in dogs by hypotonic solution transmission of ehrlichia canis to dogs by ticks (rhipicephalus sanguineus) textbook of veterinary internal medicine diseases of the liver and their treatment cyclic thrombocytopenia induced by a rickettsia-like agent in dogs shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs textbook of veterinary internal medicine canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats thiacetarsamide and its adverse effects infectious diseases of the dog and cat pediatrics: puppies and kittens canine viral diseases textbook of veterinary internal medicine antibodies to ehrlichia canis, ehrlichia platys, and spotted fever group rickettsia in louisiana dogs mastocytoma and gastroduodenal ulceration complications in the use of indwelling vascular catheters in laboratory animals deionised water as an adjunct to surgery for the treatment of canine cutaneous mast cell tumours helicobacter infection textbook of veterinary internal medicine textbook of veterinary internal medicine hygroma of the elbow in dogs thermal injuries dirofilaria immitis: do filarial cyclooxygenase products depress endothelium-dependent relaxation in the in vitro rat aorta? depression of endotheliumdependent relaxation by filarial parasite products three cases of canine leptospirosis in quebec cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs role of bordetella bronchiseptica in infectious tracheobronchitis in dogs kirk's current veterinary therapy : small animal practice the fire of life kirk's current veterinary therapy : small animal practice coinfection with multiple tick-borne pathogens in a walker hound kennel in north carolina tarsal joint contracture in dogs with golden retriever muscular dystrophy clinical and hematological findings in canine ehrlichiosis early spay-neuter in the dog and cat textbook of veterinary internal medicine chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs the brown dog tick rhipicephalus sanguineus and the dog as experimental hosts of ehrlicha canis the clinical chemistry of laboratory animals double-blinded crossover study with marine-oil supplementation containing high-dose eicosapentaenoic acid for the treatment of canine pruritic skin disease thomson's special veterinary pathology effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs transmissible venereal tumors kirk's current veterinary therapy : small animal practice soft tissue sarcomas tumors of the mammary gland canine lymphoma and lymphoid leukemias kirk's current veterinary therapy : small animal practice effect of heartworm infection on in vitro contractile responses of canine pulmonary artery and vein tick paralysis in north america and australia saunders manual of small animal practice thyroid gland and arterial lesions of beagles with familial hypothyroidism and hypedipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think urea protects helicobacter (campylobacter) pylori from the bactericidal effect of acid characterization of a new isolate of ehrlichia platys using electron microscopy and polymerase chain reaction decreased pulmonary arterial endothelium-dependent relaxation in heartworm-infected dogs with pulmonary hypertension vaccination against canine bordetellosis: protection from contact challenge dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs clinical trial of dvm derm caps in the treatment of allergic diseases in dogs: a nonblinded study diagnosis of neoplasia tumors of the mammary gland dirofilaria immitis: heartworm infection alters pulmonary artery endothelial cell behavior survey of conjunctival flora in dogs with clinical signs of external eye disease hyperoxia exacerbates microvascular injury following acid aspiration nutrient requirements of dogs surgical closure of elbow hygroma in the dog tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases walker's mammals of the world tumors of the endocrine system beta hemolytic streptococcus isolated from the canine vagina comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs clinical behavioral medicine for small animals hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs joint diseases of dogs and cats target imbalance: disparity of borrelia burgdorferi genetic material in synovial fluid from lyme arthritis patients textbook of veterinary internal medicine tumors of the skin and soft tissue thomson's special veterinary pathology canine leptospirosis: a retrospective study of cases dogs and cats as laboratory animals effects of chlorhexidine diacetate and povidoneiodine on wound healing in dogs epidemiology of thyroid diseases of dogs and cats factors influencing canine mammary cancer development and postsurgical survival muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology textbook of veterinary internal medicine canine infectious tracheobronchitis (kennel cough complex) saunders manual of small animal practice serum protein alterations in canine erhlichiosis bacterial factors and immune pathogenesis in helicobacter pylori evaluation of rhipicephalus sanguineus as a potential biologic vector of ehrlichia platys role of the eastern chipmunk (tamias striatus) in the epizootiology of lyme borreliosis in northwestern illinois evaluation of risk factors for degenerative joint disease associated with hip dysplasia in dogs pathologic features of naturally occurring juvenile polyarteritis in beagle dogs textbook of veterinary internal medicine clinical manifestations, pathogenesis, and effect of antibiotic treatment on lyme borreliosis in dogs streptococcus zooepidemicus as the cause of septicemia in racing greyhounds trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs portosystemic shunts textbook of veterinary internal medicine lumbosacral stenosis in dogs experimental respiratory disease in dogs due to bordetella bronchiseptica dirofilaria immitis: depression of endothelium-dependent relaxation of canine femoral artery seen in vivo does not persist in vitro thyroiditis in a group of laboratory dogs: a study of beagles of agriculture, animal and plant health inspection service thomson's special veterinary pathology a retrospective study of cases of naturally occurring canine ehrlichiosis role of canine parainfluenza virus and bortedella bronchiseptica in kennel cough management of superficial skin wounds serum concentrations of thyroxine and , , '-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs ehrlichia platys in a michigan dog ehrlichial diseases of dogs canine ehrlichiosis. miss albert, r. e., benjamin, s. a., and shukla, r. ( ). life span and cancer mortality in the beagle dog and human. key: cord- -lahg xlm authors: parent, joane m title: the cat with a head tilt, vestibular ataxia or nystagmus date: - - journal: problem-based feline medicine doi: . /b - - - - . - sha: doc_id: cord_uid: lahg xlm nan the presence of a head tilt, ipsilateral to the lesion, is the salient feature of vestibular disease. nystagmus and/or vestibular ataxia may accompany the head tilt. the more acute the disease process, the more likely that nystagmus and ataxia are present. in vestibular ataxia, the animal leans, drifts, falls or rolls toward the side of the lesion. the nystagmus can be resting or positional (induced by holding the head in full extension), and, vertical, horizontal or rotatory in direction. the direction may change in time. pendular nystagmus (oscillatory with rapid, short excursions) is not a sign of vestibular disease. occasionally, there is bilateral vestibular disease. the animal may be reluctant to move because of severe disorientation. if able to walk, there are characteristic exaggerated motions of the head and neck and the cat moves in a crouched posture close to the ground. physiological nystagmus is poor to absent. in these instances, a head tilt may not be present, or if present, is subtle on the most affected side. circling is not a feature of vestibular disease but is frequent with thalamic and cerebral diseases. the most important diagnostic step is to differentiate if the disease involves the peripheral or the central portion of the vestibular system. • the head tilt is toward the side of the lesion. the nystagmus is horizontal or rotatory but never vertical, and never changes in direction. the ataxia, if present, is on the side of the head tilt. • by close proximity, the neurological structures associated with the middle ear may be affected leading to facial nerve paresis/paralysis, dry eye from decreased to absent lacrimation, and/or acute to peracute non-progressive onset of a head tilt with ipsilateral falling or rolling in an otherwise healthy cat. the disorientation may be severe. if the disease is bilateral, the head tilt is not evident, the physiological nystagmus is poor to absent, and the cat moves in a crouched posture, low to the ground. the head swings from side to side in exaggerated motions. • aminoglycosides (p ) acute onset of peripheral vestibular signs uni-or bilaterally following systemic or topical therapy with aminoglycosides. deafness may accompany the vestibular signs. • ear flush (p ) peripheral vestibular signs that appear immediately or in the hours following an ear flush. • blue-tailed lizard ingestion (p ) in southeastern united states, ingestion of a blue-tailed lizard is thought to be the cause of an acute unilateral peripheral vestibular syndrome. • fracture of the petrous temporal bone or tympanic bulla, and ethmoid fracture (base of the skull) (p ) peripheral and central vestibular signs following a road accident. horner's syndrome, partial or complete. the cochlear part of the vestibulo-cochlear nerve may be affected leading to ipsilateral deafness. • if the disease is bilateral, there is no head tilt or only a mild one on the side that is the most severely affected. the more acute the disease, the more severe the disorientation. the cat is reluctant to walk and has a crouched posture, low to the ground. the head characteristically moves from side to side in exaggerated motions and there is often ventroflexion of the head and neck. the physiological nystagmus (normal involuntary rhythmic typewriter-like movements of the eyes initiated by side-to-side movements of the head) is poor to absent. • the most consistent feature indicating central vestibular disease is the concomitant presence of somnolence, or quietness of the animal. this may or may not be obvious at time of examination, but will be evident with good history taking. • the head tilt is toward the side of the lesion. the nystagmus is horizontal, rotatory or vertical and may change in direction. • due to their close proximity, other central nervous system structures may be involved ipsilaterally. these include the ascending reticular activating system or aras (somnolence) and the ipsilateral trigeminal nerve (loss of facial sensation and/or masticatory muscle atrophy), abducent nerve (strabismus), facial nerve (facial paresis/paralysis), cerebellum (tremors, hypermetria), ascending sensory pathways (proprioceptive deficits) and descending motor pathways (upper motor neuron weakness). • bilateral involvement of the central vestibular system appears clinically similar to bilateral peripheral vestibular disorders in the early phase, except that the animal is more quiet or somnolent. if the cause is not corrected, involvement of other structures of the brainstem and/or cerebellum ensues. paradoxical vestibular syndrome. • in this rare syndrome of central vestibular disease, the head tilt is contralateral to the lesion. the diagnosis is made on the presence of cerebellar signs or postural reaction deficits ipsilateral to the lesion but contralateral to the head tilt. peripheral vestibular disease results from a problem in the petrosal bone or tympanic bulla. • the peripheral part of the vestibular apparatus (receptors and nerve) is situated in the inner ear, in close proximity to the middle ear. the inner and middle ear are located within the petrosal bone or tympanic bulla. the facial nerve, the parasympathetic innervation of the lacrimal glands and the sympathetic nerve for pupillary dilatation are the neurological structures associated with the middle ear. • diseases of the inner ear can by extension reach the middle ear and vice versa. • the neurologic structures of the middle ear are more resilient to insult than the receptors in the inner ear (receptors vs axons). as a result, middle ear disease may be present without neurological deficits. with time, facial paresis/paralysis and/or horner's syndrome may appear. • the auditory receptors are situated in the inner ear as well. unilateral deafness goes unnoticed clinically but is diagnosed with electrodiagnostic testing (brain auditory-evoked potentials). central vestibular disease results from an intracranial problem in the brainstem, at the level of the rostral medulla. this area is in close proximity to the cerebellum and pons. this anatomical location is called the cerebello-ponto-medullary angle. the most common causes of vestibular disease are peripheral and include: • idiopathic vestibular disease. • otitis media-interna. • less common causes are the middle ear polyps and tumors. central vestibular diseases are not as frequent as peripheral diseases. • inflammatory diseases are the most common, with the clinical signs directly related to the location and severity of the inflammation. although inflammatory diseases are multifocal or diffuse pathologically, this is often not the case clinically, where the neurological deficits in most cases can be assigned to one location. diagnosis is based on careful history taking (to disclose if there is somnolence or quietness of the animal), physical, neurological, otoscopic and ophthalmoscopic (including schirmer tear test) examinations, serum protein concentration, cerebrospinal fluid analysis (csf), csf anti-coronavirus igg titer, electrodiagnostic testing (brain auditoryevoked responses), bullae radiography, computed tomography (ct) and magnetic resonance imaging (mri) scan. reference ranges for feline csf. • rbc < . × /l ( /μl) • wbc ≤ . × /l ( /μl) • cytology (%) -monocytoid cells - % -lymphocytes - % -neutrophils - % -eosinophils -macrophages (large foamy mononuclear cells) - % • protein . g/l ( mg/dl) it is good practice to request electrodiagnostic testing (baer) to evaluate for deafness. if deafness is concomitantly present, it is an indication of a more aggressive disorder and a thorough diagnostic work up should be pursued. when a head tilt is present, or when there is facial paresis/paralysis or a horner's syndrome, bulla radiography is recommended to evaluate the middle ear cavities. an open mouth view is best to compare the density between the bullae. bulla radiography is not a sensitive tool. it may be normal despite the presence of disease. computed tomography (ct) scan of the bullae is superior and should be done when available. regardless of the cause of the vestibular signs, nystagmus and vestibular ataxia usually resolve, but the head tilt usually persists for the life of the animal. it may be barely noticeable but may be exacerbated after a general anesthetic or when the cat is ill. idiopathic vestibular syndrome*** classical signs • acute to subacute onset of a head tilt, falling, rolling and nystagmus. • disorientation can be severe with the cat unwilling to move and crying out with anxiety. • otherwise healthy cat. unknown. possibly higher incidence in july and august in northeast united states. cats of any age or sex, median age is years old. acute, non-progressive, unilateral and occasionally bilateral, peripheral vestibular disturbance. the head tilt, ataxia and nystagmus (most often horizontal) can be severe with the animal crying out with anxiety, reluctant to walk, remaining in a crouched posture or with wide abduction of the limbs. rarely, can be bilateral. vomiting occasionally occurs, usually soon after onset of signs. affected cats are otherwise healthy. history of an acute to subacute onset, in a previously healthy cat, of severe disorientation, falling and rolling, that improves rapidly (a few days to weeks) and without treatment. the cat is otherwise healthy and has no other neurological signs. the otoscopic examination is unremarkable. diagnosis is usually by exclusion of other diseases causing similar acute signs. with currently available diagnostic techniques it may not be possible initially to differentiate this syndrome from otitis media-interna, but the vestibular signs in otitis media-interna do not usually occur so acutely and severely. it is advisable to perform baers. if there is concomitant deafness, the idiopathic syndrome is ruled out as only the vestibular system should be affected. otitis media-interna usually has a more progressive history. on otoscopic examination there may be otitis externa, mite infestation and/or a ruptured tympanic membrane. signs of middle ear disease such as facial paresis/paralysis, decreased lacrimation and/or horner's syndrome are often present. radiographic changes in the tympanic bulla are rarely observed. computed tomography and mri scans are more sensitive for detecting changes. middle ear polyps can cause peripheral vestibular disturbance uni-or bilaterally but since the polyps originate from the middle ear, facial and/or sympathetic nerve deficits are present. the onset is mild and progressive. blue-tailed lizard ingestion is believed to cause similar signs in the southeastern united states. vomiting, salivation, irritability and trembling are also observed. signs may be indistinguishable from idiopathic vestibular syndrome. aminoglycoside toxicity, especially topical streptomycin, can cause uni-or bilateral peripheral vestibular disturbance but the history reveals use of the drug. supportive. in a few cats, fluid therapy, intravenously or subcutaneously, may be necessary initially. sedation (acepromazine . - . mg/kg im, sc, iv to a maximum of mg) may occasionally be required if the rolling is severe. antibiotics such as amoxicillin or cephalosporine for days are indicated if otitis media-interna cannot be ruled out. glucocorticoids are not indicated. excellent. rapid improvement of the clinical signs within the first weeks in all cats. most cats recover entirely but frequently have a mild residual head tilt. • acute to chronic history. • variable degree of peripheral vestibular disturbance. • signs of otitis externa often present. • facial paresis/paralysis, decreased lacrimation and/or horner's syndrome often present. the infection extends from an otitis externa or from the oro-and nasopharynx by way of the eustachian tubes, or hematogenously. most infections are caused by staphylococcus spp., streptococcus spp., proteus spp., pseudomonas spp. or escherichia coli. frequently, mites are the instigating factor for the otitis externa, which leads to secondary microbial infection with spread to the middle ear. the diagnosis of otitis media-interna is difficult as, except for surgical exploration of the tympanic bulla, the tests performed have a low diagnostic yield. the cat may have a history of chronic otitis externa. the ear may be sore to touch. there may be head shaking or scratching and frequent pawing at the ear. there may be difficulty in prehension or chewing food due to pressure on the petrosal bone region upon jaw opening as the temporo-mandibular joint is in the vicinity of the petrosal bone. the corner of the mouth may be wet with saliva. on otoscopic examination, the external ear canal may be red and inflamed with a brownish discharge. mites may be present. the tympanic membrane may be red and bulging or perforated. the onset and the neurological deficits depend on the extension and severity of the infection. the onset of the vestibular signs may be acute with a marked head tilt, disorientation and nystagmus, or, chronic with nothing else other than a mild head tilt. the concomitant presence of facial nerve paresis/ paralysis, decreased lacrimation and/or horner's syndrome confirms involvement of the middle ear. the facial weakness may be subtle. look for a complete closure of the lids following a single stroke of the finger on both lids at once. look for saliva-stained hair at the commissure of the lips or for a droop of the lips on the side affected. the symmetry of the pupils is best assessed in a dark room to evaluate for the presence of a partial horner's syndrome. facial nerve paralysis may be accompanied by keratoconjunctivitis sicca because innervation of the lacrimal glands is carried by the facial nerve. a schirmer tear test should be routinely done in these cases to avoid the formation of corneal ulcer. unilateral deafness is often present, but can be substantiated only with electrodiagnostic testing (baer). otoscopic examination may disclose an otitis externa, mite infestation or tympanic membrane bulging/perforation. if there is an otitis externa, cytology and culture with sensitivity testing should be done. the same organism may be causing the middle-inner ear infection. the presence of facial paresis/paralysis, decreased to absence of lacrimation or horner's syndrome is a strong indicator of concomitant middle ear disease, but is not specific for infection. on physical examination, there may be pain on opening the mouth or pain upon manipulation of the head. • concomitant deafness indicates a more significant disease process such as in middle-inner ear infection, but is not specific for the disease. it is an indi-cation that a more thorough diagnostic work up should be performed. • normal hearing does not rule out middle-inner ear infection because the auditory receptors are in the inner ear. • it is important to obtain an open mouth view to visualize both bullae simultaneously for comparison. • a normal study does not rule out middle-inner ear disease. • the tympanic bulla may appear denser if there is an effusion. • thickening of the bulla or bone lysis may be observed in more severe and chronic infections. cytological examination and culture with sensitivity testing of the middle ear fluid is indicated when an effusion is suspected: • the myringotomy is done using an otoscope to guide a ⁄ " spinal needle through the tympanic membrane. ct or mri scans are superior to radiographs to confirm tympanic bulla disease, but the changes detected are not specific for infection. surgical exploration of the middle ear with cytological examination and culture with sensitivity testing is the only definitive diagnostic tool in middle-inner ear disease. idiopathic vestibular disease has an acute to per-acute onset in a cat that is otherwise healthy. there is no facial paresis/paralysis, decreased lacrimation, deafness or horner's syndrome because the disease is limited to the vestibular part of the inner ear. signs resolve over - weeks without treatment. neoplasia and middle ear polyps can be difficult to differentiate from middle-inner ear infection without an exploratory bulla osteotomy. if a polyp is visible in the external ear canal or in the oro-nasopharynx, a diagnosis of middle ear polyp is likely. if a bacterial middle-inner infection is suspected, early and aggressive treatment with antibiotics is best. if cul-ture and sensitivity are not available, a broad-spectrum antibiotic is chosen such as trimethoprim-sulfa, cephalosporin or a penicillinase-resistant penicillin (enrofloxacin). antibiotic treatment should be continued for - weeks. if otitis externa is present, the antibiotic is chosen based on the culture and sensitivity of the external ear canal. the otitis externa should be treated as well. systemic treatment is preferable if the tympanic membrane is perforated or cannot be visualized. avoid topical drugs that are toxic for the vestibular and/or auditory system (gentamycin, neomycin). selamectin (revolution®) or systemic ivermectin ( - μg/kg im, sc, po) is preferable as a miticide rather than topical treatment. cats with radiographic changes in the bullae are treated with surgical curettage of the tympanic bulla to allow drainage, followed by long-term antibiotherapy based on the culture and sensitivity obtained from the sample collected at time of surgery. if the culture is negative, a broad-spectrum antibiotic as listed above is administered for - weeks. if there is chronic or recurrent otitis externa, ablation of the external ear canal is indicated. in the presence of paralysis of the eyelids, artificial tears are unnecessary if tear secretion is normal as the spread of the tear film is taken over by the third eyelid. artificial tears should be administered - times daily in the affected eye, if the schirmer tear test is abnormal (< mm/minute), to avoid development of a corneal ulcer. facial paralysis and partial horner's syndrome often remain, although the severity is much improved. an ipsilateral mild deviation of the face occurs over time. the vestibular signs resolve in most cases, except for the head tilt, which frequently remains for the life of the animal. the cases managed medically may recur and require surgical curettage at later date. treat otitis externa effectively when it occurs. treat ear mites. • mild and slowly progressive head tilt. • +/respiratory signs. • +/signs of otitis externa. • +/facial paresis/paralysis, +/-horner's syndrome +/keratoconjunctivitis sicca, +/deafness. the pathogenesis is incompletely understood. the polyp originates from the middle ear cavity and is composed of inflammatory granulation tissue covered by respiratory epithelium. the instigating cause is not exactly known. some believe that it is congenital, to explain its frequency in very young cats. others postulate that it results from chronic inflammatory middle ear disease, secondary to upper respiratory infections. the respiratory infection would create abnormalities in the eustachian tube epithelium, resulting in poor middle ear ventilation and secondary inflammation. most cats with middle ear polyps have a bacterial infection. the most common bacterial isolates are pasteurella multocida, streptococcus, staphylococcus, bacteroides and pseudomonas. the polypoid growth has a tendency to exit the middle ear cavity. it can do so by rupturing the tympanic membrane and emerging into the external ear canal, causing characteristic signs of otitis externa, or it enters the eustachian tube to exit into the nasopharynx with subsequent upper respiratory signs. the frequency of each end location versus the other is unknown. the polypoid growth within the middle ear cavity may encroach on the inner ear leading to vestibular signs. typically occurs in young cats, but any age can be affected (mean age - years; range months to years). the most common signs associated with a middle ear polyp, which extends into the nasopharynx, are upper respiratory signs including noisy breathing, dyspnea with or without nasal discharge, sneezing or coughing and gagging. less often are signs characteristic of otitis externa. rarely a head tilt may be present. the head tilt may occur without other signs, or with upper respiratory signs, or with a visible external ear canal polyp. since the polyp originates within the middle ear cavity, the neurological structures of the middle ear may be affected causing facial paresis/paralysis, keratoconjunctivitis sicca and/or partial or complete horner's syndrome. these can also occur without other signs. a mild and transient head tilt may be the only abnormality on neurological examination. the concomitant presence of a fibrous mass in the external ear canal, or upper respiratory signs, is pathognomonic for middle ear polyp. the concomitant presence of facial paresis/paralysis, decreased lacrimation and horner's syndrome with a head tilt is indicative of middle and inner ear disease but is not diagnostic for the type of disease. if there are upper respiratory signs, visualization of the polyp in the nasopharynx under anesthesia is the most efficient way of reaching a diagnosis. • a glistening, red or pinkish mass filling the nasopharynx, or originating from the eustachian tube may be found. otoscopic examination, which may necessitate sedation/anesthesia to evaluate both ear canals and tympanic membranes should be performed. • aural inflammation may be present. • a pedunculated red, pink or grayish mass may be observed in the ear canal. the polyp may also affect hearing. presence of deafness is evaluated by doing baers. when a head tilt is present and there is facial paresis/ paralysis, decreased lacrimation or a horner's syndrome, bulla radiography is recommended to evaluate the middle ear cavities. an open mouth view is best to compare the density between the bullae. bulla radiography is not a sensitive tool. it may be normal despite the presence of uni-or bilateral polyps. ct and mri scans are superior in the diagnosis of middle ear cavities, and one of these scans should be performed if available. ct and mri scans are the most sensitive imaging techniques to reach a diagnosis of middle ear disease, but are not specific for the type of disease. the final diagnosis is obtained at the time of surgery. surgical exploration of the middle ear is the only reliable test to reach a diagnosis. cytology and culture and sensitivity of the curetted tissue should always be done. if the disease process involves the middle ear, otitis media-interna, middle ear polyp and neoplasia of the middle ear cavity cannot be differentiated without biopsies taken at the time of surgical bulla exploration. removal of the polyp from its origin through a ventral bulla osteotomy is the best therapeutic approach to avoid recurrence. removal by grasping the polyp and cutting it at the base from the nasopharyngeal or aural location leads to a high recurrence rate. horner's syndrome is a frequent complication of bulla osteotomy. it resolves in most cases in - weeks, but even if it remains, it does not alter the patient's quality of life. the head tilt, if present at onset, usually remains for the life of the patient. occasional transient vestibular ataxia is rarely reported as a surgical sequella of polyp removal. the cat should be treated for - weeks with an antibiotherapy based on the culture and sensitivity done at the time of surgery. minimizes the risk of recurrence. recurrence is frequent using oral or aural access to the polyp. the head tilt usually remains but the other vestibular signs (ataxia and nystagmus) usually resolve. if facial paralysis is present, ipsilateral deviation of the face ensues. if a horner's syndrome is present, it usually improves and the residual deficit (aniscoria) is of no clinical significance. • head tilt in a cat systemically ill for a few weeks. • non-specific systemic signs such as fever, weight loss and lethargy. • chronic progressive disease. • there may be neurological deficits other than a head tilt. see main references on page for details (the pyrexic cat). the causative virus is a macrophage-tropic mutant of the ubiquitous feline enteric coronavirus. the clinical disease results from an immune-mediated response of the host to macrophage-infected feline infectious peritonitis virus (fipv). the severity of the disease is based on host susceptibility and strain virulence. affected cats are usually less than years of age and from large multiple-cat households or breeders. the most common non-specific systemic signs are fever, weight loss and lethargy. thirty-five percent of the cats with fip have neurological signs. these vary with the lesion location. behavioral changes, head tremor, seizures, depression, compulsive walking and decreased menace are some of the signs that may be observed. clinically, the disease frequently appears to be focal, although this is not the case at postmortem. the head tilt is always associated with somnolence, with or without cerebellar signs, because the cerebellum is closely situated. chorioretinitis may be present (see the blind cat or cat with retinal lesions). the history of a young cat originating from a multicat household or breeder, with a protracted disease, vague systemic signs and neurological abnormalities raises the index of suspicion for fip. the cerebrospinal fluid analysis on its own is not sensitive for a diagnosis of fip. • cell counts and protein concentration can be within reference range especially if the disease is focal. typically, there is a moderate to severe pleocytosis, with mononuclear cells or neutrophils as the predominating cell type, and a marked increase in protein concentration. • protein concentration > g/l increases the likelihood of fip. mri scan of the brain is helpful, as frequently there is periventricular enhancement suggestive of ependymitis, and hydrocephalus with ventricular dilatation. the mri findings are more representative of the neuropathological extent of the disease than is the clinical presentation, which is often focal in nature. hematology and chemistry abnormalities are nonspecific except for a high serum total protein concentration, which is frequent. the anti-coronavirus igg titer in csf is consistently positive. the polymerase chain reaction (pcr) test detects the presence of feline coronaviruses but is not specific to fip coronavirus. immunohistochemistry and immunocytochemistry are techniques which appear promising for diagnosis of fip. they use monoclonal antibody targeted against feline coronavirus to demonstrate coronavirus within macrophages in tissue or effusions. the concentration of infected monocytes in csf fluid may make the test less sensitive for diagnosis of fip than if used for tissue sections. at this point in time, no single test is diagnostic for the neurological form of fip, but when the history, signalment, serum protein concentration, csf results, csf serology and mri findings are combined, an antemortem diagnosis can be reached. other inflammatory infectious diseases of the central nervous system may produce similar neurological abnormalities. however, no other central nervous system infections are typically presented with the gamut of abnormalities mentioned above. there is no effective treatment for fip. patients almost invariably die. therapy is based on supportive care. immunomodulating and antiviral agents seem promising in vitro but have not shown good results in cats. poor. most cats die of their disease from weeks to months after the onset of the neurological signs. since most cats with fip are from a multiple-cat household or breeder, adequate cleanliness is essential to prevent fecal-oral spread of virus. vaccination may be preventive. • otitis externa initially. • subsequent development of peripheral vestibular signs. • swollen face. • facial nerve paresis/paralysis and horner's syndrome are frequent. the most common tumor affecting the middle-inner ear is the squamous cell carcinoma. the tumor arises from the epithelial lining of the ear canal and is usually aggressive, invading the adjacent tissue (middle and inner ears) then the skull. squamous cell carcinomas of the ear canal are most commonly presented with neurological signs. ceruminous gland adenocarcinomas are also reported with some frequency in the middle-inner ear. middle-aged to older cats. initially, the signs relate to otitis externa. there is a more or less rapid progression depending on the tumor type, to cause peripheral vestibular signs, i.e., a head tilt, with or without nystagmus or vestibular ataxia. due to the invading nature of the squamous cell carcinoma and the ceruminous gland adenocarcinoma, the facial nerve and the sympathetic chain in the middle ear are involved leading to facial paralysis, decreased lacrimation and horner's syndrome. there is pain when the mouth is open, which may result from involvement of the temporo-mandibular joint, soft tissue pain, microfractures (pathological), bone pain from lysis or involvement of the bulla structures. the face may be swollen and firm on palpation. the history of an older cat presented with a rapid onset of neurological signs relating to the inner (vestibular signs and deafness) and middle ear (facial paralysis, decreased lacrimation and horner's syndrome) with pain upon jaw opening and a swollen face increases the index of suspicion. in most cases, bulla radiography is diagnostic for the presence of a destructive process. there is opacity in the tympanic bulla with sclerosis and lysis of the bone. depending on the tumor and how invasive it is, adjacent bony tissue such as the temporo-mandibular joint and the zygomatic arch may also be affected. as in most cases of middle-inner ear disease, if there is no swelling of the face, surgical exploration of the bulla is the only reliable approach to reach a definitive diagnosis. middle ear polyp is usually in young cats, but can be in older cats. typically, the progression is slower. the polyp may be visible in the ear canal or oro-nasopharynx. there is no pain, and no swelling of the face. aggressive excision including ear canal ablation and lateral bulla osteotomy is the treatment of choice for malignant ear canal tumors. if excision is incomplete, radiation therapy can be a useful adjunct to surgery. prognosis is guarded because of the invasive nature of the tumor and advanced stage of the disease by the time neurologic signs are present. cats with ceruminous adenocarcinoma have a % -year survival rate following aggressive ear ablation and bulla osteotomy compared to a % -year survival after conservative surgical resection. the prognosis is more guarded for squamous cell carcinoma. • acute onset of peripheral vestibular signs that develop immediately or in the hours following an ear flush. the exact pathogenesis is unknown, but several mechanisms have been postulated to be involved. an ear flush that leads to such consequences is usually done under anesthesia or heavy sedation. if the signs develop immediately after the flush, it is possibly due to: ( ) a change in the temperature of the endolymph; ( ) flooding of the inner ear through a perforated tympanic membrane; or ( ) a toxic effect on the vestibu-lar receptors by the product used to flush the ear (e.g. chlorhexidine, quaternary ammonium compounds). hours following an ear flush, an inner ear infection is suspected. bacteria may have been introduced through a perforated tympanic membrane at the time of the flush. the ear flush may not be vigorous for this to occur because the tympanic membrane may have already been ruptured secondary to an otitis externa. alternatively, topical (e.g. gentamycin-containing ear drops) or systemic treatment administered after the ear flush may be toxic to the vestibular receptors. acute onset of peripheral vestibular signs in the hours following an ear flush. the flush may have been an elective procedure or therapeutic for a severe otitis externa. diagnosis is based on the history of acute vestibular signs following a recent ear flush. idiopathic vestibular syndrome cannot be differentiated from the iatrogenic cause if the signs appear - days following the flush. whenever acute vestibular signs appear to be associated with an ear flush, a broad-spectrum antibiotic such as trimethoprim-sulfa, a cephalosporine or amoxicillin should be administered for - weeks. if the onset was immediate and due to a change in the temperature or flooding of the inner ear, then the signs may disappear within a few hours. if the signs are the result of toxicity, the head tilt may remain. if the signs appeared - days later, most vestibular signs resolve except for the head tilt, which usually persists for the life of the animal. if an elective ear flush is to be performed under anesthesia or with heavy sedation, ensure the tympanic membrane is intact prior to the procedure. if the tympanic membrane cannot be visualized, use only sterile normal saline, as most ear-cleaning solutions contain components that are potentially ototoxic, such as propylene glycol, salicylic acid, malic acid, lactic acid detergents and dioctols. perform ear flush with extreme care. cleansing the ear with rubber bulb syringes, plastic syringes or waterjet appliances may rupture the tympanic membrane. if the ear needs cleaning in the consultation room, avoid using solutions that are acidic or potentially ototoxic. instead of removing the debris manually, let the animal shake its head and dislodge the debris itself after the medication had been put and massaged into the external ear canal. • lethargic cat with a head tilt, +/ataxia, +/nystagmus. typically, the cat has usually been ill for a few weeks prior to the development of the neurological signs. the systemic signs may have been vague such as decreased appetite, weight loss, lethargy, or signs may relate to a specific system such as the upper respiratory tract, the skin or a combination of both. chorioretinitis may be present (see the blind cat or cat with retinal lesions). the development of central nervous system signs in a cat systemically ill for a few weeks or having upper respiratory tract signs and/or draining skin lesions should raise the index of suspicion for this infection. a latex agglutination test measuring the cryptococcal polysaccharide capsular antigen can be performed on the serum, cerebrospinal fluid or urine. however, negative results do not exclude the possibility of disease. the csf analysis is often diagnostic because the characteristic budding yeast forms are visible. this is best seen using india ink, but new methylene blue, diff quick, and gram's stain preparation are adequate. sometimes organisms are not evident, but grow when the csf is cultured. • the csf inflammatory response associated with cryptococcus neoformans varies greatly. the csf may be normal to grossly abnormal with wbc counts ranging from to > cells × /l ( - . × /l). • in mild inflammation, lymphocytes and monocytes predominate. in severe inflammatory responses, neutrophils and occasionally eosinophils are present. • the protein concentration varies from mildly to markedly increased (> g/l). if the disease is suspected, but the organism is not visible in the csf, csf culture and/or serology may provide a definitive diagnosis. cats may be simultaneously positive for feline leukemia virus (felv) and/or feline immunodeficiency virus (fiv), increasing their susceptibility to infection. cryptococcosis cannot be differentiated from feline infectious peritonitis in cats presented with a protracted illness and vague systemic signs on history, physical and neurological examination alone. if cryptococcus organisms are not visible, culture or serology may be necessary to establish the diagnosis. fluconazole is the drug of choice because of its broad antifungal spectrum, its meningeal penetration even in the absence of inflammation, and the fact that serious side effects are uncommon. the recommended dose is mg/kg/day divided twice daily for a period that extends beyond the resolution of all signs by - months. the drug should be given with food. ketoconazole should not be used. it does not penetrate the blood-brain barrier effectively and is hepatotoxic. see page (the cat with signs of chronic nasal disease) for further details on treatment of cryptococcosis. • bilateral vestibular signs, i.e., a characteristic ventro-flexion of the neck associated with a crouched body posture and reluctance to move. • bilateral pupillary dilatation with poor light reflexes. thiamine is a co-enzyme in the oxidative metabolism for energy production in the central nervous system. thiamine deficiency typically produces lesions (polioencephalomalacia) in the brainstem gray matter and more specifically in the vestibular, oculomotor and lateral geniculate nuclei. focal, bilaterally symmetric hemorrhages are present in affected areas. the deficiency occurs in cats that are fed an uncooked all-fish diet (due to the thiaminase content of the viscera), diets entirely made of cooked meat (where the thiamine is destroyed by heating), poor-quality thiamine-deficient commercial diets, or commercial food stored for long periods of time or in excessively hot conditions. in addition, the meat preserver sodium metabisulfite releases sulfur dioxide, which destroys thiamine. pet mince meat containing this preserver is thiamine-deficient, and even when mixed with other food may have sufficient preserver to destroy all dietary thiamine. anorexia in a sick cat especially associated with polydipsia and polyuria or fluid diuresis may precipitate thiamine deficiency and complicate the primary illness. lethargy and decreased appetite, sometimes with increased salivation, occur after - weeks on a deficient diet. the cat has an inability or a reluctance to walk and therefore may appear weak. this is associated with a characteristic rigid ventro-flexion of the neck, a crouched body posture and loss of righting responses. there is bilateral mydriasis with poor light reflexes from the involvement of the oculomotor and geniculate lateral nuclei. physiologic nystagmus is poor to absent. brief episodes of opisthotonus or neck ventroflexing and muscle rigidity may appear like seizures. bradycardia, and marked sinus arrhythmia may also occur. if untreated, the cat becomes comatose and dies. the time of death varies from week to a few weeks to months depending on the health status of the animal and the amount of thiamine in the diet. diagnosis is based on a combination of a history that the cat has been quiet and anorexic, signs of a characteristic posture with poor to absent physiologic nystagmus indicative of bilateral vestibular disease and documentation of a thiamine-deficient diet (usually large quantities of uncooked fish). rapid response (within hours) to treatment confirms the diagnosis. in bilateral idiopathic vestibular syndrome, the cat is healthy just prior to the development of the clinical signs. thiamine-deficient cats are typically lethargic and anorexic. injectable thiamine at - mg intramuscularly. for supplementation, give - mg/cat/day po to a maximum of mg/cat/day. prognosis is good if treatment is given early when the only clinical signs are bilateral vestibular signs. prognosis is poor in the late stage of the disease when the animal's consciousness is significantly altered. • siamese, himalayan and white tiger cats. • pendular nystagmus, i.e., the phase of the nystagmus is equal on both sides. • present from birth. typically, the nystagmus has a rapid, short and oscillatory motion that is equal bilaterally. it is observed especially when the cat is fixing its gaze. the defect is within the visual pathways and not the vestibular pathways. no obvious visual impairment is present. the nystagmus is always congenital and is evident in the first few weeks of life. it occurs primarily in siamese, himalayan and white tiger cats. medial strabismus is usually simultaneously present. diagnosis is based on the characteristic oscillatory nystagmus. • altered consciousness and head tilt after a recent accident. • +/-facial nerve paresis/paralysis and horner's syndrome. • +/-signs of brainstem involvement such as proprioceptive deficits. sudden onset of head tilt associated with somnolence or stupor following a road accident or other trauma that caused a fracture at the base of the skull affecting the petrosal and ethmoid bones. facial paresis/paralysis and/or horner's syndrome may be present if there is hemorrhage in the middle ear. if the injury is primarily intracranial, facial paresis/paralysis and/or ipsilateral proprioceptive deficits may be present. on otoscopic examination, blood may be observed in the ear canal. history of a road accident. brain auditory-evoked responses (baer) may be helpful in localizing the lesion to mainly a peripheral or central location. survey radiographs of the skull are difficult to interpret because of the juxtaposition of multiple structures. • unilateral or bilateral peripheral vestibular signs, following the use of drugs that are toxic for the vestibular receptors. • concomitant deafness is frequent. multiple drugs cause damage to the vestibular and/or auditory receptors separately or simultaneously. aminoglycosides especially streptomycin, chloramphenicol, chlorhexidine, cisplatin, furosemide, salicylates and ceruminolytic agents are a few of a long list of agents that may cause damage to these receptors. toxicity occurs following systemic or topical administration. the vestibular signs are peripheral and can be unilateral, or bilateral. the signs may develop acutely following exposure to a high dose or after prolonged administration (> days). cats with renal dysfunction are at risk because decreased renal excretion of many drugs results in higher plasma concentrations. the vestibular signs may disappear following discontinuation of the drug. the history of use of a drug potentially toxic for the vestibular receptors, topically or systemically. the vestibular signs may appear immediately following an ear flush, or following topical treatment with a toxic medication, in a cat that has a perforated tympanic membrane. avoid using any medication in the external ear canal if the tympanic membrane is perforated or cannot be visualized. this includes ceruminolytic agents and detergents. • unilateral or bilateral vestibular signs present at birth or developing in the first weeks of life. • siamese, burmese and tonkinese cats. • head tilt and tipping or rolling. reported in siamese, burmese and tonkinese cats. usually unilateral vestibular signs, but can be bilateral. head tilt with tipping, rolling or falling developing at birth or in the first weeks of life. the head tilt can be marked and may vary with time. the clinical signs are non-progressive and decrease in severity with time, but usually persist for life. diagnosis is based on characteristic signs in a kitten from a susceptible breed. • southeastern united states. • unilateral peripheral vestibular signs following the ingestion of a blue-tailed lizard. • the cat also salivates, vomits, trembles and is irritable. acute onset of unilateral peripheral vestibular disturbance following ingestion of the blue-tailed lizard. the syndrome has not been well substantiated. varying degrees of vestibular signs associated with salivation, vomiting, trembling and irritability. death may occur. acute onset of unilateral peripheral vestibular signs in a cat living in southeastern united states with access to lizards. signs are indistinguishable from the idiopathic vestibular syndrome although salivation, vomiting and trembling are less common with the idiopathic disease. review of idiopathic feline vestibular syndrome in cats results of ventral bulla osteotomy for treatment of middle ear polyps in cats diagnostic features of clinical neurologic feline infectious peritonitis a comparison of the rostrocaudal open mouth and rostro v entro-caudodorsal oblique radiographic views for imaging fluid in the feline tympanic bulla jr (ed) consultations in feline medicine, rd edn evaluation of dogs and cats with tumors of the ear canal: cases ( - ) reference intervals for feline cerebrospinal fluid: cell counts and cytologic features reference intervals for feline cerebrospinal fluid: biochemical and serologic variables, igg concentration, and electrophoretic fractionation a comparison of radiographic versus surgical diagnosis of otitis media key: cord- - mzr s authors: kanazawa, nobuo title: c-type lectin receptors date: - - journal: immunology of the skin doi: . / - - - - _ sha: doc_id: cord_uid: mzr s c-type lectins, originally defined as proteins binding carbohydrates in a ca( +)-dependent manner, form a large family containing soluble and membrane-bound proteins. among them, those expressed on phagocytes and working as pathogen pattern-recognition receptors were designated as c-type lectin receptors (clrs), in accordance with toll-like receptors (tlrs), nod-like receptors (nlrs), and rig-i–like receptors (rlrs). most of the genes for clrs are clustered in human chromosome close to the natural killer gene complex. similar to the killer lectin-like receptors whose genes are clustered in this complex, most of the clrs induce activating or regulatory signal cascades in response to distinct pathogen- or self-derived components, through the immunoreceptor tyrosine-based activating or inhibitory motif, respectively. in this chapter, some representative clrs are picked up and their structural features leading to the functional consequences are discussed, especially on the signaling cascades and pathogen interactions, including some impacts on cutaneous pathophysiology. these clrs should provide targets to develop effective vaccination and therapeutics for distinct infectious and autoimmune/inflammatory diseases. sugar-binding capacity, whose amino acid sequence shows incomplete conservation of the common residues in the crd [ ] . it should be noted that the term ctld is also used for the broad definition containing all ctls irrespective of carbohydrate recognition [ ] . based on the domain structure, vertebrate ctls were divided into (i-vii) or, more recently, (i-xvii) groups including the traditional seven groups [ , ] . among them, group i (lecticans: aggrecan, brevican, versican, and neurocan), iii (collectins: mannose-binding protein and pulmonary surfactant proteins), and vii (reg proteins) are soluble, whereas group ii (asialoglycoprotein receptor family), iv (selectins), v (natural killer receptor family), and vi (mannose receptor family) are bound to the cell membrane ( fig. . ). the natural killer (nk) receptor family (group v) contains a series of type ii transmembrane proteins with a single ctld expressed on nk cells, whose genes are clustered in the nk gene complex (nkc) in chromosome [ ] . they are collectively called killer lectin-like receptors (klrs) and are equipped to regulate the killer activity of their expressing cells. among them, heterodimers of cd and activating/inhibitory nkg receptors, as well as the activating nkg d homodimer, recognize major histocompatibility complex (mhc) class i or related molecules, to distinguish target cells appropriately. the ctlds included in these receptors have no capacity to bind carbohydrate. phagocytes, such as macrophages and dendritic cells (dcs), also express various kinds of ctl receptors on their cell surface for antigen capture. the mannose receptor (mr) family (group vi) contains type i transmembrane proteins with multiple ctlds, such as the mr (cd ) and dec- (cd ) [ ] . they commonly consist of an n-terminal cysteine-rich domain and a fibronectin type ii domain as well as eight or ten ctlds. in contrast, the asialoglycoprotein receptor family (group ii) contains type ii transmembrane proteins with a single ctld, such as dc-specific icam -grabbing nonintegrin (dc-sign, cd ), dectin- , dectin- , dc immunoreceptor (dcir), and macrophage-inducible c-type lectin (mincle) [ , , , , ] . notably, expression of some receptors is specific and they can be markers for distinct dc subsets; langerin (cd ) on langerhans cells (lcs) and blood dc antigen (bdca)- (cd ) on plasmacytoid dcs (pdcs) [ , ] . most of the genes for these group ii receptors are also clustered in or next to the nkc ( fig. . ) [ ] . in contrast to klrs, most receptors of both groups contain fully conserved crds binding carbohydrates, and are considered to work as pattern recognition receptors (prrs) recognizing various pathogen-associated molecular patterns (pamps). especially, dectin- is critical for recognition of fungal β-glucan and has a collaborative effect with toll-like receptor (tlr) on yeast-induced activation signals, whereas dc-sign is involved in the recognition of a variety of microorganisms including viruses, bacteria, fungi, and parasites, and suppresses the immune activation signals induced by these pathogens [ , , ] . for these pathogen-recognizing ctl receptors, "clrs" have been designated analogous to other prrs, such as tlrs, nod-like receptors (nlrs), and rig-i-like receptors (rlrs) [ ] . notably, most of the clrs are linked to the signaling cascade through the immunoreceptor tyrosine-based activating or inhibitory motif (itam or itim, respectively), which was first identified in paired klrs [ , ] . [ , ] . in addition, some of them are also able to bind endogenous self-molecules and are involved in some pathophysiological aspects. for example, dc-sign have a role in cellular trafficking, dc-sign and dectin- can mediate an interaction between dc and t cells, and dectin- is involved in ultraviolet (uv)-induced tolerance [ , , , ] . furthermore, dectin- and dcir are involved in the development of autoimmunity in mice, and snps in clec a and clec e encoding dcir and mincle, respectively, are reportedly associated with autoimmune arthritis [ , , , , ] . in this chapter, some representative clrs are picked up and their structural features leading to the functional consequences are discussed, especially on the signaling cascades and pathogen interactions, including some impacts on cutaneous pathophysiology. regarding the nomenclature, genes for the clrs have formally been designated by the symbol "clec (c-type lectin domain containing)", which is approved by the human genome organization gene nomenclature committee (hgnc) (http://www.genenames.org/genefamilies/clec). langerhans cells (lcs) are immature dcs resident in the epidermis (see chap. ). as sentinels, they form a honeycomb-like network and spread dendrites to the skin surface through epidermal tight junctions. once they capture and internalize antigens, they modulate expression of adhesion molecules and chemokine receptors to migrate through dermal lymphatics into the draining lymph node and there, after maturation, they present processed antigens to the specific t cells. lcs have been defined electron-microscopically by the presence of birbeck granules (bgs) with a "tennis-racket"-like appearance, and immunohistochemically with bg-specific lag antibody [ , ] . langerin has been designated as the first surface lc marker recognized by the lc-specific monoclonal antibody (mab) dcgm , and subsequently isolated by the expression cloning strategy [ , ] . recent structural analysis has revealed that langerin stands as a stable homotrimer via the coiled-coil interaction of its neck region [ ] . langerin has no signaling motif other than the proline-rich domain for internalization in its intracellular domain. however, the presence of such an intracellular domain provides a possibility that langerin interacts with other proteins to modulate or mobilize cellular machanisms required for defense against pathogens, such as interfering viral tlr signaling [ ] . as predicted by the presence of epn (glutamate-proline-asparagine) motif in its crd, langerin can bind mannose, fucose, and n-acetylglucosamine structures and this binding leads to internalization and transfer of the antigen into bgs, which consist of superimposed and zippered plasma membrane [ , ] . langerin deficiency by the w r point mutation in humans abolished the bg formation without apparent immunodeficiency [ ] . notably, langerin expression is not specific in lcs but is also detected in a part of dermal dcs in mice [ , , ] . by analysis of langerin-dtr mice, in which langerin-expressing cells were transiently depleted by diphteria toxin administration, distinct ontogeny of langerin þ dermal dcs from migrating lcs have been revealed. langerin recognizes human immunodeficiency virus (hiv)- through highmannose structures in its envelope glycoprotein gp [ ] . in contrast to dermal/submucosal dcs, which transmit hiv- to t cells through dc-sign, epidermal/mucosal lcs clear hiv- and prevent its infection to t cells through langerin without inflammation. immunoelectron-microscopic analysis showed that hiv- was captured by langerin and internalized into bgs, to be finally degraded. therefore, langerin on epidermal lcs is considered a natural barrier to hiv- infection. as inhibition of langerin caused hiv- infection in lcs and its subsequent transmission to t cells, anti-hiv therapeutics should not interfere with langerin expression or functions. langerin also recognizes candida and malassezia species through cell wall mannose structures and β-glucans [ , ] . mycobacterium species are also recognized and the langerin binding to their components such as lipoarabinomannan (lam) leads to its internalization into bgs and efficient loading to cd a for presentation to t cells [ ] . therefore, bgs might be an organelle specialized to load glycolipid antigens to cd a. although dc-sign has been designated by its pivotal role on dc-t cell interaction, it is well-known as a prr for a variety of pathogens. indeed, it was first cloned from placenta as a hiv- gp -binding protein independent of cd [ ] . dc-sign stands as a tetramer and contains dileucine (ll) and yxxl internalization motif in its intracellular domain [ ] . its binding to gp was inhibited by mannan, d-mannose, and l-fucose and, after binding, gp was immediately internalized [ ] . dc-sign does not allow hiv- entry into dcs, but promotes its efficient infection in trans of cells expressing cd and chemokine receptors [ ] . dc-sign also functions for capturing dengue virus, a mosquito-mediated flavivirus that causes hemorrhagic fever, and is indispensable for its infection to dcs [ , ] . interestingly, the cytoplasmic tail of dc-sign is not essential for, but enhances dengue virus infection to dcs. dc-sign is also involved in infection of other viruses such as ebola, cytomegaloviruses, human c-type hepatitis virus (hcv), measles, and severe acute respiratory syndrome (sars) coronavirus, bacteria such as helicobacter pylori and klebsiela pneumonae, fungi such as candida albicans, and parasites such as leishmania pifanoi and schistosoma mansoni [ , ] . dc-sign further works for capturing and internalizing mycobacterium species (m. tuberculosis and bcg) through the mannose-capped mycobacterial cell wall lam (manlam) [ ] . in immature dcs, internalized mycobacteria or manlam are transported into lysosome, where they are colocalized with lamp . notably, targeting dc-sign with soluble manlam inhibits mycobacteria-or lipopolysaccharide (lps)-induced dc maturation and induces il- production, suggesting that dc-sign signaling interferes with the tlr signaling to cause an antiinflammatory effect. such an effect has been shown to be mediated by a serine/ threonine kinase raf signaling, which does not require the cytoplasmic yxxl motif of dc-sign but involves formation of a distinct protein complex (lsp , ksr, cnk, larg, and rhoa) with dc-sign [ ] . raf signaling does not induce dc activation but modulates tlr-mediated nuclear factor (nf)-κb activation, resulting in increased and prolonged transcription of il- and some other cytokines. by analysis of bronchoalveolar lavage cells, alveolar macrophages from tuberculosis patients specifically expressed dc-sign, and its expression was induced by m. tuberculosis independently of tlr , il- , and il- [ ] . indeed, accumulation of m. tuberculosis in dc-sign-expressing alveolar macrophages was immunohistochemically shown. by the case-control studies, a single nucleotide polymorphism (snp) in the promoter region of dc-sign, À c has reportedly been involved in a risk for parenteral hiv- infection in european americans [ ] . it has also been reported that À snp is associated with severity of dengue diseases, including severe dengue fever and dengue hemorrhagic fever [ ] . in tuberculosis, association of À snp has further been reported by analysis of a south african population living in the areas showing a high incidence rate of tuberculosis [ ] . À g and À a are associated with protection against tuberculosis, and their combination showed a higher frequency in non-african populations, possibly as a result of genetic adaptation to a longer history of exposure to tuberculosis. recently, it has been reported that the anti-inflammatory capacity of intravenous immunoglobulin (ig) is recapitulated in mice expressing human dc-sign, by transfer of bone-marrow-derived macrophages or dcs treated with the fc fragment of igg containing glycans terminating in sialic acids (igg-sfc) [ ] . treatment with igg-sfc induced il- production in macrophages/dcs and caused expansion of il- -producing basophils in their administered mice, which in turn increased expression of an inhibitory fc receptor, fcγriib, on effector macrophages. such a novel igg-sfc-induced dc-sign-mediated th /regulatory pathway might be an endogenous mechanism for immunological homeostasis and provide a line of evidence for therapeutic effects of intravenous ig administration on various inflammatory diseases such as kawasaki disease, autoimmune dermatomyositis/vasculitis/neuropathy, myasthenia gravis, pemphigus vulgaris, and severe drug eruptions [ ] . dectin- was originally reported as a dc-specific lectin that had been cloned from a cdna library of murine xs cells after subtraction of j macrophage library [ ] . at first, dectin- was considered a costimulatory molecule because soluble recombinant dectin- stimulated t-cell proliferation. however, subsequently, dectin- was identified as a β-glucan receptor on macrophages with a different binding site from that for t cells [ ] . dectin- is capable of binding a major yeast cell wall component zymosan and a variety of β-glucans from fungi and plants, however, it does not recognize carbohydrates with other linkages. although the clec a gene is located within the nkc, dectin- expression is not observed in nk cells, but in myeloid cells including monocytes, dc, macrophages, and neutrophils [ , ] . dectin- has a cooperative role with tlr on zymosan-induced macrophage activation [ ] . dectin- and tlr are colocalized on the surface of zymosantreated macrophages and the zymosan-induced activation signals require dectin- as well as tlr . notably, the yxxl hemi-itam (hemitam) in the cytoplasmic tail of dectin- is shown to be essential for this activation. upon zymosan-binding, this hemitam directly recruits and activates spleen tyrosine kinase (syk), probably dependent on the receptor dimerization, which induces nf-κb activation through interaction with caspase recruitment domain (card) nine coupled with the bcl -malt complex [ , ] . recently, protein kinase (pk) c-δ was identified to link syk activation and card signaling [ ] . this syk activation also induces il- β secretion, through pro-il- β transcription and processing, mediated by the card -bcl -malt and the malt -asc-caspase complex, respectively [ ] . the latter complex is called noncanonical caspase -dependent inflammasome. additionally, zymosan-induced dectin- -dependent signals reportedly include phospholipase (pl) cγ -dependent ca þ influx leading to activation of mitogenactivated protein kinases (mapks) as well as nf-κb [ ] . furthermore, nlrp inflammasome is activated through dectin- -syk signaling in mycobacterium abscessus (mabc)-activated human macrophages, whereas both tlr and dectin- are required for mabc-induced il- β secretion [ ] . a cytoplasmic scaffold protein p /sqstm mediates this dectin -syk-nlrp pathway, at least partly through production of reactive oxygen species (ros). finally, raf signaling, which regulates nf-κb activation independently of syk, is also involved in the dectin- activation, as observed in case of the dc-sign activation [ ] . among syk-induced cytokines, expressions of il- β, il- , il- , and il- are enhanced but the il- expression is impaired by raf signaling, and, therefore, th responses become dominant by dectin- activation. notably, although both soluble monomer and particulate polymer of β-glucan can bind dectin- , only the polymer can activate dectin- signaling by forming a so-called "phagocytic synapse," by not only clustering the receptor but also excluding inhibitory receptors with tyrosine phosphatase activities, cd and cd [ ] . interestingly, zap -deficient skg mice reportedly developed autoimmune arthritis only in the presence of dectin- agonist, indicating a role of dectin- signaling as a trigger for manifestation of subclinical autoimmune phenotype [ ] . furthermore, zymosan-mediated dectin- signaling induced il- production and exacerbated autoimmunity through th /th responses in tlr -deficient mice, whereas zymosan-mediated tlr signaling activated foxp þ regulatory t (treg) cells through induction of il- and raldh , an essential enzyme in retinoic acid metabolism [ ] . thus, antifungal dectin- signaling is considered rather aggressive and regulated by the simultaneous tlr signaling to prevent autoimmunity. notably, dectn- -deficient mice were shown to be impaired in zymosan recognition and increased in susceptibility to candida albicans infection, whereas, in another report, they were rather more susceptible to pneumocystis carini infection [ , ] . in , a nonsense mutation (y x) in the clec a gene was identified in a dutch family with chronic mucocutaneous candidiasis (omim# ) [ ] . the mutation was homozygous in three sisters with vulvovaginal candidiasis and/or onychomycosis and heterozygous in their parents with onychomycosis. the mutated dectin- , which lacks c-terminal amino acids, showed defective β-glucan binding and induction of cytokine productions after stimulation with β-glucan or candida albicans. however, fungal phagocytosis and killing were shown to be unaffected and, therefore, invasive fungal infection was not observed in the patients. the allele frequency of the y x variant is reportedly . in the healthy dutch population and identified carriers are all heterozygous, and the variant has never been found in the chinese population [ ] . in addition, the same variant was reported to be a risk factor of increased susceptibility to invasive aspergillosis in hematopoietic stem cell transplantation, with the highest risk in cases where the variant is present in both donors and recipients [ ] . dectin- was reported as another dc-specific lectin that had been isolated by subtraction cloning from murine xs cells, whereas its human homologue was originally cloned by the database search of human chromosomal sequences homologous to the exon sequences of murine dc immunoactivating receptor (dcar) [ , ] . dcar was identified as the putative activating partner of dc immunoreceptor (dcir), and the overall amino acid sequence of murine dcar is highly homologous ( % identity) to murine dectin- [ ] . although the gene for dectin- (clec a) is clustered with those for dcir (clec a) and murine dcar (clec b) and located next to the nkc, dectin- expression is observed in various myeloid cells including monocytes, macrophages, neutrophils, and dc subsets such as lcs [ ] . indeed, murine dcar is the first reported dc/macrophage clr that associates with an yxxlx - yxxl itam-bearing adaptor molecule, the γ chain of fcr (fcrγ), whereas the direct human orthologue of dcar is absent and the functional significance of this molecule is still unknown [ ] . similar to murine dcar, dectin- with a short cytoplasmic domain associates with fcrγ, a type i membrane protein with a short ectodomain that contains itam intracellularly and provides a docking site for syk [ ] . dectin- stimulation activates the card -bcl -malt pathway through pkcδ and induces various cytokines and chemokines mediated by nf-κb activation [ , ] . additionally, dectin- signaling activates a mapks pathway through plcγ , as is the case with dectin- [ ] . most importantly, through malt -mediated selective c-rel nf-κb subunit activation, dectin- , but not dectin- , signaling leads to th responses [ ] . as predicted by the presence of epn motif in its crd, dectin- binds structures with high mannose [ ] . actually, a number of pathogens including fungi such as candida albicans, saccharomyces cerevisiae, aspergilus fumigatus, cryptococcus neoformans, paracoccidioides brasiliensis, microsporum audouinii, and trichophyton rubrum, as well as parasites such as histoplasma capsulatum and schistosoma mansoni, and mycobacterium tuberculosis are recognized by dectin- [ ] . notably, the yeast and hyphal forms of candida albicans induce different responses through dectin- [ , ] . most impressively, it has been shown by analyses of dectin- -deficient mice that dectin- is essential for protective th response to intravenous infection with candida albicans [ ] . by stimulation of murine bone-marrow-derived dcs with soluble schistosomal egg antigens following pretreatment with tlr ligands, nlrp -dependent il- β secretion was increased through dectin- -fcrγ-syk signaling and ros production [ ] . furthermore, house dust mites are also recognized by dectin- and induce eicosanoid production, leading to the th responses [ , ] . interestingly, murine dectin- is reportedly involved in ultraviolet (uv)-induced immune regulation [ ] . although injection of soluble ectodomain of dectin- (sdec ) has no effect on induction of contact hypersensitivity (chs), it inhibits uv-mediated suppression of chs and tolerance induction. sdec -binding cd þ cd þ t cells were shown to produce il- , il- , and tgfβ upon hapten stimulation and to transfer the uv-induced tolerance. dcir was originally identified as a molecule homologous to hepatic asialoglycoprotein receptors and macrophage lectin, by selecting sequences in dc libraries following the database search of human genomic sequences using the tblastn algorithm [ ] . dcir is unique among known dc/macrophage clrs because of the presence of i/vxxyxxv/l immunoreceptor tyrosine-based inhibitory motif (itim) in its cytoplasmic portion. as the itim-related signaling capacity of dcir, sh domain-containing tyrosine phosphatase (shp)- and shp- can be recruited to human dcir upon stimulation [ , ] . furthermore, using murine b cells expressing a chimeric receptor containing a cytoplasmic portion of dcir and ectodomain of fcγriib (dcir-fcr), coligation of dcir-fcr and b cell receptor (bcr) showed dcir-itim-dependent inhibitory effects on bcr-mediated calcium influx and tyrosine phosphorylation of cellular proteins [ ] . on the other hand, murine dcar was identified as a molecule whose crd shows % amino acid identity with that of dcir [ ] . dcar interacts with fcrγ to stand efficiently on the cell surface and ligation of the chimeric dcar-fcr induced activation signals into its expressing cells dependent on the itam in fcrγ. thus, dcir and dcar are equipped as structurally and functionally paired immunoreceptors in mice, although the biological significance of their pairing has been undefined [ ] . among in vitro generated human dc subsets, a higher expression of dcir was observed in cd þ -derived dermal-type dcs than in cd a þ -derived lc-type dcs [ ] . in monocyte-derived dcs (modcs), its expression did not change during differentiation from monocytes to immature dc, but remarkably decreased after stimulation with lps or cd ligand. notably, dcir expression was detected not only in myeloid dcs, but also in monocytes, macrophages, b cells, neutrophils, and plasmacytoid dcs (pdcs) [ , ] . by reverse transcription (rt)-pcr, the only reliable method to distinguish expression of dcir and dcar, dcar was rather limitedly observed, strongly in spleen and lung and weakly in skin and lymph nodes, whereas dcir was detected ubiquitously. both increased during the bonemarrow-derived dc development, but increase of the dcar expression preceded that of dcir [ ] . notably, murine dc-specific mab d recognized dcir , which can be a specific marker for cd -dcs located in the red pulp and marginal zone of the spleen [ ] . despite the presence of an eps (glutamate-proline-serine residues) motif conserved in dcir and dcar, which is postulated to recognize galactose residues [ ] , their recognizing carbohydrates have not been clarified. notably, dcir reportedly participates in capturing hiv- and its transmission to cd þ t cells [ ] . hiv- captured by surface dcir is stored as intact virion and replicates de novo intracellularly in infected dcs and, subsequently, is transmitted to cd þ t cells through induced dcir expression on them, where hiv- replication is enhanced [ , ] . these processes have been shown to be mediated by the itim signaling. furthermore, dcir can act as an attachment factor for hcv on pdcs [ ] . notably, the binding of hcv to dcir on pdcs induced specific inhibition of tlr -induced ifnα production, in contrast to the case of dcir activation on dcs by anti-dcir monoclonal antibody (mab), which induced specific inhibition of tlr -induced il- and tnfα production [ , , ] . as predicted by the presence of a tyrosine-based internalization motif overlapping with the itim, targeting dcir on dcs with antigen-conjugated abs induced clathrin-dependent receptor internalization, its trafficking into lysosomal compartments, and efficient antigen cross-presentation to cd þ t cells [ , ] . in contrast, targeting murine dcir induced class ii presentation to cd þ t cells, including stimulation of foxp þ treg cells [ , ] . the most impressive results on in vivo significance of dcir have been obtained by analyses of dcir -deficient mice. they spontaneously developed sialadenitis and enthesisis and exacerbated collagen-induced arthritis with marked expansion of dcs [ ] . furthermore, in humans, by the case-control study, a snp in the clec a gene was shown to be associated with the susceptibility to anticyclic citrulinated peptide ab (acpa)-negative rheumatoid arthritis (ra) [ , ] . among bdcas, which were designated for a series of antigens recognized by mabs raised against human cd þ lineageblood dcs, bdca- is unique by its restricted expression on cd c À cd (il- receptor) bright pdcs in fresh human blood without expression on immature modcs or cd þ -derived dcs [ ] . although murine dectin- was once proposed as the murine homologue of human bdca- , its proper murine orthologue is still undefined and murine dcar, which lacks a human orthologue, is rather considered closely related to human bdca- on structural and functional aspects [ ] . in dc subpopulations, bdca- expression was detected strongly in pdcs and very weakly in immature modcs, and its expression level was downregulated after culture with il- in pdcs and after maturation with lps in modcs [ , , ] . in tonsil, bdca- expression was observed on cd þ pdcs that were primarily present in t-cell areas but not in the germinal center. similar to dcar and dectin- , bdca- associates with fcrγ and cross-linking of bdca- on pdcs results in intracellular ca þ mobilization depending on src-family protein tyrosine kinases and on tyrosine phosphorylation of cellular proteins [ , ] . however, unlike other fcrγ-coupled clrs, bdca- signaling does not lead to nf-κb activation through the syk-pkcδ-card pathway, but passes through the syk-blnk-plcγ pathway [ ] . indeed, contrary to its predicted immunoactivating property, ligation of bdca- suppressed the production of type i ifn and the secretion of tnf-related apoptosis-inducing ligand (trail) in pdcs induced by various tlr agonists [ , ] . furthermore, inhibition of type i ifn production enhances il- production in pdcs to polarize immune responses towards th [ ] . bdca- -mediated suppression of type i ifn production by cpg oligonucleotides or by influenza virus suggests that viruses target bdca- for immune escape. on the other hand, the same phenomenon induced by antidouble-stranded dna mab plus plasmid dna or by sera from patients of systemic lupus erythematosus (sle) suggests that targeting bdca- is beneficial for treatment of sle. as predicted by the presence of the triglutamate (eee) late endosomal sorting motif in the cytoplasmic portion of bdca- , it was reported that targeting bdca- with mab induced internalization of the ab complexes through phosphorylation of actin, tubulin, and clathrin, resulting in antigen presentation to cd þ t cells [ ] . however, more recently, bdca- signaling has rather been shown to inhibit antigen processing and presentation to t cells by pdcs, and, furthermore, to suppress the induction of costimulatory molecules on cpg-stimulated pdcs [ ] . although bdca- contains the epn motif in its crd similar to dectin- , it has recently been reported that bdca- recognizes asialo-galactosyl-oligosaccharides with terminal β-galactose residues and that pdcs bind to subsets of cd þ monocytes, modcs, and several tumor cell lines via recognition of asialo-galactosyloligosaccharides by bdca- [ ] . accordingly, these cells can introduce the suppression signal into their binding pdcs through bdca- , especially on type i ifn secretion. furthermore, bdca- can bind gp of hiv- and hcv glycoprotein e , both of which can also suppress the type i ifn secretion by pdcs [ , ] . mincle was originally identified from murine peritoneal macrophages by subtraction cloning as a molecule specifically induced by activation of nf-il transcription factor [ ] . mincle is expressed on monocytes, macrophages, neutrophils, myeloid dcs, and subsets of b cells, but not on t cells, pdcs, and nk cells [ ] . mincle associates with fcrγ dependently on the positively charged arginine residue in the transmembrane portion of mincle, as is the case with murine dcar but is not the case with dectin- [ ] . mincle-fcrγ activation induces signaling through syk-pkcδ-card -nf-κb and mapk pathways, resulting in the induction of various cytokines and chemokines. as predicted by the presence of the epn motif in its crd, mincle can bind carbohydrates containing α-mannose or other structures, and fungal, mycobacterial, and necrotic cell components have been identified as ligands for mincle [ , , ] . the candida albicans cell wall component was one of the first identified ligands and mincle was shown to mediate the protective antifungal immunity [ ] . malassezia is also recognized by mincle and induces cytokine and chemokine production and inflammatory cell recruitment through mincle [ ] . mincle also recognizes fonsecaea pedrosoi, which was revealed to cause chromomycosis in the skin due to insufficient costimulation of tlr on mincle signaling [ ] . mincle also recognizes mycobacterial trehalose dimycolate (tdm), which is an immunomodulatory glycolipid component and has been called mycobacterial cord factor, and mediates tdm or synthetic trehalose dibehenate-induced inflammatory responses including production of cytokines and nitric oxide, nlrp inflammasome activation, th and th activation, and granuloma formation [ , , , , ] . however, in mincle-deficient mice, variable inflammatory responses were observed dependent on the species of infected mycobacteria, probably because they contained different combinations of pamps [ , ] . interestingly, mycobacteriainduced granuloma formation was unaffected irrespective of mycobacteria species. also interesting is that the significance of mincle expression was reportedly different in monocytes and neutrophils. mincle expression was associated with cytokine production in monocytes, whereas it was rather associated with fungal uptake and killing in neutrophils [ ] . furthermore, the expression of mincle was reciprocally detected between monocytes and neutrophils in individuals. mincle also works as a sensor for necrosis by recognizing spliceosomeassociated protein (sap) released from necrotic cells [ ] . notably, mincle has been shown to use a different binding site for sap from that used for carbohydrate recognition. although it is predictable that mincle is involved in the regulation of autoimmunity, an autoimmune phenotype of mincle-deficient mice has never been reported. in contrast, it has recently been reported that a snp in the clec e gene is associated with acpa-positive ra [ ] . as described in this chapter, critical roles of the representative clrs have been revealed in the responses for viral, fungal, and mycobacterial infections, as well as in the maintenance of immunological homeostasis (table . ). as a surface barrier to environmental stimuli including invasive pathogens, skin provides the first battlefield and dermatologists should prepare more and better ways to combat pathogens and keep homeostasis. clrs should certainly provide targets to develop effective vaccination and therapeutics for distinct infectious and autoimmune/ inflammatory diseases. intravenous gammaglobulin suppresses inflammation through a novel th pathway involvement of dectin- in ultraviolet radiation-induced tolerance molecular and genomic characterization of human dlec, a novel member of the c-type lectin receptor gene family preferentially expressed on monocyte-derived dendritic cells cloning of a second dendritic cell-associated c-type lectin (dectin- ) and its alternatively spliced isoforms identification of a novel, dendritic cellassociated molecule, dectin- , by subtractive cdna cloning promoter variation in the dc-signencoding cd is associated with tuberculosis dectin- recognition of house dust mite triggers cysteinyl leukotriene generation by dendritic cells dectin- mediates th immunity through the generation of cysteinyl leukotrienes apcs express dcir, a novel c-type lectin surface receptor containing an immunoreceptor tyrosine-based inhibitory motif role of mincle in alveolar macrophagedependent innate immunity against mycobacterial infections in mice card mediates dectin- -induced iκbα kinase ubiquitination leading to activation of nf-κb in response to stimulation by the hyphal form of candida albicans electron microscopy of melanocytes a new receptor for beta-glucans identification of a novel population of langerin þ dendritic cells bdca /fc epsilon ri gamma complex signals through a novel bcr-like pathway in human plasmacytoid dendritic cells dectin- y x polymorphism associates with susceptibility to invasive aspergillosis in hematopoietic transplantation through impairment of both recipient-and donor-dependent mechanism of antifungal immunity sequence and expression of a membraneassociated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp c-type lectin langerin is a beta-glucan receptor on human langerhans cells that recognizes opportunistic and pathogenic fungi langerin is a natural barrier to hiv- transmission by langerhans cells two distinct classes of carbohydrate-recognition domains in animal lectins evolution of ca þ -dependent animal lectins drickamer k ( ) c-type lectin-like domains differential antigen processing by dendritic cell subsets in vivo bdca- , bdca- , and bdca- : three markers for distinct subsets of dendritic cells in human peripheral blood bdca- , a novel plasmacytoid dendritic cellspecific type ii c-type lectin, mediates antigen capture and is a potent inhibitor of interferon α/β induction plasmacytoid dendritic cells: from specific surface markers to specific cellular functions the mannose receptor family trimeric structure of langerin human dectin- deficiency and mucocutaneous fungal infections hcv glycoprotein e is a novel bdca- ligand and acts as in inhibitor of ifn production by plasmacytoid dendritic cells dcir deficiency causes development of autoimmune diseases in mice due to excess expansion of dendritic cells collaborative induction of inflammatory responses by dectin- and toll-like receptor identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells dc-sign-icam- interaction mediates dendritic cell trafficking mycobacteria target dc-sign to suppress dendritic cell function sef-and nonself-recognition by c-type lectins on dendritic cells immunoreceptor tyrosine-based inhibition motif-bearing receptors regulate the immunoreceptor tyrosine-based activation motif-induced activation of immune competent cells blood-derived dermal langerin þ dendritic cells survey the skin in the steady state activation of the innate immune receptor dectin- upon formation of a 'phagocytic synapse' phospholipase cγ (plcγ ) is key component in dectin- signaling pathway, mediating anti-fungal innate immune responses c-type lectin dc-sign modulates tolllike receptor signaling via raf- kinase-dependent acetylation of transcription factor nf-kappab dectin- directs t helper cell differentiation by controlling noncanonical nf-κb activation through raf- and syk selective c-rel activation via malt controls anti-fungal t(h)- immunity by dectin- and dectin- dectin- is an extracellular pathogen sensor for the induction and processing of il- -beta via a noncanonical caspase- -inflammasome card controls a non-tlr signaling pathway for innate anti-fungal immunity a replication study confirms the association of dendritic cell immunoreceptor (dcir) polymorphisms with acpa-negative ra in a large asian cohort cloning and characterization of a novel itim containing lectin-like immunoreceptor llir and its two transmembrane region deletion variants langerhans cells utilizes cd a and langerin to efficiently present nonpeptide antigens to t cells direct recognition of the mycobacterial glycolipid, treharose dimycolate, by c-type lectin mincle bdca- signaling inhibits tlr -agonistinduced plasmacytoid dendritic cell activation and antigen presentation dcir acts as an inhibitory receptor depending on its immunoreceptor tyrosine-based inhibitory motif dendritic cell immunoactivating receptor, a novel c-type lectin immunoreceptor, acts as an activating receptor through association with fc receptor γ chain molecular cloning of human dectin- dendritic cell immunoreceptors: c-type lectin receptors for pattern recognition and signaling on antigen-presenting cells letterer-siwe disease: immunopathologic study with a new monoclonal antibody the dectin- family of c-type lectin-like receptors: an update animal lectins: a historical introduction and overview cross-priming cd þ t cells by targeting antigens to human dendritic cells through dcir the c-type lectin surface receptor dcir acts as a new attachment factor for hiv- in dendritic cells and contributes to trans-and cis-infection pathways hiv- induces dcir expression in cd þ t cells dcir-mediated enhancement of hiv- infection requires the itim-associated signal transduction pathway mycobacterium abscessus activates the nlrp inflammasome via dectin- -syk and p /sqstm neutrophils promote mycobacterial treharose dimycolate-induced lung inflammation via the mincle pathway association of arthritis with a gene complex encoding c-type lectin-like receptors toll-like receptor -dependent induction of vitamin a-metabolizing enzymes in dendritic cells promotes t regulatory responses and inhibits autoimmunity hiv- go inhibits tlr -mediated activation and ifn-α secretion in plasmacytoid dendritic cells association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection a novel lps-inducible c-type lectin is a transcriptional target of nf-il in macrophages the carbohydrate-recognition domain of dectin- is a c-type lectin with specificity for high mannose targeting dcir on human plasmacytoid dendritic cells results in antigen presentation and inhibits ifn-alpha production dcir is endocytosed into human dendritic cells and inhibits tlr -mediated cytokine production dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infectionof human dendritic cells by mosquito-cell-derived dengue viruses myeloid c-type lectin receptors in pathogen recognition and host defense the dermis contains langerin þ dendritic cells that develop and function independently of epidermal langerhans cells engagement of bdca- blocks trailmediated cytotoxic activity of plasmacytoid dendritic cells human c-type lectin domain family , member c (clec c/bdca- /cd ) is a receptor for asialo-galactosyl-oligosaccharides granulocyte macrophage-colony stimulating factor reduces the affinity of shp- for the itim of clecsf in neutrophils: a new mechanism of action for shp- schistosoma mansoni triggers dectin- , which activates the nlrp inflammasome and alters adaptive immune responses dectin- is a syk-coupled pattern recognition receptor crucial for th responses to fungal infection bdca- ) signals in plasmacytoid dendritic cells via a bcr-like signalosome involving syk, slp and plcγ syk-dependent cytokine induction by dectin- reveals a novel pattern recognition pathway for c type lectins dectin- is required for host defense against pneumocystis carinii but not against candida albicans dectin- recognition of alpha-mannans and induction of th cell differentiation is essential for host defense against candida albicans dectin- is a pattern recognition receptor for fungi that couples with the fc receptor gamma chain to induce innate immune responses a variant in the cd promoter is associated with severity of dengue disease mincle is essential for recognition and adjuvanticity of the mycobacterial cord factor and its synthetic analog treharose-dibehenate intravenous immunoglobulin therapy: how does igg modulate the immune system? the mycobacterial cord factor adjuvant analogue treharose- , -dibehenate (tdb) activates the nlrp inflammasome dc-sign; a related gene, dc-signr; and cd form a cluster on p restoration of pattern recognition receptor costimulation to treat chromoblastomycosis, a chronic fungal infection of the skin characterization of carbohydrate recognition by langerin, a c-type lectin of langerhans cells syk kinase-coupled c-type lectin receptors engage protein kinase c-δ to elicit card adaptor-mediated innate immunity dectin- is required for beta-glucan recognition and control of fungal infection dc-sign (cd ) mediates dengue virus infection of human dendritic cells dual specificity of langerin to sulfated and mannosylated glycans via a single c-type carbohydrate recognition domain dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells the monoclonal antibody dcgm recognizes langerin, a protein specific of langerhans cells, and is rapidly internalized from the cell surface langerin, a novel c-type lectin specific to langerhans cells, is an endocytic receptor that induces the formation of birbeck granules langerin functions as an antiviral receptor on langerhans cells dc-sign: escape mechanism for pathogens conservation of structural features reveals the existence of a large family of inhibitory cell surface receptors and noninhibitory/activatory counterparts a lack of birbeck granules in langerhans cells is associated with a naturally occurring point mutation in the human langerin gene mincle polarizes human monocyte and neutrophi responses to candida albicans drickamer k ( ) the c-type lectin superfamily in the immune system the macrophage-inducible c-type lectin, mincle, is an essential component of the innate immune response to candida albicans adjuvanticity of a synthetic cord factor analogue for subunit mycobacterium tuberculosis vaccination requires fcrgamma-syk-card -dependent innate immune activation macrophage-inducible c-type lectin is associated with anti-cyclic citrullinated peptide antibodies-positive rheumatoid arthritis in men phospholipase cγ is critical for dectin- -mediated ca þ flux and cytokine production in dendritic cells mincle is an itam-coupled activating receptor that senses damaged cells c-type lectin mincle is an activating receptor for pathogenic fungus cd þ cd þ splenic dendritic cells are specialized to induce foxp þ regulatory t cells immune functions encoded by the natural killer gene complex identification of a human homologue of the dendritic cell-associated c-type lectin- , dectin- a role for fungal beta-glucans and their receptor dectin- in the induction of autoimmune arthritis in genetically susceptible mice the c-type lectin-like domain superfamily key: cord- -gxbrgy g authors: lee, choongho title: griffithsin, a highly potent broad-spectrum antiviral lectin from red algae: from discovery to clinical application date: - - journal: mar drugs doi: . /md sha: doc_id: cord_uid: gxbrgy g virus entry into a susceptible host cell is the first step in the formation of all viral diseases. controlling viral infections by disrupting viral entry is advantageous for antibody-mediated neutralization by the host’s immune system and as a preventive and therapeutic antiviral strategy. recently, several plant-derived carbohydrate-binding proteins (lectins) have emerged as a new class of antiviral biologics by taking advantage of a unique glycosylation pattern only found on the surface of viruses. in particular, a red algae-derived griffithsin (grft) protein has demonstrated superior in vitro and in vivo antiviral activity with minimum host toxicity against a variety of clinically relevant, enveloped viruses. this review examines the structural characteristics of grft, focusing on its carbohydrate-binding capability. its in vitro antiviral profiles against human immunodeficiency virus (hiv) are also discussed followed by a description of the results from a combination study using anti-hiv drugs. the results of several studies regarding its novel antiviral mechanism of action are provided in conjunction with an explanation of viral resistance profiles to grft. in addition, its in vitro and in vivo host toxicity profiles are summarized with its pharmacokinetic behavior using in vivo efficacy study results. also, a large-scale production and formulation strategy, as well as a drug delivery strategy, for grft as a new class of broad-spectrum microbicides is discussed. finally, results from two ongoing clinical studies examining grft’s effects on viruses are presented. every virus starts its life cycle by entering a susceptible host cell. the host cell-targeting ability of a virus is mainly determined by the presence of appropriate host cell receptors which are engaged by a virus. for successful host cell entry, enveloped viruses have evolved a diverse array of envelope glycoproteins on their membrane with different receptor association capabilities. the best-characterized example of this viral glycoprotein-host cell receptor relationship might be the interaction between the human immunodeficiency virus (hiv) glycoprotein gp and the human t lymphocyte cd molecule. due to its essential role in the overall virus life cycle, viral entry has been an attractive target for both vaccine and antiviral drug development with the goal of disrupting the binding of viral glycoproteins to host cell receptors. in addition, heavy and unique glycosylation patterns found only on viral glycoproteins provide another level of virus specificity, which could increase the selectivity of virus-targeting therapeutics and prophylactics. in this regard, the use of carbohydrate-binding proteins, collectively called "lectins", has been explored as a new therapeutic antiviral strategy. recently discovered virus-targeting lectins include banana lectin [ ] , cyanovirin [ ] , microvirin [ ] , scytovirin [ ] , and griffithsin (grft) [ ] . among them, grft has been studied extensively for the development of corresponding secondary -sheet structures are marked with yellow arrows based on the crystal structure of a grft dimer with mannose at a resolution of . Å [ ] . tyrosine and aspartate residues in three carbohydrate-binding domains, which are essential for mannose-binding, are noted with blue and orange arrows, respectively. wildtype grft has a non-standard amino acid at position , but it is replaced by alanine (colored in red) in recombinant grft. the sequence display was generated by the rcsb protein data bank website (www.rcsb.org). the amino acid sequence of griffithsin (grft). corresponding secondary β-sheet structures are marked with yellow arrows based on the crystal structure of a grft dimer with mannose at a resolution of . Å [ ] . tyrosine and aspartate residues in three carbohydrate-binding domains, which are essential for mannose-binding, are noted with blue and orange arrows, respectively. wild-type grft has a non-standard amino acid at position , but it is replaced by alanine (colored in red) in recombinant grft. the sequence display was generated by the rcsb protein data bank website (www.rcsb.org). marine drug , x, x; doi: www.mdpi.com/journal/marinedrugs resolution of . Å [ ] . tyrosine and aspartate residues in three carbohydrate-binding domains, which are essential for mannose-binding, are noted with blue and orange arrows, respectively. wildtype grft has a non-standard amino acid at position , but it is replaced by alanine (colored in red) in recombinant grft. the sequence display was generated by the rcsb protein data bank website (www.rcsb.org). grft is a carbohydrate-binding protein made of amino acids and is . kda in size [ ] . it has a unique amino acid residue at position which does not match any of the standard amino acids [ ] (figure ). it was initially isolated from an aqueous extract of the red algae griffithsia sp. collected from waters off the shores of new zealand. researchers at the u.s. national cancer institute first reported its potent cytoprotective activity against hiv- in t-lymphoblastoid cells [ ] . five research papers reported structural results on grft by using either x-ray crystallography or nuclear magnetic resonance (nmr) techniques (table ) [ ] [ ] [ ] [ ] [ ] . in terms of structural classification, grft is a jacalin-related lectin harboring three repeats of an antiparallel four-stranded -sheet with a triangular prism shape ( figure ) [ ] . it is called a domain-swapped dimer because two -strands from one protein forming the dimer display domain-exchanging properties with the same two strands of its counterpart [ ] . it has three identical carbohydrate-binding sites (located within residues - , - , and - ) on each monomer with three conserved glycine-rich repeats (ggsgg) [ ] . it is estimated that as many as high-mannose oligosaccharides are present on one hiv- gp protein. therefore, grft's multivalent interactions with gp via these three carbohydrate-binding domains seem to be essential for its high-affinity and anti-hiv- potency at figure . the crystal structure of a grft dimer with six mannoses at a resolution of . Å [ ] . each grft monomer is presented in either red or blue. six bound mannose molecules are shown with ball and stick models. this image was created with pdb id and associated publication, ngl viewer (as rose et al. ( ) ngl viewer: web-based molecular graphics for large complexes. bioinformatics doi: . /bioinformatics/bty ), and rcsb pdb. grft is a carbohydrate-binding protein made of amino acids and is . kda in size [ ] . it has a unique amino acid residue at position which does not match any of the standard amino acids [ ] ( figure ). it was initially isolated from an aqueous extract of the red algae griffithsia sp. collected from waters off the shores of new zealand. researchers at the u.s. national cancer institute first reported its potent cytoprotective activity against hiv- in t-lymphoblastoid cells [ ] . five research papers reported structural results on grft by using either x-ray crystallography or nuclear magnetic resonance (nmr) techniques (table ) [ ] [ ] [ ] [ ] [ ] . in terms of structural classification, grft is a jacalin-related lectin harboring three repeats of an antiparallel four-stranded β-sheet with a triangular prism shape ( figure ) [ ] . it is called a domain-swapped dimer because two β-strands from one protein forming the dimer display domain-exchanging properties with the same two β-strands of its counterpart [ ] . it has three identical carbohydrate-binding sites (located within residues - , - , and - ) on each monomer with three conserved glycine-rich repeats (ggsgg) [ ] . it is estimated that as many as high-mannose oligosaccharides are present on one hiv- gp protein. therefore, grft's multivalent interactions with gp via these three carbohydrate-binding domains seem to be essential for its high-affinity and anti-hiv- potency at low concentrations (picomolar range) [ ] . in particular, three aspartate residues in these carbohydrate-binding sites (asp , asp , and asp ) play a critical role in the interaction of grft with high-mannose type oligosaccharides such as man glcnac , which is composed of nine mannose molecules and two n-acetyl glucosamines [ ] . this was supported by a study demonstrating that grft point mutations (d a, d a, or d a) partially inhibit its ability to bind to gp , and a grft mutant with all three mutations loses almost all of its binding capacity [ ] . in addition, tyrosine residues such as tyr , tyr , and tyr are necessary for grft to bind to carbohydrates [ ] . however, the apparent disparity between the gp -binding ability and hiv- inhibitory potency for these grft variants indicates the existence of another antiviral mechanism beyond simple gp -grft binding [ ] . with regard to carbohydrate-binding specificity, all six grft dimer carbohydrate-binding sites can be occupied by mannose, glucose, n-acetyl glucosamine, and - α-mannobiose with similar affinities [ ] . to determine whether grft must form a dimer to exert its antiviral activity, a monomeric form of grft (mgrft) was created by inserting either two or four amino acids at its dimerization interface. mgrft possesses greatly reduced antiviral activity against hiv- in spite of its comparable association with high-mannose oligosaccharides, since mgrft possesses all three carbohydrate-binding sites [ ] . this suggests that the intact dimeric form of grft is required to efficiently disrupt hiv- infectivity [ ] . table . five structural studies involving grft. the structure resolution of grft and its major characteristics are presented. grft alone or with mannose . and . a domain-swapped dimer, a jacalin-related lectin with a β-prism motif, and three identical mannose-binding sites on each monomer [ ] grft with glucose or n-acetylglucosamine - . and - . all six monosaccharide binding sites of grft are occupied by both glucose and n-acetylglucosamine with a mode of binding similar to that of mannose [ ] grft with - α-mannobiose or maltose . and . the binding of - α-mannobiose is similar to that of mannose and the binding of maltose is weaker than that of mannose [ ] monomeric grft - . reduced activity against hiv- due to a loss of multivalent interaction and the binding of a monomeric grft to two different nona-mannosides [ ] grft with a disrupted carbohydrate-binding site nmr study reduced binding to mannose and a weaker correlation between anti-hiv- activity and gp binding [ ] several studies have described the in vitro anti-hiv activities and cytotoxicity of grft (table ) . in their publication, they observed that grft inhibited soluble gp from binding to cd receptor-expressing cells [ ] . cell-to-cell fusion and transmission of hiv- infection were also blocked by grft at similar concentrations [ ] . in addition, the coadministration of monosaccharides such as glucose, mannose, and n-acetyl glucosamine hindered glycosylation-dependent binding of grft to soluble gp [ ]. in parallel with this observation, emau et al. also demonstrated that grft could block the infectivity of cxcr -and ccr -tropic hiv viruses at picomolar concentrations. they also confirmed the long-term stability of grft in a cervical/vaginal lavage [ ] . in order to harness the hiv-inactivating power of grft, a new peptide called grifonin- was designed based on the three carbohydrate-binding amino acid sequences of grft. its sub-micromolar anti-hiv activity was confirmed using an in vitro tzm-bl cell line and p gag antigen release assays [ ] . in addition to its ability to suppress hiv- transmission in cd + t-lymphocytes, grft also subverted dc-sign (dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin)-mediated hiv capture [ ] . mechanistically, a binding competition between grft and gp for dc-sign was proposed as a potential mode of action for grft-mediated inhibition of hiv- capture by dc-sign [ ] . corresponding to this dc-sign-dependent antiviral activity, grft also potently inhibited giant cell formation between persistently hiv-infected t cells and noninfected cd + target t cells. this led to the suppression of hiv transmission, cd + t-cell destruction, and ultimately viral replication through the dc-sign mediated pathway [ ] . this efficient blockage of the binding of dc-sign to immobilized gp by grft was further confirmed [ ] . this observation was in harmony with the grft-mediated expulsion of gp from the gp /dc-sign complex [ ] . interestingly, this highly potent inhibition of dc-sign-mediated capture and transmission by grft was markedly impaired when grft was mutated in one of its three carbohydrate-binding sites (d a, d a, or d a) [ ] , further implicating its carbohydrate-binding sites as critical determinants for anti-hiv- activity. regarding its cytotoxicity, two studies demonstrated that the cc concentration for grft was several hundred nanomolar [ , ], thousands of times higher than its reported antiviral concentrations. four studies have described the combination effects of grft with either current anti-hiv drugs or potential therapeutics under development (table ) [ , , , ] . grft showed synergistic antiviral activity with tenofovir, maraviroc (a ccr antagonist), and enfuvirtide (a gp fusion peptide inhibitor) [ ] . the different glycosylation patterns on the viral envelope of clade b and clade c gp had no observable effect on their synergistic antiviral action [ ] . covalently linking grft to the gp -binding peptide c also exhibited a potency several-fold greater than that of grft alone in inhibiting hiv env-mediated fusion in a ccr -tropic cell-cell fusion assay [ ] . in line with this observation, all grft/antiretroviral drug (entry inhibitors, reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors) combinations displayed either synergistic or additive effects in inhibiting cell-cell fusion and protected against target cd + t cell destruction [ ] . grft/antiretroviral combinations also potently inhibited short-term viral replication in t-cells via dc-sign-mediated transmission [ ] . combinations of grft and other carbohydrate-binding agents (cbas) including hippeastrum hybrid agglutinin, galanthus nivalis agglutinin, a mannose-specific monoclonal antibody (mab) ( g ), microvirin, and banana lectin also showed synergistic activity against hiv- , hiv- , and even against certain cba-resistant hiv- strains [ ] . none of the cbas competed with each other's glycan-binding sites on gp since they have distinct binding patterns on the gp envelope [ ] . in addition to antiviral synergy, gp -grft complexes showed higher immunogenicity than the individual proteins per se. this suggests that removing the mannose moieties on monomeric gp improves the humoral immune response to this protein [ ] . six different antiviral mechanisms of action against hiv- by grft have been proposed (table ) [ , , [ ] [ ] [ ] [ ] . a carbohydrate binding-dependent antiviral mechanism of grft was explored by using hiv-specific neutralizing monoclonal antibodies (mabs) such as g , d, b , and b [ , ] . grft preferentially inhibited gp from binding to the g mab, which targets n-linked glycans at positions , , and on gp . this suggests an overlapping binding specificity with the g mab [ ] . in addition, grft increased the interaction between gp and the d mab, which recognizes a cd -induced epitope [ ] . grft also enhanced the binding of hiv- to plates coated with b and b mabs [ ] . this indicates that the binding of grft to gp triggers the exposure of the cd -binding site on gp . in particular, the glycan at position , which shields the cd binding domain of gp , is also involved in the grft-mediated binding enhancements and the neutralization synergy between grft and b [ ] . in addition, a synergy between grft and b was also exhibited when hiv- isolates became more sensitive to neutralization upon increasing hiv- binding of grft to b and b mabs [ ] . together, these data suggest that grft-mediated blockage of a post-cd receptor binding event, such as the coreceptor binding with gp , might be another plausible mechanism through which grft inhibits hiv- infection [ ] . since the glycans on hiv- gp play an important role in shielding neutralization-sensitive epitopes from antibody recognition [ ] , disruption of the mannose molecules on gp by grft may also increase antibody-dependent neutralization of hiv- particles. although the multivalent interaction of grft with high-mannose oligosaccharides is believed to account for most of its picomolar antiviral potency, the looser correlation between gp -binding ability and hiv inhibitory potency for the binding site mutants of grft suggests the possibility of another unknown antiviral mechanism of grft that is not based on simple gp binding [ ] . according to an isothermal titration calorimetry binding study, grft bound to glucose and n-acetyl glucosamine in a similar fashion to that of mannose, demonstrating its flexible specificity in binding carbohydrates [ ] . this binding flexibility might have an implication for its broad antiviral spectrum. to study the potential role of the grft dimer in the suppression of hiv- infectivity, either two or four amino acids were inserted at the dimerization interface of grft. this resulted in a monomeric form of grft (mgrft) with greatly reduced antiviral activity against hiv- . these results further emphasize the importance of multivalent interactions between dimeric grft and oligosaccharides present on hiv envelope glycoproteins for the successful cross-linking and aggregation of viral particles [ ] . interestingly, an obligate dimer of grft with a peptide linker between the two subunits altered the structure of gp by exposing the cd binding site. however, grft-linker-grft with mutated carbohydrate-binding sites largely lost this ability [ ] . on the other hand, the glycan-specific dc-sign receptor binds the virus and mediates its transfer to cd + cells [ ] . in this regard, grft's ability to partially block gp from binding to human dc-sign [ ] and its potent inhibition of dc-sign-dependent transfer of hiv- [ ] could synergize with its antiviral action by blocking viral entry. to maximize the antiviral synergy caused by grft multimerization, tandem repeats of mgrft (mgrft tandemers) were engineered. they displayed picomolar-level antiviral activity in whole-cell anti-hiv assays [ ] . however, since mgrft tandemers could not aggregate hiv virions, moulaei et al. suggested the intra-virion crosslinking of hiv envelope glycoproteins may be more integral to their antiviral activity [ ] . inter-virion aggregation or clustering of hiv- gp on the viral membrane was found to be related to neutralization potency [ ] . table . grft anti-hiv- mechanisms of action. ref. exposure of the cd binding site of gp through the glycan at position and blockage of coreceptor binding step [ ] inhibition of mannose-binding to gp and improvement of the humoral immune response to gp [ ] inhibition of gp binding to dc-sign and expulsion of gp from the gp /dc-sign complex [ ] alteration of gp structure through the exposure of the cd binding site [ ] intra-virion crosslinking of gp [ ] inter-virion aggregation or clustering of gp [ ] five studies have characterized viral resistance profiles caused by chronic grft treatment (table ) [ , , , , ] . since the antiviral activity of grft mainly depends on disrupting the biological functions of multiple mannose molecules on viral glycoproteins, a reduction in the glycosylation levels of a target protein can lead to grft resistance. for example, the and glycosylation sites are involved in grft-induced hiv- neutralization since a concomitant lack of glycans at both positions resulted in natural grft resistance [ ] . conversely, introducing glycosylation sites at n and n in hiv- clade c virus increased grft antiviral potency [ ] . in line with these observations, deglycosylation at position or decreased the sensitivity of a single transmitter/founder hiv env to grft [ ] . since n and n are grft-specific, high-mannose, n-linked glycosylation sites on gp , a single deglycosylation at n or n in primary or t-cell-line-adapted hiv- isolates also resulted in marked resistance to grft [ ] . furthermore, glycosylation sites at positions , , , and located in the c region and , , and in the c -c region were also implicated in grft resistance [ ] . a loss of glycosylation sites on gp as well as a rearrangement of glycans in v also led to hiv- subtype c resistance against grft [ ] . in the case of dc-sign-dependent antiviral activity of grft, the effects of extra glycosylation seem to be dependent on the location of the glycosylation. for example, the introduction of the glycosylation site abolished hiv- sensitivity to lectin's ability to inhibit binding to dc-sign and virus transfer [ ] . however, the addition of the glycosylation site enhanced the inhibition of dc-sign-dependent hiv- transfer by grft [ ] . given the overlapping nature of the binding specificity displayed by grft and neutralizing antibodies against hiv- , grft resistance could also affect hiv- sensitivity to antibody-dependent neutralization. the ubiquitous nature of glycosylated proteins in the body raises concerns that grft may cause toxicity by interacting with glycosylated host proteins. for this reason, the effects of grft on host cells and animals have been studied extensively. according to five previous reports, grft does not exhibit any toxicity at its active antiviral concentrations (table ) [ , [ ] [ ] [ ] [ ] [ ] . grft induced only minimal changes in the secretion of inflammatory cytokines and chemokines by epithelial cells and human peripheral blood mononuclear cells (pbmcs) [ ] . in addition, it had no measurable effect on cell viability or the levels of t-cell activation markers [ ] . grft treatment induced only minimal alterations in the gene expression profile of human ectocervical cells [ ] . it also caused no significant cell death, mitogenicity, activation, or cytokine release in mouse pbmcs [ ] . no obvious changes were observed in animal fitness, blood chemistry, or complete blood count parameters in grft-treated mice [ ] . interestingly, the pbmc-bound form of grft was still able to maintain its antiviral activity, raising the potential of its versatile in vivo antiviral activity [ ] . chronic intravaginal or systemic administration of mg/kg of grft was also non-toxic in mice [ ] . grft, when administered in gel form, was not associated with any changes in the rectal proteome [ ] . an increased abundance of two common and beneficial microbial taxa after grft treatment was due to placebo formulations and not to grft, itself [ ] . this association between the placebo gel and changes in the rectal proteome and microbiota indicates the need to alter the components of the placebo gel in future studies [ ] . grft was also well tolerated after subcutaneous administration in guinea pig and mouse models [ ] . in addition, grft was found to be non-irritating and non-inflammatory in human cervical explants and in an in vivo rabbit vaginal irritation model [ ] . in this study, no mitogenic activity was reported in cultured human lymphocytes treated with grft [ ] . however, following grft treatment, reversible splenomegaly was observed with the activation of certain spleen b and t cells [ ] . this grft-associated increase in spleen and liver mass was also noted in another study [ ] . therefore, an immune response elicited by grft treatment should be controlled in order to avoid potential grft immune-related toxicity [ ] . table . toxicity studies of grft in various cell and animal models. dose tested effects of grft ref. no effect on the production of proinflammatory cytokines and chemokines. no vaginal irritation in rabbits. [ ] human cervical epithelial cells, cervicovaginal cells, and pbmcs up to µm minimal changes in secretion of inflammatory cytokines and chemokines no measurable effect on cell viability and t-cell activation markers. [ ] guinea pig balb/c mice single mg/kg and daily subcutaneous injections of mg/kg minimal overall toxicity. well tolerated. increase in spleen and liver mass. [ ] rhesus macaques intravaginal . % gels no change in rental proteome or microbiome [ ] three studies were conducted to examine the in vivo antiviral efficacy and pharmacokinetic behavior of grft using various small animal models (table ) [ , , ] . subcutaneous injections of grft into guinea pigs and mice were very well tolerated, resulting in the accumulation of grft up to relevant therapeutic concentrations [ ] . the serum from grft-treated animals was found to retain antiviral activity against hiv- -enveloped pseudoviruses in a cell-based neutralization assay [ ] . in addition, active grft, which is capable of neutralizing hiv-env pseudoviruses, was also detected in rat fecal extracts after chronic oral dosing [ ] . the in vivo efficacy of grft was also demonstrated in the humanized bone marrow-liver-thymus mice which were protected from vaginal infection with hiv- after being treated with recombinant c. crescentus expressing grft [ ] . however, grft was not orally bioavailable even after chronic treatment [ ] . table . in vivo anti-hiv- activity of grft in animal models. dose tested effects of grft ref. guinea pig balb/c mice single mg/kg and daily subcutaneous injections of mg/kg. retention of antiviral activity in serum [ ] sprague dawley (sd) rats a single dose of mg/ml intravenously or subcutaneously. ten mg/kg doses for days. neutralization activity found in fecal extracts [ ] humanized bone marrow-liver-thymus mice grft-expressing recombinant c. crescentus intravaginally. protection against hiv- infection [ ] the clinical application of protein drugs requires a cost-effective large-scale production procedure to meet the high volume needed in a clinical setting. for efficient grft production, seven different production methods have been developed and optimized (table ) [ , [ ] [ ] [ ] [ ] [ ] [ ] . giomarelli et al. used a fermenter and a rich, auto-inducing medium, which led to an approximately -fold increase in the total amount of grft per liter with approximately % of the protein expressed in the soluble fraction [ ] . o'keefe et al. were able to produce grft in multigram quantities by using nicotiana benthamiana plants transduced with a tobacco mosaic virus vector expressing grft [ ] . hahn et al. employed a simple spraying method to introduce agrobacterium vectors into nicotiana plants in the presence of a surfactant as a substitute for vacuum inoculation [ ] . they found that recombinant grft is stable during the storage of plant biomass as silage, which is suitable for mass production of cost-sensitive products [ ] . fuqua et al., however, developed a simplified grft purification method [ ] . they achieved > % pure grft by generating the initial green juice extract in ph buffer, heating the extract to • c, incubating it overnight with a bentonite mgcl mixture, and purifying it via chromatography [ ] . vamvaka et al. successfully produced grft by using the endosperm of transgenic rice plants (oryza sativa) [ ] . they found that both crude and pure grft had potent anti-hiv activity, and the crude extracts were not toxic in human cell lines [ ] . this suggests that crude grft with minimal processing could be administered to reduce costs associated with purification [ ] . petrova et al. expressed grft in the probiotic strains lactobacillus rhamnosus gg and l. rhamnosus gr- for gastrointestinal and vaginal mucosal delivery, respectively [ ] . hoelscher et al. used stably transformed tobacco chloroplasts to produce grft, which accounted for up to % of the total soluble protein of the plant [ ] . they also found that the tobacco, when dried, provides a storable source of grft that can be purified at a later date [ ] . however, when produced in nicotiana benthamiana, kim et al. found that grft had pathologic effects on plants because it was directed to the apoplast during production, resulting in necrotic symptoms associated with hypersensitive response (hr)-like cell death [ ] . they found that a specific interaction between grft and an apoplast-located endogenous glycoprotein, xyl , initiated the hr response. to suppress grft-induced cell death in nicotiana benthamiana, exogenous expression of naphthalene hydroxylase was suggested by the authors [ ] . to avoid the emergence of hiv- strains resistant to single microbicides, vamvaka et al. expressed components of multiple anti-hiv proteins including grft, g mab, and cyanovirin-n in rice endosperm [ ] . they found that extracts from plants expressing all three proteins showed enhanced in vitro binding to gp and synergistic hiv- neutralization [ ] . unexpectedly, they also found that synergistic hiv neutralization caused by the triple microbicide was enhanced by production in rice endosperm because rice globulin protein enhances gp binding in the expressed proteins [ ] . table . large-scale grft production methods. n/a indicates "not applicable." ref. transformation and the use of an autoinduction fermentor escherichia coli -fold increase [ ] transduction with tobacco mosaic virus nicotiana benthamiana multigram quantity [ ] agrobacterium vectors nicotiana benthamiana % of the leaf cells and % of the total soluble protein [ ] transduction with tobacco mosaic virus in ph buffer, heating the extract to • c, a bentonite mgcl mixture, and chromatography. nicotiana benthamiana % ± % of griffithsin from the initial extract [ ] particle bombardment rice endosperm µg/g dry seed weight [ ] use of probiotic microorganisms lactobacillus rhamnosus gg and l. rhamnosus gr- n/a [ ] chloroplast transformation nicotiana tabacum µg of pure griffithsin per gram [ ] protein stability is critical for long-term maintenance of pharmacological activity. for this reason, the susceptibility of grft to proteolytic digestion should be considered. grft was resistant to digestion by eight different proteases including pepsin, papain, leucine aminopeptidase, pronase, α-chymotrypsin, proteinase k, endoproteinase, and trypsin [ ] . a number of different nanoparticle drug delivery systems have been tested to provide sustained and controlled delivery of grft, improve its solubility, protect its payloads, and enhance its mucosal permeability (table ) [ ] [ ] [ ] [ ] . plga (poly lactic-co-glycolic acid) nanoparticles with a diameter of approximately - nm were successfully used in the co-delivery of grft and dapivirine in vitro [ ] . both drugs showed a biphasic release with an initial burst phase followed by a sustained release phase [ ] . grooms et al. successfully generated grft-modified electrospun fibers to inactivate hiv prior to cellular entry [ ] . furthermore, lal et al. developed a self-administered, vaginal fast-dissolving insert (fdi) produced by freeze-drying that delivered safe and effective amounts of grft and carrageenan (gc), a sulfated polysaccharide extracted from seaweed [ ] . fibers comprised of methoxy polyethylene glycol-plga: poly n-butyl acrylate-co-acrylic acid (mpeg-plga: pba-co-paa) were able to achieve high grft loading. these grft-loaded fibers were well maintained within a simulated vaginal fluid (svf) and showed ph-dependent release upon exposure to buffered svf and simulated semen solutions [ ] . table . pharmaceutical formulations for the efficient delivery of grft. delivery route effects on delivery ref. a biphasic release with an initial burst phase followed by a sustained release phase [ ] electrospun fibers in vitro maintenance of antiviral efficacy [ ] fdi comprised of vaginal good friability, hardness, and stability [ ] mpeg-plga:pba-co-paa vaginal high grft loading and ph-dependent release [ ] to date, two phase-one clinical studies have been initiated to investigate the potential toxicity of grft in healthy populations [ , ] . the first study aimed to evaluate the safety of grft in a cg gel used vaginally for a single dose and then for consecutive days in healthy women [ ] . this was a two-part study with the first part consisting of a single-dose and an open-label design, and the second part consisted of a multiple-dose, randomized, placebo-controlled study design. during the second part, subjects were administered a placebo and subjects were given the grft gel. in this study, the pharmacokinetic behavior of grft was also analyzed by forming a time-concentration curve of grft in blood samples. however, rising dose tolerance was not studied because of the minimal systemic absorbance of grft, which was reported in preclinical studies. although the official study results are not available yet, the population council website reported grft to be safe for vaginal use for up to days with potent anti-hiv activity in cell-based assays and cervical explants up to h after receiving the dose [ ] . in , another phase-one clinical study for grft started as an integrated preclinical/clinical program. this program, which was named prevent (pre-exposure prevention of viral entry), aimed to provide a comprehensive set of data to facilitate an informed decision on whether grft should progress within the topical microbicides pipeline [ ] . this was a randomized, double-blind phase-one safety and pharmacokinetic study of grft enema administered rectally to hiv- seronegative adults who practice urai. the number and frequency of adverse events, the blood concentration of grft, and changes in humoral antibody response were analyzed. this on-going clinical study will be completed in . grft not only displays antiviral activity against hiv but also for other enveloped viruses such as sars-cov [ ] , mers-cov [ ] , hcv [ , ] , hsv [ , , ] , jev [ , ] , and pedv [ ] . even human papillomavirus (hpv), which is a non-enveloped virus, was inactivated by grft via a glycosylation-independent mechanism [ ] . grft specifically bound to the sars-cov spike glycoprotein and inhibited viral entry [ ] . grft also inhibited particles pseudotyped with the mers-cov spike protein from entering host cells [ ] . preincubation of hcv particles with grft prevented infection of huh- hepatoma cells [ ] . furthermore, grft was able to interfere with the direct cell-to-cell transmission of hcv [ ] . this anti-hcv activity was further demonstrated in vivo when hcv infection was mitigated in chimeric mice [ ] . in this report, grft was readily bioavailable after subcutaneous injection, and it showed significant in vivo efficacy by reducing hcv viral titers in a mouse model system with engrafted human hepatocytes [ ] . in contrast to hiv, hcv resistance to grft was not directly conferred by mutations in the envelope protein genes, but it could occur through an indirect mechanism involving mutations in other viral proteins [ ] . grft displayed modest inhibitory activity against hsv- if it was present during viral entry, but if it was present post-entry, it completely blocked plaque formation, reduced plaque size, and prevented cell-to-cell propagation [ ] . these in vitro findings translated into significant protection against genital herpes in mice treated with a . % griffithsin gel [ ] . the in vivo anti-hsv activity of grft was further demonstrated when murine model test subjects were protected by c. crescentus expressing grft after intravaginal infection with hsv- [ ] . levendosky et al. explored the antiviral properties of a combination product composed of grft and cg against hsv- and hpv. they found that grft was able to block the entry of hsv- and hpv into target cells but not the adsorption of hsv- and hpv onto target cells [ ] . this grft/cg combination was also tested as a freeze-dried, fdi formulation. this product protected rhesus macaques against a high-dose vaginal simian hiv challenge h after fdi insertion [ ] . furthermore, this grft/cg fdi also protected mice, vaginally, against hsv- and hpv pseudovirus [ ] . in vitro experiments showed that the treatment of jev with grft before inoculation into bhk- cells inhibited infection in a dose-dependent manner [ ] . in vivo experiments showed that grft ( mg/kg) administered intraperitoneally before virus infection prevented mortality in mice challenged intraperitoneally with a lethal dose of jev [ ] . with regard to its antiviral mechanism, grft was shown to bind to jev glycosylated viral proteins, specifically the enveloped and pre-mature glycoproteins [ ] . in addition, grft was able to reduce pedv infection in vero cells [ ] . due to its novel carbohydrate-targeting antiviral mechanism of action, superior anti-hiv- efficacy, excellent host toxicity profile, synergistic interaction with other antiretroviral drugs, optimized large-scale production methodology, and various formulation methods for its efficient delivery, grft holds great promise as the first topical protein-based anti-hiv pre-exposure prophylactic. even though a potentially adverse immunotoxicity issue observed during preclinical animal studies needs to be resolved, its broad antiviral spectrum, which is applicable to other enveloped viruses, could make grft the first universal antiviral therapeutic that can specifically target virus carbohydrates. favorable outcomes from two ongoing phase-one clinical trials will accelerate the drug development process. if approved, grft will provide a superior way to prevent many transmissible viral infections. a lectin isolated from bananas is a potent inhibitor of hiv replication potent anti-influenza activity of cyanovirin-n and interactions with viral hemagglutinin microvirin, a novel alpha( , )-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv- activity comparable with that of cyanovirin-n but a much higher safety profile bioengineered intravaginal isolate of lactobacillus plantarum expresses algal lectin scytovirin demonstrating anti-hiv- activity isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia sp toxicity of antiretroviral therapy and implications for drug development effectiveness and safety of tenofovir gel, an antiretroviral microbicide, for the prevention of hiv infection in women global epidemiology of hiv infection in men who have sex with men griffithsin: an antiviral lectin with outstanding therapeutic potential broad-spectrum in vitro activity and in vivo efficacy of the antiviral protein griffithsin against emerging viruses of the family coronaviridae middle east respiratory syndrome coronavirus infection is inhibited by griffithsin antiviral lectins from red and blue-green algae show potent in vitro and in vivo activity against hepatitis c virus griffithsin has antiviral activity against hepatitis c virus griffithsin protects mice from genital herpes by preventing cell-to-cell spread griffithsin and carrageenan combination to target herpes simplex virus and human papillomavirus griffithsin binds to the glycosylated proteins (e and prm) of japanese encephalitis virus and inhibit its infection griffithsin inhibits japanese encephalitis virus infection in vitro and in vivo in vitro antiviral activity of griffithsin against porcine epidemic diarrhea virus domain-swapped structure of the potent antiviral protein griffithsin and its mode of carbohydrate binding monomerization of viral entry inhibitor griffithsin elucidates the relationship between multivalent binding to carbohydrates and anti-hiv activity the role of individual carbohydrate-binding sites in the function of the potent anti-hiv lectin griffithsin crystallographic, thermodynamic, and molecular modeling studies of the mode of binding of oligosaccharides to the potent antiviral protein griffithsin crystallographic studies of the complexes of antiviral protein griffithsin with glucose and n-acetylglucosamine grifonin- : a small hiv- entry inhibitor derived from the algal lectin, griffithsin mannose-rich glycosylation patterns on hiv- subtype c gp and sensitivity to the lectins, griffithsin, cyanovirin-n and scytovirin binding of the mannose-specific lectin, griffithsin, to hiv- gp exposes the cd -binding site griffithsin, a potent hiv entry inhibitor, is an excellent candidate for anti-hiv microbicide combinations of griffithsin with other carbohydrate-binding agents demonstrate superior activity against hiv type , hiv type , and selected carbohydrate-binding agent-resistant hiv type strains synergistic activity profile of griffithsin in combination with tenofovir, maraviroc and enfuvirtide against hiv- clade c role of the carbohydrate-binding sites of griffithsin in the prevention of dc-sign-mediated capture and transmission of hiv- sensitivity of transmitted and founder human immunodeficiency virus type envelopes to carbohydrate-binding agents griffithsin, cyanovirin-n and galanthus nivalis agglutinin potent strategy to inhibit hiv- by binding both gp and gp griffithsin, alone and combined with all classes of antiretroviral drugs, potently inhibits hiv cell-cell transmission and destruction of cd + t cells occluding the mannose moieties on human immunodeficiency virus type gp with griffithsin improves the antibody responses to both proteins in mice binding site geometry and subdomain valency control effects of neutralizing lectins on hiv- viral particles griffithsin tandemers: flexible and potent lectin inhibitors of the human immunodeficiency virus the griffithsin dimer is required for high-potency inhibition of hiv- : evidence for manipulation of the structure of gp as part of the griffithsin dimer mechanism the lectins griffithsin, cyanovirin-n and scytovirin inhibit hiv- binding to the dc-sign receptor and transfer to cd (+) cells mechanisms of hiv- subtype c resistance to grft, cv-n and svn removal of two high-mannose n-linked glycans on gp renders human immunodeficiency virus largely resistant to the carbohydrate-binding agent griffithsin activity of and effect of subcutaneous treatment with the broad-spectrum antiviral lectin griffithsin in two laboratory rodent models impact of the griffithsin anti-hiv microbicide and placebo gels on the rectal mucosal proteome and microbiome in non-human primates investigation of griffithsin's interactions with human cells confirms its outstanding safety and efficacy profile as a microbicide candidate studies in a murine model confirm the safety of griffithsin and advocate its further development as a microbicide targeting hiv- and other enveloped viruses scaleable manufacture of hiv- entry inhibitor griffithsin and validation of its safety and efficacy as a topical microbicide component pharmacokinetics of the antiviral lectin griffithsin administered by different routes indicates multiple potential uses a caulobacter crescentus microbicide protects from vaginal infection with hiv- jr-csf in humanized bone marrow-liver-thymus mice improving the large scale purification of the hiv microbicide, griffithsin recombinant production of anti-hiv protein, griffithsin, by auto-induction in a fermentor culture a novel and fully scalable agrobacterium spray-based process for manufacturing cellulases and other cost-sensitive proteins in plants engineering lactobacillus rhamnosus gg and gr- to express hiv-inhibiting griffithsin rice endosperm is cost-effective for the production of recombinant griffithsin with potent activity against hiv high-level expression of the hiv entry inhibitor griffithsin from the plastid genome and retention of biological activity in dried tobacco leaves characterization of the hypersensitive response-like cell death phenomenon induced by targeting antiviral lectin griffithsin to the secretory pathway unexpected synergistic hiv neutralization by a triple microbicide produced in rice endosperm degradation of naturally occurring and engineered antimicrobial peptides by proteases griffithsin-modified electrospun fibers as a delivery scaffold to prevent hiv infection development of a vaginal fast-dissolving insert combining griffithsin and carrageenan for potential use against sexually transmitted infections ph-responsive delivery of griffithsin from electrospun fibers design of poly(lactic-co-glycolic acid) (plga) nanoparticles for vaginal co-delivery of griffithsin and dapivirine and their synergistic effect for hiv prophylaxis study to evaluate the safety of griffithsin in a carrageenan gel in healthy women griffithsin-based rectal microbicides for prevention of viral entry (prevent) developing and testing a griffithsin (non-arv) microbicide generation of a dual-target, safe, inexpensive microbicide that protects against hiv- and hsv- disease hepatitis c virus resistance to carbohydrate-binding agents griffithsin carrageenan fast dissolving inserts prevent shiv hsv- and hpv infections in vivo this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord- -m u ax k authors: jin, jun; gao, de-hong; mo, xin; tan, si-ping; kou, zhen-xia; chen, yi-bo; cao, jin-bo; chen, wen-jing; zhang, ya-ming; li, bing-qing; huang, kuan-long; xu, bing-ren; tang, xiao-li; wang, yu-li title: analysis of imaging features in patients with covid- date: - - journal: bmc med imaging doi: . /s - - - sha: doc_id: cord_uid: m u ax k background: the aim of this was to analyze chest ct imaging features of patients with coronavirus disease (covid- ) in shenzhen, china so as to improve the diagnosis of covid- . methods: chest ct of patients with covid- confirmed by the nucleic acid test (nat) were retrospectively analyzed. analyses were performed to investigate the pathological basis of four imaging features(“feather sign”,“dandelion sign”,“pomegranate sign”, and “rime sign”) and to summarize the follow-up results. results: there were patients ( . %) with typical “feather sign”and ( . %) with “dandelion sign”, while few patients had “pomegranate sign” and “rime sign”. the “feather sign” and “dandelion sign” were composed of stripe or round ground-glass opacity (ggo), thickened blood vessels, and small-thickened interlobular septa. the “pomegranate sign” was characterized as follows: the increased range of ggo, the significant thickening of the interlobular septum, complicated with a small amount of punctate alveolar hemorrhage. the “rime sign” was characterized by numerous alveolar edemas. microscopically, the wall thickening, small vascular proliferation, luminal stenosis, and occlusion, accompanied by interstitial infiltration of inflammatory cells, as well as numerous pulmonary interstitial fibrosis and partial hyaline degeneration were observed. repeated chest ct revealed the mediastinal lymphadenectasis in one patient. re-examination of the nat showed another positive anal swab in two patients. conclusion: “feather sign” and “dandelion sign” were typical chest ct features in patients withcovid- ; “pomegranate sign” was an atypical feature, and “rime sign” was a severe feature. in clinical work, accurate identification of various chest ct signs can help to improve the diagnostic accuracy of covid- and reduce the misdiagnosis or missed diagnosis rate. sars-cov- is still not available, researchers are currently working on creating vaccines and investigating clinical features of the infected population. recently, wang et al examined the ct images of asymptomatic infected patients with covid- and found that chest ct scans have an essential role in the screening of the population suspected of having infection [ ] . besides, a previous report suggested that ct imaging may be very useful in the diagnosis of covid- in patients with negative nat [ ] . in the present study, we analyzed clinical data and ct images of covid- . these data could contribute to timely and accurate identification of the clinical features, laboratory test results , and ct imaging findings of covid- , thereby resulting in early diagnosis, quarantine, and treatment. this study had no potential risks for patients, and there was no direct relationship between researchers and patients. the study was conducted according to the principles of the helsinki declaration. the ethics committee of shekou people's hospital waived the signing of informed consent for this retrospective study. patients with laboratory-confirmed covid- (confirmed by a reverse transcription polymerase chain reaction, rt-pcr) were recruited from three hospitals between january , and february , . the number of cases included from each hospital is shown in supplementary table . age, gender, epidemiological features, and clinical symptoms were collected from all patients. all patients underwent a chest ct scan, which was performed using ge -row revolution ct, ge light speed -slice spiral ct, siemens somatom emotion -row spiral ct, and ge -row vct, all of which were end-inspiratory scans. for the axial-section, the slice thickness was mm, and the reconstruction slice thickness was . or . mm. two radiologists (with more than ten years of work experience) analyzed all images. five patients who received chest radiography and four with normal chest ct images were excluded. finally, patients were included in the study. fig. a older female patient with a long residence history in an epidemic area experienced fever( . °c) and sore muscle for day. a feather hand painting. b baseline chest ct image demonstrated ggo in the right middle lobe, showing a "feathery sign" (red arrow). the nucleic acid test was negative for the first time. c follow-up ct scan after days showed that the lesions were significantly enlarged. ggo in the lungs was multiple (white arrow). the second test was positive for nucleic acid fig. a older female patient with a long history of living in an epidemic area experiencing fever and sore throat for days. a dandelion hand painting. b non-contrast enhanced coronal ct image shows that the right lower lobe with a circular ggo,vascular thickening, and bronchiectasis, showing a "dandelion sign" (red arrow), and multiple small patchy ggo in the left lung (white arrow). c axial thin-section un-enhanced ct image shows a round-like ggo (red arrow) in the right lower lobe, a small piece of ggo in the right middle lobe, and the unclear border (white arrow) the following ct image features were observed for each patient: (a) the location, extent, and a number of lesions; (b) type of lesions (ggo, vascular thickening, pulmonary consolidation, pulmonary fibrous, interlobular septum, and solid nodules); (c) specific signs ("air bronchogram sign", "feather sign", "dandelion sign", "pomegranate sign", "rime sign"); (d) other signs (pleural effusion, mediastinal lymphadenectasis,etc). the ct images of solid nodule and pulmonary consolidation showed the density of lesions covering the vascular and bronchial shadows resembling [ ] ; "feather sign" or "dandelion sign" ,which was defined as the exudative lesion and thickened blood vessels forming a strip or round high-density shadow, which was very similar to the shape of feathers or dandelions (figs. and ); "pomegranate sign", which was defined as an exudative lesion accompanied by a small amount of bleeding, showing round and imbricate arrangement that was similar to a pomegranate (fig. ) ; "rime sign", which was defined as multiple exudative and punctate hemorrhage in the lesion accompanied with extensive interstitial fibrosis forming large white lung, that was similar to white rime attached to the branches (fig. ). fourteen out of patients underwent chest ct reexamination to days after being cured. two senior radiologists compared ct images for two or more times. the remaining patients did not undergo ct scanning due to the quarantine period. there were patients in this group, including males ( . %) and females ( . %). predominantly, these were middle-aged and elderly patients, with a mean age of . ± . years (range, - years). patient characteristics are showed in table . a baseline chest ct scan was abnormal in patients ( table ) ; ( . %) showed lesions involving both lungs, and ( . %) reported bilateral multifocal fig. a older female patient experiencing fever and cough for day,,and who had days of travel history in the epidemic area before onset. a pomegranate hand painting. b non-contrast enhanced chest ct scan shows that the ggo in the posterior basal segment of the left lower lobe and vascular thickening, bronchiectasis, and interlobular septal thickening, showing a "pomegranate sign" (red arrow). c a partially enlarged image at the same level as in figure b, suggesting that the ggo in the posterior basal segment of the left lower lobe showed a "pomegranate sign" (red arrow) fig. a older male patient who experienced constipation, and anorexia lasting for week, and who had no epidemiological history. a rime hand painting. b coronal ct image of the chest, showing diffuse ggo in the lungs, vascular thickening, interlobular septal thickening, showing "rime sign" (red arrow). c on the th day after admission, bedside portable chest radiograph showed diffuse high-density shadows in both lung fields, and the lesions significantly progressed compared with the previous ones lesions, with the predominant lower lobe. in addition, there were patients ( . %) with ggo and vascular thickening in the lesion, ( . %) with air bronchogram sign, ( . %) with interlobular septal thickening, ( . %) with "feather signs", ( . %) with "dandelion sign" and ( . %) with pulmonary fibrous tissue proliferation. the partial consolidation of the lesion, solid nodules, "pomegranate sign", and "rime sign" were rare. one ( . %) patient had pleural effusion and mediastinal lymphadenectasis. also, only five patients underwent chest x-ray; two showed a positive result for x-ray; four patients reported negative results for chest ct scan, and two of them showed multiple ggo during reexamination after three days. all patients were followed up. among the hospitalized patients, two showed rapid progression during the hospital stay and were in critical condition. thirty patients were cured and discharged. four patients had cough and chest distress on re-examination after - days. laboratory re-examination revealed that four patients had elevated t-lymphocyte counts, accompanied by elevated alanine aminotransferase and creatinine levels. ct re-examination indicated the following: two patients had no obvious changes; eight reported improved absorption; four reported that lesions were completely absorbed, and one had mediastinal lymphadenectasis. nat was performed in patients. two patients were positive for anal swabs, while they were negative for nasal and throat swabs; the remaining patients were normal, as shown in table . in the present study, we found that the most common ct imaging features in patients with covid- were: bilateral, multifocal ggo, peripheral distribution; the predominant lower lobe; pleural effusion and lymphadenectasis were rare, which is consistent with previous reports [ ] [ ] [ ] . in addition, "feather sign"was found in patients ( . %), "dandelion sign" in ( . %), "pomegranate sign" in nine ( . %), and "rime sign" in seven ( . %) patients, which could be considered as new features in patients with covid- . the "feather sign" and "dandelion sign" on the ct image included stripe or round ggo, thickened blood vessels, and smallthickened interlobular septa. ggo shows diffuse alveolar damage under the microscope, which is histologically caused by alveoli filled with blood, pus, water, or cells [ , ] . the reason for the thickening of blood vessels in the lesion may be the following:under the effect of inflammatory factors, the increased permeability of the vascular wall may lead to the dilation of capillaries and the corresponding thickening of the pulmonary artery [ ] . the incidence of "feather sign" and "dandelion sign" in this study was . % and . %, respectively. in this study, nine patients ( . %) presented with "pomegranate sign", which is an atypical chest ct feature of covid- . a "pomegranate sign" can be characterized as a further increase of the range of ground-glass opacity that occupies part of the lung sub-segment, the more significant thickening of the interlobular septum, complicated with a small amount of punctate alveolar hemorrhage [ , ] , and lesions that are in imbricate arrangement, and are similar to a pomegranate. moreover, among seven patients ( . %) who developed a "rime sign", two were critically ill. a "rime sign" is characterized by numerous alveolar edemas. hemorrhagic necrosis can be observed in some alveoli. moreover, mucus and hemorrhagic exudate diffusely cover the bronchiole wall. microscopically, the wall thickening, small vascular proliferation, luminal stenosis, and occlusion, accompanied by interstitial infiltration of inflammatory cells, such as lymphocytes, plasma cells, and monocytes [ ] , as well as numerous pulmonary interstitial fibrosis and partial hyaline degeneration are observed. this type of lesion has a wide range and looks like a white rime attached to abranch. all patients were followed up for two weeks. among hospitalized patients, had stable conditions and gradually recovered, two reported rapid progression during the hospital stay and were in critical condition, and underwent extracorporeal membrane oxygenation (ecmo). thirty patients were cured and discharged. fourteen underwent re-examination after - days;four had a cough, stomachache and chest distress. very few patients developed weakness and shortness of breath. laboratory tests revealed a significant increase in t-lymphocyte counts in four patients (flow cytometry: /ul, /ul, respectively; the normal value was ), accompanied by a significant increase in absolute counts of helper and cytotoxic tlymphocytes, while alanine aminotransferase, creatinine and total bilirubin levels were increased to varying degrees, which suggested that the patient's immune function was deteriorated, and liver and renal functions were impaired; these data were consistent with previous reports [ ] . these findings suggest that liver injury may be caused by sars-cov- infection or induced by drug treatment during hospitalization. among patients who underwent ct re-examination, two patients showed no obvious changes; in four patients, lesions were completely absorbed; in eight cases, lesions were partially absorbed; and one had mediastinal lymphadenectasis. these data suggest that ct can be used to monitor changes during disease progression, which is consistent with the findings of hosseiny et al [ ] . multiple nat (including nasal, anal, and throat swabs) were performed after - days. in two patients, the results of nat of nasal and throat swabs were negative, while the result of nat of the anal swab was positive, which is why the patients were immediately readmitted to the hospital for treatment. the remaining patients reported negative results for multiple nat. a recent study [ ] revealed that four patients with covid- showed "positive results" for nucleic acid test - days after discharge. this suggested that current discharge standards should be revised; nasal, anal, and throat swabs should be combined, as well as supplemented by a variety of other testing methods. covid- patients should be monitored during treatment, rehabilitation, and quarantine, so as to fundamentally control the occurrence of the "positive results" after discharge [ ] . this study had some limitations. firstly, no children are enrolled in this study, and the clinical, epidemiological, and imaging features of children with covid- are lacking. secondly,the number of patients collected in this study is so small that study results have certain limitations. the reliability of the conclusion needs to be further expanded to verify the sample size. thirdly, sufficient pathological specimens are currently unavailable for comparison with imaging features. we will collect more patients data and pathological specimens to observe the evolution and outcome of the disease and determine the correlation between the imaging and pathology. our data suggested that "feather sign" and "dandelion sign" were typical chest ct features of covid- . "pomegranate sign" was an atypical feature, and "rime sign" was a severe feature, which suggested poor prognosis. in clinical work, accurate identification of various chest ct signs in combination with epidemiological history, clinical features, multiple nucleic acid tests, and other testing methods can help improve the diagnostic accuracy of covid- and reduce the misdiagnosis or missed diagnosis rate. supplementary information accompanies this paper at https://doi.org/ . /s - - - . abbreviations covid- : coronavirus disease ; rt-pcr: reverse transcription polymerase chain reaction; nat: nucleic acid test; ggo: ground-glass opacity a novel coronavirus from patients with pneumonia in china coronaviridae study group of the international committee on taxonomy of viruses. the species severe acute respiratory syndrome-related coronavirus: classifying -ncov and naming it sars-cov- coronavirus disease (covid- ): role of chest ct in diagnosis and management chest ct for typical -ncov pneumonia: relationship to negative rt-pcr testing fleischner society: glossary of terms for thoracic imaging epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with novel coronavirus in wuhan. china the lancet clinical and ct features in pediatric patients with covid- infection: different points from adults. pediatr pulmonology imaging and clinical features of patients with novel coronavirus sars-cov- herpes simplex virus pneumonia: high-resolution ct findings radiological findings from patients with covid- pneumonia in wuhan,china:a descriptive study clinical pathology of critical patient with novel coronavirus pneumonia ( covid- ) clinical features of covid- -related liver damage. available at ssrn radiology perspective of coronavirus disease (covid- ): lessons from severe acute respiratory syndrome and middle east respiratory syndrome positive rt-pcr test results in patients recovered from covid- ct imaging features of novel coronavirus ( -ncov) publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are grateful for the high-quality hand-painting made by feng xuan and haifeng tang from fine arts, dafen oil painting exchange square, shenzhen. the authors acknowledge the valuable suggestions and advice from prof. yudong zhang from the department of radiology, the first affiliated hospital of nanjing medical university. the study was jointly designed by xt, dg, jj and yw; jj was the main contributor to the manuscript writing; yc, wc, yz, bx, st collected clinical data and ct images; kh, jc, bl analyzed the data; xm and zk performed statistical analysis; all authors reviewed and agreed with the content of this article. no funding is provided in this study. the datasets used and analyzed during the current study are available from the corresponding author on reasonable request. according to the principles of the helsinki declaration, this study had no potential risks for patients, and there was no direct relationship between researchers and patients. ethics committee of shekou people's hospital of shenzhen waived the signing of informed consent for this retrospective study. not applicable. the authors declare no competing interests. key: cord- - r aokh authors: barreiro, luis b.; quach, hélène; krahenbuhl, james; khaliq, shagufta; mohyuddin, aisha; mehdi, s. qasim; gicquel, brigitte; neyrolles, olivier; quintana-murci, lluís title: dc-sign interacts with mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the pakistani population date: - - journal: hum immunol doi: . /j.humimm. . . sha: doc_id: cord_uid: r aokh the c-type lectin dc-sign is involved in early interactions between human innate immune cells and a variety of pathogens. here we sought to evaluate whether dc-sign interacts with the leprosy bacillus, mycobacterium leprae, and whether dc-sign genetic variation influences the susceptibility and/or pathogenesis of the disease. a case–control study conducted in a cohort of individuals revealed no association between dc-sign variation and leprosy. however, our results clearly show that dc-sign recognizes m. leprae, indicating that mycobacteria recognition by this lectin is not as narrowly restricted to the mycobacterium tuberculosis complex as previously thought. altogether, our results provide further elucidation of m. leprae interactions with the host innate immune cells and emphasize the importance of dc-sign in the early interactions between the human host and the infectious agents. leprosy is a chronic granulomatous disease caused by mycobacterium leprae affecting essentially the superficial peripheral nerves, the skin, and the mucosal membranes of the upper respiratory tract. depending on the degree to which cell-mediated immunity is expressed and on the extent of spread and multiplication of the bacilli, infection can result in a broad spectrum of clinical manifestations and outcomes. at one pole of the disease, patients with tuberculoid leprosy (tt) develop a strong cellmediated immune response that contains the infection in few localized lesions with low bacillary counts and that often progresses to self-healing. at the opposite pole, lepromatous leprosy (ll) patients develop a weak cellular response and suffer multiple lesions with high bacillary loads. intermediary types of leprosy, namely borderline tuberculoid (bt), borderline borderline (bb), and borderline lepromatous (bl), with various clinical manifestations and bacillary counts, are classified in between tt and ll types. although factors influencing the type of leprosy developed upon infection remain poorly under-stood, genetic host factors have long been suspected to play a major role in the clinical outcome of the infection [ ] . indeed, polymorphisms in genes encoding important mediators of the immune response, such as toll-like receptor , tumor necrosis factor-␣, interleukin- , nramp , vitamin d receptor, and other genes, such as the parkinson-related genes park and pacrg, have been reported to be involved in susceptibility to leprosy and/or to preferential development of either type of the disease (see [ ] for a review). in the context of host factors influencing infectious disease susceptibility or outcome, the innate immunity system may play a critical role. polarization of the t lymphocyte response is tightly linked to early recognition of the pathogen by innate immunity cells, such as dendritic cells (dcs) and macrophages (ms), and to subsequent signaling events resulting in cytokine secretion and antigen presentation. thus, genetic variation in host genes whose products are involved in the early steps of pathogen recognition may have a broad range of influence in the pathogenesis of leprosy. in this context, c-type lectins play a crucial role in detecting pathogens by their characteristic carbohydrate structures and internalizing them for further antigen processing and presentation, inducing therefore adaptive immunity [ ] . we and others have recently shown that the prototypic ctype lectin dendritic cell-specific intercellular adhesion molecule- grabbing nonintegrin dc-sign (also known as cd ) is a major receptor for mycobacterium tuberculosis in human dcs [ , ] and in alveolar ms in patients with tuberculosis [ ] . dc-sign not only mediates internalization of the bacillus by dcs but may also transduce intracellular signals leading to secretion of il- and to partial dc deactivation upon recognition of the microbe [ ] . in this view, dc-sign may be a key element in shaping an appropriate t-cell response against m. tuberculosis and possibly other mycobacteria, such as m. leprae. our most recent results show that nucleotide variation in the dc-sign promoter region is associated to susceptibility to tuberculosis [ ] . here we sought to evaluate whether dc-sign interacts with the leprosy bacillus, m. leprae, and whether dc-sign genetic variation has an influence on the susceptibility and/or pathogenesis of the disease. the bacilli m. tuberculosis h rv and mycobacterium smegmatis mc harboring the pluxgfp plasmid (kind gift from g. r. stewart, london, uk) were cultivated in h medium containing adc supplement (difco) and g/ml hygromycin. suspensions of fresh, viable, nude mouse-derived thai- strain m. leprae were obtained from the national hansen's disease programs laboratory at louisiana state university in baton rouge (la, usa). this isolate of leprosy bacilli is maintained in programmed serial passage in the foot pads of athymic nu/nu mice infected with ϫ freshly harvested m. leprae. briefly, bacilli were harvested from the foot pads - months after infection (at mid-log growth), washed by centrifugation in middlebrook h medium ( , g for minutes) and enumerated by direct count according to shepard's method. the relative viability of m. leprae in a suspension was evaluated using the live/dead baclight bacterial viability kit (molecular probes). for the present study pure preparations of bacilli, free of mouse footpad tissue, were obtained by treating the footpad suspension with . m naoh for minutes followed by neutralization with . m hcl and three washes with phosphate-buffered saline. the cell membranes of these pure bacilli were stained with green pkh dye (sigma) according to the manufacturer's recommendations, recounted by the shepard technique, resuspended in rpmi- at ϫ m. leprae per milliliter, and stored at °c. hela and dc-signexpressing hela cells (hela::dc-sign) were cultivated in rpmi- (invitrogen) supplemented with % fetal calf serum (dutcher). for binding experiments, cells were cultivated in six-well plates (bd-falcon) until % confluency and infected with the bacilli at a multiplicity of infection of bacterium/cell for hours at °c. after three washes in rpmi, cells were gently collected, fixed in % paraformaldehyde, and analyzed by flow cytometry for green fluorescence using a facscalibur apparatus (becton). four independent experiments were conducted to assess the ability of m. leprae to bind to dc-sign. in two of these experiments, m. tuberculosis and m. smegmatis were included as controls. the study cohort of the present study consisted of adult pakistani individuals, including patients with leprosy and ethnically matched healthy individuals. all individuals were volunteers from whom informed consent was obtained. disease evaluation was based on clinical, bacteriological, and histological data and determined according to the presence and number of bacteria observed in skin smears taken from the right and left ears, right eyebrow, and right and left middle fingers. the bacteria were detected using afb staining. the clinical forms of leprosy were classified in accordance with the ridley and jopling classification [ ] . the leprosy individuals included patients with ll, with bl, with tt, and with bt. given the absence of significant differences between ll versus bl and between tt versus bt, individuals were grouped into lepromatous patients (bl ϩ ll; n ϭ ) and tuberculoid patients (bt ϩ tt; n ϭ ). the control sample consisted of unrelated healthy individuals belonging to the hospital staff and, therefore, in frequent contact with both types of leprosy patients. genomic dna was extracted from peripheral blood cells according to standard procedures. to identify informative dc-sign single nucleotide polymorphisms (snps) and to avoid ascertainment bias in the choice of markers to be tested, we first sequenced the whole dc-sign genomic region (seven coding exons, flanking intronic regions, and bp situated = of the start codon) in randomly chosen individuals ( chromosomes). pcr and sequence reactions of the dc-sign region were performed as previously described [ ] . using polymorphisms with a minimum allele frequency of . , unphased genotypic data were converted into haplotypes using the accelerated expectation maximization algorithm implemented in haploview v . [ ] . to define a minimal number of snps explaining most haplotypic diversity, we used the best v . software [ ] . seven haplotype-tagging snps were then selected to genotype the entire cohort. dna samples were then genotyped by either fluorescence-polarization (victor- tm technology) or taqman (abi prism- sequence detection system) assays. statistical testing for genotypic and haplotypic associations was performed using haploview v . [ ] . to evaluate whether dc-sign recognizes m. leprae, we performed cold binding assays using fluorescently labeled bacilli and dc-sign-expressing recombinant hela cells as previously described [ , ] . green fluorescent protein-expressing m. tuberculosis and m. smegmatis were included as controls because it has been previously shown that dc-sign preferentially binds to species of the m. tuberculosis complex, such as m. tuberculosis, as compared to other mycobacterial species including fast-growers such as m. smegmatis [ , ] . such preferential recognition may rely on the differential presence of mannose capping residues on the cell surfaceexposed lipoarabinomannan among the different mycobacterial species [ ] and on the presence of dc-signspecific ligands within the cell wall of species of the m. tuberculosis complex [ ] . binding of m. leprae to dc-sign-expressing cells was . (Ϯ . )-fold higher than that in control hela cells. as expected, binding of m. tuberculosis and m. smegmatis to dc-sign-expressing cells was . (Ϯ . )-and . (Ϯ . )-fold higher than that in control hela cells, respectively (figure ). in light of the observed recognition of m. leprae by dc-sign, we subsequently explored the relationship between dc-sign polymorphisms with susceptibility to leprosy per se and disease outcome in a cohort of pakistani origin. to uncover polymorphic positions in our study population, we first adopted a sequencing strategy of the ϳ . -kb dc-sign genomic region, including the seven coding exons, all introns, and ϳ bp situated = of the start codon, in randomly chosen individuals ( chromosomes). this initial resequencing step revealed polymorphisms. using polymorphisms with a minimum allele frequency of . , we reconstructed haplotypes over the entire gene region and defined the minimum number of snps explaining most haplotype diversity (haplotype-tagging snps: htsnps). seven htsnps were then selected and genotyped in the entire panel of individuals. all these htsnps were found to be in hardy-weinberg equilibrium. table reports the al- lelic frequencies of the seven htsnps in the different study groups and the comparisons between leprosy patients and controls and between patients presenting the two polarities of the disease (i.e., bt ϩ tt versus bl ϩ ll). although some variation in allelic frequencies was observed, no significant differences either between patients and controls or between the two groups of leprosy patients were detected. likewise, when performing the analysis at the haplotype level (results not shown), a test revealed no statistical differences in the global distribution of haplotype frequencies in any of the groups' comparisons (all p values being Ͼ . ). these results based on cold binding assays show that dc-sign preferentially recognizes m. leprae and m. tuberculosis, as compared to m. smegmatis. these observations strongly suggest that mycobacteria recognition by the lectin is not as narrowly restricted to the m. tuberculosis complex as previously proposed [ , ] but ex-tends to the leprosy bacillus. recognition of m. leprae by dc-sign may depend on accessibility of the lipoarabinomannan mannose capping moieties in the cell wall of this species, as suggested for the m. tuberculosis complex [ ] . in addition, other ligands within the m. leprae envelope may participate in dc-sign binding. in particular, we have recently suggested that the o-glycosylated -and -kda antigens may constitute dc-sign ligands in the m. tuberculosis envelope [ ] . the possible participation of the m. leprae homologues of these antigens to dc-sign binding will require further investigation. a recent in vivo study has reported physical association between m. leprae and dc-sign-expressing ms in tissues from patients with ll [ ] , suggesting that m. leprae interactions with dc-sign may occur during the natural course of the disease. our results strengthen this hypothesis and raise the question whether genetic variation in this lectin-coding gene could have an impact in susceptibility and/or severity of leprosy. indeed, polymorphisms in dc-sign, particularly in its = untranslated region corresponding to the promoter region, have been recently associated with susceptibility to hiv [ ] and m. tuberculosis [ ] and to severity of dengue fever [ ] . in addition, using an evolutionary approach we have recently shown that dc-sign has been under a strong selective constraint overtime that has prevented accumulation of any amino acid changes, highlighting again the importance of this gene in immunity and health throughout human history [ ] . in this light, to investigate whether variation in the coding and/or the cis-regulatory regions of dc-sign is involved in susceptibility to and clinical outcome of leprosy, we conducted an association (case/control) study based on a sequencing/genotyping strategy in a cohort of patients presenting the two polarities of the disease and a group of healthy controls. no significant differences were observed between patients and controls, either when patients were analyzed as a single group or when they were classified according to the two polarities of the disease (tuberculoid versus lepromatoid patients). thus, although the sample size in the present study may have provided limited power to detect minor effects of genetic variation, the lack of association observed in our study suggests that dc-sign variation does not constitute a major genetic risk factor for the predisposition to and final outcome of leprosy, at least in our pakistani cohort. in conclusion, the interaction of dc-sign with m. leprae illustrated in the present study emphasizes the importance of this lectin in the very first interactions between host innate immune cells and infectious agents. indeed, dc-sign has been shown to mediate interactions with a plethora of pathogens other than m. leprae (this study) and m. tuberculosis [ , ] including bacteria such as helicobacter pylori and certain strains of klebsiella pneumoniae, viruses such as hiv- , ebola, cytomegalovirus, hepatitis-c, dengue, and sars-cov, and parasites such as leishmania pifanoi and schistosoma mansoni (see [ ] for a review). in the context of leprosy, the clear interaction between dc-sign and m. leprae revealed here stresses the need for future experimental studies to better elucidate the functional role of dc-sign and other genes involved in the dc-sign signaling pathway in the pathogenesis of leprosy. acknowledgments luis b. barreiro was supported by a "fundação para a ciência e a tecnologia" fellowship (sfrh/bd/ / ). we acknowledge o. schwartz (paris) for kindly providing us with hela::dc-sign cells and g.r. stewart (london) for the pluxgfp plasmid. we thank the doctors and staff at the rawalpindi leprosy hospital and the marie adelaide leprosy centre in karachi and the leprosy centre in lahore for sample collection and m. nasir, n. saba, a. abbasi, and s. qamar for their valuable help. genetic dissection of immunity to mycobacteria: the human model genetic dissection of immunity in leprosy pattern recognition receptors: doubling up for the innate immune response mycobacteria target dc-sign to suppress dendritic cell function neyrolles o: dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells dc-sign induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis (cd ) promoter variation is associated with tuberculosis classification of leprosy according to immunity: a five-group system haploview: analysis and visualization of ld and haplotype maps minimal haplotype tagging deciphering the molecular bases of mycobacterium tuberculosis binding to dc-sign reveals an underestimated complexity the cell surface receptor dc-sign discriminates between mycobacterium species through selective recognition of the mannose caps on lipoarabinomannan tlr activation triggers the rapid differentiation of monocytes into macrophages and dendritic cells association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection a variant in the cd promoter is associated with severity of dengue disease the heritage of pathogen pressures and ancient demography in the human innate-immunity cd /cd l region distinct functions of dc-sign and its homologues l-sign (dc-signr) and msignr in pathogen recognition and immune regulation key: cord- -waz k ms authors: shu, chang; wang, shanchen; xu, tianjun title: characterization of the duplicate l-sign and dc-sign genes in miiuy croaker and evolutionary analysis of l-sign in fishes date: - - journal: dev comp immunol doi: . /j.dci. . . sha: doc_id: cord_uid: waz k ms dendritic cell-specific icam- -grabbing non-integrin (dc-sign/cd ) and liver/lymph node-specific icam-grabbing non-integrin (l-sign/cd ) which are homologues of dc-sign are important members in c-type lectin receptors family as key molecules to recognize and eliminate pathogens in the innate immune system. dc-sign and l-sign have become hot topics in recent studies which both served as cell adhesion and phagocytic pathogen recognition receptors in mammals. however, there have been almost no studies of dc-sign and l-sign structure and characters in fish, only dc-sign in the zebrafish had been studied. in our study, we identified and characterized the full-length miiuy croaker (miichthys miiuy) dc-sign (mmdc-sign) and l-sign (mml-sign) genes. the sequence analysis results showed that mmdc-sign and mml-sign have the same domains with other vertebrates except primates, and share some conserved motifs in crd among all the vertebrates which play a crucial role in interacting with ca( +) and for recognizing mannose-containing motifs. gene synteny of dc-sign and l-sign were analyzed for the first time and gene synteny of l-sign was conserved among the five fishes. interestingly, one gene next to l-sign from gene synteny had high similarity with l-sign gene that was described as l-sign-like in fish species. while only one l-sign gene existed in other vertebrates, two l-sign in fish may be in consequence of the fish-specific genome duplication to adapt the specific environment. the evolutionary analysis showed that the ancestral lineages of l-sign gene in fishes experienced purifying selection and the current lineages of l-sign gene in fishes underwent positive selection, indicating that the ancestral lineages and current lineages of l-sign gene in fishes underwent different evolutionary patterns. both mmdc-sign and mml-sign were expressed in all tested tissues and ubiquitously up-regulated in infected liver, spleen and kidney at different sampling time points, indicating that the mmdc-sign and mml-sign participated in the immune response to defense against bacteria infection. innate immunity acts in the first line of immune defense mechanisms, having an important function against foreign pathogens in vertebrates and invertebrates. in teleosts, the innate immune system plays a critical role in protecting organism against invading pathogens (aoki et al., ) . pattern recognition receptors (prrs) including toll-like receptors (tlrs), c-type lectin receptors (clrs), nod-like receptors (nlrs) and rig-i like receptors (rlrs) on antigenpresenting cells play a significant role in innate immunity which recognize pathogen associated molecular patterns (pamps) (zhu et al., a) . c-type lectin receptors (clrs) characterized by the presence of one or more homologous carbohydrate-recognition domain (crd) are a large family of ca + -dependent carbohydrate binding proteins which can specifically recognize a number of pamps including bacteria, viruses, parasites and fungi (lepenies et al., ) . the clrs including the collectins, selectins, lymphocyte lectins and proteoglycans were initially divided into two types based on the quantity of conserved crds: type i clrs such as the macrophagemannose receptor, dec and selectins have multiple crds at their nh terminus; and type ii clrs such as hepatic asialoglycoprotein receptors, macrophage lectin, dc-specific icam -grabbing nonintegrin (dc-sign), langerin, dc-associated c-type lectin (dectin- ) and dc immunoreceptor (dcir) have a single crd at the coohterminus (redelinghuys and brown, ) . dendritic cell-specific intercellular adhesion molecule grabbing non-integrin, dc-sign (cd ) and its homolog, liver/lymphnode-specific intercellular adhesion molecule grabbing non-integrin, l-sign also known as dc-sign-related receptor (dc-signr), are two kinds of the type ii clrs sharing similar structure and both serve as cell adhesion and phagocytic pathogen recognition receptors (khoo et al., ) . characters and functions of dc-sign and l-sign in human had been studied, that both can bind various viruses such as hiv- , ebola virus, cytomegalovirus, hepatitis c virus, dengue virus, and sars-coronavirus (alvarez et al., ; geijtenbeek et al., ; lozach et al., ) . meanwhile, dc-sign and l-sign also have different physiological functions and ligand-binding properties that dc-sign prior to binding to fucose whilst l-sign to mannose to recognize different pathogens (khoo et al., ) . differences are also seen in their tissue distribution that dc-sign is expressed on dendritic cells and some types of macrophages, while l-sign is expressed on sinusoidal endothelial cells in the liver, lymph node sinusoids and in the placenta (boily-larouche et al., ) . human dc-sign and l-sign structures are composed of domains: a transmembrane domain, crd and a neck-region which is highly conserved. the two genes were coded by amino acids and amino acids separately sharing % identical at the amino acid level (khoo et al., ; li et al., ) . the dileucine motif, the tri-acidic cluster and an epn sequence motif are conserved in both human dc-sign and l-sign (khoo et al., ) . but the structures, genomic organizations and functional motifs in teleost are not very clear. until now, there have been almost no studies of dc-sign and l-sign structure and characters in teleost; only dc-sign in the zebrafish had been studied which showed that dc-sign in zebrafish has conserved domains with other vertebrates and plays a crucial role for the initiation and development of adaptive immunity in zebrafish (lin et al., ) . in this study, we analyzed genomic organizations, gene structures, and synteny, evolutionary process and expression of miiuy croaker dc-sign (mmdc-sign) and l-sign (mml-sign). we are the first to comprehensively analyze l-sign in fish and gene synteny on genomic level of dc-sign and l-sign in teleost. to obtain the dc-sign and l-sign from miiuy croaker, the blastp and tblastn programs with a query set of the previously characterized dc-sign and l-sign were used to search the miiuy croaker transcriptome (che et al., ) and whole genome database (unpublished data). two corresponding scaffolds were identified which have a strong identity with sequences previously accepted as dc-sign and l-sign respectively. miiuy croaker dc-sign and l-sign have been identified and the nucleotide sequences have been deposited in genbank database under the accession numbers km and km . the orf of mmdc-sign and mml-sign were predicted using the open reading frame finder (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html) and genscan (burge and karlin, ) . the potential protein domains were characterized by the smart program (letunic et al., ) . all the other species dc-sign and l-sign cdna sequences were obtained from the genbank (http:// www.ncbi.nlm.nih.gov/genbank/) and ensemble genome browser (http://www.ensembl.org/) databases (supplementary table s ). mega software (tamura et al., ) and dnaman were used for the multiple alignment and dnastar was used to gain the divergence and percent identity values of all the known dc-sign and l-sign amino acid sequences (thompson et al., ) . a phylogenetic tree of several kinds clrs (cd , clec g, cd , asgr , cd , dc-sign, l-sign and l-sign-like) was constructed by the neighbor-joining (nj) method with bootstrapped times using the poisson model in the mega program. while another phylogenetic tree of all the known l-sign sequences was established via the bayesian approach using mrbayes v . which is running , , generation with % of trees burned (ronquist and huelsenbeck, ) and the resulting tree was visualized and edited by treeview (page, ) . the neighboring genes of mmdc-sign and mml-sign were confirmed in genomic regions by blast programs and the neighboring genes locations of l-sign in other fishes were confirmed by genomicus (muffato et al., ) . the nonsynonymous and synonymous rate ratio ω (dn/ds) stands for the change of selective pressures. in short, ω = , < , and > were indicative of neutral evolution, purifying selection and positive selection. to test the evolution of l-sign gene in fishes, the codeml of paml software (yang, ) and the hyphy package of data monkey web server (pond and frost, ) were used to estimate the ratio of ω. in both codeml and the data monkey web server, several models were carried out as described by zhu et al. ( b) . healthy miiuy croakers individuals were obtained from zhoushan fisheries research institute (zhejiang, china), temporarily cultured in the seawater tank of °c to adapt to the ambient and evaluate of the fish health under a natural photoperiod for two weeks. after acclimatizing, ten healthy tissues (liver, spleen, kidney, intestines, heart, muscle, gill, brain, eye, and fin) were obtained from the uninfected control fishes and kept at − °c. the challenge experiments of miiuy croaker with a common bacterial pathogen, vibrio anguillarum, were performed as previously described . fish samples were randomly divided into two groups, injection and control groups. in injection group, fishes were injected with ml suspension ( . × cfu/ml). at h, h, h, h, h and h post-injection, fish samples were killed and three tissues (liver, kidney and spleen) of infection were removed and stored at − °c. total rna was extracted from the various tissues samples by trizol reagent (qiagen) following the manufacturer's instructions and cdna was synthesized using quantscript rt kit (tiangen) according to the manufacturer's protocol which stored at − °c for further experiment. three pairs of primers (dc-sign-rt-f/r, l-sign-rt-f/r,β-actin-rt-f/r) were designed to study the expression level of mmdc-sign and mml-sign (supplementary table s ). real time quantitative pcr(qrt-pcr) was used to test dc-sign and l-sign genes expression level in ten uninfected tissues (liver, spleen, kidney, intestines, heart, muscle, eye, brain, fin, gill) and in three infected tissues (liver, spleen and kidney) the rt-pcr was executed on a real time pcr system (applied biosystems, usa) and every expression was performed in three independent replicates in the same condition. the conditions of the rt-pcr was carried out as described by xu et al. ( ) . significance test of the expression level was analyzed by a duncan test. expression differences were considered at a significance level when p < . and all data were expressed as the mean ± se (standard error). the full length mmdc-sign and mml-sign genes were successfully obtained. the cdna sequence of mmdc-sign which had exons and introns was bp containing a '-utr of bp, a '-utr of bp and an orf of bp coding amino acids (fig. a) . the cdna sequence of mml-sign which had exons and introns was bp containing a '-utr of bp, a '-utr of bp with polyadenylation signal (aataaa) at bp downstream of the translation termination codon and an orf of bp coding amino acids (fig. b) . introns in both mmdc-sign and mml-sign appeared the pattern of gt/ag known by comparison with other vertebrates dc-sign and l-sign sequences separately, while comparison of the deduced amino acid sequences showed that mmdc-sign shared . % to . % identity with dc-sign from other species and mml-sign shared . % to . % identity with other species separately (supplementary table s ). both mmdc-sign and mml-sign amino acid sequences had two domains: a transmembrane region and a crd differing from primates which had specific internal repeat domain (fig. c, d) . in the dc-sign crd domain, epd motif and wyd motif existed in miiuy croaker and large yellow croaker instead of epn motif and wnd motif in all other species for determining the carbohydrate binding specificity (kong et al., ) ; epn motif, wnd motif and four amino acids (glu , asn , glu and asn ) existing in crd domain of mml-sign are well conserved from fish to human. all of the conserved motifs are essential for interacting with ca + for recognizing carbohydrate ligand (khoo et al., ) . the tri-acidic cluster is con-served in human dc-sign (svajger et al., ) and l-sign (koppel et al., ) , but not in miiuy croaker. mm-dc-sign and mm-l-sign both have four conserved cysteine residues in positions cys , cys , cys , cys and positions cys , cys , cys , cys to form the internal disulfide bridges revealed by multiple sequence alignment which also existed in other known dc-sign and l-sign sequences of vertebrate (huang and meng, ; lin et al., ) . miiuy croaker dc-sign and l-sign had the same domains with other fish, amphibian and bird that showed both dc-sign and l-sign had conserved domains from fish to mammals except primates. interestingly, when we analyzed genomic synteny of l-sign in teleost, one gene next to l-sign which has high similarity with l-sign gene and clustered with l-sign in the phylogenetic tree of clrs ( fig. a) was named as l-sign-like gene tentatively. l-sign-like gene was obtained from miiuy croaker, tilapia, medaka, table s . stickleback from the genome databases. meanwhile, only one l-sign gene existed in human, chimpanzee, rat and xenopus known from genome databases in ensemble, but there are l-sign and l-signlike in some fishes maybe in consequence of the fish-specific genome duplication (fsgd or r) to adapt to the specific environment. only one l-sign was found in platyfish maybe because the l-sign-like gene was lost after l-sign duplicated in platyfish (fig. b) . the l-sign-like genes with l-sign genes formed a cluster that the genes were located in scaffold , scaffold gl . , chromosome , scaffold jh . , and group xv in miiuy croaker, tilapia, medaka, platyfish and stickleback genomes separately. sequence alignment of l-sign and l-sign-like from several fishes was carried out by dnaman software to show that l-sign-like shared high similarity with l-sign genes containing conserved cysteine residues, a relatively conservative epn, wnd motifs (fig. e) . this finding provide more information to reveal the adaptive evolution and selection of l-sign genes. information from genome was used to analyze genomic synteny of mmdc-sign (fig. f ) and l-sign among several fishes (fig. g ) to extend their evolutionary analysis. we confirmed mmdc-sign located in scaffold and mml-sign located in scaffold of miiuy croaker genome, and also confirmed some genes surrounding mmdc-sign and mml-sign to propose a linear framework of dc-sign and l-sign in teleost (fig. f, g) which showed mmdc-sign located between clec e and cd and no znf , znf and lrrc e genes surrounding the mmdc-sign which surrounded dc-sign in a chromosome of human and zebrafish separately (lin et al., ) . miiuy croaker l-sign was located between l-sign-like and ttc , and also differences happened among the linear organization of the five fishes in basic chromosome number and the amount of surrounding genes. both miiuy croaker and tilapia formed a cluster containing five c-type lectin receptors, but only two c-type lectin receptors were clustered together in other three fishes. there was one l-sign and one l-sign-like in four fishes except platyfish (fig. g ). among these fish genomes, genes from ttc to entpd located downstream of l-sign had conserved synteny only differing in no rabepk gene in miiuy croaker and no rabepk and aldh a in stickleback. upstream of l-sign gene, only miiuy croaker had c orf and medaka had two nek which were the biggest difference among these fishes. stickleback was lacking in acyp , different from all other fishes (fig. f ). among the clr members, two genes (l-sign-like and mrc ) in tilapia had different orientation from other fishes. but synteny analysis still revealed that the gene loci for l-sign in teleost were quite conserved. the phylogenetic tree of members belong to clrs ( fig. a) showed that each kind clr gathered together separately except dc-sign, l-sign and l-sign-like which gathered together that perhaps because of the genes with similarity among species, branches of cd , clec g, cd , dc-sign, l-sign and l-sign-like grouped together showing that these genes had a closer evolution position and had a relatively further evolution distance to asgr and cd . in branch of dc-sign and l-sign, l-sign-like sequences grouped with l-sign sequences closely which showed l-sign-like share high similarity with l-sign; all fishes and mammal sequences grouped separately that mmdc-sign grouped with large yellow croaker and mml-sign grouped with zebra mbuna and tilapia l-sign-like. another phylogenetic tree of l-sign genes constructed by bayesian approach (fig. c ) was used to test the positive selection in ancestral lineages of fishes. firstly, the value of ω was . in the one-ratio model which assumed that one unique ω in all branches of the tree (p = . , supplementary table s ) , showing that the entire l-sign genes underwent the purifying selection. secondly, the freeratio model fitted the data significantly better than the one ratiomodel (p = . , supplementary table s ). finally, the branch-site model was used to detect whether positive selection sites existed in ancestral lineages of fishes. no positive selection sites were found in ancestral lineages of fishes (supplementary table s ). meanwhile, multiple ml methods were carried out to explore the selective pressures on l-sign genes of fishes. for fishes l-sign genes, no positive selection genes were detected in m a and m model (table ) , but other ml methods detected positive selection sites in this group (table ) . to improve the accuracy of positive selection sites, we considered that the site under positive selection should be detected in at least three of the ml methods. so we found one positive selection site ( , table ) on fishes l-sign genes. the result showed that the ancestral lineages of fishes underwent purifying selection, but the current lineages of fishes underwent positive selection. the fishes had lived in aquatic environment which was filled with countless types and numerous amounts of pathogens. as the years went by, the aquatic environment had changed greatly and the pathogen significantly differs from before. due to the change of aquatic environment, the fishes must further evolve to adapt to the environment. so we detected positive selection sites in current fishes of l-sign genes. in order to explore expression patterns of dc-sign and l-sign genes, the expression level of mmdc-sign and mml-sign genes in different tissues were confirmed by qrt-pcr. we set gill as calibrator to normalize dc-sign and l-sign expression level in ten uninfected tissues. the highest expression level of mmdc-sign was in liver, muscle and intestines, while the expression in gill and heart was weak. miiuy croaker l-sign have a very high level expression in intestines and spleen and moderate expression in kidney and liver, while the expression level in heart, muscle, eye, gill, fin and brain was much lower than other tested tissues (fig. a) . in vibiro anguillarum challenged groups, the tissue distribution and temporal response levels of mmdc-sign and mml-sign at different hours were detected in the infected liver, kidney and spleen. in infected liver group, both dc-sign and l-sign have a sharp up-regulation in h and floating up and down from h to h, then reaching the peak at h and sharply decreasing in h (fig. b ). in spleen, the expression of dc-sign was volatile from h to h; the highest expression level and lowest expression level were at h and h. the expression of l-sign floats up and down from h to h, then increased gradually and reached its peak at h (fig. c ). in the kidney, the expression of dc-sign increased and reached its peak at h and the lowest expression level was at h, while the highest expression level and lowest expression level of l-sign occurred in h and h, respectively (fig. d ). both the expressions of mmdc-sign and mml-sign were up-regulated in infected liver, spleen and kidney at different sampling time points, and mml-sign had the same trend with mmdc-sign in floating up and down in liver and kidney. these results indicated that the mmdc-sign and mml-sign participated in the immune response to defend against bacterial infection. in this study, dc-sign and l-sign genes were identified from the miiuy croaker genome and also analyzed the characters of both genes. miiuy croaker dc-sign and l-sign had similar domains with other fishes. we proposed a linear framework of dc-sign and l-sign in teleost and confirmed that mmdc-sign had no conserved genomic synteny with human and zebrafish, and l-sign genes in teleost had conserved synteny and underwent positive selection to adapt to the aquatic environment. we also confirmed that the two genes participated in the immune response in teleost. most important of all, it is the first time to comprehensively analyze l-sign in fish and comparative genomics of gene synteny in dc-sign and l-sign to provide more information on the study of dc-sign family genes. l-sign-like gene which has high similarity with l-sign gene was obtained in miiuy croaker, tilapia, medaka, stickleback from gene synteny and clustered with l-sign in phylogenetic tree. but only one l-sign gene was found in mammals and frog. two l-sign genes existed in some fishes maybe because of fsgd. from gene synteny, the l-sign and l-sign-like clustered together maybe to perform more functions to recognize and defend pathogens. the information can reveal the adaptive evolution and selection in the changing environment of l-sign genes. our study will provide more information in studying the evolutionary process and expression patterns of immune genes. c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans innate immunity of finfish: primordial conservation and function of viral rna sensors in teleosts dc-sign and dc-signr genetic diversity among different ethnic populations: potential implications for pathogen recognition and disease susceptibility prediction of complete gene structures in human genomic dna characterization of the miiuy croaker (miichthys miiuy) transcriptome and development of immune-relevant genes and molecular markers identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses identification of a porcine dc-sign-related c-type lectin, porcine clec g (lsectin), and its order of intron removal during splicing: comparative genomic analyses of the cluster of genes cd /clec g/dc-sign among mammalian species dc-sign and l-sign: the signs for infection a novel c-type lectin from bay scallop argopecten irradians (aictl- ) agglutinating fungi with mannose specificity distinct functions of dc-sign and its homologues l-sign (dcsignr) and msignr in pathogen recognition and immune regulation targeting c-type lectin receptors with multivalent carbohydrate ligands smart : domains in the context of genomes and networks the vntr polymorphism of the l-sign gene and susceptibility to hiv- infection in han chinese population the dc-sign of zebrafish: insights into the existence of a cd homologue in a lower vertebrate and its involvement in adaptive immunity dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e genomicus: a database and a browser to study gene synteny in modern and ancestral genomes treeview: an application to display phylogenetic trees on personal computers datamonkey: rapid detection of selective pressure on individual sites of codon alignments inhibitory c-type lectin receptors in myeloid cells mrbayes : bayesian phylogenetic inference under mixed models c-type lectin dc-sign: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice characterization and expression of the cxcr and cxcr in miiuy croaker and evolutionary analysis shows the strong positive selection pressures imposed in mammal cxcr paml : phylogenetic analysis by maximum likelihood advances in research of fish immune-relevant genes: a comparative overview of innate and adaptive immunity in teleosts characterization of the ccr and ccr genes in miiuy croaker and different selection pressures imposed on different domains between mammals and teleosts this study was supported by national natural science foundation of china ( ) and zhejiang province natural science foundation of distinguished young scientists (lr c ). supplementary data to this article can be found online at doi: . /j.dci. . . . key: cord- -ebj v o authors: marini, robert p.; otto, glen; erdman, susan; palley, lori; fox, james g. title: biology and diseases of ferrets date: - - journal: laboratory animal medicine doi: . /b - - / - sha: doc_id: cord_uid: ebj v o nan ferrets (mustela putorius furo) belong to the ancient family mustelidae, which is believed to date back to the eocene period, some million years ago. the taxonomic groups in the family mustelidae, as recognized by corbet and hill ( ) , include species from north, central, and south america, eurasia, and africa. no other carnivore shows such diversity of adaptation, being found in a wide variety of ecosystems ranging from earlier references to ferrets are probably the basis of the belief that ferrets originated in north africa (thomson, ) . evidently they were bred specifically for rabbiting (rabbit hunting) and were muzzled before being sent into rabbit burrows. this practice was later introduced into europe, asia, and the british isles, where the sport is still practiced today. although the ferret has been historically used for hunting, more recently it has been increasingly used in biomedical research and is popular in north america as a pet. it is most likely a domesticated version of the wild european ferret or polecat (m. putorius or m. furo) (thomson, ) . alternatively, it may be related to the steppe polecat (m. eversmanni), which it closely resembles in skull morphology (walton, ) . the domesticated ferret, although introduced to north america by the early english settlers some years ago, has not established feral colonies on this continent. the ferret was not recognized as having potential as an animal model for biomedical research until the s. early studies utilized the ferret in classic experiments with influenza virus pathogenesis (pyle, ) . its use was cited infrequently; an article published in , detailing the use of ferrets in research, cited only publications (pyle, ) . literature reviews undertaken in , , , and , however, revealed an increasing appreciation for the ferret's usefulness and versatility in the study of human physiologic, anatomic, and disease mechanisms (hahn and wester, ; marshall and marshall, ; shump et al., ; frederick and babish, ) . in , a bibliography containing "selected" literature citations on the ferret and its use in biomedical research was published (clingerman et al., ) . the document was designed to serve as a reference tool for individuals involved in the care or use of ferrets in the laboratory setting. although not comprehensive, the document provides extensive coverage of ferret biology, diseases, and use as an animal model. the domesticated ferret has been and continues to be used extensively in studies involving virology, reproductive physiology, anatomy, and endocrinology, as well as other areas of biomedical research (morgan and travers, ) . the ferret is also being used to replace the cat in some types of neuroendocrinology, neuroanatomy, and cardiology experiments. the ferret's increasing popularity in research and as a pet is mainly a result of large-scale commercial production. for example, commercial farms have been raising ferrets for almost years. biomedical researchers in the united states can request animals of a specific sex, weight, and age for individual experiments. investigators in other countries may acquire fer-rets from fur operations or may make arrangements with commercial vendors in the united states. even though the ferret is nonstandardized with regard to exact genotype and pedigree, its routine availability in a clinically healthy state has aided immeasurably its acceptance as a research animal. readily available commercial stocks, based on coat color, are albino, sable (or fitch), siamese, silver mitt, and siamese-silver mitt (siamese with white chest and feet) (mclain et al., ) . the fitch or so-called wild coat color is the most common, recognized by yellow-buff fur with patches of black or dark brown, particularly on the tail and limbs (andrews and illman, ) . the production of ferrets by large commercial operations has raised concern by some that inbreeding of these animals has made the ferret more susceptible to diseases, e.g., endocrinerelated disorders. this topic is covered in more detail in chapter . housing of ferrets in a research facility is similar to that of other small carnivores such as cats (fox, c) . ferrets tolerate low temperatures well and high temperatures poorly; the recommended temperature range for juvenile and adult animals is - ~ (hammond and chesterman, ) . ferrets less than weeks of age should be housed at > ~ c. kits under this age require a heat source if separated from the dam; older kits that are group-housed do not. elevated temperatures (> ~ cannot be tolerated by ferrets, because they have poorly developed sweat glands and are susceptible to heat prostration. signs of hyperthermia include panting, flaccidity, and vomiting. the preferred humidity is - %. for nonbreeding animals that will remain in the facility for a short time, a conventional dark-light cycle at : hr is adequate. lighting may be altered to control breeding cycles. breeding and lactating jills should be exposed to hr of light daily. ferrets that are maintained for breeding or for use beyond months should be exposed to "winter" lightw weeks per year of hr of dark dailywto maintain physiologic normalcy. it is also essential that researchers receiving time-pregnant jills preserve the photoperiod to which jills were exposed prior to shipment. failure to do so may cause inappetence, with subsequent negative energy balance and pregnancy toxemia. similar to other laboratory animal species, ferrets should be housed with - air changes per hour (usdhhs, ) . it is important to use nonrecirculated air because of the strong odor of ferrets and the susceptibility to respiratory tract infections. the ferret odor should not overlap into any rodent housing areas, because rodents have an instinctive fear of ferrets, and the ferret scent can disrupt rodent breeding and physiology (fox, c) . female ferrets can be housed singly or in groups, but estrous females that are cohoused may become pseudopregnant (beck et al., ) . males should be housed individually after weeks of age. molded plastic caging used to house rabbits works very well for ferrets. the solid bottom is perforated with holes and is readily sanitizable. an absorbable paper liner may be used in the pan beneath the cage to facilitate daily disposal of urine and feces. in a research setting, the plastic caging should be washed weekly to avoid excessive soiling. the spacing of grid walls should be . x . inches apart, or . inch if using wire mesh. ferrets like to lick and bite at their enclosures, so sharp edges and galvanized metal should be avoided. zinc toxicosis has been reported from licking galvanized bars from which metals had leached during steam sterilization (straube and walden, ) (table i) . ferrets can be trained to use a litter box because they repeatedly urinate or defecate in one corner of the cage. clay litters have been reported to cause chronic upper respiratory irritation parameter (jenkins and brown, ) . ferrets prefer sleeping in a soft isolated area, and in a research facility this can be accomplished by providing a washable "snooze tube" (fox, c) . the thorax of the ferret is narrow and elongated, and as a result the trachea is proportionally long. this makes the ferret an ideal species for studies of tracheal physiology. the tracheal size and laryngeal anatomy make endotracheal intubation somewhat challenging, and as a result the ferret has been advocated as a species suitable for use in pediatric intubation training (powell et al., ) . the lungs are relatively large, and the total lung capacity is nearly times that which would be predicted based on body size, as compared with other mammals. this characteristic, together with a higher degree of bronchiolar branching and more extensive bronchial submucosal glands (as compared with the dog), makes the ferret an attractive model for pulmonary research studies (vinegar et al., ) . although a previous report (willis and barrow, ) commented that the carotid arterial branching pattern in the ferret is unusual, it is actually typical for a carnivore. as is the case in the dog and the cat, the paired common carotid arteries arise from the brachiocephalic trunk (sometimes called the innominate artery) at the level of the thoracic inlet (andrews et al., b) . the ferret's gastrointestinal tract is specialized to fit its carnivorous nature. the simple monogastric stomach is similar to that of the dog. there is no cecum present, and the indistinct ileocecal transition makes it difficult to identify the junction of the small and large intestines during a gross examination. the overall length of the alimentary tract is very short relative to the body size, resulting in a gastrointestinal transit time as short as hr (bleavins and aulerich, ) . as in other mustelids, the paired anal scent glands of the ferret are well developed. although not as potent as those of the skunk, the secretions of the ferret are sufficiently odoriferous that many pet or research ferrets are descented. surgical techniques for this procedure have been described (creed and kainer, ; mullen, ) . ferrets, especially intact males and estrous jills, may possess a distinctive musky odor even after a successful descenting, because of normal sebaceous secretions. ferrets lack well-developed sweat glands for use in thermal regulation, and as a result they are predisposed to heat prostration when ambient temperatures reach ~ ( ~ f) (ryland et al., ) . extramedullary hematopoiesis is commonly found during histological examination of the spleen, and in some cases it may result in a grossly evident splenomegaly (erdman et al., ) . this must be differentiated from splenomegaly that can arise from a variety of pathologic conditions or from isoflurane administration (see section iii,e). experimental evidence suggests that ferrets have no naturally occurring antibodies against unmatched erythrocyte antigens, and that none develop even in the face of repeated transfusions . ferrets are seasonal breeders, and the resulting pronounced physiological variations in body weight, behavior, and gametogenesis are utilized in scientific studies of photoperiod responses and neuroendocrine control. prolonged estrus in unbred females can cause an aplastic anemia, an effect that can be reproduced with exogenous estrogen administration . the male has a radiographically evident os penis, and, contrary to some earlier reports, a prostate gland is present in males (evans and an, ) . newborn ferret kits weigh - gm at birth and will grow to gm by the time they are weaned at - weeks (shump and shump, ) . in sexually intact populations, males ( . - . kg) can be twice the size of females ( . - . kg). the adult weight of nonobese male and female ferrets that have been gonadectomized prior to weaning and raised in captivity will generally fall between . and . kg (brown, a) . adult animals (especially those that are sexually intact) may be subject to seasonal fluctuations in body fat percentage, which can cause body weight to fluctuate by - % (fox and bell, ) . the approximate life span for the ferret is - years, but on rare occasions they may live as long as years (table ii) . normal hematology and serum chemistry values have been reported for the ferret (thornton et al., ; lee et al., ; fox, e) . these values are not greatly dissimilar from those of other domestic carnivores. one distinctive hematological characteristic of the ferret is the presence of a relatively robust erythron, characterized by hematocrit, hemoglobin, and total erythrocyte and reticulocyte counts that are generally higher than those of the dog or cat. reported neutrophil-lymphocyte ratios range from . : to . : . representative hematology and chemistry ranges from one of our studies (fox et al., b) are shown in tables iii and iv, but for diagnostic purposes any laboratory that evaluates ferret samples should develop its own set of specific normal ranges. a low-grade proteinuria may be identified by urinalysis in normal, healthy ferrets (thornton et al., ) (table v) . ferret diets have been formulated both empirically and based upon the nutrient requirements of other mustelids (fox and mclain, ) . specific requirements for various life-cycle stages have not been determined experimentally. available commercial diets are certainly capable of supporting growth, reproduction, and maintenance in conventional settings. in the (fox et al., b) . bfour-to -month-old ferrets (loeb and quimby, ) . cnd, not done. absence of careful analysis, however, it is uncertain whether the proportion and quantity of ingredients in these diets is optimal. ferrets are strict carnivores with a high requirement for dietary fat and protein. their short digestive tract and rapid gastrointestinal transit time ( - hr) require protein to be readily digestible. there is general agreement that ferrets should not be given diets high in complex carbohydrates or fiber. diets that are high in fish products are also not recommended for ferrets (fox and mclain, ) . the use of any raw chicken, beef, or other meats is strongly discouraged because of the potential contamination by campylobacter, salmonella, listeria, mycobacterium, and streptococcus (fox, a) . daily maintenance energy consumption for ferrets is - kcal/kg body weight. calorie-percent protein ratios have been determined for mink (mustela vison) kits up to and after weeks of age (sinclair et al., ; allen et al., ) . a ratio of and a caloric density of kcal/ gm of feed, corresponding to % protein, provided optimum growth for male kits up to weeks. after weeks, ratios of and , corresponding to % and % protein, respectively, were recommended. diets containing - % fat and - % carbohydrate have been used successfully to maintain ferrets. one author recommends - % protein and - % fat for adult, nonbreeding animals and a minimum of % protein and % fat for reproductively active animals and those that have notreached sexualmaturity (brown, a) . the long-term impact of diets containing high levels of fat and protein are unknown. ferrets have been used to investigate the absorption, metabolism, and interaction of the dietary micronutrients [ -carotene and vitamin e. ferrets, like humans, convert [ -carotene to vitamin a in the gut and absorb ~-carotene intact (fox and mclain, ) . in intestinal perfusion experiments in ferrets, it was demonstrated that [ -carotene, retinol, and retinyl esters are absorbed intact into lymph and that cleavage products, including [ -apo- '-carotenal, [ -apo- '-carotenal, and retinoids, accumulate in the intestinal mucosa (wang et al., ) . the intestinal mucosa is capable of converting [ -carotene into retinoic acid and other polar metabolites, which are then transported via the portal vein to the liver (wang et al., ) . [ -carotene absorption is enhanced by co-perfusion with a-tocopherol, and the perfusion of the latter is unaltered by the presence of [ -carotene. the conversion of [ -carotene into retinol is also enhanced by the presence of a-tocopherol (wang et al., ) . these and other findings have established the ferret as an important model for the study of these antioxidants. adult ferrets drink - ml of water daily, depending on the dry-matter content of the feed (andrews and illman, ) . fresh water can be provided ad libitum in stainless steel bowls or water bottles with sipper tubes. ferrets are playful and will overturn bowls or water bottles that are not well secured. features of ferret reproduction may be found in table vi for males, corresponding temporally to increasing day length. ferrets born in the late spring or early summer and maintained under natural lighting will not assume an adult pattern of gonadal activity (i.e., puberty) until the following season (baum, ) . under artificial illumination, jills that are maintained at hr light- hr dark reach puberty at - months. stimulatory photoperiods may be used, however, in the laboratory or intensive production setting, as a method of breeding ferrets out of the natural season. however, the transfer from short to long photoperiods should not occur prior to days of age, because jills that are prematurely transferred will remain anestrous (hammond and chesterman, ) . management practices in one breeding facility are such that jills commence breeding at - months, average . litters a year, and are cycled out of reproduction after litters. in another strategy, ferrets are exposed to a : hr photoperiod at weeks of age, are bred at weeks during their first estrus, and whelp at v months. vulvar swelling is the hallmark of estrus in jills. the ease with which estrus is detected in the ferret, as well as the size of the ferret and ease of its maintenance in captivity have made the ferret a model for study of neuroendocrine events and their gonadal correlates. along with the hamster, the ferret has contributed extensively to an understanding of the photoperiodic influences on the hypothalamic-pituitary-gonadal axis (baum, ) . as in females of other species, estradiol concentrations are responsible for controlling the development of the female reproductive tract and secondary sexual characteristics, and the tonic inhibition of luteinizing hormone (lh) secretion by the anterior pituitary during both prepubertal life and anestrus. the sensitivity of the hypothalamic gonadostat to negative feedback inhibition by estradiol changes at the time of puberty, and under the influence of increasing light exposure, lh levels rise despite estradiol (ryan, ) . similarly, age differences in the sensitivity of negative feedback inhibition of the hypothalamic secretion of gonadotropin-releasing hormone (gnrh) by testosterone, or to estrogenic compounds derived from the aromatization of testosterone, appear to be essential in determining puberty and seasonality of reproduction in the male (baum, ) . estrus in jills is characterized by dramatic vulvar swelling from an anestrous diameter of - mm to an estrous diameter of - mm. changes in vaginal cytology have also been described for the ferret and other mustelid species, but these changes are seldom used to determine onset of estrus or to schedule breeding (williams et al., ) . after a -to -week proestrus, estrus occurs. estrus onset is not associated with elevated serum fsh in the ferret, as it is in the rodent. once estrus has occurred, it may terminate in coitus-induced ovulation and pregnancy, pseudopregnancy after infertile mating, pharmacologic termination (by injection of human chorionic gonadotropin (hcg) or gnrh), death due to estrogen-induced aplastic anemia, or spontaneous remission and anestrus due to reduced photoperiod. waves of follicular development occur in estrus, and - ova are ovulated approximately - hr after coitus. female ferrets are brought to the male approximately days after vulvar enlargement. females and males copulate many times and for prolonged periods of time; they are typically left together for days. both intromission and neck restraint by the male are apparently required for induction of ovulation (baum, ). an lh surge accompanies coitus in females, but the same is not true of males (carroll et al., ) . implantation occurs days after mating; both a functional corpus luteum and the anterior pituitary are required for implantation and maintenance of pregnancy. placentation is typical of carnivores and is zonary and endotheliochorial (morrow, ) . pregnancy may be detected by ultrasonographic demonstration of - discrete nonechogenic structures as early as day (peter et al., ) , by palpation as early as day , or by radiographic demonstra-tion of calcified fetal skeletons at approximately days of gestation. jills within weeks of parturition should be singly housed and provided with a secluded place in which to deliver their kits. when rabbit cages are used for housing, nest boxes may take the form of polypropylene rat cages or other plastic boxes (cat litter box or dishpan). nest boxes should have bedding provided for warmth and comfort. materials suitable for bedding include pieces of fabric (towels), ripped cageboard, shredded paper, or cotton batting. the nest box should be at least inches deep and should prevent the kits from wandering from the jill. entrance to the nest box should be smooth, to avoid injury to the teats and mammary gland. at our institution, jills are provided a stainless steel rectangular box with a smooth-surfaced plastic entrance ( fig. ) . a retractable steel roof panel and a guillotine side panel exposing a plexiglas sidewall allow access to the jill and permit observation with minimal disturbance. one major supplier of ferrets uses sunken tubs filled with bedding to promote a sense of security and isolation of the jill. most jills will leave the nest box to eat and drink. if the jill will not leave, however, low-sided food bowls should be placed within the nest box. parturition occurs rapidly in ferrets and may last as little as - hr. primiparous jills typically deliver on day of gestation whereas multiparous jills deliver on day . there are few signs of impending parturition, although abdominal enlargement and mammary development do occur in the last week or two. small litters (fewer than three) may result in inadequate stimulus for parturition. jills that pass their due date without delivery should be palpated for fetuses. kits remaining in utero beyond the rd day typically die; kits with congenital malformations such as cyclopia and exencephaly may also delay the initiation of labor. dystocia is common in ferrets because of positional abnormalities and fetal oversize and should be treated by cesarean section. jills tolerate cesareans well and will nurse kits delivered in this way. if small littel: size is responsible for delayed parturition, prostaglandins ( . - . mg lutalyse) may be used, followed by . ml oxytocin ( u) after hr (fox and bell, ) . failure to deliver within hr of administration of prostaglandin is an indication for cesarean section. jills should be provided heat, energy, hydration, and analgesia following cesarean. kits will attempt to nurse soon after parturition, but jills experiencing difficult labor may not allow them to nurse until all kits are delivered. jills that are not attentive to their kits should be palpated for the presence of additional, undelivered kits. oxytocin may be used to facilitate delivery of remaining kits. offering the jill regular chow mixed with warm water may promote maternal acceptance. kits should be kept warm pending acceptance by the jill. jills should be left undisturbed for the first several days postpartum to avoid their cannibalizing the litter. cross-fostering to other jills may be successfully accomplished, provided that the kits are warm and that the foster jill has kits of similar age. kits to be fostered should be allowed to mingle with the foster jill's own kits while their dam is absent so that rejection due to olfaction will not occur. kits are born in an altricial state, covered by lanugo hair and with their eyes closed. by days of age, albino ferrets retain their white hair whereas pigmented ferrets acquire a gray coat. they are completely dependent on the jill for the first weeks of life. defecation and urination are stimulated by jills through anogenital licking of the kits. kits are born weighing - gm, double their weight in days, and triple it in days to a weight of gm. the -week-old male kit should weigh at least gm. sexual dimorphism in size is apparent by week and persists into adulthood. developmental landmarks include ability to hear at days, opening of the eyes at days, eruption of deciduous teeth at days, eruption of permanent canines at - days, and displacement of deciduous canines by - days (fox and bell, ) . gender may be distinguished in neonatal ferrets, as in other species, by anogenital distance, with the distance being much shorter in females than in males. in males, the urogenital opening is seen just caudal to the umbilicus. the prominent midline raphe penis overlying the palpable os penis is also a distinctive feature in the male. ferrets are typically weaned at weeks of age. early weaning may be encouraged by making a slurry of the jill's chow available at - weeks; fat may be added to achieve a fat content of %. the fatty acid supplement linatone (lambert kay, cranberry, new jersey) is recommended by one author (brown, a) . the diet should contain approximately % fat and % protein. the slurry should be fed twice daily for a restricted time and then removed to avoid having kits walking through and defecating in the diet. unthrifty kits over days of age may be supplemented with canine or feline milk replacers administered per os by tygon-tipped pasteur pipette . weaned ferrets are best housed in groups until sexually mature. males over weeks old may begin to fight if exposed to greater than hr light per day. jills may return to estrus during the second or third week of lactation if they have fewer than kits or weeks after weaning if the litter is of normal size. jills should be rebred or administered hcg to terminate estrus, even if still lactating. a highquality, calorie-dense diet is required for lactation and to maintain pregnancy. if maintained on a stimulatory photoperiod and adequate nutrition, jills may have - liters of or more kits yearly until they are years old (fox and bell, ) . a nonstimulatory photoperiod should be used weeks per year to rest the ferret and preserve maximum fertility; a maintenance diet can be given at this time. jills return to estrus approximately weeks after reinstitution of the longer photoperiod. artificial insemination is not commonly performed in ferrets but has been studied in the context of providing strategies for species perpetuation of the endangered black-footed ferret (wildt et al., ) . synchronization of estrus as practiced in rodent production is not used as a tool of reproductive management in the ferret. synchronization ofjills may be approximated, however, by manipulation of photoperiod. with natural illumination in outdoor housing, jills all come into estrus within a -to -week period (baum, ) . in the laboratory setting, when jills are maintained in a nonstimulatory photoperiod ( hr light- hr dark) for - weeks, followed by reversal of the cycle ( hr light- hr dark), estrus will follow in weeks (immature jills) or weeks (mature jills) after the change (carroll et al., ) . this correlates with follicular development and increased plasma estradiol. the occurrence of infectious disease affects animal health and well-being and may complicate research efforts. a program combining good animal husbandry, optimal nutrition, health monitoring practices, and clinical care is essential to maintaining a healthy ferret colony. etiology. the etiologic agent is clostridium perfringens type a (clostridium welchii). epizootiology and transmission. clostridium perfringens is ubiquitous and is present in the intestinal contents of humans and animals. clostridium perfringens type a has been associated with the occurrence of acute abdominal distension, dyspnea, and cyanosis in weanling ferrets (field and laboratory service veterinary staff, ) and an outbreak of gastroenteritis in weanling black-footed ferrets (schulman et al., ) . the exact cause of these conditions is uncertain, but predisposing factors such as overeating, sudden changes in diet, the prolifer-ation of c. perfringens type a, and the production of overwhelming amounts of toxins are suspected (field and laboratory service veterinary staff, ; schulman et al., ) . the alpha toxin is the principal lethal toxin. it is hemolytic and necrotizing and possesses the ability to split lecithin or lecithinprotein complexes, leading to destruction of cell membranes and subsequent necrosis. reported cases have involved weanling animals exclusively. clinical signs. ferrets may present with acute abdominal distension, dyspnea, and cyanosis or may be found dead and bloated (field and laboratory service veterinary staff, ; schulman et al., ) . diagnosis. isolation of c. perfringens type a from gastric and small-intestinal contents is required. toxin identification may be performed by the use of a mouse protection assay (smith, ) . necropsy findings. gross findings include markedly distended stomachs and intestines containing a large amount of gas and a moderate amount of brown, semiliquid ingesta, and subcutaneous emphysema with minimal or no putrefaction (field and laboratory service veterinary staff, ; schulman et al, ) . histologic findings observed in weanling black-footed ferret cases included the observation of abundant gram-positive bacilli in smears of gastric and intestinal contents. other findings included varying degrees of gastrointestinal mucosal necrosis, numerous gram-positive bacilli lining the denuded mucosal surface and extending into the gastric glands and intestinal crypts; lymphoid necrosis of lymph nodes, spleen, and thymus; mild to moderate dilatation of central hepatic sinusoids with mild, acute, centrilobular hepatocellular dissociation and multifocal aggregates of small numbers of necrotic neutrophils within portal areas (schulman et al., ) . and feeding practices is the primary means of control. in the reported cases of c. perfringens type a-associated gastroenteritis in black-footed ferret weanlings, supportive care and gastric trocharization were unrewarding. the occurrence of the condition was eliminated by restricting feeding of weanlings to twice a day instead of times daily. etiology. campylobacteriosis is caused by infection with campylobacter jejuni. epizootiology and transmission. campylobacter jejuni is a gram-negative, spirally curved microaerophilic bacterium that is recognized as a significant cause of human enteritis and is as-sociated with diarrheic illness in several animal species, including dogs, cats, cows, goats, pigs, mink, ferrets, and sheep (carter et al., ) . it also known to cause mastitis in cows, infectious hepatitis of chickens, and abortion in cattle, sheep, goats, dogs, and mink (carter et al., ) . the organism may also be cultured from the feces of normal asymptomatic dogs, cats, and ferrets carter et al., ) . transmission occurs by ingestion of organisms through direct contact with feces or contaminated food and water (carter et al., ) . there have been reports linking the disease in humans to pets. many of these outbreaks were associated with dogs, puppies, and kittens recently obtained from animal shelters or pounds and displaying diarrhea before the human illness occurred . isolation of campylobacter jejuni from asymptomatic ferrets also implies a potential for zoonotic transmission (fox et al., . clinical signs. experimental oral inoculation of ferret kits with various strains of c. jejuni produced a self-limiting diarrhea that ranged in character from very mild to watery manning, a, ) . the presence of mucus and/or blood was also noted in the feces of affected animals. anorexia, dehydration, and tenesmus with watery diarrhea were also observed. intravenous inoculation of pregnant mink and pregnant ferrets resulted in reproductive failure, ranging from fetal resorption to expulsion of dead or premature living kits . oral inoculation resulted in abortion in a majority of the infected animals . diagnosis. diagnosis is based on history, clinical signs, and culture of affected animals. reports of spontaneous cases in ferrets require diagnostic confirmation and differentiation from cases of proliferative bowel disease and other infectious and noninfectious causes of diarrhea. campylobacter jejuni grows slowly and has specific culture requirements that involve the use of selective media or filtration techniques, and a requirement for thermophilic ( ~ and microaerophilic conditions (fox, a) . cultures should be examined every hours for round, raised, translucent, and sometimes mucoid colonies (fox, a) . campylobacter jejuni revealed small focal neutrophilic infiltrates in the lamina propria of the colon of relatively few infected animals . bell and manning ( ) noted mild to moderate enterocolitis with neutrophilic infiltration of the lamina propria, which was most severe in kits with concurrent cryptosporidiosis. placentitis was the most notable histologic finding in pregnant ferrets and mink after experimental inoculation of a strain of an abortion storm-associated isolate of c. jejuni . erythromycin is the drug of choice for treatment of human campylobacteriosis (fox, a) . in a study to eliminate the carrier state in ferrets, erythromycin was ineffective even though in vitro isolates of c. jejuni were sensitive to the antibiotic . according to the author, reasons for therapeutic failure included dose selection, interspecies differences in pharmacokinetics and possible reinfection. supportive care should be instituted, and choice of antibiotic therapy in confirmed diarrheic cases should be based on culture and sensitivity. in addition, because of its zoonotic potential, isolation of affected animals and good hygienic practices are recommended. reculture of animals after treatment to ensure elimination of the organism is recommended. epizootiology and transmission. in , a gastric helicobacter-like organism was isolated from the margins of a duodenal ulcer of a ferret and named helicobacter mustelae (fox et al., a (fox et al., , a . subsequently, in the united states, gastritis and peptic ulcers have been routinely reported in ferrets colonized with h. mustelae (fox et al., b (fox et al., , a . every ferret with chronic gastritis is infected with h. mustelae, whereas specific pathogen-free (spf) ferrets not infected with h. mustelae do not have gastritis, gastric ulcers, or detectable igg antibody to the organism (fox et al., , a . helicobacter mustelae has also been isolated from the stomachs of ferrets living in england, canada, australia and, most recently, from ferrets in new zealand (forester et al., ; tompkins et al., ) . koch's postulates have been fulfilled: by oral inoculation of h. mustelae into naive ferrets uninfected with h. mustelae, the infection induced a chronic, persistent gastritis similar to that observed in ferrets naturally infected with h. mustelae (fox et al., b) . it is now known that h. mustelae colonizes nearly % of ferrets shortly after weaning. feces from weanling and adult ferrets have been screened for the presence of h. mustelae to determine whether fecal transmission could explain the % prevalence observed in weanling and older ferrets (fox et al., b (fox et al., , b . helicobacter mustelae was isolated from the feces of of nine-week-old and of eight-month-old ferrets. ferrets placed on proton pump inhibitors, which raise gastric ph, have a statistically higher recovery of h. mustelae from feces when compared with age-matched untreated control ferrets . clinial signs and pathology. helicobacter mustelae-infected ferrets examined in our laboratory are usually asymptomatic. ferrets with gastric or duodenal ulcers can be recognized clinically by vomiting, melena, chronic weight loss, and lowered hematocrit. clinical signs in ferrets with h. mustelae-associated gastric adenocarcinoma have consisted of vomiting, anorexia, and weight loss, signs that may be confused with gastric foreign body. diagnosis. gastric and duodenal ulcers are observable endoscopically. it is interesting that the ferret is the only domesticated animal to date that has naturally occurring helicobacterassociated ulcer disease. the h. mustelae isolated from ferrets has similar but not identical biochemical features to those of h. pylori, particularly in regard to the production of large amounts of urease. gastric samples collected by endoscopy or necropsy are minced with sterile scalpel blades and inoculated onto blood agar plates supplemented with trimethoprim, vancomycin, and polymixin b (remel, lenexa, kansas). the plates are incubated at ~ or ~ in a microaerobic atmosphere ( % n , % h , and % co ) in vented jars for - days. bacteria are identified as h. mustelae on the basis of gram-stain morphology; production of urease, catalase, and oxidase; resistance to cephalothin; and sensitivity to nalidixic acid. necropsy and findings. the histopathological changes occurring in the stomach closely coincided in topography with the presence of h. mustelae . a superficial gastritis present in the body of the stomach showed that h. mustelae was located on the surface of the mucosa but not in the crypts. inflammation occupied the full thickness of the distal antral mucosa, the so-called diffuse antral gastritis described in humans (fig. a,b) . in this location, h. mustelae was seen at the surface, in the pits, and on the superficial portion of the glands. in the proximal antrum and the transitional mucosa, focal glandular atrophy, a precancerous lesion, and regeneration were present, in addition to those lesions seen in the distal antrum. also, deep colonization of h. mustelae was observed focally in the affected antral glands. animals infected with helicobacter spp. may also be susceptible to gastric cancer yu et al., ) . there is recent documentation of the presence of argyrophilic bacteria, compatible in location and morphology to h. mustelae, within the pyloric mucosa of male ferrets with pyloric adenocarcinoma . in humans, epidemiologic data strongly support the association between h. pylori and development of gastric adenocarcinoma. similarly, we have recently documented a series of h. mustelae-infected ferrets with gastric mucosa-associated lymphoid tissue (malt) lymphoma that parallels the same syndi'ome found in humans. lymphoma was diagnosed in the wall of the lesser curvature of the pyloric antrum, corresponding to the predominant focus ofh. mustelaeinduced gastritis in ferrets. gastric lymphomas demonstrated characteristic lymphoepithelial lesions, and the lymphoid cells were igg positive in all ferrets (erdman et al., ) . these findings and their parallels in h. pylori-infected humans implicate the involvement of h. mustelae in the pathogenesis of gastric cancer in ferrets. treatment. studies in ferrets indicate that triple therapy consisting of oral amoxicillin ( mg/kg), metronidazole ( mg/ kg), and bismuth subsalicylate ( . mg/kg) (pepto-bismol original formula, procter and gamble) times a day for - weeks has successfully eradicated h. mustelae . clinical improvement, including increased appetite and resolution of melena, may occur within hr of initiation of triple therapy. a new treatment regimen being used to eradicate h. pylori in humans has also been used successfully for eradication of h. mustelae from ferrets . ferrets received mg/kg ranitidine bismuth and . mg/kg clarithromycin per os times daily for weeks. culture of tissue collected by gastric endoscopic biopsy at , , and weeks after termination of treatment indicated that long-term eradication was achieved in all ferrets. eradication was associated with decrease in anti-h, mustelae igg antibody titers, results that are consistent with findings in humans after h. pylori eradication. omeprazole in ferrets at an oral dose of . mg/kg once daily effectively induces hypochlorhydria and may be used in conjunction with antibiotics to treat h. mustelae-associated duodenal or gastric ulcers. cimetidine at mg/kg tid per os can also be used to suppress acid secretion. acute bleeding ulcers must be treated as emergencies, and fluid and blood transfusions are essential. etiology. proliferative bowel disease is caused by intracellular campylobacter-like organisms, closely related to desulfovibrio spp., that are now classified as lawsonia intracellularis in proliferative enteropathy of swine (fox, a) . the organisms are gram-negative, comma-to spiral-shaped bacteria. epizootiology and transmission. proliferative bowel disease is a common clinical disease observed in young ferrets. fecaloral spread is suspected. the disease typically involves the large bowel, although it has been observed to affect the small bowel (rosenthal, ) . campylobacter species, coccidia, and chlamydia have been isolated from some cases of proliferative bowel disease in ferrets (li et al., b) . the role, if any, of copathogens in this disease is unclear. clinical signs. clinical signs include chronic diarrhea, lethargy, anorexia, weight loss (which is often marked), and dehydration. diarrhea may be blood-tinged, may contain mucus, and is often green in color. rectal prolapse may be observed in affected animals. ataxia and muscle tremors have also been observed (fox et al., ) . diagnosis. diagnosis is based on clinical signs, a palpably thickened colon, and colonic biopsy. it is important to rule out other causes of diarrhea and weight loss through diagnostic tests that include but are not limited to a complete blood count, chemistry profile, radiographs, and fecal analysis and culture. necropsy findings. gross findings include a segmented, thickened lower bowel, usually the terminal colon but occasionally including the ileum and rectum (fox et al., ; fox, a) . histologic examination consistently reveals marked mucosal proliferation and intracytoplasmic l. intracellularis demonstrated with silver stain within the apical portion of epithelial cells in the hyperplastic epithelial cells (fox et al., ; fox, a) (fig. a,b) . other common histologic changes observed include the presence of a mixed inflammatory infiltrate that is variable in severity, reduced goblet cell production, hyperplasia of the glandular epithelium, glandular irregularity with penetration of the mucosal glands through the muscularis mucosa, and an increase in thickness of the tunica muscularis (fox et al., ; fox, a) . translocation of proliferating glandular tissue to extraintestinal sites, including regional lymph nodes and liver, has been described in two ferrets (fox et al., b) . differential diagnosis. proliferative bowel disease should be differentiated from other diseases that may cause diarrhea and wasting, including dietary changes, eosinophilic gastroenteritis, gastric foreign bodies, lymphoma, aleutian disease, and gastric ulcers (bell, b) . a complete physical exam that includes palpation of the abdomen should reveal a palpably thickened intestine in cases of proliferative bowel disease. treatment and control. supportive care, including fluid therapy and nutritional support, should be provided. treatment with chloramphenicol ( mg/kg bid po, sq, im) or metronidazole ( m/kg bid po) for weeks is reported to be effective (krueger et al., ; bell, b) . clinical improvement may be apparent within hr. etiology. tuberculosis can be caused by a variety of mycobacteria, including mycobacterium bovis, m. avium, and m. tuberculosis. epizootiology and transmission. mycobacteria are aerobic, gram-positive, nonbranching, non-spore-forming, acid-fast rods. natural infections with mycobacterium bovis and m. avium have been reported in the ferret. ferrets are also susceptible to experimental infection with human tubercle bacillus. most reports of tuberculosis in ferrets are in animals that were used for research in england and the rest of europe between the years of to and were likely related to the feeding of raw poultry, raw meat, and unpasteurized milk to ferrets during this time (fox, a) . the feeding of commercially prepared diets and widespread tuberculosis testing and elimination in livestock and poultry have resulted in the reduced incidence of the disease in ferrets. mycobacterium avium-infected wild clinical signs and necropsy findings. clinical signs and lesions are dependent on the infective strain. systemic infection with the bovine strain in ferrets results in disseminated disease with weight loss, anorexia, lethargy, death, and miliary lesions involving the lungs and other viscera (fox, a) . progressive paralysis has also been reported in a case of spontaneously occurring bovine tuberculosis in a ferret (symmers and thomson, ) . mycobacterium bovis lesions contain numerous acid-fast bacilli within macrophages with little cellular reaction (fox, a) . in contrast, infection of ferrets with the human tubercle bacilli results in localized infection, often confined to the site of injection and adjacent lymph nodes; microscopically few organisms are observed. an impaired cell-mediated response may account for the large number of organisms observed in m. bovis lesions. vomiting, diarrhea, anorexia, and weight loss were observed in a pet ferret with granulomatous enteritis caused by m. avium (schultheiss and dolginow, ) . granulomatous inflammation characterized by large numbers of epithelioid macrophages containing numerous acid-fast bacilli were present in the lamina propria and submucosa of the jejunum and pylorus. other sites of granulomatous inflammation included peripancreatic adipose tissue, mesenteric lymph nodes, spleen, and liver. a source of infection was not identified in this report. pulmonary infection with m. avium has also been reported in ferrets in a zoo in france (viallier et al., ) . diagnosis. definitive diagnosis of tuberculosis requires isolation and identification of the organism from suspect tissue specimens. great care should be exercised in handling suspect clinical specimens, and an appropriately equipped laboratory should be identified for culture and identification of the organism. although there has been some experimental work in the area of the intradermal tuberculin skin-test response in ferrets and its apparent use in controlling tuberculosis in a breeding colony of ferrets, a tuberculin skin-testing regimen, including dose and type, has not been definitively characterized for clinical use in ferrets (kauffman, ) . treatment and control. because of the zoonotic risk, ferrets infected with m. bovis and m. tuberculosis should be euthanized (fox, a) . recurrent m. bovis infection involving the palmar aspect of the wrist of a -year-old man, which developed after he was bitten by a ferret at the age of , was reported and demonstrates the zoonotic potential (jones et al., ) . mycobacterium avium infection is not reportable but may pose a risk to immunocompromised patients (fox, a) . personnel at risk should be followed up by a physician for appropriate diagnostic testing (fox, a) . etiology. salmonellosis is caused by infection with organisms of the genus salmonella. epizootiology and transmission. salmonella is a gram-negative, non-spore-forming, facultative anaerobic rod in the family enterobacteriaceae (carter et al., ) . infection is by the oral route. transmission may be direct from infected carrier animals or humans or through contaminated food products or water (carter et al., ) . several salmonella serovars have been isolated from mink with gastroenteritis and abortion (gorham et al., ) . contaminated raw meat products were suspected as the source in one outbreak. salmonella typhimurium was isolated in ferrets in an outbreak of clinical disease (coburn and morris, ) and several serotypes including s. hadar, s. enteritidis, s. kentucky, and s. typhimurium were isolated from the feces of ferrets surveyed in a research colony (fox et al., a) . clinical signs and necropsy findings. clinical signs of an outbreak of s. typhimurium in ferrets included conjunctivitis, rapid weight loss, tarry stools, and febrile temperature fluctuations (coburn and morris, ) . gross findings in ferrets days after inoculation with s. typhimurium of ferret origin included marked tissue pallor, petechiae in the gastric mucosa, and the presence of melena in one and a dark-colored fibrinous exudate in the large intestine of the other ferret (coburn and morris, ) . studies involving experimental inoculation with s. enteritidis, s. newport, and s. choleraesuis via the oral route to healthy, distemper-infected, and feed-depleted ferrets and mink showed a fairly high resistance to infection (gorham et al., ) . only animals of in the diet-restricted group-- ferret and minknshowed clinical signs of infection after feeding s. newport culture. signs included lethargy, anorexia, trembling, and fecal blood. the gastrointestinal tract showed a large amount of mucus containing red blood cells; bits of desquamated epithelium and few mononuclear cells overlying the gastric mucosa; an exudate in the small intestine consisting of mu-coid material, red blood cells, and desquamated small intestinal villi; edematous villi in the ileum; and a diffuse infiltrate of the small intestinal mucosa with lymphocytes and macrophages. necrotic foci in the liver, spleen, and, less commonly, the kidney, as well as splenomegaly and visceral lymphadenopathy, were observed in chronic fatal infections (coburn and morris, ) . abortion and gastroenteritis have been reported in mink (gorham et al., ) . diagnosis. diagnosis is based on history, clinical signs, and isolation of the organism. the organism can be cultured on enrichment and selective media and then characterized serologically. samples of blood, feces, exudates, tissues, and intestinal material may be cultured. treatment and control. coburn and morris ( ) treated of ferrets experimentally infected with s. typhimurium with sulfathalidine in the feed (coburn and morris, ) . salmonella typhimurium was isolated in of control animals and none of the treated animals days after the administration of the last dose. sulfathalidine was administered by the same authors to a colony of ferrets in which an outbreak of salmonella occurred. the group was surveyed days after sulfathalidine treatment and showed weight gain, improvement in condition, and a reduction in the number of salmonella-infected ferrets (coburn and morris, ) . salmonella spp. isolated from ferrets may show resistance to a number of antibiotics (fox, a) . treatment includes appropriate use of antimicrobials and supportive care, which may include fluid therapy, nutritional support, maintenance of electrolyte balance, treatment of concurrent diseases, recognition of and attention to shock, and reduction of stress (fox, a) . etiology. streptococcus zooepidemicus and other group c and g streptococci, escherichia coli, klebsiella pneumoniae, pseudomonas aeruginosa, and bordetella bronchiseptica have been reported as primary and secondary bacterial pathogens in pneumonia in ferrets (fox, a) . epizootiology and transmission. bacterial pneumonia may occur secondary to megaesophagus in the ferret. an influenza virus-bacteria synergism has been the subject of several studies in ferrets (fox, a) . debilitated and immunosuppressed animals and animals with concurrent diseases such as influenza may be more susceptible to bacterial pneumonias (fox, a) . clinical signs. clinical signs may include nasal discharge, dyspnea, lethargy, anorexia, increased lung sounds, cyanosis, and fever (rosenthal, ) . fulminant pneumonia may progress to sepsis and death (fox, a) . diagnosis. diagnosis is based on history, clinical findings, a complete blood count, culture and cytology of a tracheal wash or lung wash, and radiographs (rosenthal, ) . differential diagnosis. diagnostic rule-outs include dilatative cardimyopathy, heartworm disease, mycotic pneumonia, pneumocystis pneumonia in immunosuppressed animals, neoplasia, and influenza. treatment and control. treatment should consist of appropriate antimicrobial therapy and supportive care, which may include the administration of oxygen, fluid therapy, and force feeding (rosenthal, ) . etiology. a variety of bacteria have been associated with abscesses and localized infection of the lung, liver, uterus, vulva, skin, mammary glands, and oral cavity. these include staphylococcus spp., streptococcus spp., corynebacterium spp., pasteurella, actinomyces, hemolytic escherichia coli, and aeromonas spp. (fox, a) . epizootiology and transmission. abscesses in ferrets may result from wounds that are inflicted secondary to biting during fighting, playing, mating, or chewing sharp objects. clinical signs. localized or subcutaneous abscesses present as swellings with or without draining tracts. the swelling may be fluctuant. in most cases, the abscess is walled off and does not result in systemic signs (fox, a) . abscesses or infection involving visceral organs may give rise to organ-specific and/or systemic signs. diagnosis. cytologic and gram staining of an aspirate of a suspect subcutaneous swelling will aid in the definitive diagnosis. culture and sensitivity of the aspirate should also be performed to identify the causative organism and guide appropriate antibiotic therapy. differential diagnosis. differential diagnosis of a subcutaneous swelling in a ferret should include myiasis, granuloma, hematoma, and neoplasia. treatment and control. prevention of ferrets from exposure to sharp objects in the cage and feed, and limiting the exposure of male and female during breeding, can minimize the occurrence of abscesses. treatment of localized abscesses should include appropriate antibiotic therapy and establishment of drainage and debridement if necessary. bacterial culture and sensitivity of the exudate should be performed. a broad-spectrum antimi-crobial may be used pending results of culture and sensitivity (orcutt, ) . etiology. gram-positive cocci such as streptococcus spp., staphylococcus aureus, and coliforms such as hemolytic e. coli are the most frequently associated organisms (bernard et al., ; bell, a) . although the exact pathogenesis of mastitis in ferrets is not clear, a number of factors may play a role and include the stress of lactation, injury to mammary glands by the kits' teeth, environmental contamination, and the virulence of the organism. in one report, the causative organism, hemolytic e. coli, was cultured from the feces of mastitic and healthy ferrets and the oral cavity of suckling kits (liberson et al., ) . the high level of perineal contamination and the presence of the organism in the oral cavity of suckling kits may enhance transmission and introduction of this organism into mammary tissue. in another outbreak, the causative organisms were cultured from bovine meat fed prior to the outbreak, and the meat was suspected as a possible source. clinical signs. mastitis occurs in nursing jills and has been characterized as acute or chronic (bell, a) . the acute form is reported to occur soon after parturition or after the third week of lactation. examination of affected jills reveals swollen, firm, red or purple, and painful glands. affected glands may quickly become gangrenous. the chronic form, which may occur when kits are weeks old or as a sequela to the acute form, is characterized by glands that are firm but not painful or discolored. diagnosis. diagnosis is based on history, clinical signs, physical examination findings, and isolation of the causative organism. necropsy findings. in acute mastitis, grossly affected glands are swollen, and the skin overlying the gland may be discolored. surgical biopsies and necropsies of ferrets with mastitis caused by hemolytic e. coli (liberson et al., ) revealed extensive edema, hemorrhage, and coagulative and liquefactive necrosis involving the glandular tissue as well as surrounding subcutaneous tissue. other findings included the presence of a mixed leukocytic infiltrate composed primarily of polymorphonuclear leukocytes; large numbers of bacteria; and thrombosis and necrosis of vessels within and immediately adjacent to areas of inflammation (liberson et al., ) . in an outbreak of mastitis in mink due to staphylococcus aureus and escherichia coli, histologic examination of affected glands revealed an acute suppurative mastitis with desquamation of alveolar epithelium, edema of the connective tissue stroma, alveoli filled with neutrophils and cellular debris, and lactiferous ducts filled with purulent exudate and mats of bacteria within lobules (trautwein and helmboldt, ) . treatment. broad-spectrum antibiotic therapy may be instituted pending culture and sensitivity results of the milk. enrofloxacin ( . mg/kg bid po after a loading dose of . mg/kg im) is often effective. jills may require aggressive care, because acute mastitis may progress rapidly and animals may become septicemic and moribund (liberson et al., ) . oral antibiotic administration to kits nursing affected jills is recommended (bell, a) . surgical resection and debridement of affected glands and supportive care may be necessary for jills with acute mastitis. supplementation of kits with milk replacer may also be necessary, because jills with acute mastitis are reluctant to nurse, and jills with the chronic form have diminished lactation as milk-producing tissue is replaced by scar tissue (bell, a) . maintaining thorough personal hygiene practices when handling affected jills is important in minimizing spread to other lactating jills. cross-fostering kits may be required; however, kits may spread infection to healthy jills. it is reported that jills with the chronic form of mastitis should be culled (bell, a ). etiology. canine distemper (cd) is caused by a paramyxovirus of genus morbillivirus that is related to measles and rinderpest (budd, ) . there are several strains, including a ferret-adapted strain of canine distemper virus (cdv), that vary in incubation, clinical signs, and duration . the virus can be inactivated by heat, light, and various chemicals, including phenol, roccal, sodium hydroxide, and formalin (shen and gorham, ; budd, ) . infectious virions have been recovered from fomites after min at room temperature. canine distemper is the most serious viral infection of ferrets. mortality approaches %, making appropriate husbandry and vaccination imperative. the disease has a catarrhal phase and a neurological, or central nervous system (cns), phase. the catarrhal phase is - days postinfection and involves anorexia, pyrexia, photosensitivity, and serous nasal discharge. an erythematous pruritic rash spreads from the chin to the inguinal region. it is suspected that the rash results from cell-mediated immunity to infected endothelial cells, similar to the response seen in humans with measles (norrby and oxman, ) . hyperkeratosis of footpads, called hard pad, is an inconsistent feature. secondary bacterial infections result in mucopurulent ocular and nasal discharge and possibly bacterial pneumonia. the cns phase, with ataxia, tremors, and paralysis, may or may not be preceded by the catarrhal phase. death occurs in - days from ferret strains of cdv and up to days with canine strains. infection is uniformly fatal. epizootiology and transmission. virus is shed from infected hosts from conjunctival, nasal, and oral exudates, urine, feces, and sloughed skin (gorham and brandly, ) . transplacental infection is not reported in ferrets. attenuated cdv vaccine strains have not been recovered from the body secretions of ferrets following vaccination (shen et al., ) . unvaccinated dogs and other canids, mustelids, and procyonids may serve as reservoirs of infection. viremia is detectable days postinfection and persists until the ferret dies or mounts a neutralizing antibody response (liu and coffin, ) . the primary site of replication is the respiratory and lymphatic systems, and cdv has been recovered from the nasal secretions of ferrets - days postinfection. a decrease in lymphocyte subsets is detectable - days postinfection. clinical signs and necropsy findings. histologically, intracytoplasmic and intranuclear inclusion bodies may be observed in tracheal, bronchial, epithelia, and bile duct as well as transitional epithelium in the bladder (liu and coffin, ) (fig. ) . the eosinophilic (hematoxylin-eosin) inclusions appear orange using pollack's trichrome stain. diagnosis and differential diagnoses. presumptive diagnosis is based on clinical observation, questionable vaccination history, and exposure. a fluorescent antibody test can be used on peripheral blood and conjunctival mononuclear cells to detect infection. reverse transcriptase-polymerase chain reaction (rt-pcr) has also been used to detect experimental infection (stephensen et al., ) . differential diagnoses should include infection with influenza virus or bordetella bronchiseptica. influenza does not rapidly progress to mucopurulent ocular and nasal discharge as cd does. during an outbreak, clinically affected ferrets should be isolated and the remainder of the colony vaccinated. distemper infection can be prevented by vaccination with modified live vaccine of chicken embryo tissue culture origin (cetco) administered subcutaneously or intramuscularly. kits should be vaccinated every - weeks, starting at age weeks, until weeks and annually thereafter . it is important to adhere to the prescribed vaccination protocol, because ferret deaths have been reported following double-dose vaccination (carpenter et al., ) . inactivated distemper vaccines do not elicit consistent, effective immunity and are not recommended. it is important to know the vaccination schedule of your ferret supplier and to vaccinate supplementally as appropriate. new ferrets should be held in quarantine for weeks prior to introduction into the resident colony. ferrets have been experimentally infected with feline panleukopenia, canine parvovirus, canine parainfluenza virus, mink enteritis virus, respiratory syncytial virus, transmissible mink encephalopathy, and pseudorabies, but natural infection with these viruses has not been reported . etiology. aleutian disease virus (adv) is a parvovirus with strains of varying virulence and immunogenicity. mink-derived strains are more virulent to mink than are ferret-derived strains . epizootiology and transmission. aleutian disease (ad) is a chronic progressive illness that was first described in mink (oxenham, ) . it was originally named hypergammaglobulinemia (hgg) because of this remarkable finding. infection may be subclinical for years. because the immunomodulation associated with adv infection is disruptive to biomedical research, it is important to seek sources of adv-free ferrets . transmission between ferrets may be direct or via aerosol of urine, saliva, blood, feces, and fomites (kenyon et al., ; gorham et al., ) . vertical transmission is established in mink but is unproven in ferrets. clinical signs. ferrets infected with adv as adults develop persistent infection but rarely disease, although chronic progressive weight loss, cachexia, malaise, and melena have been described (porter et al., ) . ad may also cause ataxia, paral-ysis, tremors, and convulsions (oxenham, ; welchman et al., ) . the lesions are typically immune-mediated, and there is elevation of the gammaglobulins to generally greater than % of the total proteins (porter et al., ; fig. ). the precise mechanism of immunomodulation is unknown, but in mink there is depression of b-and t-cell responses. necropsy. ferrets may have no lesions upon necropsy, or infrequently they may have hepatosplenomegaly and lymphadenopathy. the most consistent histological finding is periportal lymphocytic infiltrates (fig. ). bile duct hyperplasia and periportal fibrosis have also been reported. membranous glomerulonephritis has been described (ohshima et al., ) . although lesions are subtle, use of adv-infected ferrets in biomedical research is contraindicated because histological lesions interfere with the interpretation of study results . diagnosis and differential diagnoses. presumptive diagnosis is based on hgg and chronic weight loss. diagnosis is confirmed by immunofluorescent antibody (ifa) or counterimmunoelectrophoresis (ciep) for antibody to adv antigen (palley et al., ) . pcr-based assays have also been used (erdman et al., b; saifuddin and fox, ; erdman et al., ) . differential diagnoses include the neurotropic form of cd, as well as chronic wasting diseases such as neoplasia, malabsorption, maldigestion, and bacterial enteritis . vaccination against adv would be contraindicated because of the immune-mediated reaction, and a vaccine is not available. chemical disinfection may be achieved tp - . g/dl /globulin = . g% , .. with formalin, sodium hydroxide, and phenolics (shen et al, ) . there is no treatment for ad, and infected ferrets should be culled from the colony. etiology. influenza is caused by an orthomyxovirus that is transmissible from humans to ferrets and ferrets to humans (smith and stuart-harris, ) . human influenza viruses a and b are pathogenic to ferrets . ferrets are also susceptible to avian, phocine, equine, and swine influenza, although only porcine influenza causes clinical signs. because the viruses can be readily transmitted from humans to ferrets, handling precautions such as wearing masks and gloves should be in place to minimize transmission. epizootiology, transmission, and clinical signs. influenza virus generally remains localized in nasal epithelium in ferrets but may cause pneumonia. clinical signs appear hr postinfection and include anorexia, fever, sneezing, and serous nasal discharge. conjunctivitis, photosensitivity, and otitis are also sometimes seen . secondary bacterial infection by streptococcus sp. and occasionally bordetella bronchiseptica may prolong recovery. transmission occurs via aerosol and direct contact. diagnosis. diagnosis is based on typical clinical presentation and recovery within days, unlike with cd, which progresses to more severe disease and death. hemagglutination inhibition antibody titers on acute and convalescent sera are rarely needed. treatment and control. antibiotic therapy may be instituted to preclude secondary bacterial infection. animal technicians and investigators suffering from influenza should avoid contact with ferrets. ferrets have been used extensively as a model for influenza research because the biological response to infection is similar to that in humans . ferrets have been used in influenza a research to study pathogenesis, to investigate reye's syndrome, and to evaluate vaccine trials (deshmukh, ; sweet et al., ) . etiology. rabies is caused by a rhabdovirus. rabies infection is infrequently reported in ferrets, and until recently, research on rabies in ferrets was lacking . ferrets in a well-managed facility would have low risk of exposure to rabies virus. a usda-approved, killed rabies vaccine given subcutaneously at ages months and year and annually thereafter is recommended to protect ferrets against rabies (rupprecht et al., ) . modified live vaccine (mlv) is not recommended, because there is at least one case of rabies in a ferret that was vaccinated with mlv rabies vaccine . there is no treatment for rabies. clinical signs and pathogenesis. clinical signs of rabies infection in ferrets may include anxiety, lethargy, and posterior paresis. in one experimental infection, of ferrets died, and negri bodies were seen in the brain of only of the (blancou et al., ) . there is conflicting data on the isolation of rabies virus from the salivary glands following experimental infection. in one study using raccoon variant of rabies for infection, more than half of the ferrets had rabies isolated from the salivary glands . ferrets at risk for exposure to rabies virus that bite or scratch a human should be placed under quarantine for not less than days of observation. veterinarians and facility managers should seek assistance from state public health officials. diagnosis and differential diagnoses. differential diagnosis includes the neurotropic form of cd. diagnosis is based on direct ifa of brain tissue. because rabies in ferrets is poorly understood, the head from ferrets that exhibit signs compatible with rabies and that have exposure histories that raise concerns about rabies should be shipped to the state public health authority for confirmation. etiology. rotaviruses cause diarrhea in young of many species, including humans, calves, pigs, sheep, and rats. diarrhea in ferret kits is thought to be caused by a poorly characterized, atypical rotavirus that has not been cultivated in vitro (torres-medina, ) . atypical rotaviruses lack the rotavirus common antigen. epizootiology, transmission and clinical signs. clinical disease may occur in kits as young as - days old or in older animals up to weeks of age. diarrhea soils the perineum and possibly the fur and nest material. mortality rates are agedependent, with high mortality occurring in young kits and lower mortality occurring in kits over days of age (bell, a; fox et al., b) . secondary bacterial infection may influence the severity of diarrhea. necropsy andpathogenesis. lesions are restricted to the gastrointestinal tract. yellow-green liquid or mucous feces may be seen in the terminal colon on necropsy. subtle small-intestinal villous atrophy and epithelial cell vacuolation are detectable histologically. diagnosis and differential diagnoses. clinical diagnosis can be confirmed by using clarified and ultracentrifuged fecal pel-lets for electron microscopy. the ferret rotavirus does not crossreact with commercially available enzyme immunoassays (torres-medina, ) . it is desirable to avoid sources that are known to be infected with ferret rotavirus. affected kits may be supplemented with kitten milk replacer, using a medicine dropper. mortality is reduced if the kits continue nursing. treatment of secondary bacterial infections may reduce severity of the diarrhea, and supportive care, including subcutaneous fluid administration for young kits, may be required . jills develop immunity to rotavirus infection, and subsequent litters are protected. infectious bovine rhinotracheitis (ibr) was isolated from the liver, spleen, and lung of clinically normal ferrets (porter et al., ) . raw beef was suspected as the source of infection, reinforcing the need to exclude raw meat products from the diet of ferrets used for research. ibr does cause significant respiratory pathology in experimentally infected ferrets (porter et al., ) . a transmissible diarrhea, referred to as epizootic catarrhal enteritis, has been observed in adult ferrets several days after direct contact and fomite exposure to affected ferrets . clinically the diarrhea is green and bile-tinged, and the ferrets become rapidly dehydrated. mortality is low. some ferrets develop elevated liver enzymes. treatment involves aggressive oral and systemic fluid therapy. a recent study implicates a coronavirus as the cause of this disease (williams et al., ) . a. protozoa i. enteric coccidiosis etiology. three species of the genera isospora and eimeria have been reported to infect the ferret: isospora laidlawi, eimeria furonis, and e. ictidea (blankenship-paris et al., ) . tion of sporulated oocysts. clinical signs. coccidiosis in ferrets is usually subclinical but has been reported to be associated with diarrhea, lethargy, and dehydration in one ferret (blankenship-paris et al., ) . clinical signs are often seen in young, newly acquired ferrets and are more common after a stressful event (rosenthal, ) . rectal prolapse can also develop in association with coccidial infection (rosenthal, ) . diagnosis. diagnosis is generally made by any of the fecal flotation methods commonly used in veterinary practice or by direct wet mount of feces and microscopic examination for sporulated or unsporulated oocysts. because coccidial oocysts are small, slides should be examined under higher magnification. necropsy findings. diagnosis is usually performed antemortem. pathologic lesions associated with enteric coccidiosis in a laboratory-reared ferret that was euthanized were described in one published report (blankenship-paris et al., ) . microscopic lesions were confined to the jejunum and ileum and consisted of villous and epithelial thickening. parasitic cysts and microorganisms within epithelium, and a mild granulomatous inflammation in the villar lamina propria, were also observed. a recent report documents clinical and anatomic pathology associated with biliary coccidiosis in a weanling ferret (williams et al., ) . differential diagnosis. diarrhea may be observed in ferrets that present with gastroenteritis secondary to gastrointestinal foreign bodies and dietary indiscretion, as well as other nutritional, inflammatory, infectious, or other systemic diseases. infectious causes such as proliferative colitis, salmonellosis, giardiasis, rotavirus, and campylobacteriosis should be considered. diarrhea may also be seen in eosinophilic gastroenteritis, an uncommonly reported condition in ferrets. good husbandry practices that include sanitation and frequent disposal of feces reduce the number of oocysts in the environment. cleaning cages with a strong ammonium hydroxide solution is reported to be effective (kirkpatrick and dubey, ) . heat treatment of surfaces and utensils may also be effective (kirkpatrick and dubey, ) . treatment of ferrets with sulfadimethoxine at mg/kg orally once and then mg/kg orally every hr for days is recommended (rosenthal, ) . as in dogs and cats, the complete elimination of a coccidial infection requires an immunocompetent host. ii. cryptosporidiosis etiology. cryptosporidiosis is caused by infection with cryptosporidium spp. epizootiology and transmission. cryptosporidium is a protozoan in the class sporozoa, subclass coccidia, that inhabits the respiratory and intestinal epithelium of birds, reptiles, mammals, and fish (regh et al., ) . it is known to cause gastrointestinal tract disease in many species, including rodents, dogs, cats, calves, and people (hill and lappin, ) . it has a life cycle similar to other coccidian parasites and is transmitted by ingestion of sporulated oocysts. autoinfection is also a characteristic of the life cycle. transmission may occur through consumption of contaminated food or water. cattle, dogs, and cats, shedding oocysts, are reported to be potential sources of human infection (hill and lappin, ; fox, g) . immunosuppressed people are at greatest risk of developing severe fulminating gastrointestinal disease (hill and lappin, ) . the finding of cryptosporidiosis in two ferrets that died from unrelated causes in one animal facility resulted in a survey of the existing ferret population and new arrivals into the facility to determine the prevalence and incidence of infection (regh et al., ) . findings indicated that % of the resident population and - % of new arrivals had oocysts in their feces but showed no clinical signs. clinical signs. only subclinical infection has been reported in both immunocompetent and immunosuppressed ferrets (regh et al., ) . diagnosis. diagnosis is based on the identification of the organism in feces. the oocysts are small when compared with other coccidia and may be overlooked or mistaken for yeasts (kirkpatrick and dubey, ) . yeasts are oval, whereas cryptosporidium oocysts are spherical or ellipsoidal. additionally, yeasts will stain with iodine and are not acid-fast, whereas cryptosporidium has the opposite staining characteristics. the oocyst residuum is seen as a refractive dot under phase-contrast microscopy, a structure lacking in yeast (kirkpatrick and dubey, ) . sugar-solution centrifugation and fecal sedimentation using formalin-ether or formalin-ethyl acetate are effective di-agnostic concentration techniques (hill and lappin, ) . oocysts may then be viewed with phase-contrast or bright-field microscopy of specimens stained with an acid-fast method. a direct fecal smear may be methanol-or heat-fixed and stained with an acid-fast method (hill and lappin, ) . necropsy findings. histologic evaluation reveals the presence of organisl ns, spherical to ovoid in shape and from to ~tm in diameter, associated with the brush border of the villi. a mild eosinophilic infiltrate was observed in the lamina propria of the small intestine in most animals. the ileum was the most common and heavily infected section of small intestine (regh et al., ) . there is no known definitive treatment for cryptosporidiosis (fox, g) . supportive and symptomatic care should be provided in clinical cryptosporidiosis. infections are self-limiting in immunocompetent patients (fox, g) . control is aimed at eliminating or reducing infective oocysts in the environment and avoidance of contact with known sources. because of the potential for zoonotic transmission, restricting contact of children and immunosuppressed individuals with infected ferrets and practicing good hygiene may help reduce the potential for infection. drying, freeze-thawing, and steam cleaning inactivate the organism (hill and lappin, ) . there are few effective commercial disinfectants. i. sarcoptic mange etiology. sarcoptic mange is caused by infection with sarcoptes scabiei. epizootiology and transmission. transmission occurs through direct contact with infected hosts or contact with fomites. this parasitic infection is rare under research conditions. clinical signs. infection of ferrets with s. scabiei may occur in a generalized or a pedal form (bernard et al., ) . in the generalized form, lesions consist of focal or generalized alopecia with intense pruritus. in the pedal form, lesions are confined to the toes and feet, which become swollen and encrusted with scabs. nails may be deformed or lost if the condition is left untreated. diagnosis. diagnosis is made by finding the mites in skin scrapings or removing crusts, breaking them up, and clearing with % koh for microscopic examination (phillips et al., ) . false-negative results are possible; multiple scrapings may be necessary. differential diagnosis. differential diagnosis should include other pruritic external parasitic conditions, including flea infes-tation. demodicosis has been reported to cause mild pruritus and alopecia in ferrets (noli et al., ) . in the pedal form, treatment consists of trimming the claws and removing the scabs after softening them in warm water (bernard et al., ) . treatments that have been used include ivermectin, . - . mg/kg, administered subcutaneously and repeated every - days until mites are gone; shampoos or soaks to reduce the pruritus; and topical or systemic antibiotic administration for treatment of secondary bacterial dermatitis (hillyer and quesenberry, b) . alternatively, weekly dips in % lime sulfur until weeks after clinical cure have been shown to be effective (fox, a) . treatment of all affected animals as well as contact animals, and decontamination of enclosures and bedding, are recommended. ii. demodicosis etiology. demodicosis is caused by infection by demodex spp. epizootiology and transmission. the parasite is found in normal skin of almost all dogs and is not considered contagious. predisposing factors such as immunologic or genetic conditions have been suggested (kwochka, ) . one clinical report describes demodicosis in two adult ferrets that had been treated with an ear ointment containing triamcinolone acetonide for recurrent ear infections daily for periods of months each during the course of a year (noli et al., ) . clinical signs. in the report mentioned above, the ferrets presented with alopecia, pruritus, and orange discoloration of the skin behind the ears and on the ventral surface of the abdomen and an accompanying seborrhea (noli et al., ) . diagnosis. deep skin scrapings should be performed to demonstrate mites. finding a large number of live adult mites or immature forms and eggs is necessary to confirm the diagnosis. in very chronic cases, the skin may be so thickened that scrapings may be unrewarding. in these cases, a skin biopsy may be diagnostic (kwochka, ) . necropsy findings. histologic evaluation of skin biopsies obtained in the case report described above revealed mites with a short, blunted abdomen similar to that of demodex criceti and located in the infundibulum of hairs. the epidermis was slightly hypertrophic, and there was a mild superficial orthokefatotic hyperkeratosis. a very mild superficial and perivascular mixed cellular infiltrate was also observed in the dermis. differential diagnosis. generalized demodicosis should be differentiated from sarcoptic mange and flea infestation. primary or secondary bacterial dermatitis or pyoderma should also be considered. treatment and control. the ferrets in the above-mentioned clinical report were treated initially with a suspension of . % amitraz applied as a dip times at -day intervals for treatments. two drops of the same solution were applied in each ear every other day. after the initial treatment, the ferrets were reexamined, and treatment was continued with the same concentration of solution applied once every days, while the tail was washed with a higher concentration of amitraz ( . %) once every other day. thereafter, final treatments with . % amitraz every days for the body, and every other day for the ears and tail, were administered. the ferrets were evaluated and skin scrapings were performed regularly during treatment and posttreatment to monitor response to therapy. treatment of any associated pyodermas, systemic illnesses, or management problems should also be included as part of the therapeutic regimen. iii. ear mites etiology. the ear mite, otodectes cynotis, which commonly infects dogs and cats, is also a common clinical problem in ferrets (fox, g) . epizootiology and transmission. ear mites are transmitted through direct contact with infested ferrets, dogs, or cats (fox, g) . the entire life cycle is completed in weeks. clinical signs. ear mite infestation in the ferret is usually asymptomatic (orcutt, ) . however, clinical signs may include head shaking; mild to severe pruritus with inflammation and excoriation; secondary otitis interna with ataxia; circling; torticollis; and horner's syndrome (orcutt, ; fox, g) . a brownish black waxy discharge is often present. diagnosis. diagnosis is based on direct observation of mites via otoscopic examination or microscopic identification of the ear mite or any of the life-cycle stages of the mite in exudate from the ear canal. treatment and control. several treatment regimens, including topical and injectable mitocidal treatments, have been recommended (orcutt, ; fox, g) . a recent study using three treatment regimens--two topical and one injectable--revealed that topical treatments were more efficacious than the injectable in reducing or eradicating ear mites (patterson et al., ) . efficacy was evaluated by microscopic evidence of ear mites in debris from aural swabs taken weekly for an -week period. topical % ivermectin (ivomec, merck agvet division, rahway, new jersey), diluted " in propylene glycol at a dosage of ~tg/kg body weight divided equally between the two ear canals and administered on days and of the study, was the most effective treatment. all susceptible animals in a household should be treated. ears should be gently cleaned prior to initiating treatment (orcutt, ) . high doses of injectable iver-mectin ( . ml of % ivermectin) administered to jills at - weeks of gestation resulted in high rates of congenital defects (orcutt, ) . iv. fleas etiology. ctenocephalides species can infest ferrets. epizootiology and transmission. transmission requires direct contact with another infested animal or a flea-infested environment. clinical signs. flea infestation may be asymptomatic or may cause mild to intense pruritus and alopecia of the dorsal thorax and neck (timm, ) . differential diagnosis. sarcoptic and demodectic mange should be included in the differential diagnosis of pruritic skin disease in the ferret. close examination of the pelage for fleas or flea excrement should be performed. skin scrapings may be indicated. as with flea infestation in dogs and cats, concurrent treatment of the environment, as well as all animals in the household, is essential for effective flea control. compounds approved for flea control in cats such as rotenone or pyrethrin powders or sprays may be used in ferrets (hillyer and quesenberry, a) . ferrets may develop systemic disease from blastomyces, coccidioides, cryptococcus, and histoplasma. the reservoir of most of these fungi is the soil, however, making infection unlikely in a research facility. in production facilities, exposure can be minimized through careful selection of source animals, appropriate sanitation, and control of pests, particularly birds. pneumocystis carinii has been recently reclassified as a fungus. although p. carinii inhabits the lungs of many different species, recent transmission studies suggest that these fungi are highly species-specific (gigliotti et al., ; fox et al., b) . clinical disease is evident only in immunocompromised ferrets and can be induced using high doses of exogenous steroids (stokes et al., ) . lesions include interstitial pneumonitis with mononuclear cell infiltrates; cysts and trophozooites are evident with gomori methanamine-silver nitrate and giemsa on bronchoalveolar lavage. treatment with trimethoprim sul-famethoxazole probably controls but does not eliminate infection . ferrets are susceptible to secondary fungal infection of the outer ear canal with absida corymbifera or malassezia spp. (dinsdale and rest, ; fox, d) . the fungi are widespread in the environment and will cause a secondary fungal infection in the ears of ferrets infested with otodectes cynotis. the yeasts can be visualized by impressions of ear exudates. treatment involves eradication of the underlying mite infestation followed by oral and topical ketoconazole, miconazole, and polymyxin b. dermatomycoses in ferrets are caused by microsporum canis and trichophyton mentagrophytes. dermatophytes are transmissible to humans and are a zoonosis; thus affected animals should be quarantined and removed from the facility to minimize risk (dinsdale and rest, ; scott et al., ; fox et al., b) . control of infection includes general disinfection and destruction of contaminated bedding. lesions are circumscribed areas of alopecia and inflammation, which begin as small papules that spread peripherally in a scaly inflamed ring. the yellow-green fluorescence of m. canis under ultraviolet light helps distinguish it from t. mentagrophytes. skin scrapings digested with % potassium hydroxide reveal characteristic arthrospores. treatment with griseofulvin causes clinical remission but may not clear infection. other ectoparasitic infections observed to occur in ferrets include cutaneous myiasis and tick infestation. granulomatous masses in the cervical region caused by the larval stage of hypoderma bovis have been reported in ferrets (fox, g) . cuterebra larvae, although uncommonly observed in ferrets, may cause subdermal cysts found in the subcutis of the neck (orcutt, ) . infestation with the flesh fly has been reported as a problem in commercially reared mink and ferrets housed outdoors (fox, g) . ticks may be found on ferrets housed outdoors or on those used for hunting rabbits (fox, g) . ticks should be removed carefully with hemostats or tweezers, ensuring that the entire head and mouthparts are removed from the skin. appropriate caution should be exercised in tick removal, because ticks are responsible for transmission of various zoonotic pathogens; gloves should be worn. etiology. the ferret is susceptible to natural and experimental infection with dirofilaria immitis. epizootiology and transmission. dirofilaria immitis is a filarial parasite that is transmitted by mosquitoes, which serve as the intermediate host and vector. microfilaria are ingested by mosquitoes and, after two molts, become infective third-stage larvae. infective larvae are deposited onto the skin when mosquitoes feed, and larvae find their way into the body of the final host through the bite wound and migrate subcutaneously to the thorax and eventually to the heart (knight, ) . the primary reservoir of infection is dogs, but heartworm may be found in a variety of mammals, including humans. all other species except wild and domestic canids, domestic felines, ferrets, and the california sea lion are considered aberrant hosts (knight, ) . clinical signs. the following clinical signs have been reported in clinical reports describing cases of d. immitis in the ferret: weakness, lethargy, depression, dyspnea, cyanosis, anorexia, dehydration, cough, and pale mucous membranes (miller and merton, ; parrott et al., ; moreland et al., ) . moist lung sounds and/or muffled heart sounds were revealed by thoracic auscultation in many of these cases. pleura/or abdominal effusion may be observed radiologically. the ferrets described in these cases were housed outdoors and either died or were euthanized. diagnosis. diagnosis of heartworm is based on clinical signs, radiographic findings, and testing for circulating microfilariae and heartworm antigen. microfilaremia is not consistently observed in naturally occurring and experimental cases of heartworm infection in ferrets (fox, g) . testing for heartworm antigen appears to be more diagnostically useful (stamoulis et al., ) . in a study to determine the minimum oral dose of ivermectin needed for monthly heartworm prophylaxis in ferrets, the use of an antigen test (uni-tec canine heartworm test, pitman-moore co., mundelein, illinois) detected infection in more untreated control animals than did the modified knott test for detection of circulating microfilaria in the same ferrets (supakorndej et al., ) . necropsy findings. cardiomegaly, pleural and/or abdominal fluid, and pulmonary congestion are common findings at necropsy. grossly, adult worms have been observed in the right atrium, right ventricle, pulmonary artery, and cranial and caudal vena cava. microscopically, microfilaria may be seen in small and large vessels of the lung. differential diagnosis. differential diagnosis should include primary cardiac diseases, such as dilatative cardiomyopathy, and other systemic or pulmonary diseases. control is best directed at prevention through the administration of heartworm preventative and it is recommended that ferrets in heartworm-endemic areas receive monthly oral ivermectin throughout the year (stamoulis et al., ; fox, g) . the dosage recommended for ferrets by the american heartworm society is . mg/kg body weight monthly (fox, g) . housing ferrets indoors, particularly during the mosquito season, would help minimize exposure. successful adulticide treatment in ferrets has been described and includes the administration of thiacetarsemide, with the same precautions used in dogs: antithrombotic therapy, treatment for heart failure, and strict cage confinement (stamoulis et al., ) . one should follow up with heartworm antigen tests until negative and resume heartworm prevention month after adulticide treatment (stamoulis et al., ) . ferrets are also susceptible to infection with the following nematodes: toxascaris leonina; toxocara cati; ancylostoma spp.; dipylidium caninum; mesocestoides spp.; atriotaenia procyonis; trichinella spiralis; filaroides martis; and spiroptera nasicola (rosenthal, ; fox, g) . a day of their due date should include cesarean section and intensive postoperative support, including force-feeding a gruel of high-quality cat food and ferret chow, nutritive pastes, intravenous fluids containing glucose, and supplemental heat. cesarean section should be performed under isoflurane anesthesia because hepatic dysfunction prolongs the metabolism of injectable agents. agalactia is common after cesarean section, and kits may require hand feeding with kitten or puppy milk replacers, administered per os by fine-tipped syringe times daily for the first hr. cross fostering is an effective method of enhancing kit survival; hand rearing of kits if the jill fails to nurse within a day postoperatively is energy-consuming and generally unrewarding. for jills that develop pregnancy toxemia before day of gestation, fluids and nutritional support must be provided until viable kits can be delivered by cesarean. pregnancy toxemia may be avoided by close monitoring of appetite of jills in late gestation, provision of a highly palatable diet with > % fat and > % crude protein, and avoidance of stress and dietary change. water should be made available in both bowls and water bottles, and food should be provided ad libitum in several bowls. pregnancy toxemia in the ferret occurs predominantly in primiparous jills carrying large litters. an inadvertent fast in late gestation is sometimes implicated. at least % of jills carrying more than kits will develop pregnancy toxemia if subjected to hr of food withdrawal in late gestation (bell, a; batchelder et al., ) . any jill with or more kits may develop pregnancy toxemia because abdominal space is not adequate for both the gravid uterus and the volume of food required to support it. pregnancy toxemia of the ferret is of the metabolic type and shares features with similar conditions in pregnant sheep, obese cattle, pregnant camelids, obese guinea pigs, and starved pregnant rats, as well as with the condition feline idiopathic hepatic lipidosis. it is characterized by abnormal energy metabolism with consequent hyperlipidemia, hypoglycemia, ketosis, and hepatic lipidosis. in this condition, energy demand exceeds intake, leading to excessive mobilization of free fatty acids and a chain of metabolic events that culminates in a shift from fatty acid metabolism and export to ketosis and hepatic lipidosis. clinical signs include anorexia, lethargy, melena, dehydration, and easily epilated hair. differentials include dystocia, metritis, pyometra, septicemia, renal failure, and helicobacter mustelae-induced gastric ulcer. in a recent study of ferrets with pregnancy toxemia, consistent clinical chemistry abnormalities included azotemia ( %), hypocalcemia ( %), hypoproteinemia ( %), and elevated liver enzymes ( %) (batchelder et al., ) . anemia was found in % of ferrets tested. necropsy findings include tan or yellow discolored liver, gastric hemorrhage, and gravid uterus. treatment forjills within ferrets are induced ovulators and may remain in persistent estrus if they are not bred or if estrus is not terminated chemically or via ovariohysterectomy (bell, a) . jills that remain in estrus for more than month are at risk for developing estrogeninduced anemia. hyperestrogenism from persistent estrus causes bone marrow hypoplasia of all cell lines in approximately half of ferrets in prolonged estrus (ryland et al., ) . clinical signs include vulvar enlargement, bilaterally symmetric alopecia of the tail and abdomen, weakness, anorexia, depression, lethargy, weight loss, bacterial infection, and mucopurulent vaginal discharge. hematology findings may vary from an initial neutrophilia and thrombocytosis early in the disease course to lymphopenia, thrombocytopenia, neutropenia, and anemia. the anemia begins as normocytic normochromic but progresses to macrocytic hypochromic (sherrill and gorham, ) . coagulopathy associated with hepatic dysfunction and thrombocytopenia combine to produce extensive manifestations of bleeding, pallor, melena, petechiation or ecchymosis, subdural hematoma, and hematomyelia (hart, ; fox and bell, ) . at necropsy, tissue pallor, light tan to pale pink bone marrow, hemorrhage, bronchopneumonia, hydrometra, pyometra, and mucopurulent vaginitis may be seen. histopathology may reveal cystic endometrial hypoplasia, hemosiderosis, diminished splenic extramedullary hematopoiesis, and mild to moderate hepatic lipidosis (sherrill and gorham, ; bell, a) . treatment consists of terminating estrus while supporting the animal with antibiotics, blood transfusion, b vitamins, and nutritional supplementation. estrus may be terminated by injection with - iu of human chorionic gonadotropin (hcg) or ~tg of gonadotropin-releasing hormone (gnrh), repeated week after initial injection if required. ovariohysterectomy may be considered for ferrets that are stable and have adequate numbers of platelets and red cells. ferrets with a packed cell volume (pcv) of % or greater have a good prognosis and require only termination of estrus for resolution of aplastic anemia. jills with a pcv of - % may require blood transfusions and have a guarded prognosis. ferrets with a pcv of less than % have a poor prognosis and require aggressive therapy with multiple transfusions. the lack of identifiable blood groups in ferrets makes multiple transfusions uncomplicated by potential transfusion reactions . estrogen-induced anemia may be avoided by ovariohysterectomy. of nonbreeding females, use of vasectomized hobs, or pharmacologic termination of estrus initiated days after estrus onset. a -to -day pseudopregnancy then follows, except in the case of ovariohysterectomy. repeated administration of hcg may result in sensitization and anaphylaxis. after several administrations, hcg is unlikely to be effective in termination of estrus. anaphylaxis is manifest as incoordination, tremor, vomiting, and diarrhea and may be reversed by prompt administration of diphenhydramine. arginine-free diets are unlikely to be fed in the laboratory setting, but administration of such a diet to young ferrets fasted for hr leads to hyperammonemia and encephalopathy within - hr (thomas and desmukh, ) . exacerbation of signs may be achieved by challenging young ferrets with influenza virus and aspirin (desmukh et al., ) and constitutes a model of reye's syndrome in children. lethargy and aggressiveness yield to prostration, coma, and death in affected ferrets. hyperammonemia presumably occurs because of the inability of ferrets to produce adequate amounts of ornithine from non-arginine precursors. detoxification of ammonia is thereby compromised. ferrets more than months old are unaffected by arginine-free diets. ferrets of all ages are susceptible to zinc toxicosis, and the condition has been documented in two ferret farms in new zealand (straube and walden, ) . leaching of zinc from steam-sterilized galvanized food and water bowls was implicated. clinical signs included pallor, posterior weakness, and lethargy. definitive diagnosis requires demonstration of elevated concentrations of zinc in kidney and liver. at necropsy, kidneys are enlarged, pale, and soft; livers are orange, and gastric hemorrhage may be seen. histopathology reveals glomerular collapse, tubular dilation, tubular proteinaceous debris, focal cortical fibrosis, hepatic periacinar infiltration, and depression of the erythroid series. avoidance of galvanized materials precludes the development of zinc toxicosis. umbilical entanglement may occur in ferrets on the day of parturition and has been associated with fine-particle bedding, large litters, and short kit-birth intervals (bell, a; fox et al., a) . jills may neglect to clean placentas from their kits, or kits may be born so rapidly that there is not adequate time for the jill to clean the kits of placental membranes, thereby predisposing to entanglement. entangled kits may succumb to dehydration, hypothermia, and hypoglycemia because they are unable to nurse and the jill cannot curl around them. detailed dissection with fine scissors and forceps under a heat lamp or on a heated surface can free the kits. occasionally, kits may need to be rotated on their umbilical pedicle to achieve adequate clearance to cut the cord; cords should be cut as far from the umbilicus as possible. the use of warm saline or water may help soften the mass. some kits in the tangle may present with dark, swollen extremities or prolapsed umbilical cords and may require euthanasia. parturition should be supervised, if possible, to avoid umbilical entanglement. hydronephrosis may occasionally occur in the ferret and is most commonly associated with inadvertent ligation of the ureter during ovariohysterectomy. ovarian remnants are another potential sequela to ovariohysterectomy. ovarian remnants in ferrets may be associated with estrus, vulvar enlargement, and alopecia. appropriate diagnostic procedures include ultrasonography and plain and contrast radiography for hydronephrosis and ultrasonography and serum hormone concentrations for ovarian remnants. exploratory celiotomy confirms the diagnosis, and unilateral nephrectomy or ovariectomy is indicated if the remaining kidney is normal and the ferret is otherwise healthy. over the last few decades, increasing numbers of ferrets have been used in research or kept as pets, and as these animals have received veterinary care, it has become evident that ferrets are subject to a wide variety of neoplastic conditions . however, four categories of cancer account for the majority of ferret neoplasms: pancreatic islet cell tumors, adrenocortical cell tumors, lymphoma, and skin cancers. functional pancreatic islet cell tumors (insulinomas) are the most common neoplasm diagnosed in ferrets . disease may be evident in ferrets as young as years old, but later onset (at - years of age) is typical (caplan et al., ; ehrhart et al., ) . nonspecific presenting signs include weight loss, vomiting, and ataxia. weakness is often evident, ranging from lethargy to posterior paresis or outright collapse (caplan et al., ) . hypoglycemia caused by excess production of insulin by neoplastic cells may cause tremors, disorientation, or seizures (fox and marini, ) . excessive salivation (ptyalism) or pawing at the mouth is a frequent finding. clinical signs are often intermittent or episodic. other common findings include splenomegaly and lymphocytosis. presumptive diagnosis is made based on clinical signs in conjunction with the demonstration of hypoglycemia. blood glucose determinations for the diagnosis of insulinoma are most useful when taken after a hr fasting period. fasting glucose concentrations below may be diagnostic for the condition (quesenberry and rosenthal, ) , whereas values between and are suspect and the test should be repeated (fox and marini, ) . other potential causes for hypoglycemia should be ruled out, including anorexia, starvation, hepatic disease, sepsis, and nonpancreatic neoplasia (antinoff, ) . demonstration of concurrent hyperinsulinemia aids the diagnosis (caplan et al., ) . medical management using prednisone and/or diazoxide along with dietary modification such as frequent feeding of high-protein meals can minimize or control clinical signs but will not affect the underlying tumor (quesenberry and rosenthai, ) . surgical exploration of the pancreas and tumor excision are recommended for animals that are healthy enough to be subjected to anesthesia and surgery. histological examination of the tissue removed can provide a definitive diagnosis, and although the effect may be transient, clinical signs are often reduced or eliminated after surgical debulking (figs. and ) (ehrhart et al., ) . histologically, these tumors reveal ma-lignant proliferation of pancreatic cells, and local recurrence or metastasis to lymph nodes, mesentery, spleen, or liver may occur (caplan et al., ) . adrenocortical cell tumor is the second most common type of neoplasia in ferrets and is generally diagnosed between and years of age. if clinical signs are present, they often include weight loss and a bilateral, symmetric alopecia. pruritus is a variable finding (quesenberry and rosenthal, ) . although ferrets with this syndrome have been called "cushingoid," it is rare to diagnose elevated resting levels of glucocorticoids or an abnormal response to adrenocorticotropic hormone (acth) stimulation or dexamethasone suppression testing. elevation of adrenal sex hormones (e.g., androstenedione, -hydroxyprogesterone, and/or estradiol) is more likely, and these may lead to characteristic changes such as estruslike vulvar swelling in spayed females and prostatic changes in males coleman et al., ) . rule-outs for enlarged vulva include estrus in an intact female or functional ovarian remnants in a spayed female. abdominal palpation may reveal cranial abdominal masses, and ultrasound may be useful (barthez et al., ) . serum assay for abnormal levels of the sex hormones listed above should be considered (lipman et al., ; wagner and dorn, ; rosenthal and peterson, ) . in many cases the alopecia begins as a seasonally intermittent partial hair loss that becomes more severe as time goes on (fig. ) . even severe manifestations of this endocrine alopecia can spontaneously reverse in the absence of specific therapy, as demonstrated in a group of ferrets referred to our facility for diagnostic workup. in each of these ferrets, near total alopecia resolved within a few months of being housed in a research environment. despite being asymptomatic at the end of the study, all were shown to have histologic evidence of adrenocortical neoplasia. although this phenomenon is mediated by hormonal effects, anecdotal reports such as this suggest that the alopecia may be significantly modulated by environmental factors (e.g., photoperiod or diet). surgical exploration and removal of enlarged adrenals are commonly performed to establish the diagnosis and to remove hyperfunctional tissue. unilateral adrenalectomy early in the disease may be curative, but because bilateral neoplastic involvement is not uncommon, full or partial removal of both glands may be required. adrenolytic agents such as mitotane have been used with limited success (quesenberry and rosenthai, ) . histologically, adrenocortical adenomas are generally cm or less in diameter and are composed of well-differentiated cells with a granular or vacuolated cytoplasm. adrenal cell carcinomas are less commonly found and are larger, with a more pleomorphic and invasive character . metastasis to nearby tissues can occur. in our experience, adrenal cortical hyperplasia with or without neoplasia is an extremely common finding in aging ferrets, even in those not showing clinical signs. in one retrospective survey of our necropsy records it was found that more than % of ferrets greater than years of age had hyperplastic or neoplastic adrenal changes when examined (data not shown). for this reason, careful considerations of other possible disease processes should be made before attributing clinical signs solely to adrenal enlargement. lymphoma can affect ferrets of almost any age. ferrets younger than years of age often present with mediastinal lymphoma and/or leukemia, whereas those older than years of age often develop multicentric solid tumors . the early age of onset in some ferrets and reports of case clustering have led to investigation into potential infectious etiologies for lymphoma in the ferret (erdman et al., b) . earlier reports of feline leukemia virus (felv) seroconversion in affected animals have not been substantiated. however, experimental and epidemiological evidence suggests that a retrovirus that is distinct from felv may be involved . in one study, whole or filtered lymphoma cells from a -year-old ferret with spontaneous lymphoma were injected ip into recipient ferrets . two of the ferrets were euthanized after months, but the remaining developed splenomegaly, lymphocytosis, and lymphoma. one ferret that received cell-free materials developed multicentric lymphoma with prominent cutaneous lymphoma nodules. elevated reverse transcriptase activity and retrovirus-like particles evident by electron microscopy were seen in the donor and all of the affected recipient ferrets. other potential etiologies that have been considered include two infectious agents that are known to cause chronic immune stimulation in affected ferrets, the aleutian disease virus (adv) and helicobacter mustelae. a link with adv has not been proven, but h. mustelae seems to be responsible for the development of a very specific type of gastric b-cell lymphoma (erdman et al., ) . affected ferrets may exhibit localizing signs (e.g., dyspnea in a ferret with mediastinal involvement or peripheral lymphadenopathy in an animal with a multicentric distribution) but as is the case in many species, lymphoma is a "masquerader," and affected ferrets often present with chronic, nonspecific signs. weight loss, anorexia, and lethargy are often reported. splenic and/or hepatic enlargement may be evident. cutaneous involvement has been documented (li et al., ; rosenbaum et al., ) . although hematological examination typically reveals anemia and lymphopenia, lymphocytosis may be found, especially in younger ferrets. atypical lymphocytes are identified in the circulation in some cases. antemortem definitive diagnosis of lymphoma can be made by cytological examination of specimens obtained via fine-needle aspiration or excisional biopsy. tan-colored masses involving lymph nodes, spleen, liver, or other organs are commonly found at necropsy (fig. ) . diffuse involvement may lead to uniform enlargement of these organs or to a thickening of the wall of the stomach or intestines. as in other species, histological evaluation reveals neoplastic lymphocytes in affected tissues, generally evident as a monomorphic population (fig. ) . although surgery and radiation therapy may be useful in certain cases, most attempts to treat ferret lymphoma have utilized chemotherapeutic regimens with dosages extrapolated from other domestic animals or humans. treatment generally results in a remission that may last from months to years (brown, b; erdman et al., ) . mast cell tumors are among the most commonly reported integumentary tumors in ferrets (parker and picut, ; . cutaneous mastocytomas may occur anywhere on the body and present as firm, nodular skin lesions - mm in size that are often associated with alopecia or crusty ulceration of the overlying skin. pruritis is common (stauber et al., ) . histologically, they are composed of well-differentiated mast cells with metachromatic cytoplasmic granules that may be difficult to detect in sections stained with hematoxylin-eosin, but are more evident in toluidine bluestained sections. a variety of tumors of epithelial origin occur in ferrets, and they can appear at any site on the body. the most common are the basal cell tumors, which present as firm plaques or pedunculated nodules that are white or pink (parker and picut, ) . they may grow rapidly and become ulcerated. the percentage of basiloid cells present in these tumors, and the degree of associated squamous or sebaceous differentiation can vary, resulting in a spectrum of tumor subtypes and associated histological diagnoses (orcutt, ) . however, as is the case with mastocytomas, most are benign and will not recur after excision. resected tumors should be examined histologically to rule out less common tumors that might have a more guarded prognosis, such as squamous cell carcinoma or apocrine gland adenocarcinoma. chordomas are not epithelial tumors, but they often present as readily evident firm masses on the tail that may cause ulceration of the overlying skin. these neoplasms arise along the axial skeleton from notochord remnants and are typically slow-growing (dunn et al., ) . tumors involving the tail generally do not recur after amputation of the affected region, but a wide surgical margin should be maintained by removing several vertebrae proximal to the tumor. the prognosis is guarded for those rare chordomas that arise in the cervical region, and metastasis has been documented (williams et al., ) . congenital defects identified in ferrets include a variety of neural tube defects, gastroschisis, cleft palate, amelia, corneal dermoids, cataracts, and supernumerary incisors (willis and barrow, ; ryland and gorham, ; mclain et al., ; besch-williford, ) . cystic or polycystic kidneys have been observed (andrews et al., a; dillberger, ) . cystic genitourinary anomalies associated with the prostate, bladder, and/or proximal urethra most likely develop secondary to aberrant hormone secretion by adrenocortical tumors coleman et al., ) . newborn ferrets are normally born with a closed orbital fissure and are prone to developing subpalpebral conjunctival abscesses. treatment involves surgically opening the lids (a minor procedure) to establish drainage and to allow topical antibiotics to be administered (bell, a) . cardiomyopathy is a common cause of disease in aging ferrets. the dilatative form of disease is most commonly diagnosed. affected animals commonly present with lethargy, weight loss, and anorexia. physical examination may reveal signs of congestive heart failure such as hypothermia, tachycardia, cyanosis, jugular distension, and respiratory distress (lipman et al., ) . auscultation may reveal a heart murmur and/or muffled cardiac sounds. hepatomegaly and splenomegaly are often identified. radiographs may reveal an enlarged cardiac silhouette and evidence of pulmonary edema or pleural effusion (greenlee and stephens, ) . electrocardiography and echocardiography can help make the definitive diagnosis. medical therapy (supportive care, diuretics, and inotropic drugs) may relieve clinical signs and improve the qual-ity of life for a period of months (stamoulis et al., ) . the long-term prognosis for survival is guarded to poor. splenomegaly is a common finding in ferrets. in many cases the enlarged spleen appears to be a secondary manifestation of another disease (e.g., insulinoma, cardiomyopathy, or adrenal tumor) and is of unknown significance (stamoulis et al., ) . histologic examination of affected organs has revealed that the most common cause for splenic enlargement (in the absence of a neoplastic infiltrate) is extramedullary hematopoiesis (emh) (erdman et al., ) . this may be an incidental finding, but it has been suggested that in some cases a pathologically enlarged spleen may play a role in chronic anemia that may respond to splenectomy, a syndrome known as hypersplenism (ferguson, ) . splenomegaly can also be commonly found in conjunction with lymphoma, with or without intrasplenic neoplastic lymphoid accumulations. in anesthetized ferrets, splenomegaly may be caused by splenic sequestration of erythrocytes (marini et al., (marini et al., , . because this is a transient effect, the normalization of splenic size upon recovery from anesthesia can help in the differentiation of anesthetic-induced splenomegaly from that due to other causes. eosinophilic gastroenteritis is an idiopathic disorder characterized by peripheral eosinophilia ( - % of circulating leukocytes), hypoalbuminemia, and diffuse infiltration of the gastrointestinal tract with eosinophils (fox et al., a) . presenting signs for this syndrome generally include chronic weight loss, anorexia, diarrhea, and occasionally vomiting. eosinophilic granulomas have been found in the mesenteric lymph nodes of most affected ferrets, and in some cases other organs (e.g., lung or liver) may be involved. an interesting finding in many ferrets is the presence of splendore-hoeppli material in the inflamed lymph nodes, a histological phenomenon that has been associated in other species with helminths, bacteria, fungi, and foreign bodies (fig. ). an etiological agent has not been identified; consequently, therapy consists largely of supportive care to treat the chronic enteritis (fox, b) . based on the biology of eosinophils, however, the use of corticosteroids or ivermectin has been attempted and may be beneficial (bell, b) . megaesophagus has been diagnosed in ferrets presenting with a variety of signs, including weight loss, anorexia, difficulty in eating, or repeated regurgitation. the cause is generally unknown, and the prognosis is poor, despite efforts at supportive care (blanco et al., ) . gray, yellow, or white small raised lesions may be found on the surface of ferret lungs at gross examination. histologically, these lesions are composed of a superficial thickening of the lung tissue with mononuclear cell infiltration and varying degrees of fibrosis, with or without cholesterol-like clefts. the etiology of this condition (known as subpleural histiocytosis, pleural lipidosis, or lipid pneumonia) is unknown, and it appears to be an incidental lesion (fox, f) . energy: protein relationships in the diets of growing mink. can the phylogeny of mustelids and the systematics of ferrets some observations of anatomical abnormalities and disease states in a population of ferrets (mustela furo) some aspects of the physiology and anatomy of the cardiovascular system of the ferret, mustela putorius furo ufaw handbook on the care and management of laboratory animals musculoskeletal and neurological diseases ultrasonography of the adrenal glands in the dog, cat, and ferret pregnancy toxemia in the european ferret (mustela putorius furo) use of the ferret in reproductive neuroendocrinology comparison of the teratogenic effects of mustine hydrochloride in rats and ferrets. the value of the ferret as an experimental animal in teratology periparturient and neonatal diseases helicobacter mustelae gastritis, proliferative bowel disease, and eosinophilic gastroenteritis a domestic ferret model of immunity to campylobacter jejuni-induced enteric disease reproductive failure in mink and ferrets after intravenous or oral inoculation of campylobacterjejuni evaluation of campylobacterjejuni colonization of the domestic ferret intestine as a model of proliferative colitis estrogen-induced bone marrow depression in ferrets biology and diseases of ferrets biology and medicine of the ferret megaesophagus in nine ferrets rage experimetale du furet (mustela putorius furo) enteric coccidiosis in a ferret feed consumption and food passage time in mink (mustela vison) and european ferrets (mustela putoriusfuro) basic anatomy, physiology, and husbandry neoplasia. in "ferrets, rabbits, and rodents: clinical medicine and surgery infectious diseases of wild mammals diagnosis and treatment of insulin-secreting pancreatic islet cell tumors in ferrets: cases ( - ) fatal vaccineinduced canine distemper virus infection in black-footed ferrets coital stimuli controlling luteinizing hormone secretion and ovulation in the female ferret sex difference in the effect of mating on the pulsatile secretion of luteinizing hormone in a reflex ovulator, the ferret essentials of veterinary microbiology ferrets as laboratory animals: a bibliography the treatment of salmonella typhimurium infection in ferrets cystic prostatic disease associated with adrenocortical lesions in the ferret (mustela putorius furo) a world list of mammalian species surgical extirpation and related anatomy of anal sacs of the ferret serum glutamatedehydrogenase and ornithine carbamyl transferase in reye's syndrome polycystic kidneys in a ferret yeast infection in ferrets a histomorphologic and immunohistochemical study of chordoma in ferrets (mustela putorius furo) pancreatic beta cell tumor in ferrets: cases ( - ) transmission of a chronic lymphoproliferative syndrome in ferrets clinical and pathogic findings in ferrets with lymphoma: cases ( - ) clusters of malignant lymphoma in ferrets helicobacter mustelae-associated gastric mucosa associated lymphoid tissue (malt) lymphoma in ferrets hematopoietic diseases anatomy of the ferret idiopathic hypersplenism in a ferret diseases of the fitch. surveillance isolation of helicobacter mustelae from ferrets in new zealand biology and diseases of the ferret diseases of the gastrointestinal system biology and diseases of the ferret normal clinical and biologic parameters biology and diseases of the ferret biology and diseases of the ferret biology and diseases of the ferret biology and diseases of the ferret proliferative colitis in ferrets ferret as a potential reservoir for human campylobacteriosis campylobacter-like organisms isolated from gastric mucosa of ferrets serum chemistry and hematology reference values in the ferret (mustela putoriusfuro) campylobacter jejuni infection in the ferret: an animal model of human campylobacteriosis zoonoses in ferrets gastric colonization of campylobacterpylori subsp, mustelae in ferrets campylobacter mustelae, a new species resulting from the elevation of campylobacter pylori subsp, mustelae to species status proliferative colitis in ferrets: epithelial dysplasia and translocation helicobacter mustelae-associated gastritis in ferrets: an animal model of helicobacter pylori gastritis in humans gastric colonization of the ferret with helicobacter species: natural and experimental infections helicobacter mustelae-induced gastritis and elevated gastric ph in the ferret (mustela putorius furo) eosinophilic gastroenteritis with splendore-hoeppli material in the ferret (mustela putorius furo) helicobacter mustelae isolation from feces of ferrets: evidence to support fecal-oral transmission of a gastric helicobacter role of gastric ph in isolation of helicobacter mustelae from the feces of ferrets animal models for helicobacterinduced gastric and hepatic cancer helicobacter mustelae-associated gastric adenocarcinoma in ferrets (mustela putorius furo) diseases of the genitourinary system viral diseases compendium of recent literature on the ferret pneumocystis carinii is not universally transmissible between mammalian species the transmission of distemper among ferrets and mink. paper presented at the th meeting of the american veterinary medical association salmonella infections in mink and ferrets the experimental transmission of a virus causing hypergammagloblinemia in mink: sources and modes in infection meningeal cryptococcosis and congestive cardiomyopathy in a ferret the biomedical use of ferrets in research the universities federation for animal welfare handbook on the care and management of laboratory animals endocrine factors in hematological changes seen in dogs and ferrets given estrogens cryptosporidiosis in the dog and cat dematologic diseases ferrets, rabbits, and rodents: clinical medicine and surgery a practitioner's guide to rabbits and ferrets recurrent mycobacterium bovis infection following a ferret bite cell mediated immunity in ferrets: delayed dermal hypersensitivity, lymphocyte transformation, and macrophage migration inhibitory factor production experimental transmission of aleutian disease with urine enteric coccidial infections heartworm infections treatment of proliferative colitis in ferrets current veterinary therapy haematological and serum chemistry profiles of ferrets (mustela putorius furo) biology and diseases of the ferret cutaneous lymphoma in a ferret (mustela putoriusfuro) cystic urogenital anomalies in the ferret (mustela putorius furo) coinfection with intracellular desulfovibrio species and coccidia in ferrets with proliferative bowel disease neoplastic diseases in ferrets: cases ( - ) mastitis caused by hemolytic escherichia coli in the ferret clinical, functional, and pathologic changes associated with a case of dilatative cardiomyopathy in a ferret estradiol- [ -secreting adrenocortical tumor in a ferret studies on canine distemper infection by means of fluorescein-labeled antibody. . the pathogenesis, pathology, and diagnosis of the disease in experimentally infected ferrets the clinical chemistry of laboratory animals derivation of gnotobiotic ferrets: perinatal diet and hand-rearing requirements lack of detectable blood groups in domestic ferrets: implications for transfusion distribution of technetium m-labeled red blood cells during isoflurane anesthesia in ferrets ranitidine-bismuth citrate and clarithromycin, alone or in combination, for eradication of helicobacter mustelae infection in ferrets the effect of isoflurane on hematologic variables in ferrets the biomedical use of ferrets in research congenital malformations and variations in reproductive performance in the ferret: effects of maternal age, color, and parity dirofilariasis in a ferret dirofilariasis in a ferret use of the ferret in cardiovascular research current therapy in theriogenology soft tissue surgery demodiciasis in ferrets (mustela putorius furo) measles virus comparison of the lesions of aleutian disease in mink and hypergammaglobulinemia in ferrets dermatologic diseases eradication of helicobacter mustelae from the ferret stomach: an animal model of helicobacter (campylobacter) pylori chemotherapy parvovirus-associated syndrome (aleutian disease) in two ferrets histopathologic features and post-surgical sequelae of cutaneous neoplasms in ferrets (mustela putorius furo l.) dirofilaria immitis in three ferrets clinical trial to control ear mites in a ferret colony real-time ultrasonography determination of pregnancy and gestational age in ferrets pedal sarcoptes scabiei infestation in ferrets (mustela putorius furo) isolation of infectious bovine rhinotracheitis virus from mustelidae aleutian disease in ferrets use of the ferret as a model for pediatric endotrocheal intubation training use of ferrets in laboratory work and research investigators endocrine diseases cryptosporidiosis in ferrets cutaneous epitheliotropic lymphoma in a ferret ferrets, rabbits, and rodents: clinical medicine and surgery evaluation of plasma androgen and estrogen concentrations in ferrets with hyperadrenocorticism evaluation of an inactivated rabies virus vaccine in domestic ferrets hormonal correlates of photoperiod-induced puberty in a reflex ovulator, the female ferret the ferret and its diseases a clinical guide to the pet ferret identification of a dna segment in ferret aleutian disease virus similar to hypervariable capsid region of mink aleutian disease parvovirus gastroenteritis associated with clostridium perfringens type a in black-footed ferrets (mustela nigripes) granulomatous enteritis caused by mycobacterium avium in a ferret small animal dermatology survival of pathogenic distemper virus at ~ and ~ vet using jet injection to vaccinate mink and ferrets against canine distemper, mink viral enteritis, and botulism, type c bone marow hypoplasia associated with estrus in ferrets growth and development of the european ferret (mustela putorius) a bibliography of mustelids. part : ferrets and polecats the influence of apparent digestible energy and apparent digestible nitrogen in the diet on weight gain, feed consumption, and nitrogen retention of growing mink influenza infection of man from the ferret cardiovascular diseases mast cell tumors in three ferrets canine distemper virus (cdv) infection of ferrets as a model for testing morbillivirus vaccine strategies: nyvac-and alvac-based cdv recombinants protect against symptomatic infection experimental pneumocystis carinii pneumonia in the ferret zinc poisoning in ferrets (mustela putorius furo) biology, diagnosis, and prevention of heartworm infection in ferrets production of passive immunity of neonatal ferrets following maternal vaccination with killed influenza a virus vaccines observations on tuberculosis in the ferret (mustela furo l.) effect of arginine-free diet on ammonia metabolism in young and adult ferrets the ferret, mustela putoriusfuro, as a new species in toxicology pruritis in rabbits, rodents, and ferrets the characterization and pathological significance of gastric campyiobacter-like organisms in the ferret: a model of chronic gastritis? isolation of atypical rotavirus causing diarrhea in neonatal ferrets mastitis in mink due to staphylococcus aureus and escherichia coli guide for the care and use of laboratory animals place de mycobacterium avium dans l'epidemiologies mycobacterienne actuelle chez les animaux domestiques et savages. sci pulmonary physiology as a model for inhalation toxicology evaluation of serum estradiol concentrations in alopecic ferrets with adrenal gland tumors the ferret intestinal uptake and lymphatic absorption of [ -carotene in ferrets: a model for human [ -carotene metabolism intestinal perfusion of [ -carotene in the ferret raised retinoic acid level in portal blood vitamin e enhances the lymphatic transport of [ -carotene and its conversion to vitamin a in the ferret aleutian disease in domestic ferrets: diagnostic findings and survey results semen characteristics and testosterone profiles in ferrets kept in a long-day photoperiod, and the influence of hcg timing and sperm dilution medium on pregnancy rate after laporoscopic insemination biliary coccidiosis in a ferret (mustela putoriusfuro) cervical chordoma in two ferrets (mustela putorius furo) coronavirus-associated epizootic catarrhal enteritis in ferrets comparative vaginal cytology of the estrous cycle of black-footed ferrets (mustela nigripes) the ferret (mustela putorius furo) as a laboratory animal effect of helicobacter mustelae infection on epithelial cell proliferation in ferret gastric tissues key: cord- -ridm qd authors: martínez, maría guadalupe; prado acosta, mariano; candurra, nélida a.; ruzal, sandra m. title: s-layer proteins of lactobacillus acidophilus inhibits junv infection date: - - journal: biochem biophys res commun doi: . /j.bbrc. . . sha: doc_id: cord_uid: ridm qd it has been previously described that s-layer binds to the c-type lectin dc-specific icam- -grabbing nonintegrin (dc-sign, cd ). it was also shown that dc-sign is a cell-surface adhesion factor that enhances viral entry of several virus families. among those, junin virus (junv) entry is enhanced in cells expressing dc-sign and for that reason surface-layer protein (s-layer) of lactobacillus acidophilus atcc was evaluated as a possible junv inhibitor. experiments using t cells stably expressing dc-sign, showed an almost complete inhibition of junv infection when they were treated with s-layer in a similar extend as the inhibition shown by mannan. however no inhibition effect was observed in t wild type cells or in t cells expressing liver/lymph node-specific icam- grabbing nonintegrin (l-sign or dc-signr or cd l). treatments with s-layer during different times in the infection demonstrated that inhibition was only observed when s-layer was presented in early stages of the viral infection. this inhibition does not involve the classic recognition of mannose by this c-type lectin as the s-layer showed no evidence to be glycosylated. in fact, the highly basic nature of the s-layer (pi > . ) seems to be involved in electrostatic interactions between dc-sign and s-layer, since high ph abolished the inhibitory effect on infection cause by the s-layer. in silico analysis predicts a ca( +)-dependant carbohydrate recognition domain in the slpa protein. this novel characteristic of the s-layer, a gras status protein, contribute to the pathogen exclusion reported for this probiotic strain and may be applied as an antiviral agent to inhibit several kinds of viruses. many species of the genus lactobacillus possess surface layer (s-layer) proteins which are arrays of a single protein noncovalently bound that constitute the outermost cell envelope also found in several members of bacteria and archaea. one of the main characteristics of lactobacillus s-layer proteins is their high isoelectric point (pi) showing them as highly basic [ ] . lactobacillus acidophilus is one of the major species found in human intestines and some strains have probiotic characteristics making them gras (generally recognized as safe). probiotics are live microorganisms, usually contained in food, traditionally regarded as safe for human consume that, when ingested in sufficient number, play an important role in control of the host intestinal microbiota and in the modulation of the host immune response. l. acidophilus atcc has been described as a probiotic strain [ ] with a kda s-layer protein highly basic with a calculated pi of . , (expasy) that was recently showed to have an antibacterial endopeptidase activity [ , ] . s-layers have also been considered to function as protective coats, maintenance of cell shape and ion exchangers and be involved in adhesion to biotic and abiotic surfaces [ , ] . although many studies focus on the structure of the s-layer, their biological functions remains poorly understood. s-layer would account for the probiotic properties of some strains [ ] being capable of influencing the immune response [ ] and favoring cell adhesion [ ] [ ] [ ] [ ] . several reports have involved the s-layer proteins in the enteric pathogen exclusion phenomena [ , ] . in the gastro-intestinal tract (git)l. acidophilus regularly encounters many antigens presenting cell, such as dendritic cells (dcs) [ ] . these cells express dc-specific icam- -grabbing nonintegrin protein (dc-sign), which is a cell-surface receptor, that is express mainly in dcs and recognize mannose and fructose glycans that are present on microbial and viral surfaces. dcs play a very important role in the innate and adaptive immune response [ ] . it has been shown that dc-sign can act enhancing viral entry of different viruses such as hiv type , hepatitis c, ebola, dengue and sars [ ] [ ] [ ] . it was also shown that dcs interact with l. acidophilus; this contact involve the dc-sign and the s-layer presented on the bacterial cell-envelope, and regulates the induction of a number of cytokines involved in cellular immune regulation [ , ] . junv, a new world arenavirus, is the etiological agent of the argentine hemorrhagic fever (ahf) [ ] , and endemo-epidemic disease that causes hemorrhagic and neurological complications. ahf presents mortality rates from - % and mainly affects population on the fertile farming land of argentina [ ] . it has been shown that junv uses the human transferrin receptor (tfr ) to infect human cells [ ] . in addition to its primary receptor (tfr ), junv virus infection was shown to be enhanced by c-type lectins (martinez & candurra, unpublished results). among the c-type lectins dc-sign and the liver/lymph node-specific icam- -grabbing nonintegrin (l-sign) were studied. dc-sign and l-sign are human homologs, share % of the amino-acid sequence and has function similarities. the role of these receptors (dc-and l-sign) in the infection of several major importance viruses placed them as a target for antiviral agents. the most common compounds used so far are carbohydrate-binding agents that can interact with the glycoprotein present on the virus surface and prevent the infection [ ] . lactobacilli have been described to have antiviral activity [ ] however the nature of this effect has not been clearly established. in this work we evaluated the effect of the purified s-layer protein of l. acidophilus atcc on junv infection to provide evidence on its role in pathogen exclusion and in the search for new antiviral agents with gras status. we used t cells stably expressing dc-sign, or its homolog l-sign or vero cells for transient expression as in vitro models. since t cells are poorly infect by junv in the absence of expression of c-type lectins this model provides a really strong tool to study the effect of s-layer protein on the infection enhanced by dc-sign or l-sign. the s-layer proteins were extracted from overnight l. acidophilus atcc cells grown in mrs medium at °c, by using m licl, extensively dialyzed against distilled water overnight at °c and after centrifugation ( , g min) suspended in sterile pbs and store at À °c [ ]. monolayers of vero cells (atcc ccl ) were grown in mem containing % heat-inactivated fetal bovine serum (fbs) and supplemented with lg/ml gentamycin. maintenance medium (mm) consisted of mem containing . % fetal calf serum, and titration medium consisted of mem x, % fbs and methyl cellulose ( . %). t cells (atcc ccl ) and t -derived dc-sign and l-sign cells (niaid aids research and reference reagent program) were grown in mem supplemented with % fbs. in all cases, cultures were grown in % co and were supplemented with hepes ( lm). the naturally attenuated junin iv strain was used in all experiments with live virus, and propagated in vero cells. virus yields were then determined by plaque formation (pfu) assays in vero cells. the titers of the junv stock suspensions were -  pfu/ml. fitc-goat anti mouse and mannan were purchased from sigma. anti-junv monoclonal antibodies na -ag were kindly do-nated by dr. a. sanchez (centers for disease control, atlanta ga, usa). anti-hdc-sign and anti-hl-sign mabs ( , e a , eg ) were obtained from the niaid aids research and reference reagent program. confluent t dc-sign cells grown in -well plates were treated with different concentrations of s-layer for h. after the treatment period, the medium was removed and the cells were infected in the presence of the protein. after infection, the inoculum was removed and fresh mm was added. the viral supernatants were harvested after h and junv yields were determined by pfu assays. viability of cell cultures under each treatment condition was determined by the mtt assay. transfection was performed according to the lipofect-amine manufacturer (invitrogen). glycosylation detection was performed with periodic acid-schiff's reagent (pas) according to the procedure of segrest et al. [ ] and pro-q_emerald glycoprotein gel stain kit (invitrogen) on sds-polyacrylamide gels. this fluorescence-based staining method allows the detection of less than . ng glycoprotein which is times more sensitive than pas staining. free access sites were used to predict protein structure and function for the s-layer protein slpa caa . we infected t cells wt or stably expressing c-type lectins with junv. as it can be seen in fig. a t cells are poorly infected with junv in the absence of dc-sign or l-sign expression. to study the effect of s-layer protein on junv infection t cells and t -derived stably expressing dc-sign and l-sign cells were incubated with different concentrations of s-layer protein, infected with junv and then cells expressing viral nucleoprotein (np) were quantify. the results showed reduction in the infection of junv in t dc-sign cells, but none inhibition was shown in t l-sign cell (fig. a) . when we performed a similar experiment but adding the s-layer protein after the infection with junv, there was almost no effect of this treatment in cell expressing junv np. this suggests that the effect of the s-layer treatment in junv infection is only in an early step of the replication cycle, not affecting later stages (fig. b) . to provide further evidence that blocking in the infection with s-layer was due to the presence of dc-sign but not l-sign, we performed transient transfection of vero cells with plasmids expressing hdc-sign or hl-sign. after h of the transfection the cells were treated with the s-layer protein and then infected with junv (table ). this confirms that the s-layer inhibitory effect was exclusive to cells expressing dc-sign but not l-sign. to further investigate the potential of the s-layer protein to act as an inhibitor for junv infection t dc-sign cells were incubated with different concentrations of s-layer for h ant then infected with junv. then, h post infection we measure viral production by pfu assay and quantified viral np by if (fig. ) . as it is shown in fig. a when we increased the concentration of slayer we observe less junv infection. however when we exceed lg/ml, where it is likely to have auto-assembly of a crystalline s-layer array, the inhibitory effect was suppressed. virucidal effect on junv was assayed by treatment with s-layer during min at °c. no difference with the untreated control was observed by infectivity assay (fig. c ). this indicates that the s-layer protein have no effect on the viral particles. cytotoxicity effect of the protein s-layer protein in t cells was also studied: no toxicity was found in the concentration of s-layer needed for the inhibition (fig. b ). previous reports suggested that dc-sign and the s-layer protein interact [ ] . to determine glycosylation state of the s-layer preparation, two assays were performed as shown in methods (fig. ) . results showed no glycosylation therefore s-layer protein of l. acidophilus recognition of dc-sign does not involve the classic mannose interaction. to verify if the highly basic nature of the s-layer (pi > . ) was responsible for electrostatic interactions that could be responsible for the binding with dc-sign, interfering with junv interaction with the lectin, basic ph treatments were assayed. increasing the ph in the medium reduced the inhibition of infection by junv in dc-sign expressing cells, due to a change in s-layer net charge (pi . ) (fig. a ). this effect was not observed in treatments of l-sign expressing cells, where no virus inhibition was observed (fig. b) , indicating that there was not modification of the infectivity due to basic ph. chelating effects were also verified to influence the interaction, reducing the inhibitory effect on virus infection (not shown). we presumed that the c-terminal portion of the s-layer would be responsible of the interaction with the glycan strand of dc-sign, since we have already shown it to interact with cell wall peptidoglycan [ ] and it has been reported, that two repeats sequences in the c-terminal portion of the protein showed homology to carbohydrate binding domains (cbd) of clostridium difficile s-layer/ toxin [ , ] . the repeated sequences are a general theme for protein-carbohydrate interaction described for extracellular virulence factors of gram positive bacteria [ ] . slpa protein sequence launched in cdart showed that the c-terminal part, domain slap pfam , aligned with structural homology with putative cbd of a subgroup of bacterial protein homologous c-type lectin-like (clect or ctld domain), protein domains homologous to the carbohydrate-recognition domains (crds) of the c-type lectins (clect cd and clect_vcbs cd ) however bacterial ctlds within this group are functionally uncharacterized. when only the amino-acid sequence of slap pfam domain ( aa c-terminal portion) was used as query for smart analysis two internal repeats not previously revealed with the whole protein sequence, because they overlap with pfam domain, were actually found with good accuracy (e-value . e - ). the two internal repeats showed % identity (table ). total number of positively charged residues (arg + lys) within the repeats gives a predicted pi of . and . for each sequence and supports the carbohydrate binding capacity suggested (table ) . when using i-tasser for d structure and function prediction, result obtained showed similarity to ca + binding protein. the consensus prediction of gene ontology terms obtain include functions of cell adhesion, protein binding and surface expression (http://zhanglab.ccmb.med.umich.edu/i-tasser/output/s ). in summary these analyses results suggest that the s-layer of l. acidophilus is predicted to have a carbohydrate binding capacity. cells stably expressing dc-sign can be infected by junv, but an almost complete inhibition of junv infection was shown when they were treated with purified s-layer protein from l. acidophilus atcc prior to infection (fig. ) . this effect was not observed in cell lines that do not express this c-type lectin. post infection treatments with s-layer did not have an effect in viral infection, leading to the idea that the inhibition caused by s-layer was due to an early effect during viral replication cycle. since no virucidal effect was observed for the s-layer, it seems that the target is not the viral particle but the direct interaction with the dc-sign. this inhibitory effect is a novel characteristic of the s-layer protein of l. acidophilus, which could account for the pathogen exclusion effect described for this probiotic bacterium. the s-layer is a highly stable protein with no toxicity to cells as cell viability was higher than % (fig. ) . differences in binding properties between dc-sign and l-sign to its target have been reported [ ] . therefore it is not surprising the lack of an inhibitory effect in cell expressing l-sign ( fig. and table ). the mechanism of interaction involved it is not expected to be the classic mannose recognition, known for this kind of lectins. in fact no glycosylation was observed in the s-layer table transient expression of lectins on vero cells. vero cells were transfected with plasmids encoding hdc-sign or hl-sign. h post-transfection, cells were treated with s-layer ( lg/ml), mannan ( lg/ml) or left untreated (control) and then infected with junv (m.o.i. = ). cells were fixed at h p.i. and stained for if microscopy. lectin expression was verified using anti hdc-/hl-sign antibodies provided by nih ( ). data is indicated as a mean of the percentage of inhibition relative to each control. results from three independent experiments were captured and quantified and are shown. preparation (fig. ). it has been previously described that dc-sign forms a tetramer that enables multivalent interaction with pathogens. dc-sign-pathogen complex can be internalized into compartments with acid ph, and it was shown that there is a ph sensitive control of the oligomerization state of this complex [ ] . therefore we believe that the highly basic nature (pi > . ) of this particular s-layer protein might be responsible of interfering with the calcium-dependent viral interaction by structural and electrostatic forces (fig. ) . the interaction of the s-layer protein that lacks glycan strands with the dc-sign was predicted by bioinformatics methods. it is interesting to remark that this novel mechanism of interaction between slpa that lacks glycan strands and the dc-sign resembles the interaction between non glycosylated c-type animal lectins with their ligands, for example galectin- protein [ ] . this family of soluble proteins displays conserved crd that recognizes glycans in its glycoprotein ligand. the repeating sequences described in slpa (table ) are a general theme for protein-carbohydrate interaction [ ] and the s-layer protein is proposed to interact with carbohydrate moieties in a calcium dependant manner. dc-sign has been shown to determine susceptibility to hiv type , hepatitis c virus, ebola virus, cytomegalovirus, dengue virus and sars coronavirus [ ] [ ] [ ] . the results obtained in this in vitro model, suggest that s-layer could also inhibit these viruses that use dc-sign for viral entry. although a great deal of work needs to be done to address the type of interaction predicted between the s-layer protein with the dc-sign, this is the first report of a direct viral inhibition by the purified s-layer protein that support the probiotic status of the strain. table internal repeats obtained with smart within slap domain (pfam ) and clustal w alignment with consensus von eichel-streiber carbohydrate binding sites [ ] . pi was predicted with protparam (expasy). consensuses are in italic or bold letters. (k + r) indicate positively charged residues (arg + lys), n°for number of residues and position within slpa protein. symbols:''|'' identical aminoacid, '':'' indicates group similarity, ''.'' indicates low group similarity. amino acid notation according to iupac-iub-cbn. surface and adhesion properties of lactobacilli live probiotics protect intestinal epithelial cells from the effects of infection with enteroinvasive escherichia coli (eiec) murein hydrolase activity in the surface layer of lactobacillus acidophilus atcc synergistic effects of the lactobacillus acidophilus surface layer and nisin on bacterial growth s-layers as a tool kit for nanobiotechnological applications metal biosorption by surface-layer proteins from bacillus species functionality of the s-layer protein from the probiotic strain lactobacillus helveticus m s layer protein a of lactobacillus acidophilus ncfm regulates immature dendritic cell and t cell functions importance of s-layer proteins in probiotic activity of lactobacillus acidophilus m functional analysis of putative adhesion factors in lactobacillus acidophilus ncfm isolation of surface (s) layer protein carrying lactobacillus species from porcine intestine and faeces and characterization of their adhesion properties to different host tissues lactobacillus plantarum surface layer adhesive protein protects intestinal epithelial cells against tight junction injury induced by enteropathogenic escherichia coli surface-layer protein extracts from lactobacillus helveticus inhibit enterohaemorrhagic escherichia coli o h adhesion to epithelial cells lactobacillus acidophilus s-layer protein-mediated inhibition of salmonella-induced apoptosis in caco- cells intestinal lactobacilli and the dc-sign gene for their recognition by dendritic cells play a role in the aetiology of allergic manifestations dc-sign and l-sign the signs for infection differences in the mannose oligomer specificities of the closely related lectins from galanthus nivalis and zea mays strongly determine their eventual anti-hiv activity entry of hepatitis c virus and human immunodeficiency virus is selectively inhibited by carbohydratebinding agents but not by polyanions antiviral activity of carbohydrate-binding agents and the role of dc-sign in dengue virus infection lactobacillus acidophilus induces virus immune defence genes in murine dendritic cells by a toll-like receptor- -dependent mechanism concerning the epidemic outbreak in junin argentine hemorrhagic fever transferrin receptor is a cellular receptor for new world haemorrhagic fever arenaviruses targeting the glycans of glycoproteins a novel paradigm for antiviral therapy inhibition of herpes simplex virus type by vaginal lactobacilli red cell membrane glycoprotein amino acid sequence of an intramembranous region the s-layer protein of lactobacillus acidophilus atcc : identification and characterisation of domains responsible for s-protein assembly and cell wall binding one repeat of the cell wall binding domain is sufficient for anchoring the lactobacillus acidophilus surface layer protein evidence for a modular structure of the homologous repetitive c-terminal carbohydrate-binding sites of clostridium difficile toxins and streptococcus mutans glucosyltransferases molecular basis of the differences in binding properties of the highly related c-type lectins dc-sign and l-sign to lewis x trisaccharide and schistosoma mansoni egg antigens dc-sign neck domain is a phsensor controlling oligomerization saxs and hydrodynamic studies of extracellular domain protein-glycan interactions in the control of innate and adaptive immune responses this work was supported by research grants from the consejo nacional de investigaciones científicas y técnicas from argentina (conicet) and agencia nacional de promoción científica y tecnológica (anpcyt-mincyt). key: cord- -t f vr w authors: dowers, kristy l; lappin, michael r title: the pyrexic cat date: - - journal: problem-based feline medicine doi: . /b - - - - . - sha: doc_id: cord_uid: t f vr w nan • temperature > . ˚c ( . ˚f). • true fever results from a cascade of events, which starts with activation of leukocytes. pyrogenic factors released from the leukocytes increase the thermoregulatory set point in the hypothalamus. signs that may be associated with fever include: • elevated body temperature. • reluctance to move. • anorexia. • depression. • hyperpnea. • muscle or joint stiffness/discomfort. • shivering. • inflammation anywhere in the body can result in elevation of core body temperature above . ˚c ( . ˚f). • the most common etiology for fever in the cat is percutaneous cellulitis or abscess. viral diseases such as fiv, felv and fip are important diseases to consider. conjunctivitis is the predominant sign and is often initially unilateral and becomes bilateral. ocular discharge is serous initially then mucopurulent, but is usually mild. fever, anorexia and lethargy may occur. true fever must be differentiated from hyperthermia, which can be caused by increased muscle activity, increased environmental temperature and stress. true fever results from activation of leukocytes that release factors (pyrogens) such as interleukin- and tumor necrosis factor. • these factors cross the blood-brain barrier and increase the thermoregulatory set point in the hypothalamus. • leukocytes are activated by a multitude of infectious agents, neoplasia, tissue necrosis and immune-mediated diseases. fever is defined as systemic elevation of core body temperature above . ˚c ( . ˚f). the most accurate measurement of core body temperature is obtained rectally. aural temperature is approximately − . ˚c ( . ˚f) lower than the rectal temperature. fever is a general clinical sign that can be associated with many different diseases. the most common disease causing fever in the cat is percutaneous cellulites or abscess. many viral and bacterial diseases cause fever because leukocytes are recruited and activated as part of the general immune response. organ inflammation, such as pancreatitis, cholangiohepatitis and myocarditis, can be associated with an elevated temperature even when an infectious agent is not present. classical signs • fever. • anorexia (partial or complete). • reluctance to move, lethargy and depression. • pain, heat or swelling at site of abscess or cellulitis. cellulitis usually precedes an abscess, and if treated appropriately, the abscess may not even form. cellulitis may be the only evidence of a previous abscess. an abscess may rupture spontaneously, and the owner may notice foul-smelling, purulent discharge on the fur. • some abscesses resolve on their own with or without rupture, if they have been present long enough. regional lymphadenopathy may occur near the affected site. cellulitis spreads rapidly with the development of multiple fistulae and a febrile response. • lameness from septic arthritis is a common sequelae to infection with l forms. joints are affected by the hematogenous route and may be distant to the initial site. lower limbs (tarsus and carpus) are most commonly affected. the joints often ulcerate with a grayish mucinous exudate. infection remains confined to subcutaneous tissues and joints without systemic spread to internal organs. history supports access to outdoors or conflict with other cats indoors. palpation reveals a tender area or fluctuant swelling, with or without evidence of puncture wounds. microscopic examination of a fine-needle aspirate of the abscess reveals a heterogeneous population of bacteria, numerous degenerate neutrophils and intracellular bacteria. a complete blood count will generally show neutrophilia. l forms are not visible in tissue samples even with special stains, nor do they grow on culture. on electromicroscopy, organisms are visible intracellularly within phagocytes. diagnosis is often made by response to tetracyclines in a therapeutic trial (doxycycline mg/kg po, q h). response is rapid and evident within h. non-healing abscesses should have histopathology and culture of tissue. causes include nocardia, fungi, mycobacteria, and tumors. see page , the cat with non-healing wounds. in plague-endemic regions, yersinia pestis (plague) must be considered, if the swelling is predominately in the neck region and the cat's fever is in the region of . ˚c ( ˚f). cautionary measures such as gloves, masks and isolation of the suspect cat should be taken until diagnosis established. (see below for discussion of y. pestis infections). fracture. ligament/tendon injury. neoplasia. clip area looking for evidence of puncture wounds. drainage of the purulent material is the key to treatment. surgical drainage can be done under sedation or general anesthesia with a # blade. make a / - / " incision over the dependent area, or the area most likely to allow for continued drainage. flush the wound thoroughly with sterile saline or a saline/betadine mixture. explore the wound with a sterile cotton swab or hemostats to assess the extent of dead-space and to look for a possible foreign body. leave the wound open to allow drainage of further purulent material. do not suture incision closed, as this will only allow the abscess to reform. a penrose drain may be placed for - days to allow maximum drainage for abscesses that close too early. antibiotic therapy for - days directed against anaerobes: penicillins, cephalosporins, clindamycin and metronidazole are reasonable choices. most abscesses respond extremely well to drainage and amoxicillin at - mg/kg po q hours for days or amoxicillin/clavulonic acid ( . mg/kg po q hours). l-forms and mycoplasma spp. respond to doxycycline or tetracycline within hours, but not other antibiotics. if the wound is not healing well, or the cat has had recurrent abscesses, felv/fiv testing is recommended to rule out an underlying immunodeficiency. further considerations are inappropriate antibiotics (consider culture and sensitivity testing) or the presence of an undetected foreign body (consider surgical exploration of the area) or involvement of underlying bone (osteomyelitis). prognosis is good unless there is an underlying immunodeficiency. restrict the cat to an indoor environment only; although less effective, confine cat indoors at least from dusk to dawn. neuter male intact animals to decrease territorial behavior. felv and fiv serology should be repeated - months following bite wounds. classical signs acute onset of sneezing followed by oculonasal discharge. discharge progresses from serous to mucoid to mucopurulent. severe conjunctivitis with tearing, photophobia and chemosis. hypersalivation may occur as an initial sign before the classic signs of upper respiratory tract appear. punctate corneal ulcers that may coalesce to larger ulcers or perforation. fever of - days duration, anorexia and depression. retching or coughing may occur. cats with anterior uveitis have occasionally have herpesvirus in the aqueous humor. presumptive diagnosis can be made on the basis of history and clinical signs because treatment for feline herpes virus- and calicivirus are similar. ocular ulcerations and chemosis are more suggestive of fhv- . definitive diagnosis is by direct ifa of cells obtained from conjunctival or nasal scrapings, or by viral isolation or polymerase chain reaction assays from oropharyngeal or nasal swabs. sudden onset of serous ocular discharge and mild conjunctivitis; these signs may begin unilaterally, but often progress bilaterally. initial signs are rapidly followed by sneezing, which are not paroxysmal and are less prominent than in herpesvirus. nasal discharge is primarily serous to mucoid and rarely progresses to purulent. oral ulcerations are common, especially on the tongue, and may be associated with drooling or hypersalivation. ulcers may also occur at the mucocutaneous junction, hard palate and nose. fever generally spikes initially after infection prior to onset of signs, and returns with onset of clinical signs. viral pneumonia occurs occasionally with certain strains, and may produce significant mortality. death is often sudden and preceded by laboured respiration. a rare variant strain (fcv-ari) reported from the united states, produces a high fever, facial and paw edema ( % of cats), ocular and nasal discharge, conjunctivitis and ulcerative stomatitis ( % of cats), hemorrhage from the nose, git, etc. ( - % of cats), icterus ( % of cats) and rapid death. mortality is high ( - %). presumptive diagnosis can be made on basis of history and clinical signs because treatment for feline herpes virus- and calicivirus are similar. oral ulcerations or clinical signs of pneumonia are more suggestive of calicivirus. definitive diagnosis is by viral isolation or reverse transcriptase polymerase chain reaction assays from swabs taken from the oropharynx, ideally in the first week of illness. demonstration of increasing serum antibody titers to feline calicivirus in paired samples is also useful, whereas measurement of a single titer is not useful because many cats have titers from vaccination. identification of fcv-ari is based on the clinical syndrome, pathology and culture of virus from blood, nasal or ocular discharge, spleen or lungs. clinical signs are often non-specific and include fever, anorexia and weight loss. dyspnea and harsh lung sounds without coughing is common. peripheral and visceral lymphadenopathies are frequently present. pale mucous membranes, icterus, hepatomegaly or splenomegaly may be evident. ocular signs are uncommon, but can occur. gastrointestinal signs are uncommon in cats compared to dogs, and include chronic diarrhea, mesenteric lymphadenopathy and anorexia. osseous lesions produce soft tissue swelling and lameness. diagnosis is by demonstration of the organism in lymph nodes, draining tracts, bone lesions or vitreous humor. the organism has a thin capsule and is intracellular within macrophages. no reliable serologic test available. genetic predisposition appears to play a role. fip is most common in catteries and multi-cat households. there are two clinical forms of fip, effusive or wet form and non-effusive or dry form. both are characterized by a fluctuating fever unresponsive to antibiotics, anorexia, lethargy and weight loss. typical age of onset is months to years, but any age can be affected. the effusive form may have any of the following signs: • abdominal effusion that is non-painful but progressive. the amount of effusion varies from volumes causing abdominal enlargement, to amounts only detectable by abdominocentesis. fluid is straw-colored and highly viscous, like egg white. • pleural effusion resulting in dyspnea occurs in % of cats with the effusive form. pericardial fluid may be evident on ultrasound. usually it not associated with clinical signs, but occasionally can produce cardiac tamponade. • male cats may present with scrotal swelling. the non-effusive form may have any of the following signs: • ocular signs result from pyogranulomatous inflammation of the iris and ciliary body. they include bilateral uveitis, perivascular exudates (cuffing), retinal hemorrhage, retinal detachment. • neurologic signs include cerebral and cerebellarvestibular signs such as seizures, personality changes, nystagmus, head tilt, circling, head tremor and hyperesthesia. • dysfunction of any organ system may result from granuloma formation within the tissue of that organ, e.g., liver, kidney, spleen, intestines, lungs, etc., however, organ failure producing clinical signs only rarely occurs, and most dysfunction is only detected on biochemical tests. • granulomatous masses may be palpable in abdominal viscera especially mesentery, mesenteric lymph nodes and omentum as tender, irregular masses. occasionally vomiting or diarrhea results from extensive lesions on the bowel wall. jaundice may occur with either form of the disease. histopathology of affected tissues provides the only definitive antemortem diagnosis. the classic fip lesion is pyogranulomatous infiltration around venules. the following are typical abnormalities associated with fip. all asterisked items must be present for a high likelihood of fip; if any one parameter is not present, fip is unlikely. a negative coronavirus ("fip") titer suggests fip is not the cause of the fever, although a few cats with the effusive form of the disease are titer negative. lymphopenia (< . × cells/μl).* occurs in many cats with fip, and many cats without fip. except where the classical effusive fluid is present, definitive diagnosis of fip requires organ biopsy and demonstration of classical histopathological lesions. various non-specific abnormalities may be evident on laboratory tests, including increased total white cell count, mild to moderate anemia, and increased concentrations of bilirubin, liver enzymes, bun, creatinine, fibrinogen, globulin and mild proteinuria. csf typically has increased protein (> g/l) and cell counts (> cells/ml) which are predominantly nonlytic neutrophils. ocular signs: toxoplasmosis, fungal agents. neurologic signs: toxoplasmosis, neoplasia (e.g., lymphoma), trauma, congenital abnormalities in young cats. other clinical signs: rule out other diseases associated with the apparent organ dysfunction. lymphocytic, plasmocytic cholangiohepatitis occasionally produces a high protein abdominal fluid similar to that of effusive fip. fip is a fatal disease with no known treatments. the therapies listed below have been used in an attempt to slow progression and/or to improve quality of life. glucocorticoids at immunosuppressive doses (prednisolone mg/kg/day). cyclophosphamide ( - mg/m q - weeks or . mg/kg daily for days each week) or chlorambucil ( mg/m q - weeks). +/− broad-spectrum antibiotics to control secondary bacterial infections while the cat is immunosuppressed. prognosis is poor. the mortality is > %. fecal-oral transmission is most likely; transplacental transmission is rare. fomites, e.g., food bowls and litter trays, may be an important mode of transmission, as some strains of fcov survive in dried secretions for several weeks. a seronegative cat introduced into a household where coronavirus is endemic has a in chance of developing fip; a seropositive cat under the same conditions has a in chance. both young and old animals seem to be most susceptible due to vulnerable immune systems. maternal antibodies that protect kittens wane at approximately - weeks of age. reduce fecal-oral contamination by providing one litterbox for every - cats, cleaning litterboxes daily, and placing litterboxes away from feeding areas. minimize stress, especially crowding in catteries. do not introduce fcov-positive cats into a multi-cat household. wean kittens at weeks and remove from the queen's environment if she is seropositive. an intranasal vaccine is available for use in seronegative cats. however, efficacy has not yet been demonstrated against wild strains. classical signs see main reference on page for details (the anemic cat). onset of illnesses occurs over an extended period of time (months to years), although young kittens can become acutely ill. chronic, opportunistic infections occur that do not respond to appropriate antibiotic therapy and are primarily due to immunosuppression. fever may occur in any age cat but is primarily seen initially in the viremic stage or later in response to neoplastic, inflammatory or immunosuppressive effects. chronic fever occurs in later stages of disease. weight-loss/cachexia. non-regenerative anemia. thrombocytopenia. lymphoma is associated with felv-positive cats, especially thymic and multicentric forms. history and clinical signs may be suggestive. complete blood count showing anemia, thrombocytopenia, leukemias, increased mcv and leukopenia are supportive. bone marrow aspirate may show myeloproliferation and arrested erythroid differentiation. a positive felv antigen test (viral core antigen p ) on whole blood using an ifa (can also be done on bone marrow sample) or an elisa test (also on serum, plasma, saliva, tears). see page for interpretation. polymerase chain reaction is available from some laboratories. • pale mucous membranes. see main reference on page for details (the anemic cat). classical signs are pale mucous membranes and/or icterus primarily from extravascular hemolysis due to complement binding of infected erythrocytes. severe, regenerative hemolytic anemia may ensue. anorexia and depression are typical. fever occurs in % of cats in the acute phase, and may occur intermittently in chronic infections. history and clinical signs are suggestive, especially if an immunosuppressive disorder is present concurrently. diagnosis is via demonstration of the organism on the surface of erythrocytes. use a marginated blood sample for diagnosis, e.g., ear vein. multiple blood smears over a number of days may be required as most of the organisms are removed from circulation by the time clinical signs are apparent. infected cats may be coomb's positive. a polymerase chain reaction test is available in some laboratories for diagnosis. classical signs gastrointestinal signs, primarily abdominal discomfort and small bowel diarrhea, are due most likely to replication of the organism (tachyzoites) in enteroepithelial cells resulting in necrosis. clinical signs in the acute, fatal form of extraintestinal disease are caused primarily by tissue damage from the rapidly dividing tachyzoites. tachyzoites begin to disappear from tissues approximately weeks after infection. the organism may persist in tissues as tissue cysts containing bradyzoites. chronic disease may be a result of delayed hypersensitivity reactions and tissue reaction to antibody-antigen complex deposition. concomitant illness, such as felv, fiv and immunosuppression with glucocorticoids, has been reported in some cases. gastrointestinal disease. • mild, self-limiting small bowel diarrhea may occur in the definitive host (cats), but only after ingestion of tissue cysts, oocysts or sporulated oocysts. • young kittens are more likely to have gastrointestinal signs, although mild clinical disease has been reported in adult cats as well. all newborn kittens experimentally infected developed severe diarrhea - days later. • fatal extraintestinal disease is most likely to occur in transplacentally infected kittens. • kittens may be stillborn or exhibit signs that are severe and rapidly progressive and reflect involvement of the lungs, liver and cns tissues. these signs may also be observed in postnatally infected kittens and include: -a distended abdomen from an enlarged liver and/or ascites. -icterus from hepatitis or cholangiohepatitis. -dyspnea is present in most kittens and cats with signs of acute infection. -neurologic deficits; continuous vocalization; excessive sleeping. -fever, anorexia, depression often accompanies the tissue-specific signs. • cats may have a moderate fever, lethargy and depression that waxes and wanes. • hyperesthesia and stiff painful joints or shifting lameness may be evident, presumably due to an immune-mediated process. • unilateral or bilateral anterior or posterior uveitis may occur with possible sequelae of lens luxation, glaucoma or retinal detachment. • seizures and ataxia may be present if cns tissues are involved. • rarely, a toxoplasma granuloma (tissue cyst) forms in the gastrointestinal tract or pancreas causing chronic vomiting. clinical signs consistent with toxoplasmosis are suggestive, especially when other causes of the signs have been ruled out. igm titers > : and a four-fold increase in igg:igm titers within weeks correlate best with clinical toxoplasmosis. however, some cats do not develop detectable igm titers, and in other cats, positive igm titers can persist for months to years after infection. elevated ocular and csf titers relative to serum titers in cats with ocular or neurologic signs, respectively, are very suggestive. coefficient values > . are highly suspect and > . strongly suggest local production of t. gondii antibodies. response to therapy for toxoplasmosis is a useful indicator of infection. definitive diagnosis requires demonstration of the organism in inflamed tissues by histology, immunohistochemistry or polymerase chain reaction assay. rule out diseases associated with affected organs, e.g., fip for neurologic and ocular signs. clindamycin at - mg/kg orally q hours for weeks is usually effective. • cats should respond within several days of treatment. • if no response is evident after weeks of antibiotic therapy, reconsider the diagnosis. • the chronic form may recur even after successful treatment, as drugs tend to suppress replication rather than kill the parasite. other systemic drugs with potential efficacy include the trimethoprim sulfas combination, doxycycline, minocycline, azithromycin and clarythromycin. cats with ocular lesions should also be treated with corticosteroids, either topically (e.g. topical . % prednisolone acetate drops applied q - h) or systemically to control inflammation and its sequelae (glaucoma, lens luxation). gastrointestinal disease has a good prognosis, although it may lead to inflammatory bowel disease in rare cases. acute extraintestinal disease has a guarded to poor prognosis. chronic extraintestinal disease has a fair to good. • the placenta or milk with tachyzoites. • ingestion of meat infected with tissue bradyzoites, e.g., rodents. • ingestion of sporulated oocysts in food or water. t. gondii has a zoonotic potential. infection of humans can occur via: • ingestion of undercooked meat containing tissue bradyzoites (most common mode of transmission). • ingestion of sporulated oocysts from the environment. • transplacentally, if first-time exposure to the organism occurs during pregnancy. only cats host the sexual replication that results in oocysts in the feces. • oocysts are shed for - weeks. • most seropositive cats do not shed oocysts on repeat exposure. oocysts must sporulate to be infectious: • sporulation occurs - days after environmental exposure, thus handling individual cats rarely results in infection of humans. transplacental transmission occurs in cats and people after primary exposure. discourage cats from going outdoors and hunting behavior. do not feed cats undercooked meat. • cook meat at ˚c ( ˚f) for minutes. • use gloves when gardening or changing the litterbox, and wash hands well. • change litterboxes daily. use litterbox liners or clean with scalding water. • lethargy and anorexia. see main reference on page for details (the cat with depression, anorexia or dehydration). the classical signs are not as well-defined for cats as for dogs for the following reasons: • cats tend to have intermittent bouts of chronic pancreatitis. • diagnostic tests for pancreatitis are not as reliable in cats. • there is poor correlation of biochemical parameters with pancreatitis in the cat. lethargy and anorexia is variable depending on chronicity. vomiting only occurred in % of cases in one study. dehydration occurred in % of cases in the same study. abdominal pain is quite variable. fever is variably present, and generally mild. in severe acute pancreatitis it may progress to hypothermia, which is a poor prognostic sign. diagnosis is unreliable based on a biochemistry panel. lipase may be increased or normal in pancreatitis. • hyperbilirubinemia and elevated liver enzymes may be present. • hypocalcemia occurs in % (total serum calcium) or % (plasma ionized calcium concentration) of cats due to soponification of fat. cats with a plasma ionized calcium concentration < . mmol/l (< . mg/dl) have a grave prognosis ( % mortality) and aggressive medical treatment is indicated. pancreatic lipase immunoreactivity is probably a more sensitive diagnostic tool for confirming pancreatitis in cats than measurement of plasma lipase concentration or trypsin like immunoreactivity. a feline-specific assay must be used. abdominal ultrasound to visualize an enlarged pancreas or heterogeneous echogenicity in the area of the pancreas is considered by many to be most sensitive. demonstration of higher lipase levels in abdominal fluid compared to those of the serum is suggestive. diagnostic peritoneal lavage may be necessary to obtain a fluid sample. clinical signs may be acute, chronic or intermittent. typically, there is anorexia and depression together with icterus or increased bilirubin and liver enzymes on a biochemistry panel. vomiting and dehydration may be present. fever, especially in the suppurative form occurs in approximately % of the cases. chronic cholangiohepatitis may lead to end-stage liver disease and the cat may present with ascites and hepatic encephalopathy. multiple causes include bacterial, protozoal (t. gondii) and immune-mediated disease. complete blood count may show neutrophilia with a left shift, and mild non-regenerative anemia. biochemistry panel shows hyperbilirubinemia, elevated liver enzyme activities (alp, alt, ggt), +/− elevated serum bile acids. • signs of late-stage liver disease are occasionally present, such as decreased bun, glucose and albumin concentrations. abdominal ultrasound should be performed to evaluate the gall bladder and bile duct for cholelithiasis, bile sludging and cholecystitis. liver aspirates/biopsy allows for differentiation of suppurative from non-suppurative forms of cholangiohepatitis. clinical signs are primarily due to immunosuppression, i.e., chronic recurring infections that do not respond to appropriate therapy. gingivitis, stomatitis and peridontitis are more common findings in fiv infections than in felv, although one study suggests that these signs may be to an effect of age, rather than a consequence of fiv infection. fever is chronic and is related to production of tumor necrosis factor and/or il- in infected cats. weight loss/cachexia are common in the late stages of fiv, as in human hiv infections. diarrhea resembles a panleukopenia-type syndrome that may be due to actual enterocyte infection by the virus or secondary to inflammation. cats are often thin and scruffy with an unkempt haircoat, and may have miliary dermatitis. diagnosis may be suspected based on history and clinical signs, but requires antibody or antigen tests for confirmation. virus isolation and polymerase chain reaction for virus detection is available at some research facilities. cats infected with fiv can be co-infected with felv. chronic nasal discharge can be unilateral or bilateral and is generally serosanguineous. sneezing and stertorous breathing is often present. facial deformity may occur due to invasion of the surrounding bone. chronic low-grade fever may be present. depression, anorexia and weight-loss are signs of disseminated disease. neurologic signs occur via hematogenous spread or invasion into the cns through the cribiform plate but are uncommon. signs include seizures, blindness, depression and ataxia. the skin form typically produces nodules which often ulcerate. diagnosis is by demonstration of narrow-based budding yeast with a very thick capsule from affected tissue or by culture of affected tissue or csf. demonstration of cryptococcus antigen in serum, urine or csf is also diagnostic. occurs in cats of all ages, with and without outdoor access. progressive clinical signs occur over a period of - weeks. according to one study, non-supportive meningoencephalitis may be the most common cause of seizures in cats. systemic signs, which are not present in all cats, include fever, anorexia, lethargy, vomiting, diarrhea and lymphadenopathy. the condition, however, does not appear to be contagious to other cats. csf tap can be very useful to rule out other causes of cns signs, specifically toxoplasmosis and fip; csf analysis reveals a normal or mild protein elevation (typically < g/l) and/or an increased white cell count (< cells/μl). complete blood count findings are non-specific and may include leukopenia or leuko-cytosis, eosinophilia and anemia. • uniphasic or biphasic fever. • depression. • lethargy. • mild generalized lymphadenopathy. • +/-signs of cardiac failure. • viral, e.g., fip has been shown to cause cardiac infection. • trypanosoma cruzi, which causes chagas' disease in humans. • streptococcus and borrelia (lyme's disease) in certain geographic areas. no single agent has been identified, and the disease may be multifactorial. fever is biphasic in % of the cats; if biphasic: • first fever occurs approximately days after exposure, lasts - days and peaks at . - . ˚c ( . - . ˚f). • second fever occurs - weeks after the first fever (at - weeks post-exposure), lasts days and peaks at . - . ˚c ( . - . ˚f). appetite is mildly decreased in some cats, but most continue to eat and drink. some animals exhibit mild generalized lymphadenopathy. irritable disposition and hyperesthesia may occur, and are most likely due to fever and malaise. in a few case reports, cats have died from peracute cardiac failure, but this outcome is not common. myocarditis/diaphragmitis is a diagnosis of exclusion. biochemistry and complete blood counts are unremarkable, except for a mild to moderate increase in ck in less than % of experimentally infected cats. definitive diagnosis can only be made at necropsy. histopathology shows a neutrophilic infiltrate with a foci of myonecrosis in myocardium and diaphragm. any other causes of fever should be ruled out including infectious, inflammatory, immune-mediated, drugs, neoplasia and metabolic. other causes of cardiac failure that should be ruled out include congenital deformities, hypertrophic cardiomyopathy, restrictive cardiomyopathy and dilatative cardiomyopathy. supportive therapy is indicated if dehydration or cardiac disease are present. broad-spectrum antibiotics are indicated if complete blood count supports an infectious cause. fever and depression resolve spontaneously in the majority of cats. prognosis is poor if peracute cardiac failure is present with systemic signs of fever and depression. although an infectious agent is suspected, no single etiologic agent has been identified, making recommendations for prevention difficult. • cardiovascular or respiratory compromise. • external signs of injury. cardiovascular compromise may result in tachycardia, hypovolemia or hypotension. respiratory compromise may produce dyspnea/ tachypnea due to pneumothorax, hemothorax or pyothorax. internal injuries may result in abdominal pain from organ rupture, bone/joint pain or focal swelling. diagnosis is based on clinical signs and history. radiographs of the chest, abdomen and/or limbs may be required to characterize the injury. complete blood count and biochemistry panel is indicated to rule out specific organ injury and primary infection. • alert, febrile cat being treated with antibiotics or antifungal agents. history of treatment with antibiotics or antifungal agents. fever does not correspond to clinical appearance of animal. cats are bright, alert and responsive, despite a temperature in the range of . - ˚c ( - ˚f). onset of fever is idiosyncratic and variable, but the fever is generally present for the duration of the drug treatment. tetracycline is the most common antibiotic cause of drug-induced fever in cats. amphotericin b can cause fever by disrupting cell membranes and releasing pyrogens into circulation. be aware that other drugs (griseofulvin, chloramphenicol and chemotherapeutic drugs) can cause bone marrow suppression leading to a cat with fever, neutropenia and secondary bacterial infection. these cats are obviously sick, whereas the drug-induced fever animals are bright and alert in comparison. • history is of treatment with fever-inducing drugs, especially tetracycline and amphotericin b. • clinical signs are inappropriate, that is, the cat appears bright and alert although febrile. temperature normalizes after drug is discontinued. classical signs • sneezing. • conjunctivitis and ocular discharge. see main reference on page for details (the cat with acute sneezing or nasal discharge). marked conjunctivitis is the predominant sign, which often starts unilaterally, but usually progresses to both eyes. classic triad of upper respiratory infection signs including oculonasal discharge and sneezing. serous ocular discharge accompanied by blepharospasm, chemosis and conjunctival hyperemia are initial signs. discharge becomes mucopurulent over the course of the disease. mild to moderate fever can be seen in the acute phase. pneumonia is rarely associated with this infection. history and clinical signs are highly suggestive. cytology of conjunctival scrapings reveal dark blue inclusion bodies (giemsa stain). immunofluorescent antibody staining or polymerase chain reaction assay to demonstrate the organism in conjunctival scrapings is available from some laboratories. • acute onset of depression. • acute onset of vomiting. rapid onset of depression, anorexia, and vomiting especially in peracute and acute disease. fetid diarrhea (may be hemorrhagic) typically follows - days after initial onset of signs. severe dehydration and electrolyte abnormalities. initial fever followed by hypothermia as the disease progresses. high mortality rate when signs are severe. the disease should be suspected in cats less than one year of age with no history of vaccination and a rapid clinical course. panleukopenia evident on hematology. parvoviral antigen can be detected in feces using the canine parvoviral antigen tests or electron microscopy. histopathologic changes include denuded intestinal crypts and blunted villi (often a post-mortem diagnosis). classical signs francisella tularensis is a gram-negative coccobacillus. clinical signs are associated with gram-negative endotoxins and bacteremia. there are two main strains of the organism, both of which have been isolated from cats. • associated with tick-rabbit cycle. • found only in north america. • highly virulent for laboratory rabbits. • associated with more severe disease in humans. • associated with a more complex cycle involving rodents, ticks, mosquitoes, mud and water. • found throughout the northern hemisphere. • avirulent for laboratory rabbits. history of contact with rabbits, especially if the cat is a hunter. any age of cat can be infected, but younger cats are more susceptible to developing septicemia. the spectrum of illness varies from severely affected to asymptomatic. fever is generally > ˚c ( ˚f). marked depression, anorexia and lethargy, with or without vomiting are typical. on physical examination, peripheral lymphadenopathy, icterus and palpable splenomegaly and hepatomegaly are reported. oral, lingual or pharyngeal ulcers may be present. clinical signs together with a history of exposure to wild rabbits is highly suggestive. hematologic and serum biochemical abnormalities may include panleukopenia, with severe toxic changes in neutrophils, high band neutrophil count, thrombocyto-penia and hyperbilirubinemia. definitive diagnosis is via identification of the bacterial agent by ifa or bacterial culture, but should only be performed in a qualified laboratory. • samples can be obtained from affected lymph nodes, bone marrow, urine or blood. serum antibody titers > : or a four-fold increase in serum antibodies in samples collected during acute and convalescent phases ( - days) are considered diagnostic. fip, fiv, panleukopenia. toxoplasmosis. multicentric lymphoma. antimicrobial efficacy studies have not been done in the cat, therefore therapy is derived from case reports and/or human therapy regimens. enrofloxacin ( mg/kg q hours iv or po). tetracycline and chloramphenicol may be effective, but because they are bacteriostatic for f. tularensis, relapses can occur. in humans, the drugs of choice are streptomycin and gentamycin. prognosis is poor to fair as mortality rate varies across case reports. f. tularensis has a serious zoonotic potential if there is contact with infected animal tissue. bites from infected ticks, deer flies or mosquitoes are the most common method of transmission. infection can also occur via ingestion of infected meat. • this is the most common method of transmission to humans in cat-associated cases. • the infected cat may have no obvious signs of illness, but have a history of hunting wild animals, especially rabbits. inhalation of aerosolized organisms may also transmit the disease. care should be taken by veterinary and laboratory personnel handling suspected animals or samples being prepared for ifa or culture. discourage hunting behavior in cats. ectoparasite control, especially tick control. onset of illness occurs - hours after exposure to the organism. transmission to cats is either via ingestion of infected rodents or a fleabite from infected fleas. rapid multiplication of organism causes tissue damage and necrosis. the host immune response contributes to pathology. three forms of the plague exist: bubonic (local infection), bacteremic/septicemic and pneumonic. bacteremia occurs in many cases, resulting in the septicemic or pneumonic form of plague. endemic regions of the world include the western usa, south america, africa, asia, eastern europe. history of hunting rodents, especially in known endemic areas. current flea infestation is evident. acute onset of fever, anorexia, depression over a period of - days. the clinical course may last - days. submandibular or cervical swelling associated with lymph nodes (can be unilateral or bilateral). the inflamed, swollen lymph node is referred to as a bubo. subcutaneous abscessation may occur and appear similar to a cat bite abscess. in the pneumonic form (~ % of cases), upper and lower respiratory signs may be present, including sneezing, nasal discharge, coughing, dyspnea/tachypnea. initially, microscopic examination of a lymph node aspirate, especially a markedly swollen lymph node (bubo) should reveal a homogeneous population of bipolar-staining coccobacilli. • blood should be examined in cats with the bacteremic/septicemic form. fluorescent antibody testing of sample provides a definitive diagnosis. culture of organism should be performed by a qualified laboratory only. a four-fold rise in antibody titers (taken - days apart) is suggestive of plague. these results must be interpreted carefully, as high titers can persist for up to one year after infection. chest radiographs may reveal patchy, nodular lesions if the pneumonic form is present. be aware that the risk of exposing other staff members to the disease should be weighed against the benefit of the diagnostic test. reactive lymph nodes from a percutaneous abscess or tooth-root abscess. • aspirates of cat bite abscesses contain a mixed bacterial population compared to y. pestis, which is homogeneous. neoplasia, although it is less common in the us for cats with lymphoma to have peripheral lymphadenopathy. respiratory signs may be due to other upper respiratory infections (calicivirus, herpesvirus, chlamydophila) or lower respiratory disease (parenchymal lung disease, pleural disease). other diseases which cause high fever (tularemia, toxoplasmosis, fip, etc.). absolute caution must be practiced in all suspect plague cases. cautionary measures include gloves, mask, isolation of animal and limited exposure to other staff members. doxycycline/tetracycline: ( ) doxycycline at mg/kg q hours po for - days or ( ) tetracycline mg/kg q hours po. • begin treatment immediately after samples for diagnosis have been collected. • doxycycline is preferred as tetracycline has been associated with relapse. consider aminoglycosides or enrofloxacin ( mg/kg im q hours) for the first days to avoid placing hands into the cat's mouth (see transmission section below). prognosis for bubonic plague is fair to good. prognosis for the pneumonic form is guarded to fair. prognosis for septicemic form is guarded. persistent fever > ˚c ( ˚f) despite treatment is associated with a poor prognosis. y. pestis has a serious zoonotic potential, and great care should be taken in suspect cats to prevent transmission to humans and other cats. • infected cats are no longer a zoonotic risk after days of antibiotic therapy. infection can also occur via inhalation of aerosolized organism, either from aspirates of infected tissue or nasal discharge/sneezing of cats with pneumonic form. discourage hunting behavior especially during the peak flea season (april to october). provide effective flea control to prevent flea bites. • anorexia and weight loss. see main reference on page for details (the anemic cat). this disease is uncommonly reported in cats and is difficult to diagnose because of its vague and variable clinical signs. age range of cats with documented disease was - years of age, with no breed or sex predilection reported. infection has a variable effect on appetite, from mild inappetence to anorexia and mild to moderate weight loss. chronic intermittent fever in the moderate range is common. lymphadenopathy was reported in three of cats. hyperesthesia, joint pain or irritable disposition is common. complete blood counts may show a non-regenerative anemia with a leukopenia or a leukocytosis; thrombocytopenia is present in about % of the cats. biochemistry abnormalities are uncommon, except for hyperglobulinemia in about % of documented cases. a complete blood count and biochemistry panel consistent with chronic ehrlichia spp. infection is suggestive. diagnosis is by demonstrating e. canis and/or anaplasma phagocytophilum serum antibody titers or a positive ifa test. demonstration of morulae in mononuclear cells, neutrophils or eosinophils (rare) is diagnostic. pcr assays can be positive. • anorexia, lethargy, weight loss. • ± fever. • signs depend on tumor type and organ system involved. anorexia, lethargy and weight loss. poorly groomed coat. some cats have a fever associated with neoplasia, which is generally a secondary neoplastic syndrome. tumors which destroy the bone marrow and result in neutro-penia are classically associated with fever. fever may occur with other tumors via other mechanisms, including antibody stimulation from tumor antigens, and tissue necrosis which activates leukocytes to release pyrogenic factors. signs are specific to the organ system involved. lymphoma (mediastinal, gi, renal) , mammary adenocarcinoma, squamous cell carcinoma (nasal, oral) and mast cell tumor are the most common tumors in cats. hematology, biochemistry panel, radiology, ultrasound and/or bone marrow aspirates may be necessary to provide evidence that a tumor is present, especially if it involves the hematopoietic system (leukemia) or is located internally (splenic mast cell tumor). identification of the tumor type is via fine-needle aspirates and/or biopsies. organ dysfunction due to infectious or degenerative disease process. felv/fiv or other immunosuppressive illness. benign masses (granulomas, abscesses, reactive lymph nodes, benign tumors). treatment involves surgical excision of identifiable masses +/− regional lymph nodes, especially for mammary adenocarcinoma, nasal squamous cell carcinoma, splenic mast cell tumor, etc. chemotherapy may be effective and needs to be based on tumor type, e.g., cop (cyclophosphamide, vincristine, prednisolone) protocol in lymphoma cases. radiation therapy is used for local disease only, and response to radiation therapy is tumor dependent (e.g., squamous cell carcinoma). • radiation therapy is most effective after surgical debulking of the primary mass. the effectiveness of radiation therapy may be enhanced with concurrent chemotherapy. classical signs acute onset of fever and malaise are initial clinical signs. vomiting, diarrhea and abdominal pain may occur, however, approximately % do not have gastrointestinal signs. dehydration. shock may occur if septicemia/bacteremia develops. mortality rate approaches % and may be higher if the cat is concurrently immunosuppressed. typically, the cat is an outdoor cat with a history of hunting behavior, especially of birds. complete blood count and biochemistry panel supports infectious diarrhea or septicemia, e.g., neutropenia with a left shift, bacterial rods in blood leukocytes if overwhelming sepsis present, hypoglycemia, hypoproteinemia, pre-renal azotemia. blood cultures provide the best definitive diagnosis if positive. three separate samples over a - hour period should be taken during febrile episodes using aseptic techniques. fecal cultures may isolate salmonella organisms, but because many animals are subclinical carriers, positive culture does not prove that the organism is the cause of the clinical signs. • skin lesions. • respiratory signs. • ocular lesions. • fever, anorexia, depression. the geographical distribution includes south-west usa, central america and south america in areas that have sandy soil with low rainfall and high temperatures. soil is the reservoir for infection, and the highest frequency of cases occur when the soil is dry and dusty, and organisms are disseminated in the wind. most humans and animals in endemic areas become infected, but the majority of infections are subclinical or cause only mild, transient clinical signs. cats are more resistant to infection and signs are less common than in dogs. infection is contracted via inhalation, and only a few organisms are required to produce signs, which occur after - weeks. initial infection is confined to the respiratory tract, but dissemination may occur resulting in chronic disease over months or years with signs referable to bones, eyes, central nervous system and abdominal organs. localized infection following a penetrating skin wound appears to be rare. cats appear to be resistant to clinical disease. skin lesions are the most frequent types of infection in cats and were reported in % of cats in one study. • lesions begin as small bumps and progress to abscesses, ulcers or draining tracts. • in cats, underlying bone involvement is uncommon. systemic signs such as fever, anorexia and depression are commonly reported ( % of cats) and can be seen with skin lesions. respiratory signs such as coughing and wheezing are less common in cats and occur in approximately % of cases. musculoskeletal signs such as lameness, with or without painful bone swelling, were reported in % of cats. ocular lesions are seen infrequently and include chorioretinitis and anterior uveitis. ocular or cns signs were reported in % of cats. most cats have clinical signs for less than weeks prior to diagnosis. hyperproteinemia is present in approximately % of cats. definitive diagnosis is by identification of the organism via biopsy of lesions. antibody detection is available using latex agglutination (igm), agid (igm) or elisa (igm or igg). tube precipitin (tp) for igm and complement fixation (cp) for igg were previously thought to be less reliable in cats, but have been subsequently demonstrated to detect feline infections. itraconazole ( mg/kg po if possible, q h or mg/kg q h) is the treatment of choice. treatment is required for - months and must be continued for at least months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at / the dose for - days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective ( . mg/kg in ml dextrose % iv over minutes q h or given subcutaneously) -see page , for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of mg/kg is given or until bun > . mmol/l ( mg/dl). amphotericin has the disadvantage of requiring frequent parenteral or subcutaneous administration and causes significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at - mg/kg iv days per week for a total of - treatments (cumulative dose of - mg). dilute to a concentration of mg/ml in dextrose % and infuse over - hours. if the titer has decreased four-fold and there is a similar improvement in physical and radiographic signs, treatment can be stopped after - months. antibodies may persist for long periods and obtaining a zero titer is not a useful treatment goal. classical signs • fever. • respiratory signs. • ocular signs. • lymphadenopathy. the geographical distribution includes north america, central america and africa. soil is believed to be the reservoir for infection, and living near a lake or river increases the risk of infection in dogs. signs are more common in dogs than in the cats. disseminated disease is primarily contracted via inhalation. respiratory signs include coughing, dyspnea and harsh lung sounds. ocular disease, such as uveitis, glaucoma and retinal detachment, is a frequent finding. fever, anorexia, depression, weight loss and lymphadenopathy are systemic signs associated with disseminated disease. draining skin lesions may occur and are usually a manifestation of systemic disease rather than local disease. neurological signs are associated with cns involvement of the brain or spine and include circling, disorientation, anisocoria, paresis, decreased conscious proprioception, or upper motor neuron signs, hyperesthesia and extensor rigidity. definitive diagnosis is by demonstration of an extracellular, broad-based budding yeast in aspirates or biopsies from lymph nodes, draining tracts, bone lesions or vitreous humor. an antibody detection test is available, but may be negative. itraconazole ( mg/kg po if possible, q h or mg/kg q h) is the treatment of choice. treatment is required for - months and must be continued for at least months after all signs have resolved. • some cats develop anorexia, and less commonly vomiting or diarrhea. stop the drug for a few days until the cat is eating, and then restart at / the dose for - days, before increasing back to the full dose, which is usually then tolerated. amphoteracin b is also effective ( . mg/kg in ml dextrose % iv over minutes q h or given subcutaneously) -see page , for cyptococcosis in the cat with signs of chronic nasal disease. continue amphotericin b therapy until a cumulative dose of mg/kg is given or until bun > . mmol/l ( mg/dl). amphotericin has the disadvantages of requiring frequent parenteral or subcutaneous administration and causing significant nephrotoxicity. • because of its quick onset of action, amphoteracin b in combination with itraconazole is useful in cats with severe pulmonary signs that are rapidly deteriorating. if the cat survives, after a few weeks treatment can be continued with itraconazole alone. • lipid-complexed amphoteracin formulations allow higher dosages with less toxicity, and should be used in cats with severe pulmonary signs, although the cost is higher. dose at - mg/kg iv days per week for a total of - treatments (cumulative dose of - mg). dilute to a concentration of mg/ml in dextrose % and infuse over - hours. classical signs see main reference on page for details (the anemic cat). primarily found in the south-central and southeast united states. the north american bobcat is the natural host. there is usually a history of exposure to ticks in the previous - days (incubation period is - days). the clinical course of disease is approximately week and often ends in death. clinical signs are the result of an overwhelming hemolytic crisis. rapid onset of fever, dyspnea, anorexia, pale mucous membranes, icterus and dark-colored urine are typical. collapse and death occur - days after the fever peak. hypothermia occurs in the terminal stages. there appear to be non-pathogenic strains as well. a complete blood count reveals regenerative anemia, hemoglobinemia and neutrophilia or neutropenia. the biochemistry panel commonly has hyperbilirubinemia. urinalysis may show evidence of hemoglobin and bilirubin. demonstration of the organism in erythrocytes (merozoite stage) is possible only relatively late in the disease, approximately - days before death. parasitemic cats usually have only - % of rbcs affected, and up to % of cats have parasitemias that are very low or undetectable. demonstration of the organism in macrophages from bone marrow, spleen, liver or lymph node aspirates may be possible even when organisms are not evident in blood. serum antibody levels and direct fa test for detection of tissue phase are available through some labs. weight loss in spite of normal to increased appetite. polyuria/polydipsia. behavioral changes which often include hyperactivity and aggression. unkempt, rough hair coat and sometimes overgrown nails. tachycardia accompanied by a "gallop" rhythm and/or a systolic murmur. mild fever which may be intermittent in nature and reflect the increased metabolic rate in this disease. these cats are easily stressed and may present dyspneic and tachycardic with a mildly elevated temperature, usually not greater than . ˚c ( . ˚f). enlarged thyroid glands are often evident on palpation of the neck. diagnosis is based on clinical signs and history and confirmed by demonstrating increased thyroid hormone concentrations (total t , free t ). thyroid glands can be palpated in approximately % of cats with hyperthyroidism, and are unilaterally or bilaterally enlarged. • enlarged thyroid glands may not be palpable if the abnormal thyroid tissue is within the thoracic inlet. complete blood count and a biochemistry panel are required to rule out diseases such as diabetes mellitus, renal disease, etc. a trh stimulation test may be necessary when clinical signs are highly suggestive and total and free t are in the upper region of the reference range for normal cats. thyroid radionuclide uptake and imaging with pertechnetate ( m tc) is also available at some institutions. response to therapy with anti-rickettsial drugs (tetracycline or doxycycline) is highly suggestive. • subclinical or mild fever and occasional ocular signs. bartonella henselae is an intracellular bacterium within erythrocytes. bacteremia is present in many healthy cats in the population, and cats are reservoirs for infection. b. henselae is an important pathogen because of its zoonotic potential in immunocompromised humans. • humans may develop fever, malaise, lymphadenopathy and skin eruptions following cat scratches or bites. • b. henselae causes bacillary angiomatosis, bacillary peliosis and encephalitis in human aids patients. naturally infected cats usually only develop subclinical infection. mild, self-limiting fever lasting - hours has been documented in some experimentally infected cats. anterior uveitis and fever were documented in naturally exposed cats. lymphadenopathy. atypical seizures occur in some cats. antibody titers are prevalent in healthy cats, but there is a poor correlation with blood culture and pcr assay results. intermittent bacteremia may occur for longer than one year following infection, with - % of healthy cats bacteremic for up to months. the organism is present within erythrocytes, therefore, hemolyzing red blood cells increases the sensitivity of the culture. other causes of mild transient fever need to be considered, such as mild cellulitis following a cat fight. other infectious causes of anterior uveitis need to be ruled out, such as toxoplasmosis, fungal diseases, felv, fiv, cuterebra or dirofilaria. antimicrobial efficacy has not been clearly demonstrated. clinical signs of disease have resolved when the cats are administered doxycycline at - mg po q h for days. azithromycin is used in humans and is a safe alternative in cats when administered at mg/kg po q h for days fluoroquinolones also may be effective. while clinical signs resolve, bacteremia is usually only temporarily suppressed. b. henselae has very low pathogenicity in cats. once cleared of infection, cats are resistant to re-infection by innoculation, but are still susceptible if transmitted via blood transfusion. transmission is via arthropod vectors. in endemic areas, cats infested with fleas and/or ear mites are more likely to be seropositive. the organism survives in flea feces for at least days. because of the frequency of bacteremia in healthy cats, blood transfusions are a likely route of infection. primary immune-mediated disease is extremely rare in cats. stimulation from primary infectious disease antigens is the most common cause of immune-mediated disease in cats, and is most often associated with hemobartonella (mycoplasma) and calicivirus. systemic lupus erythematosus is rare in cats. a multitude of signs may occur including fever, weight loss and cutaneous lesions. immune-mediated hemolytic anemia is most commonly associated with hemobartonellosis. signs include anemia, icterus, fever and anorexia. cats with immunosuppressive disorders such as felv may be more susceptible. immune-mediated thrombocytopenia is rarely reported in cats. felv-positive cats, however, may have thrombocytopenia that is thought to be the result of an immune-mediated response. immune-mediated polyarthritis is uncommon in cats, but has been documented in kittens and adult cats with post-calicivirus vaccination. -the pyrexic cat a bacteriologic investigation of subcutaneous abscesses in cats clinical, clinicopathologic, and pathologic features of plague in cats: cases ( - ) bacterial diseases; fungal diseases; and protozoal diseases feline toxoplasmosis: interpretation of diagnostic test results bartonella spp. antibodies and dna in aqueous humor of cats feline toxoplasmosis and the importance of the toxoplasma gondii oocyst feline infectious peritonitis. part i. etiology and diagnosis feline infectious peritonitis. part ii. treatment and prevention consensus statement of ehrlichial disease of small animals from the infectious disease study group of the acvim feline infectious myocarditis/diaphragmitis diagnostic approach and medical treatment of seizure disorders an appraisal of the value of laboratory tests in the diagnosis of feline infectious peritonitis tularemia in two cats key: cord- - o yd h authors: thépaut, michel; luczkowiak, joanna; vivès, corinne; labiod, nuria; bally, isabelle; lasala, fátima; grimoire, yasmina; fenel, daphna; sattin, sara; thielens, nicole; schoehn, guy; bernardi, anna; delgado, rafael; fieschi, franck title: dc/l-sign recognition of spike glycoprotein promotes sars-cov- trans-infection and can be inhibited by a glycomimetic antagonist date: - - journal: biorxiv doi: . / . . . sha: doc_id: cord_uid: o yd h the efficient spread of sars-cov- resulted in a pandemic that is unique in modern history. despite early identification of ace as the receptor for viral spike protein, much remains to be understood about the molecular events behind viral dissemination. we evaluated the contribution of c-type lectin receptors (clrs) of antigen-presenting cells, widely present in air mucosa and lung tissue. dc-sign, l-sign, langerin and mgl bind to diverse glycans of the spike using multiple interaction areas. using pseudovirus and cells derived from monocytes or t-lymphocytes, we demonstrate that while virus capture by the clrs examined does not allow direct cell infection, dc/l-sign, among these receptors, promote virus transfer to permissive ace + cells. a glycomimetic compound designed against dc-sign, enable inhibition of this process. thus, we described a mechanism potentiating viral capture and spreading of infection. early involvement of apcs opens new avenues for understanding and treating the imbalanced innate immune response observed in covid- pathogenesis in the detection of carbohydrate-based pathogen-associated molecular patterns by antigen-presenting cells (apc), including macrophages and dendritic cells, and in the elaboration of the immune response (geijtenbeek and gringhuis, ; takeuchi and akira, ) . many innate immune cells express a wide variety of clrs, which differ between cell types, allowing specific adjustments of the immune response upon target recognition. thus, clrs such as dectin- , mincle, mgl (macrophage galactose lectin), langerin and dc-sign are major players in the recognition of pathogenic fungi, bacteria, parasites and viruses (de jong et al., ; van kooyk and geijtenbeek, ; mnich et al., ; van breedam et al., ) . the interaction of these clrs with their ligands allows dendritic cells (dc) to modulate the immune response towards either activation or tolerance. this is done in particular through antigen presentation in lymphoid organs (primary mission of apcs) but also through the release of cytokines. thus, dcs have a major role in modulating the immune response from the early stages of infection. to fulfill their sentinel function, dcs are localized at and patrol the sites of first contact with a pathogen, such as epithelia and mucous interfaces, including the pulmonary and nasopharyngeal mucosae. similarly, alveolar macrophages are found in the lung alveoli. in this battle for infection, some pathogens have evolved strategies to circumvent the initial role of clrs in activating immunity and even to divert clrs to their benefit for their infection process. many viruses associate with clrs and other host factors at the cell surface to facilitate they transfer towards their specific target receptors that will trigger fusion of viral and host membranes. this kind of viral subversion has been reported for several c-type lectin receptors, including l-sign (also called dc-signr) and especially dc associated dc-sign, which promotes cisand/or trans-infection of several viruses such as hiv, cytomegalovirus, dengue, ebola and zika viruses (alvarez et al., ; carbaugh et al., ; geijtenbeek et al., ; halary et al., ; navarro-sanchez et al., ) . in particular, dc-sign mediates direct hiv infection of dcs (cis-infection) and can also induce trans-infection of t cells, the primary target of the virus (de witte et al., ) , while in the case of dengue and ebola, dc-sign allows direct cis-infection of the receptor-carrying cells (alvarez et al., ; navarro-sanchez et al., ) . even more noteworthy nowadays, dc-sign and l-sign (herein after collectively referred to as dc/l-sign) have also been reported to be involved in the enhancement of sars-cov- infection (jeffers et al., ; marzi et al., ; yang et al., ) . in the context of the current covid- pandemic, attention is now focused on the sars-cov- virus zhou et al., ) .coronaviruses use a homotrimeric glycosylated spike (s) protein protruding from their viral envelope to interact with cell membranes and promote fusion upon proteolytic activation. in the case of sars-cov- , a first cleavage occurs within infected cells, at the level of a furin site (s /s site), generating two functional subunits s and s that remain complexed in a prefusion conformation in newly formed virus. s contains the fusion machinery of the virus, while the surface unit s contains the receptor-binding domain (rbd) and stabilizes s in its pre-fusion conformation. the s protein of both sars-cov- (hoffmann et al., ; letko et al., ; walls et al., ; zhou et al., ) and sars-cov- (li et al., ) use ace (angiotensin-converting enzyme ) as their primary receptor. for sars-cov- spike, interaction of its rbd with ace , as well as a second proteolytic cleavage at a s ' site, trigger further irreversible conformational changes in s , thus engaging the fusion process (hoffmann et al., ) . the sequence of events around the s protein/ace interaction are becoming increasingly clearer, but much remains to be unraveled about additional factors facilitating the infection such as sars-cov- delivery to the ace receptor. indeed, s proteins from both sars-cov- and sars-cov- have identical affinity for ace (walls et al., ) , but this translates to very different transmission rates. we posit that the enhanced transmission rate of sars-cov- relative to sars-cov- (hca lung biological network et al., ) might result from a more efficient viral adhesion through host-cell attachment factors, which may promote efficient infection of ace + cells. this type of mechanism is frequently exploited by viruses using alternatively heparan sulfate, glycolipids or clrs to concentrate and scan cell surface for their receptor. additionally, in the case of sars-cov- , a new paradigm is needed to untangle the complex clinical picture, resulting in a vast range of possible symptoms and in a spectrum of disease severity associated on one hand with active viral replication and cell infection through interaction with ace along the respiratory tract, and, on the other hand, to the development of excessive immune activation, i.e. the so called "cytokine storm", that is related to additional tissue damage and potential fatal outcomes. in this framework, c-type lectin prrs and the apcs displaying them, i.e. dcs and macrophages, can play a role both as viral attachment factors and in immune activation. thus, their role in sars-cov- infection deserves attention and we focused on dc-sign and l-sign because of their involvement in sars-cov- infections (jeffers et al., ; marzi et al., ; yang et al., ) . l-sign is expressed in type ii alveolar cells of human lungs as well as in endothelial cells and was identified as a cellular receptor for sars-cov- s glycoprotein (jeffers et al., ) . dc-sign was also characterized as a sars-cov- s protein receptor (marzi et al., ) able to enhance virus cellular entry by dc transfer to ace + pneumocytes (yang et al., ) . recent thorough glycan and structural analyses comparing both sars-cov- / spike glycoproteins have shown that glycosylation is mostly conserved in the two proteins, both in position and nature of the glycan exposed (watanabe et al., a (watanabe et al., , b wrapp et al., ) , creating a glycan shield which complicates neutralization by antibodies. secondly, elegant molecular dynamic simulations suggested how some of the spike glycans may directly modulate the dynamics of the interaction with ace , stabilizing the up conformation of the rbd domain (casalino et al., ; zhao et al., ) . finally, and yet unexplored, spike glycans may contribute to infectivity by acting as anchor points for dc-sign and l-sign on host cells surfaces. indeed, % of glycans are of the oligomannose-type and could therefore constitutes ligands for clrs and notably for dc-sign and l-sign. this argues also for the potential use of these clrs by sars-cov- , as do sars-cov- . additionally, some mutations modulating sars-cov- virulence have an impact on the glycosylation level of the spike. as an example, the d g mutation, which increases virulence, has been reported as potentially increasing glycosylation at neighboring asparagine (brufsky and lotze, ; jia et al., ; korber et al., ) . a recent proteomic profiling study pointed to dc-sign as a mediator of genetic risk in covid- (katz et al., ) and finally it is of note that dc/l-sign expression is induced by proinflammatory cytokines such as il- , il- , il- and il- , known to be overexpressed in severe sars and covid- cases (lucas et al., ; relloso and puig-kroger) . these observations prompted us to investigate the potential interaction of c-type lectins receptors, notably dc/l-sign with sars-cov- , through glycan recognition of the spike envelope glycoprotein, as well at their potential role in sars-cov- transmission. in order to accurately analyze the interaction properties of the spike protein from sars-cov- with c-type lectin receptors, we expressed and purified recombinant spike protein using an expression system well-characterized in term of its site-specific glycosylation. we used here the same construct that was used ) to obtain the cryoelectron microscopy structure of the structure (wrapp et al., ) and ) for extensive characterization of glycan distribution on the spike surface (watanabe et al., a) . expression was performed as reported, without using kifunensine to avoid blocking glycan processing. the spike protein was purified exploiting its xhis tag, followed by a superose size exclusion chromatography (sec). sec chromatogram deconvolution allowed to select the best fractions ( figure b ). sds-page analysis confirmed protein purity and differences in migration after reduction supported the presence of expected disulfide bridges and thus proper folding ( figure a ). furthermore, sample quality and trimeric assembly were confirmed by d class averages of the spike obtained from negatively stained sample observed under the electron microscope ( figure c ). this construct contains " p" stabilizing mutations at residues and (pallesen et al., ) , a inacivated furin cleavage site at the s /s interface, and a c-terminal sequence optimizing trimerization (wrapp et al., ) . nonetheless, we observed a limited stability over a week time scale at °c. to further improve protein stability and therefore ensure the quality of the following investigations, we optimized the storage buffer. increasing ionic strength of the purification buffer up to mm nacl proved successful, preserving the trimeric state at °c at least for three weeks ( figure d to g). this "high-salt" concentration does not modify the structural properties of the protein as shown by identical elution profile in sec ( figure b ); in addition, negativestain em images are better in "high-salt" conditions ( figure d and f). all protein samples were therefore subsequently produced in mm nacl and stored at - °c. buffer was then modified according to the analysis performed. (a) sds-page analysis ( % acrylamide gel) of µg purified sars-cov- s protein; non-reduced and reduced with mercapto-ethanol, nr and r, respectively. (b) chromatograms of gel filtration profile of sars-cov- s protein using buffer with mm nacl (green line), mm nacl (blue line) and mm nacl (thick red line). manual deconvolution of gel filtration chromatogram at mm nacl: principal peak (thin red line) and contaminants (dashed red line). collected fractions are represented by the grey area. (c) classification of particles of sars-cov- s protein after the first step of purification on histrap hp column, using relion (auto-picking mode). (d) to (g) quality control of sars-cov- s protein performed by negative staining transmission electron microscopy (tem) using uranyl acetate as stain ( % w/v). scale bar is nm. (d) and (e) sample produced in mm nacl buffer, day of production and after days at °c, respectively. (f) and (g) sample produced in mm nacl buffer, day of production and after days at °c, respectively. dc-sign and l-sign have been already described as receptor of sars-cov- and twenty out of the twenty-two sars-cov- s protein n-linked glycosylation sequons are conserved. glycan shielding represent to % of the spike surface considering the head or the stalk of the s ectodomain, respectively (casalino et al., ; sikora et al., ) . one third of n-glycans of sars-cov- spike are of the oligomannose type (watanabe et al., a) . these glycans are common ligands for dc-sign and l-sign, and also for langerin, a clr of langerhans cells, a subset of tissue-resident dcs of the skin, also present in mouth and vaginal mucosae (hussain and lehner, ) . to compare their recognition capabilities, spr interaction experiments were performed with the various clrs with immobilized sars-cov- s proteins. first, a s protein functionalized surface was generated using a standard procedure for covalent amine coupling onto the surface. the functionalization degree of this "non-oriented" surface depends upon the number of solvent exposed lysine residues (figure a ), which may be severely restricted by the glycan shield discussed above. such restricted protein orientation could preclude the accessibility of some specific n-glycan clusters, located close to the linkage site and the sensor surface, thus hampering recognition by the oligomeric clrs tested. in order to overcome these limitations, we devised and generated a so-called "oriented surface" where the s protein is captured via its cterminal streptagii extremities onto a streptactin functionalized surface ( figure b ). in this set-up, no lateral parts of the s protein are attached to, and thus masked by, the sensor surface. moreover, in the "oriented surface" set-up the spike protein is presented as it would be at the surface of the sars-cov- virus, which might better reflect the physiological interaction with host receptors. considering both surface setups for the spike, the "non-oriented" one may favor access to n-glycans of the spike's stalk domain while the "oriented" one may favor access to n-glycans of the head domain. on the c-type lectin receptors side, we tested exclusively recombinant constructs corresponding to the extracellular domains (ecd), containing both their carbohydrate recognition domain (crd) and their oligomerization domain. thus, the specific topological presentation of their crd as well as their oligomeric status is preserved for each of the clr, going from tetramers for dc-sign and l-sign to trimers for mgl and langerin, ensuring interactions with avidity properties as close as possible to the physiological conditions for each clr. sensorgrams of interaction with both types of surface for various clr are presented in figure a and b. dc-sign and l-sign, initially tested on both surfaces, recognized the spike with the same profile, whatever the set-up. thus, the next two clrs were tested only on one type of surface. langerin was found to interact with the s protein in agreement with the presence of oligomannose-type glycans. finally, mgl, a lectin that specifically recognizes glycans bearing terminal gal or galnac residues, also interacted with the s protein ( figure a ). this shows that complex n-glycans may also serve as potential anchors for the sars-cov- s protein to cell surface clrs. while all clrs tested interacted with the spike, the interactions observed are not all equivalent. unfortunately, the complexity of the process involving probably multiple binding sites per oligomeric clr prevented a kinetic fitting using classical kinetic models, which precluded the determination of kinetic rate constants. nevertheless, an apparent equilibrium dissociation constant (kd) could be obtained by steady state fitting for dc-sign, l-sign and mgl. for langerin, despite a longer injection time, a much higher range of concentration would have been required to reach the equilibrium and accurately evaluate an apparent kd. dc/l-sign and mgl showed affinities in the µm range, from around to µm (table ) , depending on the clr and the surface type, while langerin has an affinity of at least one order of magnitude lower. despite the impossibility to evaluate kinetic association and dissociation rate constants (kon and koff), visual inspection of the sensorgrams clearly reveals different behaviors between dc-sign and l-sign independent of the surface set-up. while association and dissociation seem to be very fast for dc-sign, l-sign sensorgrams suggest a much slower association and dissociation rate that compensate each other to provide a kd similar to that of dc-sign. however, while the higher kon value for dc-sign argues for a faster formation of the dc-sign/s protein complex, the lower koff value for l-sign suggests that the l-sign/s-protein complex might be more stable. finally, for dc-sign and l-sign, which have been tested both on "non-oriented" and "oriented" s surface, no obvious differences has been observed in the interaction sensorgrams. this suggests that the interaction is not restricted to a limited glycan cluster, but rather that oligomannose-type glycans are multiple, accessible and distributed over the whole s protein. figure . they are the average from to independent measurements with different s protein preparations. the spr interaction analysis argues for multiple potential binding sites for clrs on the s protein. such initial host adhesion mechanism could be essential for efficient viral capture, viral particles concentration on the cell surface and subsequent enhanced ace targeting and infection. negative stain electron microscopy was used to visualize potential dc-sign/s protein complexes. extemporaneously after a fresh purification of s protein, sec fractions corresponding to the pure trimeric spike were mixed with a dc-sign ecd preparation in a molecular ratio : (meaning trimeric spike for tetrameric dc-sign ecd). in order to enrich the proportion of complex in the sample for em observation, we directly reinjected this mix onto same sec column and recovered fractions in the elution profile corresponding to higher molecular weight, thus potentially corresponding to dc-sign/s protein complex. these fractions were immediately used to prepare and observe negatively stained electron microscopy grid (figure ) . figure showed a great infectivity of all primary cell lines. however, this infection was dc-sign independent, since anti-dc-sign antibodies did not impact the infection level ( figure a ). pseudovirions dc/l-sign are known to enhance viral uptake for direct infection in the process referred to as cis-infection and can also internalize viral particles into cells for storage in non-lysosomal compartments and subsequent transfer to susceptible cells in the process recognized as trans-infection (alvarez et al., ; geijtenbeek et al., ) . to study the potential function of dc/l-sign in sars-cov- trans-infection, mddcs were incubated with vsv/sars-cov- for h and, after extensive washing, they were placed onto susceptible vero e cells, the reference ace + cell line for sars-cov- cell culture (zhou et al., ) . interestingly, dc-sign promoted efficient sars-cov- trans-infection from mddc to vero e ( figure b ). an anti-dc-sign antibody could reduce substantially the infectivity observed ( % inhibition), confirming the role of this clr in the process of sars-cov- trans-infection. the figure c ). polyman (pm , figure a ) is a multivalent glycomimetic mannoside tailored for optimal interaction with dc-sign (ordanini et al., ) . it is known to bind dc-sign carbohydrate recognition domain (crd), eliciting a th- type response from human immature monocyte derived dendritic cells (berzi et al., ) . it also inhibits dc-sign mediated hiv infection of cd + t lymphocytes with an ic of nm (ordanini et al., ) . pm was used in spr competition experiments to inhibit dc-sign binding to immobilized spike protein, both in the oriented and non-oriented set-ups ( figure b -c). the lectin ( µm) was co-injected with variable concentrations of pm (from µm to . µm), and the results showed clear dose-dependent inhibition. no significant difference was observed between the oriented and non-oriented surface, which is consistent with the binding data previously discussed ( figure ) . thus, an ic of , ± , µm correlates with the interaction affinity between dc-sign and the spike functionalized surfaces. it suggests, in such competition test were the reporting interaction can be limiting (porkolab et al., ) , that a real higher avidity towards dc-sign can be awaited for pm ( figure c ). trans-infection, respectively, which is consistent with the results described for hiv inhibition and confirming an effective affinity in the nanomolar range for pm (ordanini et al., ) . even if viruses target mainly one specific cellular entry receptor within their infection cycle, their efficiency often largely depends upon additional binding events at the cell surface, which promote access to the so-called primary receptor. although such additional receptors may not promote any fusion step, they can drive viral internalization through endocytic processes or simply by viral adhesion to the host cell, accumulation of viral particles on the cell surface and finally engagement with the primary receptor followed by the fusion event. different types of attachment factors can be found on the host cell surface: either glycans, such as heparan-sulfate, glycolipids or protein n-glycans, often targeted by envelope viral protein with lectin-like properties (dimitrov, ) , or immune lectin-type receptors including clrs and siglecs (chiodo et al., ) . given the importance of the role played by glycan determinants in this recognition event, therefore peculiar attention must be paid to the quality of the s protein sample used. indeed, it has to be ideally as close as possible to the physiological product in terms of glycosylation pattern and distribution. in particular, the expression system considered as well as the local protein environment may have a strong impact on the type of glycan added as well as on their level of maturation. viral envelope proteins display a dense array of glycans resulting from evolutionary pressure to mask immunogenic epitopes at their surfaces. this glycan density coupled to specific structural features of envelope proteins generate steric constraints preventing proper access of glycan processing enzymes to some substrate glycans (behrens and crispin, ) . expressing the whole spike ectodomain or just the single rbd domain may therefore lead to very different n-glycan distribution, especially considering that the rbd contains only n-glycosylations sites, while up to nglycan sites are found over the whole spike protein. for these reasons, we selected the entire ectodomain of s as our model to investigate additional attachment factors for sars-cov- . we expressed the protein using the same construct enabling the spike em structure (wrapp et al., ) and its glycan profiling (watanabe et al., a) , using a hek -derived expression system known to provide glycosylation pattern similar to epithelial tissues. similarly, we expressed the entire ectodomain for the clrs as well, avoiding fc-crd constructs, in order to preserve their specific oligomeric assembly and therefore their avidity properties. using spr we showed that all the c-type lectin receptors tested interact with the spike protein. three of those, dc-sign, l-sign and langerin share the ability to recognize high-mannose oligosaccharides. in particular, l-sign is tightly specific for high-mannose, while dc-sign additionally recognizes fucose based ligands (several lewis type glycans) and langerin binds sulfated sugars as well. mgl is specific for gal and galnac terminated glycans and may bind to complex n-glycans as a function of their level of maturation (valverde et al., ) . analyzing the glycosylation pattern of the spike protein, reported in figure b , all glycosylation site depicted in green or orange are potential ligands for l-sign and langerin, with different level of probability from site to site, while mgl's ligands will be found in magenta sites. dc-sign might potentially recognize all of them. beside all considerations about specificity, the accessibility of n-glycan sites upon spike presentation at the sars-cov- virus surface is also of paramount importance for recognition. dc-sign and l-sign share the same tetrameric organization and they recognize with similar avidity the spike functionalized spr surface, suggesting that they share a primary recognition epitope -i.e. high mannose. the spr experiments described here have been performed sequentially on the same spike surfaces with the different clrs. of these, dc-sign and l-sign have similar organization and molecular weight (feinberg et al., ) , thus the difference in ru level reached by the two lectins at their equilibrium (approx. ru higher for dc-sign) suggests that there is more dc-sign binding and thus more epitopes available for it, implying that high mannose are not the unique glycan epitope used here by dc-sign. this suggest that dc-sign may be able to also bind to some o complex n-glycosylation sites (in magenta in figure b ), possibly presenting a proper fucosylation pattern that generates lewis-type epitopes. these considerations, in addition to the oligomeric state of the clrs examined, lead us to rule out a simple interaction with a single preferential epitope and a : stoichiometry in favor of a more complex picture with multiple and simultaneous binding events, much like the "velcro effect" often recalled when discussing glycan-protein interactions. this is clearly supported by the em characterization of spike/dc-sign complexes ( figure c ) that shows several interactions areas on the spike and can also explain the absence of affinity differences between non-oriented and oriented spikes surfaces in spr. the complexity of the binding event(s) described above does not allow to extract kinetic association and dissociation rates from the sensorgrams. only a global apparent kd could be inferred, giving avidity levels. however, l-sign may have a slightly better affinity (around µm, while values ranging from to µm have been obtained for dc-sign and mgl) and seems to generate more stable complexes. such µm range of affinity, as determined here for soluble forms of clr, will result in surface avidity of several order of magnitude higher at the cell membrane (porkolab et al., attachment point for viral capture (cambi et al., ) . and initial dissemination of a number of viral agents has been described in animal models for measles (mesman et al., (mesman et al., , , japanese encephalitis virus (liang et al., ) and in vivo for hiv- . the founder viruses that initiate hiv infection through mucosa exhibit higher content of high-mannose carbohydrates (go et al., ) , as well as higher binding to dcs dependent on dc-sign expression (parrish et al., ) . in the case of ebola virus, dcs and macrophages have been shown to be the initial targets of infection in macaques (geisbert et al., ; martinez et al., ) and circulating dc-sign + dcs have been shown to be the first cell subset infected upon intranasal ebov inoculation in a murine model (lüdtke et al., ) . in sars-cov- infection it was shown that dc/l-sign can enhance viral infection and dissemination (marzi et al., ; yang et al., ) and even it has been proposed that l-sign could act as an alternative cell receptor to ace (jeffers et al., ) . our work shows that dc/l-sign are important enhancers of infection mediated by the s protein of sars-cov- that greatly facilitate viral transmission to susceptible cells. in vivo, dc-sign is largely expressed in immature dendritic cells in submucosa and tissue resident macrophages, including alveolar macrophages (tailleux et al., ) whereas l-sign is highly expressed in human type ii alveolar cells and lung endothelial cells (jeffers et al., ) . infection (geijtenbeek et al., ) . an obvious increase of infection was observed when sars-cov- pseudovirions were incubated with these primary cells and then placed in contact with susceptible veroe cells ( figure b ). similar results were obtained with the t lymphocyte jurkat cell line. t lymphocytes lack ace expression (hamming et al., ) and both the parental jurkat cell line and jurkats expressing dc/l-sign were not directly infected by sars-cov- pseudovirions ( figure a ). therefore, we did not observe that these clrs can function as alternative receptors to ace in non-permissive cells such as t lymphocytes or hek (supplementary information), as it has been recently suggested by amraie et al. (amraie et al., ) . on the extracellular domains (ecd) of dc-sign (residues: - ), l-sign (residues: - ), langerin (residues: - ) and mgl (residues: - ) were produced and purified as already described (achilli et al., ; chabrol et al., ; maalej et al., ; reina et al., ) while sars-cov- spike protein was expressed and purified as follows. the mammalian expression vectors used for the s ectodomain, derived from a p h vector, was a kind gift from j. mclellan (wrapp et al., ) . this construct possesses, in its c-terminus, an xhis tag followed by streptagii. expi cells grown in expi expression medium were transiently transfected with the s ectodomain vector according to the manufacturer's protocol (thermo fisher scientific). cultures were harvested five days after transfection and the medium was negative-stain grids were prepared using the mica-carbon flotation technique (valentine et al., ) . µl of spike samples from purifications diluted at about . - . mg/ml were adsorbed on the clean side of a carbon film previously evaporated on mica and then stained using % w/v uranyl acetate for s. the sample/carbon ensemble was then fished using an em grid and air-dried. images were acquired under low dose conditions (< e−/Å ) on a tecnai fei electron microscope operated at kv using a gatan orius sc camera (gatan, inc., pleasanton, ca) at , x nominal magnification. to facilitate the visualization of the molecules, a gaussian filter was applied to the images using photoshop, then the gray levels were saturated and the background eliminated. for the d classification, images were processed with relion . (scheres, ) . ctf was estimated with ctffind- . (zhang, two types of surface plasmon resonance (spr) experiments were performed at °c on a biacore t . the first experiments with non-oriented spike surfaces were performed using a cm sensor chip, functionalized at μl/min. the second type of experiments used oriented spike surfaces and were performed using a cm sensor chip functionalized at μl/min. the procedure for oriented functionalization has been described in our recent work (porkolab et al., baby hamster kidney cells (bhk- , - - maw, kerafast, boston, ma) and african green monkey cell line (veroe ) were cultured in dulbecco´s modified eagle medium (dmem) supplemented with % heat-inactivated fetal bovine serum (fbs), μg/ml gentamycin and mm l-glutamine. jurkat, jurkat dc-sign, jurkat l-sign (alvarez et al., ) and jurkat langerin were maintained in rpmi supplemented with % heat-inactivated fbs, μg/ml gentamycin and mm l-glutamine. blood samples were obtained from healthy human donors (hospital for differentiation of m -mdms, cells were incubated at ºc with % co for days and activated with m-csf ( u/ml) (miltenyi biotec) every second day. rvsv-luc pseudotypes were generated following a published protocol ( after h incubation at ºc, cells were washed exhaustively with pbs and then dmem supplemented with % heat-inactivated fbs, μg/ml gentamycin and mm lglutamine were added. pseudotyped particles were collected - h post-inoculation, clarified from cellular debris by centrifugation and stored at - ºc (hoffmann et al., ; letko et al., ; whitt, ) . infectious titers were estimated as tissue culture infectious dose per ml by limiting dilution of rvsv-luc-pseudotypes on vero e cells. luciferase activity was determined by luciferase assay (steady-glo luciferase assay system, promega). cell lines: jurkat, jurkat dc-sign and jurkat l-sign ( x cells) or primary cells: mddcs, m -mdms ( x cells) were challenged with sars-cov- , ebov-gp or vsv-g pseudotyped recombinant viruses (moi: . - ). after h of incubation, cells were washed twice with pbs and lysed for luciferase assay. for trans-infection studies, jurkat dc-sign, jurkat l-sign, jurkat langerin ( x cells) or mddcs ( x cells) were challenged with recombinant sars-cov- , ebov-gp or vsv-g pseudotyped viruses (moi: . - ) and incubated during h at room temperature with rotation. cells were then centrifuged at rpm for minutes and washed twice with pbs supplemented with . % bovine serum albumin (bsa) and mm cacl . jurkat dc-sign and mddcs were then resuspended in rpmi medium and co-cultivated with adherent vero e cells ( . x cells/well) on a -well plate. after h, the supernatant was removed and the monolayer of vero e was washed with pbs three times and lysed for luciferase assay. polyman (pm ) is a known glycomimetic ligand of dc-sign and an antagonist of dc-sign mediated hiv trans-infection (berzi et al., ; ordanini et al., ) . it was synthesized as previously described and tested in spr studies as an inhibitor of dc-sign interaction to the spike protein of sars-cov- , using both the oriented and nonoriented s surface described above. in both cases, a µm solution of dc-sign in a running buffer composed of mm tris ph , mm nacl, mm cacl , . % p surfactant was co-injected with variable concentrations of polyman , from µm to . µm, in the same buffer. ic values were determined from the plot of pm concentration vs % inhibition by fitting four-parameter logistic model as previously described (varga et al., ) . in the infection studies, cells were first incubated with the compound pm for min at room temperature with rotation and then challenged with sars-cov- recombinant viruses (moi: . - ) during h at room temperature with rotation. the concentrations tested for compound pm were and . μm. as a control, inhibition experiment was performed in the presence of anti-dc/l-sign antibody (r&d systems). cells were then washed as described above, resuspended in rpmi medium and co-cultivated with adherent vero e cells ( . x cells/well) on a -well plate. after h, the supernatant was removed and the monolayer of vero e was washed with pbs three times and lysed for luciferase assay. the authors declare no conflict of interest. tetralec, artificial tetrameric lectins: a tool to screen ligand and pathogen interactions c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans cd l/l-sign and cd /dc-sign act as receptors for sars-cov- and are differentially expressed in lung and kidney epithelial and endothelial cells (biorxiv) is innate immunity our best weapon for flattening the curve? structural principles controlling hiv envelope glycosylation c-type lectin receptors in antiviral immunity and viral escape pseudo-mannosylated dc-sign ligands as imbalanced host response to sars-cov- drives development of covid- dc/l-signs of hope in the covid- pandemic microdomains of the c-type lectin dc-sign are portals for virus entry into dendritic cells envelope protein glycosylation mediates zika virus pathogenesis shielding and beyond: the roles of glycans in sars-cov- spike protein (biorxiv) glycosaminoglycans are interactants of langerin: comparison with gp highlights an unexpected calcium-independent binding mode novel ace -independent carbohydrate-binding of sars-cov- spike protein to host lectins and lung microbiota (biorxiv) virus entry: molecular mechanisms and biomedical applications the dc-sign-related lectin lsectin mediates antigen capture and pathogen binding by human myeloid cells structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr signalling through c-type lectin receptors: shaping immune responses dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells pathogenesis of ebola hemorrhagic fever in cynomolgus macaques characterization of glycosylation profiles transmitted/founder envelopes by mass spectrometry analysis of the sars-cov- spike protein glycan shield: implications for immune recognition human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis sars-cov- entry factors are highly expressed in nasal epithelial cells together with innate immune genes sars-cov- cell entry depends on ace and tmprss and is blocked by a clinically proven protease inhibitor clinical features of patients infected with novel coronavirus in wuhan comparative investigation of langerhans' cells and potential receptors for hiv in oral, genitourinary and rectal epithelia cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus analysis of the mutation dynamics of sars-cov- reveals the spread history and emergence of rbd mutant c-type lectin langerin is a beta-glucan receptor on human langerhans cells that recognizes opportunistic and pathogenic fungi proteomic profiling in biracial cohorts implicates dc-sign as a mediator of genetic risk in covid- dc-sign: escape mechanism for pathogens tracking changes in sars-cov- spike: evidence that d g increases infectivity of the covid- virus functional assessment of cell entry and receptor usage for sars-cov- and other lineage b betacoronaviruses angiotensin-converting enzyme is a functional receptor for the sars coronavirus dc-sign binding contributed by an extra n-linked glycosylation on japanese encephalitis virus envelope protein reduces the ability of viral brain invasion longitudinal immunological analyses reveal inflammatory misfiring in severe covid- patients ebola virus infection kinetics in chimeric mice reveal a key role of t cells as barriers for virus dissemination the human macrophage galactose-type lectin, mgl, recognizes the outer core of e. coli lipooligosaccharide. chembiochem eur the role of antigen-presenting cells in filoviral hemorrhagic fever: gaps in current knowledge dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus analysis of the interaction of ebola virus glycoprotein with dc-sign (dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin) and its homologue dc-signr a prominent role for dc-sign+ dendritic cells in initiation and dissemination of measles virus infection in non-human primates measles virus suppresses rig-i-like receptor activation in dendritic cells via dc-sign-mediated inhibition of pp phosphatases human coronavirus nl utilizes heparan sulfate proteoglycans for attachment to target cells c-type lectin receptors in host defense against bacterial pathogens dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cellderived dengue viruses designing nanomolar antagonists of dc-sign-mediated hiv infection: ligand presentation using molecular rods immunogenicity and structures of a rationally designed prefusion mers-cov spike antigen structures of mers-cov spike glycoprotein in complex with sialoside attachment receptors phenotypic properties of transmitted founder hiv- anti-siglec- antibodies block ebola viral uptake and decrease cytoplasmic viral entry development of c-type lectin-oriented surfaces for high avidity glycoconjugates: towards mimicking multivalent interactions on the cell surface docking, synthesis, and nmr studies of mannosyl trisaccharide ligands for dc-sign lectin dc-sign (cd ) expression is il- dependent and is negatively regulated by ifn, tgf-␤, and anti-inflammatory agents dc-sign (cd ) expression is il- dependent and is negatively regulated by ifn, tgf-β, and anti-inflammatory agents relion: implementation of a bayesian approach to cryo-em structure determination map of sars-cov- spike epitopes not shielded by glycans (biorxiv) dc-sign induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis pattern recognition receptors and inflammation structural basis for human coronavirus attachment to sialic acid receptors immunology of covid- : current state of the science regulation of glutamine synthetase. xii. electron microscopy of the enzyme from escherichia coli molecular recognition in c-type lectins: the cases of dc-sign, langerin, mgl, and l-sectin. chembiochem cbic bitter-sweet symphony: glycan-lectin interactions in virus biology a multivalent inhibitor of the dc-sign dependent uptake of hiv- and dengue virus function, and antigenicity of the sars-cov- spike glycoprotein site-specific glycan analysis of the sars-cov- spike vulnerabilities in coronavirus glycan shields despite extensive glycosylation generation of vsv pseudotypes using recombinant Δg-vsv for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines langerin is a natural barrier to hiv- transmission by langerhans cells distinct roles for dc-sign+-dendritic cells and langerhans cells in hiv- transmission cryo-em structure of the -ncov spike in the prefusion conformation distinct cellular interactions of secreted and transmembrane ebola virus glycoproteins covid- : immunopathogenesis and immunotherapeutics ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign gctf: real-time ctf determination and correction virus-receptor interactions of glycosylated sars-cov- spike and human ace receptor (biorxiv) a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord- - u afl authors: balzarini, jan title: targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy date: journal: nat rev microbiol doi: . /nrmicro sha: doc_id: cord_uid: u afl several chronic viral infections (such as hiv and hepatitis c virus) are highly prevalent and are a serious health risk. the adaptation of animal viruses to the human host, as recently exemplified by influenza viruses and the severe acute respiratory syndrome coronavirus, is also a continuous threat. there is a high demand, therefore, for new antiviral lead compounds and novel therapeutic concepts. in this review, an original therapeutic concept for suppressing enveloped viruses is presented that is based on a specific interaction of carbohydrate-binding agents (cbas) with the glycans present on viral-envelope glycoproteins. this approach may also be extended to other pathogens, including parasites, bacteria and fungi. any attempts to develop an efficient vaccine against chronic viruses, such as hiv and human hepatitis c virus (hcv), have so far failed. this is mainly due to the inventive immunological escape mechanisms of these viruses , , as well as a lack of efficient long-term protective vaccines that can be directed against conserved epitopes of viruses that are involved in acute infections (such as the influenza virus) . instead, a broad range of chemotherapeutic agents is available for the treatment of various viral infections, in particular for hiv . however, the appearance of long-term side effects and, in particular, the eventual emergence of viral resistance under drug pressure, often weakens the therapy and makes the drugs useless and even harmful in the long run. the targets of the currently available antiviral agents are essential virus-encoded enzymes, virus-specific structural proteins or cellular proteins (that is, viral receptors) , but the sometimes-abundant presence of glycans on viral-envelope glycoproteins has never been seriously envisaged as a therapeutic target. glycans on the viral envelope often have a crucial role in enabling an efficient transmission of the pathogen and/or entry into its susceptible target cells. moreover, it has been shown that the presence of glycans on the envelope of viruses, such as hiv and hcv, is also of crucial importance for the evasion of the immunological surveillance of the host. agents that interact with the viral-envelope glycans may, therefore, compromise the efficient entry of the virus into its susceptible target cells. such agents do not interfere with the glycosylation enzymes from the cell, but rather act by directly binding to the intact glycans on the viral envelope. perhaps more importantly, such carbohydrate-binding agents (cbas) may force the virus to delete at least part of its glycan shield to escape drug pressure ; this might result in the initiation of an immune response against uncovered immunogenic envelope epitopes. cbas may become the first chemotherapeutics with a dual mechanism of antiviral action: first, through direct antiviral activity, by binding to the glycans of the viral envelope and subsequently blocking virus entry, and second, through indirect (additional) antiviral action resulting from the progressive creation of deletions in the envelope glycan shield, thereby triggering the immune system to act against previously hidden immunogenic epitopes of the viral envelope (fig. ) . in the broader perspective, apart from viruses, other pathogens such as mycobacterium tuberculosis, helicobacter pylori and some parasites may also be susceptible to this novel therapeutic approach. this review will focus on cbas and the molecular mechanism of their antiviral activity (fig. ) . the escape mechanisms of hiv in response to cba pressure and how these escape mechanisms might involve the immune system to further combat the viral infection will also be discussed. the interference of cbas with the dendritic cell (dc)-specific intercellular adhesion molecule (icam- )-grabbing non-integrin (dc-sign)-directed capture and transmission of hiv and other pathogens will also be highlighted, and the unique features of the cba therapeutic concept and its potential pitfalls will be discussed. mannose-binding lectins (mbls) . a superfamily of strictly mannose-specific lectins, all of which consist of subunits with a similar sequence and overall threedimensional structure. glycosylation is a highly diverse co-and post-translational protein-modification reaction that can be broadly divided into two categories; o-linked and n-linked glycosylation. in o-linked glycosylation, which probably occurs in the golgi apparatus, the carbohydrate moiety is covalently linked to the hydroxyl oxygen of serine and threonine, but it can also be bound to tyrosine, or to -hydroxylysine and -hydroxyproline . o-glycosylation has various functions, such as providing ligands for selectins, resistance to proteolysis of stem regions of membrane proteins and creating specific recognition phenomena . it can also help to mask immunogenic epitopes on the protein. o-linked glycosylation usually has n-acetylgalactosamine (galnac) as the binding sugar but can also involve other sugars, such as fucose, glucose and n-acetylglucosamine (glcnac). galactose and/or sialic acid are also often found in o-glycans. the covalently n-linked glycans (fig. ) are added co-translationally to native polypeptides in the endoplasmic reticulum (er) as blocks of fourteen sugars (glc man glcnac ). these glycans are then subject to extensive modification during their transport through the er and the golgi complex before reaching their final destinations inside or outside the cell . in the er and the early secretory pathway, the oligosaccharide repertoire is still small. in the golgi complex, however, the glycans acquire complex and highly diverse structures by terminal glycosylation, which results in a tremendous heterogeneity. such diversity differs between cell types, tissues and species, and helps to further increase microheterogeneity in the presence of an identical genetic polypeptide background. this results in the creation of new functionalities and specificities , . the n-glycans may also have an important role in proper protein folding and degradation , and solubility, by avoiding the precipitation that is caused by lipophylic aminoacid stretches in the nascent polypeptide . they also control proper peptidic oligomerization and the sorting of the peptides, as well as peptide transport and trafficking (by acting as universal 'tags' for specific cellular lectins and modifying enzymes) , , . the presence of a glycan shield on the peptides also enables the efficient protection of the glycoproteins against degradation by proteases. the interactions of carbohydrates with cellular lectins are also of crucial importance for the efficient operation of the innate immune system. examples include the mannose-binding lectin (mbl) , dc-sign , defensins and macrophage mannose receptors . leukocyte interactions with endothelial cells represent a well-characterized example of a cell-adhesion event that depends on glycan-receptor interactions . cell-surface glycoproteins can, therefore, mediate cell adhesion and signalling events, as well as intercellular communication. as well as mammalian cells, many different pathogens, including viruses, bacteria, fungi and parasites, also use glycoproteins extensively for diverse functions, in part similar to eukaryotic cells. however, as the glycans on the pathogen (in particular, viral-derived glycoproteins) are produced by the cellular machinery, they are often recognized as 'self ' by the immune system. therefore, the glycans on pathogen glycoproteins in the viral envelope or bacterial cell wall help to escape recognition by the immune system, and the subsequent destruction or neutralization of the pathogen. classes of carbohydrate-binding agents arbitrarily, two different categories of cbas can be distinguished: lectins, which are proteins that specifically recognize carbohydrate (glycan) structures, and non-peptidic small-size agents that may have a good and often specific affinity for monosaccharide and/or oligosaccharide structures. exposure of cbas to the virus in cell culture has also been shown to force the virus to delete part of the protective glycan shield that is present on its envelope glycoprotein gp . it is assumed that such glycan deletions trigger an enhanced neutralizing antibody response to the previously hidden immunogenic epitopes of gp and possibly also a cellular immune response. parts c and d reproduced with permission from ref. © ( ) university of amsterdam. ccr , chemokine receptor ; lfa- , lymphocyte function-associated antigen . ∼asn-x-ser/thr∼ c syncytium a multinucleated giant cell that is formed following the fusion of infected cells expressing hiv-encoded envelope glycoproteins and uninfected cells expressing the cd co-receptor. the resulting syncytium subsequently undergoes apoptosis. (table ) . the most well-studied cba is undoubtedly cyanovirin-n (cv-n), an -kda protein (composed of amino acids consisting of two sequence repeats) originally purified from extracts of the cyanobacterium nostoc ellipsosporum . the elucidation of cv-n crystal structures revealed the existence of a domain-swapped dimer, with two primary carbohydrate-binding sites and two secondary carbohydrate-binding sites on opposite ends of the dimer [ ] [ ] [ ] . the carbohydrate-recognition sites have a binding geometry of high-mannose glycans, in particular α( , )-linked mannose oligomers , . a monomeric -kda protein isolated from the unicellular freshwater bloom-forming cyanobacterium microcystis viridis nies- strain (microcystis viridis lectin (mvl)) was also shown to be composed of two tandemly repeated homologous domains, with specificity for α( , )and possibly α( , )-mannose oligomers. its smallest target is a man glcnac tetrasaccharide core . scytovirin (svn), a . -kda peptide, with amino acids, has most recently been isolated from the cyanobacterium scytonema varium and was shown to have a pronounced affinity for α( , )-α( , )-mannose trisaccharide units . both cv-n and svn inhibit hiv infection in cell culture, at % effective concentrations of . and . nm, respectively. mvl, however, is less inhibitory against hiv . a cba derived from the sea coral gerardia savaglia (gsa) was one of the first lectins isolated from a primitive eukaryotic organism . this d-mannose-specific cba is a dimer, with each monomer being . kda, and requiring calcium to preserve full carbohydratebinding activity. actinohivin , derived from the actinomycete longisporum alba (a . -kda protein, with amino acids), and griffithsin (grft) , isolated from the red alga griffithsin spp. (a -kda protein, with amino acids), were also recently shown to recognize mannose-type glycans. interestingly, the calcium-independent grft, a dimeric protein with four α( , )-mannose carbohydrate-binding domains (cbds) separated by short linker sequences, has no homology to any other primary amino-acid sequence that has been found so far . gsa showed complete suppression of hiv- infection in the h cell line at a concentration of . µm. at the same concentration, syncytia formation between h and hiv- -persistently infected jurkat cells was blocked . actinohivin inhibits both t-cell and macrophage infection by hiv- at to nm concentrations in cell culture ; grft is exquisitely active against cxc-chemokine receptor (cxcr )-tropic hiv- (x hiv- ) and cc-chemokine receptor (ccr )-tropic hiv- (r hiv- ) isolates, with ec s ranging from . to . nm . cbas that have a broad array of carbohydrate specificities, including mannose, glucose, galactose, fucose, sialic acid, glcnac and galnac oligomers, are prevalent in many plant families. monomer and dimer forms of plant lectins predominate, but trimer, tetramer and even octamer plant lectins exist that lead to quaternary protein complexes that have relatively high molecular weights (for an overview, see refs , ) . the crystal structures of a number of plant lectins in complex with carbohydrate oligomers have been determined, such as the mannose-specific lectin from galanthus nivalis (gna) examples of the structural composition of high-mannose-type n-glycans. a | tri-antennary complex-type n-glycans. b | hybrid-type n-glycans. c | high-mannose-type n-glycans that are abundantly present on the envelope glycoprotein gp of hiv, but are rare on mammalian glycoproteins. besides high-mannose-type n-glycans, the complex-type and hybrid-type n-glycans are also present on gp . asn, asparagine; fuc, fucose; gal, galactose; glcnac, n-acetylglucosamine; man, mannose; sa, sialic acid; ser, serine; thr, threonine; x, any amino acid except proline. cbas have also been isolated from invertebrates, such as cvl from chaetopterus variapedatus or mermaid from laxus oneistus . whereas cvl has β-galactose specificity, mermaid is a calcium-dependent mannose-specific cba. interestingly, mermaid was reported to have a strong structural resemblance to mammalian dc-sign . the antiviral activity of cvl against hiv is in the range of . - . µm ; the anti-hiv activity of mermaid has not yet been reported. mammalians also have several types of cba. mbl is a calcium-dependent multimeric cba that is found in serum , and contains subunits of approximately kda. besides mannose, it also binds glcnac and fucose. mbl is part of the innate immune system and binds pathogens as the initiating step of the lectin pathway in order to opsonize the pathogen , . dc-sign is another example of a vertebrate mannose-specific lectin that is predominantly present on immature dcs . it functions in dc recognition and the uptake of pathogens (such as hiv), leading to antigen presentation to t cells . various other cbas, apart from mbl or dc-sign, are also part of the innate and/or adaptive immune system. mammalian defensins (α, β and cyclic Φ), a family of soluble glycan-binding proteins, are probably the best-studied lectins of our immune system . galectins also have a role in cell-cell recognition and the triggering of intracellular signalling cascades that lead to apoptosis . finally, the monoclonal antibody g is one of the few broadly neutralizing anti-hiv antibodies. it is directed against an epitope on the hiv envelope glycoprotein gp , that lies around the c -v region , . this epitope contains high-mannose-type glycans, which are present at several highly conserved n-glycosylation sites (specifically n , n and n in gp ) , . the predominant interaction sites of g with gp are probably the terminal α( , )mannose oligomers of the high-mannose glycans. it should be noted, however, that g specifically recognizes hiv gp glycans, but does not specifically interact with peptide moieties near the glycan structures on gp (refs - ). the g antibody was found to be inhibitory to hiv- (iii b ) in different cell types at an ec of . to . µg per ml. however, it should be noted that g activity can vary depending on the nature of the hiv- subtype isolates that are evaluated . in the course of screening for new antibiotics that are active against fungi, the actinomycete strain actinomadura hibisca was found to produce pradimicin a (prm-a) . this antibiotic has a unique non-peptidic structure that contains the amino acid d-ala and the carbohydrates d-xylose and , -dideoxy- -methylamino-d-galactose attached to a substituted , -dihydrobenzo[a]naphtacenequinone (fig. a) . prm-a binds to terminal d-mannose pyranoside and calcium to yield a ternary complex that consists of two molecules of prm-a, four molecules of mannose and one calcium atom . benanomicin a (bnm-a) (fig. b) , a closely related antibiotic with mannose specificity that is similar to prm-a, has also been isolated from the actinomycete actinomadura spadix and studied for antifungal activity , . prm-a, bnm-a and semisynthetic analogues of these compounds are the only antibiotics that are formally known to have well-defined carbohydrate-binding properties and for which the antiviral activity in cell culture has been reported (the % effective concentration against hiv- ranks in the lower micromolar range) [ ] [ ] [ ] . several research groups focus on the synthesis and characterization of synthetic cbas that bind specific oligosaccharide structures. binuclear copper (ii) complexes , acyclic pyridine-and pyrimidine-based compounds , and tetrapyrrole (porfyrin) derivatives have all been reported to have carbohydrate-binding properties. these compounds may have potential benefit as both antiviral and diagnostic agents. however, for these small-size cbas, few, if any, antiviral data are available, and their potential for cytotoxicity has not been carefully addressed so far. it would be interesting, therefore, to explore the antiviral properties of such compounds to identify novel synthetic low-molecular-weight cba lead compounds that have chemotherapeutic potential. a broad range of proteins bind high-mannose-type glycans of hiv gp . several binding modes can be distinguished . one group of lectins interact as c-type lectins via a calcium ion. two of the best-known examples of such calcium-dependent carbohydrate-binding lectins of the innate immune system are dc-sign and serum or liver mbls. another group of lectins interacts with single-terminal carbohydrates or have more intimate interactions with multiple sugar rings, without the need of a metal ion. for a third group of lectins, the interactions have not yet been resolved. the carbohydrate specificity of mbl is broad. the mbls recognize d-mannose, glcnac and l-fucose. a common motif among these sugars is defined by figure | low-size non-peptidic carbohydrate-binding antigens (cbas). structural formulae of the calciumdependent mannose-binding pradimicin a (a) and benanomicin a (b) antibiotic cbas that are produced by actinomadura hibiscus and actinomadura spadix, respectively. red represents the d-alanine moiety; black represents the dihydrobenzonaphtacenequinone core; green represents the carbohydrate part of the molecule. another example of a c-type lectin is dc-sign that has been crystallized in complex with man glcnac (ref. ) (protein data bank (pdb) id lk i) (fig. b) . interestingly, the internal α( - )-mannose (man)-linked core carbohydrate binds to the principal calcium site. the equatorial c- and c- hydroxyls coordinate the calcium ion and form hydrogen bonds with the amino acids that coordinate the calcium. uda is an example of a calcium-independent cba. a bound glcnac molecule is sandwiched between the binding sites of the high-affinity cbd of a first uda molecule and the low-affinity cbd of a second uda molecule. the high-affinity cbd is formed by the residues ser , trp , trp and tyr . the low-affinity calcium is shown as a green sphere, whereas the carbohydrate is shown in a yellow stick representation. the calcium coordination is shown in detail, with the hydrogen bonds to important coordinating residues shown as dotted spheres. the gray sphere represents a calcium site of another carbohydrate-recognition domain. c | hydrogen bonding, van der waals interactions and aromatic ring stacking of glcnac , with the glcnac-specific lectin from urtica dioica (uda) (pdb id ehh). two isolectin vi molecules are shown in blue and green. dotted lines are hydrogen-bonding contacts between uda and glcnac . sugar residues are labelled with a, b, and c from the non-reducing end. d | hydrogen-bonding pattern and van der waals contacts of the narcissus psuedonarcissus lectin (npl ) carbohydrate-binding domain complexed to manα cbd is formed by the residues ser , his , trp and tyr . of most importance for sugar recognition are the multiple hydrogen bonds, van der waals interactions and aromatic-ring stacking of glcnac with the two uda molecules (pdb id ehh) (fig. c) . indeed, at least five hydrogen bonds are formed in the carbohydrate-lectin complex, as well as several ring stackings between the individual glcnac entities and the trp , trp , trp and his residues of the uda molecules. the molecular interaction of the narcissus pseudonarcissus lectin with manα - man has also been revealed (pdb id npl) (fig. d) . the narcissus psuedonarcissus lectin (npl ) isolectin contains three cbds in one monomer. each cbd in npl seems to have equal affinity for the particular carbohydrates and are equally occupied. in addition to the three conserved cbds, a unique fourth (low-affinity) carbohydrate-binding site has been observed near the tryptophan cluster. three residues (asn , asp and gln ) create a polar patch in the cbd pocket that restricts the carbohydate ligand to an axial hydroxyl group at c- . the multi-alignment of various monocot mannosebinding lectins revealed a striking sequence identity among narcissus pneudonarcissus agglutinin (npa), narcissus hybrid cultivar agglutinin (nha), galanthus nivalis agglutinin (gna), lycoris radiata agglutinin (lra), lycoris aurea agglutinin (laa) and zephyranthus grandiflora agglutinin (zga), all of which contain three mannose-binding sites represented by the conserved qxdxnxvxy motif. this motif binds mannose through a network of four hydrogen bonds that interconnects the hydroxyls of c , c and c of mannose to the four (qdny) amino-acid residues , - . a valine present within this motif also interacts with c and c of mannose through van der waals interactions. interestingly, as also described for npa, a fourth mannose-binding site has been found in laa near the tryptophane cluster . investigation of the complex molecular interactions of cbas with their carbohydrate ligands should enable the rational design of small-size molecules, including peptidomimetics. it is obvious that such low-molecularweight cbas will, by necessity, have fewer interactions with the individual carbohydrates of the gp glycans, and thus may display a lower affinity towards the carbohydrate oligomers. however, the non-peptidic small-size antibiotics prm-a and bnm-a (fig. ) have shown that their affinity and specificity for mannose oligomers are sufficiently high to enable an efficient antiviral activity. unfortunately, no crystal structures have yet been obtained for prm-a that allow visualization of the molecular interactions of the antibiotic with its target α( , )-mannose glycan. if such information became available, further optimization of such cbas may be possible. numerous studies have shown that cbas can prevent hiv infection of cell cultures. this has been shown for many virus strains (including laboratory hiv- and hiv- strains, members of group o and different hiv- clades) that infect many different target cells (including laboratory cell lines, peripheral-blood mononuclear cells (pbmcs), macrophages and dcs). inhibition of hiv infection by cbas occurs at % effective concentrations that rank between the lower nanomolar and micromolar ranges. the antiviral activity will also depend on the nature of the cba and the virus strains that are investigated , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , , , , . although most studies have been performed with laboratory virus strains that are adapted to growth in immortalized cell lines (such as mt- , cem and molt), several other studies have shown the pronounced activity of cbas against different virus clade isolates in pbmc cultures or against the r -hiv- strain ba-l in primary macrophage cell cultures , , , . the size of the cbas and their carbohydrate specificity seem to have an important role in their eventual potency and antiviral efficacy. in general, whereas most mannose-specific cbas are inhibitory to hiv, uda is the only example of an hiv- -active glcnac-specific lectin, and poor, if any, anti-hiv activity has been reported for lectins with other carbohydrate specificities . interestingly, cbas that are assumed to have a well-defined glycan specificity may still differ considerably in antiviral potency, although this phenomenon is not well understood. it is clear, however, that subtle differences in their three-dimensional conformation, as well as the proper steric availability of the glycans that efficiently interact with cbas and the presence, or lack, of well-defined glycan conformations on the viral envelope, have an important role in the eventual antiviral efficacy of cbas. the formation of syncytial giant cells between cells persistently infected with hiv- and uninfected cells can also be efficiently prevented by cbas . inhibition of giant-cell formation in hiv- -infected and non-infected t-cell co-cultures usually requires cba concentrations that are five to tenfold higher than those needed for inhibition of cell-free virus infection of the target cells. some cbas (such as the non-peptidic prm-a), however, are equally effective in their inhibitory potential for both modes of viral transmission . recently, it was shown that cbas inhibit hiv- infection of dcs and dc-directed hiv- transfer . it has also been demonstrated that cbas have a pronounced inhibitory effect on virus capture by cells that express dc-sign and on the subsequent transmission of the virus to uninfected t cells . dc-sign is a natural c-type lectin that has a pivotal role in innate immune defence (box ; fig. ) . to eliminate the pathogen from the infected host, dc-sign captures pathogens such as hiv through its envelope glycans, and subsequently presents the pathogen or pathogen fragments to appropriate t cells . these observations are potentially important from a microbicide viewpoint, as microbicides aim to prevent virus infection that may occur after the exposure of an individual to the virus through sexual intercourse. indeed, dc-signmediated virus capture and transmission to t cells is believed to be among the first events that occur during the viral infection process through sexual contact , (box ). however, its exact role in hiv transmission remains controversial -boggiano et al. and wang et al. agents that efficiently prevent the first steps of virus propagation may qualify as promising microbicidal agents and have the potential to prevent persistent establishment of infection. the observation that cbas might compete directly with dc-sign to capture hiv deserves further investigation in terms of their impact on the efficiency of virus capture and the subsequent transmission of the virus to t cells. interestingly, it was recently reported that langerin, a c-type lectin that is present on epithelial langerhans cells (lcs), prevents hiv- transmission by lcs by internalization of captured hiv particles and subsequent intracellular degradation . although such a mechanism may be efficient for a first-line inactivation of hiv, cba-exposed hiv strains may decrease the efficiency of lcs to eliminate hiv, but at the same time may compromise the ability of the virus to be efficiently transmitted by dcs. the interactions of several cbas have been extensively investigated, including: the prokaryotic cv-n and actinohivin; a variety of plant lectins, including hippeastrum hybrid agglutinin (hha) and uda; the non-peptidic low-molecular-weight antibiotic prm-a; and the monoclonal antibody g with the hiv-envelope gp and/or several glycan structures. interestingly, deletion of the n-glycans at amino-acid positions , and/or rendered the g monoclonal antibody completely incapable of efficiently neutralizing the virus particle . the asparagines at positions , and were previously shown in crystallographic gp - g complexes to be indispensable for an efficient interaction with the g monoclonal antibody . the interaction of the monoclonal antibody g with the glycans on a well-defined gp epitope has also been revealed in several studies , , . alteration of the glycosylation pattern of hiv gp in the er by inhibiting the glycosidases i and ii prevents proper folding of gp . consequently, the interaction of gp with the er lectin calnexin is inhibited and, as a result, fewer infectious virus particles are released by the drug-treated cells. in addition, virus particles from which the glycans had been stripped became markedly less infectious. interestingly, combining cbas with the mannosidase inhibitor -deoxymannojirimycin (dmj) led to the expression of synergistic antiviral activity . exposure of glycandeleted mutant hiv- strains to dmj results in an enhanced suppression of mutant virus-induced cytopathicity in cell culture . therefore, the presence of an intact glycan shield on gp has also been proven to be indispensable for optimal infectivity and for an efficient interaction of hiv with its target cells. studies determining the direct interaction of cbas with fixed gp molecules have revealed the strong binding affinity of cv-n, hha, prm-a and actinohivin with gp . however, for certain cbas (such as cv-n and hha), a low dissociation rate was observed , . these results indicate that some of the cbas exert a strong and virtually irreversible binding to gp . this is in agreement with cellular experiments, which show that a short pre-incubation of cell-free virus particles with cbas before infection of t cells markedly increases the antiviral activity of the cbas , . the binding of the cba to gp does not prevent the initial virus-cell interaction. indeed, it was found that, in the presence of cbas, the virus could still efficiently bind cd + t cells . however, one of the next steps during the entry process (which is binding to the co-receptor(s) and/or the subsequent exposure of gp to the target-cell membrane) becomes blocked in the presence of the cba. the exact molecular mechanism of the blockade of virus entry and/or fusion has not yet been determined. however, it can be assumed that the attachment of the cba to the glycans of gp hinders or prevents the conformational changes and flexibility of gp that are required to properly interact with the cell-membrane receptor, before or during the fusion process. in a number of cases, it has even been observed that the binding of virus particles to their target cells can be enhanced in the presence of some cbas . this phenomenon may indicate that some sort of crosslinking between the virus particles and the cell-membrane glycoproteins occurs. alternatively, cba-induced conformational changes in gp may allow a more optimal interaction of the cd -binding site on gp with the cellular receptor. it should be mentioned that the cd -binding site on gp does not contain n-glycans, and deletion of n-glycans in the vicinity of the cd -binding site on gp (of simian immunodeficiency virus strain sivmac ) has been reported to increase the binding efficiency of the mutant gp with cd ; it was concluded that most pathogens that bind to dc-sign (dendritic cell (dc)-specific intercellular adhesion molecule (icam- )-grabbing non-integrin) cause long-lasting and chronic infections, and often induce tolerance or immune evasion . dc-sign is a c-type (calcium-dependent) lectin that is predominantly expressed on dcs, but also to some extent on macrophages, activated b cells, lymphoid tissues, skin dermis, placenta and the intestinal and genital mucosa. it functions as a tetramer, and consists of a cytoplasmic domain, a transmembrane domain, an exogenous (hepta) repeat domain that allows multimerization, and a terminal carbohydrate (high-mannose)-recognition domain . dcs that are present at the sites of pathogen entry (for example, at the mucosal barrier underneath the vaginal epithelia) recognize and trap the pathogen. the activated dcs then migrate to draining lymph nodes, where naive t cells are primed to eradicate the pathogen (fig. ) . one of the roles of dc-sign is to grab icam- that is present on t cells to enable close contact and allow antigen presentation to the t cells. dcs can efficiently capture hiv- particles through their c-type lectin (dc-sign) receptors. following dc contact with t cells, virus particles can be observed at the cell-cell junctions, which possibly creates an infectious synapse in which the passage of virions between the two cell types is facilitated. such a synapse depends on dc-sign expression and strong cell-cell adhesion mediated by the icam- -lfa- (lymphocyte function-associated antigen ) interaction. it has been shown that, in the case of hiv, the infectious synapse leads to an efficient transfer of the pathogen to t cells . certain glycans might be particularly important for the shielding of the cd -binding site from antibody recognition . the exposure of hiv to any antiviral drug will eventually result in the appearance of phenotypic resistance, both in cell culture and in hiv-infected individuals. antiviral drug resistance is usually due to aminoacid mutations and/or deletions in the viral target with which the particular drug directly or indirectly interacts. in many cases, one amino-acid change is sufficient to provoke a marked degree of drug resistance, although sometimes several mutations are required before pronounced phenotypic resistance becomes evident. viral fitness or infectivity is often compromised in the presence of such resistance mutations. however, compensatory mutations may also occur. these aminoacid mutations do not contribute to drug resistance, but rather are meant to restore the fitness of the virus. for hiv, exposure to cbas invariably results in the appearance and accumulation of amino-acid mutations, predominantly in the putative n-glycosylation motifs of gp (refs , , , , ( ) ( ) ( ) . either an asparagine, or a serine or threonine, are mutated, leading to the annihilation of the glycosylation site. no glycan deletions were observed, however, in the hiv- glycoprotein gp of such mutant virus strains. the hiv- gp consists of approximately - % high-mannose-type glycans (approximately out of glycans in hiv- /iii b ) , (fig. a) , which are the preferential sites for glycan deletions that occur under cba pressure (fig. b) . indeed, up to % of high-mannose-type glycan sites are affected in hiv- gp under prolonged cba pressure. interestingly, such a phenomenon consistently occurs regardless of the nature of the cba, and has been observed in the presence of the α( , )-man-specific cv-n and prm-a , the α( , )-and/or α( , )-manspecific plant lectins (hha and gna) , , the glcnacspecific plant lectin uda and the n-glycan-recognizing monoclonal antibody g (ref. ). there is a close correlation between the number of glycan deletions in the envelope of a particular hiv strain and the degree of phenotypic drug resistance and, in general, the greater the number of glycan deletions in hiv gp , the greater the degree of cba resistance , , . however, when the first glycosylation-site deletions occur under cba pressure in hiv-infected cell cultures, phenotypic resistance is rarely observed. for example, mutant virus strains have been isolated that contain at least three or more glycosylation-site mutations in gp without the appearance of visible phenotypic resistance to the glcnac-specific uda , . therefore, there seems to be a threshold for the number of glycan deletions below which no significant phenotypic cba resistance is evident. so far, many mutant hiv- strains containing up to nine glycan deletions in gp have been isolated under escalating cba pressure and such mutant virus strains can be up to -fold less sensitive to some cbas. interestingly, several of these mutant virus strains are less infective, resulting in a lower viral fitness compared with the wild-type virus . however, in a few cases, mutant virus strains have been isolated that contain to glycan deletions in gp , yet have an increased infectivity and fitness . it is unclear what structural requirements the mutant gp must fulfill to have an increased infectivity. however, such virus strains have never been observed to emerge when more than five glycans were concomitantly affected in gp . in fact, among more than fifty independent mutant virus isolates that emerged under cba pressure, only three were demonstrated to have an increased infectivity. there is much evidence indicating that the glycan shield of hiv- prevents the immune system from efficiently neutralizing the virus. hiv- strains lacking the highly conserved n-linked glycan at position (designated as in fig. owing to a different numbering of the amino acids) within the v loop of gp are highly sensitive to neutralization. bolmstedt and co-workers also showed that glycosylation at this amino-acid position shields hiv- from neutralizing antibodies. kang and colleagues recently reported that hiv env-encoded proteins, with deleted glycans in the gp domains surrounding the cd binding site, or in the gp variable loop, expose immunogenic epitopes at much higher levels than wild-type virus does, which may provide a tool for novel vaccine immunogens. specific n-linked glycosylation modifications in the envelope v domain of siv or in a siv-hiv hybrid variant have also been shown to evolve in the host and alter recognition by neutralizing antibodies , . studies with sivmac , which is highly resistant to neutralization by polyclonal antisera or monoclonal antibodies, have shown that elimination of n-glycan attachment sites in the envelope gp results in a dramatically increased sensitivity to neutralization by monoclonal antibodies . importantly, removal of specific n-glycans from v and v led to an increase in sensitivity to neutralization by antibodies recognizing epitopes from both within and outside the v -v sequence. indeed, mutations in v not only resulted in an increased antibody recognition to epitopes in v , but also in a redirection of antibody responses to the v loop, which is distant in the linear polypeptide sequence . when rhesus monkeys were infected with mutant siv strains that were lacking in combinations of the two n-glycosylation sites in gp , a marked increase in antibody binding to specific peptides derived from the glycan-deleted regions was observed, which resulted in an increased neutralizing activity. these results convincingly demonstrated that the presence of n-glycans limits the neutralizing antibody response to siv, and helps shield the virus from immune recognition . these results also illustrate that deletion of as few as two glycosylation sites in the viral env gene is sufficient to trigger a significant neutralizing antibody response. blay et al. showed that a significant divergence in the env proteins occurs over time in macaques infected with the siv strain shiv- . p (containing hiv env subtype b in a siv background). importantly, the total number of potential n-glycosylation sites did not increase over time, and there was a remarkable degree of conservation in patterns of change in env glycans. these findings suggest that the configuration of the glycan shield is under considerable constraints, which is in agreement with the findings of poon et al. , who showed that negative (exclusive) interactions occur more often between co-localized glycans, whereas positive (inclusive) interactions are restricted to more distant glycans. these data imply that the adaptive repertoire of alternative configurations in the hiv- glycan shield is limited by functional interactions between the n-glycans . therefore, it seems likely that cba exposure to siv or hiv strains would seriously compromise these constraints, by forcing the aids, acquired immunodeficiency syndrome; dc-sign, dendritic-cell-specific intercellular adhesion molecule -grabbing non-integrin; hcv, human hepatitis c virus; l-sign, liver/lymph node-sign ; sars, severe acute respiratory syndrome. virus to progressively delete envelope glycans and allowing the immune system to become actively involved in inhibiting the virus infection. in conclusion, much data are currently available to show that glycan deletions in the viral envelope uncover immunogenic epitopes that result in an increased neutralization of the mutant virus. pathogen susceptibility to cba therapy it has been shown that dc-sign can recognize and internalize numerous other viruses, bacteria and protozoa in addition to hiv (table ) . in this way, dc-sign can be considered to be a universal pathogen receptor. indeed, the recent identification of the carbohydrate specificity of the sign molecules for high-mannose-and/or fucose-containing glycans has led to the identification of various pathogens that are recognized by these receptors (table ) . dc-sign binds and internalizes dengue virus , human cytomegalovirus , hcv and ebola virus to allow efficient trans infection of the target cells. it is also well documented that hcv closely interacts with both dc-sign and liver/lymph node (l)-sign, a close homologue of dc-sign that is expressed on specialized liver and lymph-node endothelial cells that have antigen-presenting capacity . the capture of hcv by the sign-positive cells, through the highly glycosylated (high-mannose type) envelope glycoprotein e , facilitates hcv transmission to proximal hepatocytes . dc-sign and l-sign were also shown to enhance infection mediated by the marburg virus glycoprotein gp and the s protein of severe acute respiratory syndrome coronavirus (sars-cov) through ph-dependent endocytosis, and might promote virus dissemination . although m. tuberculosis primarily infects macrophages, it also binds to dcs through the interaction of its cell-wall component manlam (mannosylated lipoarabinomannan) with dc-sign , . the binding of manlam to dc-sign on dcs blocks dc maturation and induces the expression of immunosuppressive interleukin- (il- ) . as a result, m. tuberculosis enables the suppression of immune activation signals, which allows immune escape. probiotic bacteria, such as lactobacillus spp., also exert immune suppression through dc-sign by the induction of il- -producing regulatory t cells . it has recently been shown that serotypes and of streptococcus pneumoniae specifically interact with dc-sign through the capsular polysaccharide, but the immunological consequences are unclear . h. pylori and the parasite (protozoa) schistosoma mansoni were shown to bind to dc-sign through non-sialylated lewis antigens that are expressed on lipopolysaccharides of the bacterium and the cell-wall glycolipids of the parasite, respectively , . this results in immune regulation that is to the advantage of the pathogen , . leishmania mexicana has also been shown to express glycoconjugates that are recognized by dc-sign . it is clear that the often-indispensable interactions of various pathogens with dc-sign are required to allow efficient pathogen transmission to its eventual target cells and/or immune escape and successful persistence in the host. it is therefore likely that cbas, by binding to the pathogens' glycoconjugates, directly compete with the lectins of the innate immune system. the cba may prevent efficient capture and transmission of the pathogen and/or suppression of an efficient immune response against the pathogen. therefore, it can be predicted that cbas may have a more general role in abrogating successful pathogen infection and persistence. cbas should, therefore, be put in a broader microbial therapeutic context, and not be explored solely for hiv therapy. unique features of the cba concept it has been unambiguously shown that cbas efficiently inhibit virus entry by inhibiting the fusion of cell-free hiv particles with susceptible cells, and forming syncytia between persistently infected and uninfected cells . cbas also prevent the capture of virus particles by dc-sign, and the subsequent transmission of the virus to t cells . cba treatment of virus-infected cells provokes drug pressure on the virus, resulting in predominant deletions of n-glycans in the hiv gp envelope . such glycan deletions uncover previously hidden immunogenic epitopes on gp , which may give the immune system the opportunity to produce a humoral and/or cellular response (fig. ) . what are the unique features of the cba concept that differentiate this therapeutic approach from those that are currently available (box )? • the direct interaction of carbohydrate-binding agents (cbas) with the glycans of the viral envelope glycoproteins. • cba pressure forces hiv to delete n-glycans in the envelope glycoprotein gp . • multiple cbas bind to one envelope gp target molecule. • a high genetic barrier. • no cross-resistance of cba-resistant mutant virus strains to other antivirals. • altered interaction of mutant (glycan-deleted) viruses with target cells. • the cba-induced glycan deletions compromise the protective role of the intact glycan shield on hiv gp . • the eventual antiviral cba activity may combine a direct drug-mediated virus suppression and an indirect (delayed) induction of a specific antiviral immune response against the mutant gp envelope. • the cba concept may also apply to other chronic enveloped virus infections, such as the human hepatitis c virus. • other pathogens such as dendritic-cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-recognizing bacteria, fungi or parasites may also be susceptible to the cba approach. in contrast to the existing drugs for the treatment of hiv, which interact with specific amino-acid configurations on their target proteins, cbas directly interact with the glycans that are present on the envelope gp of hiv. it is important to realize that cbas do not need to be taken up by the virus-infected cell in order to exert their antiviral activity and do not interfere with the synthesis of the glycans on glycoproteins per se. in this respect, this concept differs entirely from inhibitors of cellular glycosylation, such as dmj and castanospermine, which aim to disturb the glycan formation in viral glycoproteins, but at the same time may also disturb the formation of glycans on cellular glycoproteins. cba pressure progressively forces the virus to delete n-glycans in gp . this mutational pattern is unique and does not consistently occur in the presence of other anti-hiv drugs, nor in any other known antiviral. a relatively high number of n-glycans are present on each hiv- gp molecule (approximately to glycans, depending on the nature of the virus clade and the individual virus strain) . it can be reasonably assumed that multiple individual cbas simultaneously bind to every hiv- gp molecule. by contrast, hivhiv- inhibitors other than cbas stoichiometrically bind to their target -one drug molecule interacts with one target protein molecule. the multiple bindings of cbas to single gp molecules results in the cbas having a high genetic barrier. this means that several mutations (owing to glycan deletions) need to accumulate in gp before significant phenotypic drug resistance becomes evident. with the 'traditional' current drugs, the appearance of a single mutation, or at least two or three mutations in the target protein, is usually sufficient to produce a significant drop in sensitivity of the virus to the particular drug. as cbas selectively target n-glycans on gp , cba-mutated virus strains are not likely to show crossresistance to drugs that act against other targets (such as protease, integrase and reverse transcriptase). the exception to this rule may be drugs that target the hiv- transmembrane gp . indeed, preliminary findings indicate that some mutant virus strains that contain various n-glycan deletions in gp show some diminished sensitivity to the fusion gp inhibitor enfuvirtide (also known as t or fuzeon). it remains to be seen, however, whether this is a consistent behaviour of these mutant virus strains or whether the particular glycan-deleted virus strains have an intrinsically low sensitivity to enfuvirtide that is unrelated to the absence of some of the n-glycans of gp . certain glycans may have an instrumental role in the correct folding of the protein immediately after the native peptide has been formed on the ribosomes of the er . therefore, it can be expected that correct folding can become hampered if glycans are lacking on the viral envelope after amino-acid mutation of the glycosylation motif. such compromised (or altered) gp folding may affect the efficient interaction of the mutated gp envelope with the co-receptor molecules, virus fusion efficacy and the eventual fitness of the mutant virus strains. as the transmission of hiv is believed to be mediated by carbohydrate-recognizing dc-sign-expressing cells (that is, immature dcs) that are present in the vaginal-uterine mucosa-epithelial border , it can be assumed that the mode of interaction of cells with mutated glycan-deficient hiv particles is altered owing to a changed glycan landscape on the gp envelope. the protective role of the glycan shield in hiding immunogenic epitopes on gp from the immune system may get lost, or at least compromised, on deletion of the particular glycans . such glycan deletions may trigger the production of neutralizing antibodies that are specific for those gp peptide epitopes that were previously shielded by the glycans. whether this phenomenon will occur in cba-treated hiv- -infected individuals remains to be determined, but it seems likely. it is also currently unknown how strong and efficient the neutralizing antibody response will be on the mutant virus strains that emerge under cba pressure and whether the virus can use other immunological subversions to circumvent cba drug pressure. it would also be interesting to see whether, and to what extent, a cellular immune response would be triggered under such conditions, and what contribution a provoked cellular immune response might make in the eventual inhibition of the virus infection in cba-treated individuals. none of the existing antiviral chemotherapeutics has been shown to have the potential to act in concert with the immune system to further increase the therapeutic pressure on the mutated virus. therefore, treatment of hiv with cbas may become the first strategy to combine drug-mediated virus suppression and induction of a specific antiviral immunological response . such a phenomenon may result in 'self-vaccination' of the cba-exposed hiv-infected individuals by means of a chemotherapeutic agent. if this principle of a combined concerted action between chemotherapy and the immune response proves valid, it may also be applied to other chronic infections by viruses with a highly glycosylated envelope, such as hcv , . also, more acute virus infections (for example, influenza virus, sars-cov and ebola virus) may be highly sensitive to the inhibitory action of cbas, as has been shown for certain cbas against feline and human coronaviruses , and for cv-n against ebola virus in cell culture. besides viruses, other pathogens, such as the dc-sign-recognizing m. tuberculosis and h. pylori or fungi that contain a glycan-rich cell wall (such as aspergillus spp., cryptococcus spp. and candida spp.), and even parasites, may become ideal candidate microorganisms to explore their susceptibility to the inhibitory action of cbas. therefore, the potential of cbas to selectively target some enveloped viruses may also be extended to other pathogens of an entirely different nature. if the glycans on these pathogens are sufficiently different from those of the host, an acceptable therapeutic window may be achieved. considering cbas in the larger context, beyond that of the therapeutic field of virus infections, may reveal an unprecedented therapeutic potential, and should trigger extensive efforts by both chemists and microbiologists to explore this novel therapeutic avenue in the broadest possible sense. potential pitfalls of the cba concept virtually all known cbas that are inhibitory to hiv infection (with the exception of the low-molecular-weight non-peptidic antibiotic prm-a and bmn-a analogues (fig. ) ) are proteins. such agents are expensive to produce, scale-up and purify. there may also be storage and stability problems, although some plant lectins are remarkably temperature-and ph-stable , . bioavailability is also expected to be low for peptidic cbas and, therefore, their pharmacokinetics and pharmacodynamics could be unfavourable, particularly for chronic therapeutic administration. besides a sometimes pronounced mitogenic and red-blood-cell-agglutinating activity, lectins might also be endowed with inflammatory activity and cellular toxicity . also, lectins such as cv-n have the capacity to stimulate various differentiation markers (such as cd , cd and human leukocyte antigen (hla-dr)) . again, it must be emphasized that, although these properties are unfavourable and undesirable from a therapeutic viewpoint, the number and intensity of biological side effects is highly dependent on the nature of the cba. for example, whereas cv-n was shown to display a broad variety of side effects , other cbas such as gna and hha showed much fewer, if any, side effects . moreover, some of the side effects that were observed for cv-n were shown to be independent of its carbohydrate-binding properties . therefore, the proper selection of cbas that have a high selectivity for viral glycans and minimal cellular side effects must be an achievable goal. given the proteinaceous nature of lectins, it could be assumed that repeated systemic administration of cbas will eventually elicit a specific antibody response. such a reaction by the immune system may hamper and attenuate the activity of the cba against the viral carbohydrates (making them less antivirally active). it may also provoke hyperreactivity of the immune system, which would necessitate premature abrogation of the continued administration of the cba. obviously, lowmolecular-weight non-peptidic cbas will not suffer from this potential drawback. the greatest concern, for both protein and non-peptidic cbas, is the degree of selectivity they may eventually show for the viral glycoproteins -that is, their potential to discriminate between pathogen (non-self) glycoproteins and cellular (self) glycoproteins. however, the hiv envelope gp carries a higher proportion of high-mannosetype glycans than do mammalian glycoproteins . the three-dimensional configuration of the glycans that are displayed on the glycoproteins of the pathogen has been shown to be important, which may help the cba to distinguish between 'non-self ' glycans of the pathogen and 'self ' glycans of the host. high-mannose-type glycans contain terminal α( , )-mannose oligomers that are rare on glycans of mammalian glycoproteins. in fact, mbl and dc-sign can distinguish between pathogen-derived glycoproteins and cellular glycoproteins, enabling a selective elimination of the pathogen through specific interaction with its carbohydrate configuration , . it is also important that the cba can discriminate between the glycans present on commensal bacteria and the glycans that must be targeted on the viral-envelope glycoproteins. it will be a challenging goal, but one, i believe, which is achievable, to discover or design cbas that show a marked degree of discriminating selectivity between pathogen glycans and the glycans of the host, including the glycans of commensal bacteria. although most cbas are proteins (such as prokaryotic and plant-lectin cbas), the demonstration that small-size nonpeptidic cbas (that is, those with a molecular weight of less than - kda) (fig. ) can efficiently suppress viral and fungal infections makes this class of compounds a feasible and realistic tool for pathogen inhibition in the clinical setting. however, much work still has to be done, and more synthetic low-molecular-weight compounds need to be designed or discovered to enable the efficient exploration of this novel functional class of antivirals. this is the only way to enable a careful and rational selection of cbas that have a high specificity and selectivity for the pathogen and few, if any, side effects in the host, especially when included in long-term treatment modalities. several synthetic cba lead compounds are already available , , which should trigger the synthesis of structurally related compounds, by organic and medicinal chemists, to allow extensive structure-activity relationship studies. such investigations could indicate which cbas are likely to be the most potent and selective candidates for further pre-clinical investigations. glycans on the envelope or cell wall of pathogens often seem to have similar functional roles, such as escape from recognition by the immune system or recognition by lectins from the innate immune system, that allow efficient transmission of the pathogen. the concept of interfering with and/or abrogating these protective mechanisms should, therefore, be put into a broader context than solely antiviral therapeutic intervention. many different microorganisms other than viruses (such as certain bacteria, fungi, yeasts and parasites) should be thoroughly investigated for their potential interactions with cbas. however, care should be taken to ensure that those microorganisms that have a pivotal role in maintaining the homeostasis of human functions, such as non-pathogenic lactobacilli in the vaginal environment or commensal bacteria in the intestine, are not negatively affected by the cba. including such bacterial strains in the screening of cbas may allow selection of the highest-possible pathogen-selective and specific cbas in the early stages of drug development. cbas should be considered to be valuable agents in their own right, directly suppressing or preventing pathogen infection in the host (fig. ) . however, as they can force the pathogen to mutate (or delete) its protective glycan shield to escape cba pressure this adds an exciting new dimension to cbas as a novel conceptual class of antimicrobial agents. they may indeed represent the first agents that combine direct chemotherapeutic activity and an indirect, active involvement of the immune system by triggering a humoral response (by producing neutralizing antibodies) and/or a cellular response (t-cell-based immunity). although the triggering of a cellular immune response by cbas is still to be confirmed in vivo, indirect information suggests that an immune response is to be expected after prolonged treatment of the pathogen with cbas. immunology of hepatitis b virus and hepatitis c virus infection progress and obstacles in the development of an aids vaccine viral escape by selection of cytotoxic t-cell-resistant variants in influenza a virus pneumonia antivirals and antiviral strategies the first extensive description of the new concept that cbas may have a dual mechanism of antiviral action database analysis of o-glycosylation sites in proteins intracellular functions of n-linked glycans glycosylation: heterogeneity and the d structure of proteins biological importance of glycosylation role of n-oligosaccharide endoplasmic reticulum processing reactions in glycoprotein folding and degradation hydration of a glycoprotein: relative water affinity of peptide and glycan moieties n-glycan processing in er quality control role of the mannose-binding lectin in innate immunity specificity of dc-sign for mannose-and fucose-containing glycans defensins: natural anti-hiv peptides carbohydrate recognition systems in autoimmunity adhesion molecules in leukocyte endothelial interaction discovery of cyanovirin-n, a novel human immunodeficiency virus-inactivating protein that binds viral surface envelope glycoprotein gp : potential applications to microbicide development solution structure of cyanovirin-n, a potent hiv-inactivating protein crystal structure of cyanovirin-n, a potent hiv-inactivating protein, shows unexpected domain swapping structures of the complexes of a potent anti-hiv protein cyanovirin-n and high mannose oligosaccharides solution structure of a cyanovirin-n:man α - manα complex: structural basis for high-affinity carbohydrate-mediated binding to gp cyanovirin-n defines a new class of antiviral agent targeting n-linked, highmannose glycans in an oligosaccharide-specific manner isolation and characterization of a mannan-binding lectin from the freshwater cyanobacterium (blue-green algae) microcystis viridis crystal structures of the hiv- inhibitory cyanobacterial protein mvl free and bound to man glcnac : structural basis for specificity and highaffinity binding to the core pentasaccharide from n-linked oligomannoside a potent novel anti-hiv protein from the cultured cyanobacterium scytonema varium oligosaccharide and glycoprotein microassays as tools in hiv glycobiology: glycandependent gp /protein interactions structural studies of algal lectins with anti-hiv activity a d-mannose-specific lectin from gerardia savaglia that inhibits nucleocytoplasmic transport of mrna molecular cloning of actinohivin, a novel anti-hiv protein from an actinomycete, and its expression in escherichia coli isolation and characterization of griffithsin, a novel hiv-inactivating protein, from the red alga griffithsia spp the d-mannose-specific lectin from gerardia savaglia blocks binding of human immunodeficiency virus type to h cells and human lymphocytes in vitro actinohivin, a novel anti-hiv protein from an actinomycete that inhibits syncytium formation: isolation, characterization, and biological activities handbook of plant lectins: properties and biomedical applications lectins nd edn the mannose-specific bulb lectin from galanthus nivalis (snowdrop) binds monoand dimannosides at distinct sites. structure analysis of refined complexes at . Å and . Å resolution crystal structures of urtica dioica agglutinin and its complex with triacetylchitotriose - )-and α-( - )-d-mannosespecific plant lectins are markedly inhibitory to human immunodeficiency virus and cytomegalovirus infections in vitro the mannose-specific plant lectins from cymbidium hybrid and epi pactis helleborine and the (n-acetylglucosamine) n -specific plant lectin from urtica dioica are potent and selective inhibitors of human immunodeficiency virus and cytomegalovirus replication in vitro mannose-specific plant lectins from the amaryllidaceae family qualify as efficient microbicides for prevention of human immunodeficiency virus infection carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp . a new therapeutic concept to hit the achilles heel of hiv a β-galactose-specific lectin isolated from the marine worm chaetopterus variopedatus possesses anti-hiv- activity a new c-type lectin similar to the human immunoreceptor dc-sign mediates symbiont acquisition by a marine nematode mannose-binding lectin (mbl) and hiv mannose-binding lectin: do we need it? identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-sign, a dendritic cellspecific hiv- -binding protein that enhances transinfection of t cells targeting diversity the broadly neutralizing antihuman immunodeficiency virus type antibody g recognizes a cluster of α → mannose residues on the outer face of gp human monoclonal antibody g defines a distinctive neutralization epitope on the gp glycoprotein of human immunodeficiency virus type dissection of the carbohydrate specificity of the broadly neutralizing anti-hivhiv- antibody g the mannose-dependent epitope for neutralizing antibody g on human immunodeficiency virus type glycoprotein gp antibody domain exchange is an immunological solution to carbohydrate cluster recognition resistance of hiv- to the broadly hiv- -neutralizing, anti-carbohydrate antibody g pradimicin, a novel class of potent antifungal antibiotics studies on the mode of antifungal action of pradimicin antibiotics. iii. spectrophotometric sequence analysis of the ternary complex formation of bmy- with d-mannopyranoside and calcium new antifungal antibiotics, benanomicins a and b from an actinomycete new antifungal antibiotics, benanomicins a and b inhibit infection of t-cell with human immunodeficiency virus (hiv) and syncytium formation by hiv pradimicin a inhibition of human immunodeficiency virus: attenuation by mannan pradimicin a, a carbohydratebinding nonpeptidic lead compound for treatment of infections with viruses with highly glycosylated envelopes, such as human immunodeficiency virus a sugar discriminating binuclear copper (ii) complex molecular recognition of carbohydrates with artificial receptors: mimicking the binding motifs found in the crystal structures of protein-carbohydrate complexes orientation of bound ligands in mannose-binding proteins. implications for multivalent ligand recognition an extensive and detailed overview of carbohydrate-binding proteins that interact with the hiv structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr insights into carbohydrate recognition by narcissus pseudonarcissus lectin: the crystal structure at Å resolution in complex with α - mannobiose strucure of mannose-specific snowdrop (galanthus nivalis) lectin is representative of a new plant lectin family a novel tetrameric lectin from lycoris aurea with four mannose binding sites per monomer mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type strains with n-glycan deletions in their gp envelopes role of envelope glycoprotein carbohydrate in human immunodeficiency virus (hiv- ) infectivity and virus-induced cell fusion sugar-binding proteins potently inhibit dendritic cell human immunodeficiency virus type (hiv- ) infection and dendritic-cell-directed hiv- transfer inhibition of hiv entry by carbohydratebinding proteins carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign)-directed hivhiv- - dc-sign: escape mechanism for pathogens anti-human immunodeficiency virus (hiv- ) microbicide drug candidates to prevent hiv infection maturation of blood derived dendritic cells enhances hiv- capture and transmission dendritic cell-mediated trans-enhancement of human immunodeficiency virus type infectivity is independent of dc-sign cd co-expression regulates dc-signmediated transmission of human immunodeficiency virus type langerin is a natural barrier to hiv- transmission by langerhans cells antibody domain exchange is an immunological solution to carbohydrate cluster recognition glycobiology at oxford. a personal view the α( , )-mannosidase i inhibitor -deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant hiv- strains containing glycan deletions in gp identification of two n-linked glycosylation sites within the core of the simian immunodeficiency virus glycoprotein whose removal enhances sensitivity to soluble cd profile of resistance of human immunodeficiency virus to mannose-specific plant lectins marked depletion of glycosylation sites in hiv- gp under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis resistance of human immunodeficiency virus type to the high-mannose binding agents cyanovirin n and concanavalin a assignment of intrachain disulphide bonds and characterization of potential glycosylation sites of the type recombinant human immunodeficiency virus envelope glycoprotein (gp ) expressed in chinese hamster ovary cells a general model for the surface glycoproteins of hiv- and other retroviruses enhanced immunogenicity of a human immunodeficiency virus type env dna vaccine by manipulating n-glycosylation signals. effects of elimination of the v glycan modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies specific n-linked and o-linked glycosylation modifications in the envelope vi domain of simian immunodeficiency virus variants that evolve in the host alter recognition by neutralizing antibodies selection for neutralization resistance of the simian human immunodeficiency virus shivsf a variant in vivo by virtue of sequence changes in the extracellular envelope glycoprotein that modify n-linked glycosylation assorted mutations in the envelope gene of simian immunodeficiency virus lead to loss of neutralization resistance against antibodies representing a broad spectrum of specificities removal of n-linked glycosylation sites in the v region of simian immunodeficiency virus gp results in redirection of b-cell responses to v a role for carbohydrates in immune evasion in aids consistent patterns of change during the divergence of human immunodeficiency virus type envelope from that of the inoculated virus in simian/human immunodeficiency virusinfected macaques evolutionary interactions between n-linked glycosylation sites in the hiv- envelope distinct functions of dc-sign and its homologues l-sign (dc-signr) and msignr in pathogen recognition and immune regulation dc-sign (cd ) mediates dengue virus infection of human dendritic cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection c-type lectins l-sign and dc-sign capture and transmit infectious hepatitis c virus pseudotype particles c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus mycobacteria target dc-sign to suppress dendritic cell function dc-sign is the major mycobacterium tuberculosis receptor on human dendritic cells selective probiotic bacteria induce il- -producing regulatory t cells in vitro by modulating dendritic cell function through dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin dc-sign specifically recognizes streptococcus pneumoniae serotypes and references and provide the first identification of the important role of the natural c-type lectin dc-sign in innate pathogen recognition, capture and antigen presentation to the immune system the dendritic cell-specific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x helicobacter pylori modulates the t helper cell /t helper cell balance through phase-variable interaction between lipopolysaccharide and dc-sign dc-sign mediates binding of dendritic cells to authentic pseudo-lewisy glycolipids of schistosoma mansoni cercariae, the first parasitespecific ligand of dc-sign demonstration of the crucial importance of how a continuously evolving glycan shield on the hiv envelope gp escapes immune surveillance in hiv cyanovirin-n inhibits hepatitis c virus entry by binding to envelope protein glycans the inhibition of infection and entry of the human immunodeficiency virus (hivhiv- ) and hepatitis c virus (hcv) closely correlates for carbohydrate-binding agents (cba), but not for polyanions. virology (in the press) plant lectins are potent inhibitors of coronaviruses by interfering with two targets in the viral replication cycle cyanovirin-n binds to the viral surface glycoprotein, gp , and inhibits infectivity of ebola virus safety concerns for the potential use of cyanovirin as a microbicidal anti-hiv agent in abstracts of the th conference on retroviruses and opportunistic infections glycosylation and the immune system dendritic-cell interactions with hiv: infection and viral dissemination carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? dendritic cell-mediated hiv- transmission. thesis structure of an hiv gp envelope glycoprotein in complex with the cd receptor and a neutralizing human antibody hepatitis c virus targets dc-sign and l-sign to escape lysosomal degradation west nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection the role of dc-sign and dc-signr in hiv and ebola virus infection: can potential therapeutics block virus transmission and dissemination? dc-sign is a receptor for human herpes virus on dendritic cells and macrophages measles virus targets dc-sign to enhance dendritic cell infection dc-sign interacts with mycobacterium leprae but sequence variation in this lectin is not associated with leprosy in the pakistani population role of the c-type lectins dc-sign and l-sign in leishmania interaction with host phagocytes dc-sign mediates the binding of aspergillus fumigatus and keratinophylic fungi by human dendritic cells the c-type lectin dc-sign (cd ) is an antigen-uptake receptor for candida albicans on dendritic cells the research of the author has been supported by grants from the rega center of excellence at the katholieke universiteit leuven, the geconcerteerde onderzoeksacties and the european commission. d. schols and s. liekens, rega institute, leuven, belgium, are acknowledged for the critical reading of the manuscript and valuable discussions. i thank c. callebaut and c. biernaux for their dedicated editorial help. the authors declare no competing financial interests. key: cord- -znvikfza authors: caparrós, esther; serrano, diego; puig-kröger, amaya; riol, lorena; lasala, fátima; martinez, iñigo; vidal-vanaclocha, fernando; delgado, rafael; rodríguez-fernández, josé luis; rivas, luis; corbí, angel l.; colmenares, maría title: role of the c-type lectins dc-sign and l-sign in leishmania interaction with host phagocytes date: - - journal: immunobiology doi: . /j.imbio. . . sha: doc_id: cord_uid: znvikfza leishmaniasis is a parasitic disease that courses with cutaneous or visceral clinical manifestations. the amastigote stage of the parasite infects phagocytes and modulates the effector function of the host cells. our group has described that the interaction between leishmania and immature monocyte-derived dendritic cells (dcs) takes place through dendritic cell-specific icam- -grabbing nonintegrin (dc-sign), a c-type lectin that specifically recognizes fungal, viral and bacterial pathogens. the dc-sign-mediated recognition of leishmania amastigotes does not induce dc maturation, and the dc-sign ligand/s on leishmania parasites is/are still unknown. we have also found that the dc-sign-related molecule l-sign, specifically expressed in lymph node and liver sinusoidal endothelial cells, acts as a receptor for l. infantum, the parasite responsible for visceral leishmaniasis, but does not recognize l. pifanoi, which causes the cutaneous form of the disease. therefore, dc-sign and l-sign differ in their ability to interact with leishmania species responsible for either visceral or cutaneous leishmaniasis. a deeper knowledge of the parasite-c-type lectin interaction may be helpful for the design of new dc-based therapeutic vaccines against leishmania infections. vector, while l. infantum causes vl due to parasite dissemination into internal organs. leishmania parasites exist in two developmental stages. the flagellated promastigote is transmitted with the bite of the sand fly (insect vector) to the mammalian host, where it transforms into the amastigote stage. leishmania amastigotes infect mononuclear phagocytes, where their intracellular location allows them to subvert the effector and regulatory functions of these cells (duclos and desjardins, ) . since epidermal langerhans cells and dermal dendritic cells (dcs) contribute to immunosurveillance of the skin (banchereau and steinman, ; mellman and steinman, ) and are located in proximity to the site of parasite delivery, their role in the initiation of leishmania-specific immune responses is an active area of research. to attain a successful infection, leishmania needs to subvert the host immune response from the early steps after its inoculation. in the natural course of infection, these events occur in the dermis, where dcs may act as host cells for leishmania, independently of the pathological outcome of the infection (mcdowell et al., ) . in this regard, langerhans cells within cutaneous lesions are parasitized by leishmania in vivo in both human and experimental murine cl (blank et al., ; moll, ) . studies on the interactions of leishmania with murine or human dc have not yet clearly determined the range of parasite forms that these cells can internalize (amprey et al., ; bennett et al., ; blank et al., ; konecny et al., ; marovich et al., ; moll, ; moll and flohe, ; qi et al., ; sacks and sher, ; udey et al., ; von stebut et al., von stebut et al., , or their influence on parasite survival. although leishmania species might differentially subvert dc effector function (antoine et al., ; brandonisio et al., ; chaussabel et al., ; ghosh and bandyopadhyay, ; jebbari et al., ; konecny, et al., ; ponte-sucre et al., ; scott and hunter, ; von stebut et al., ) , the receptors involved in the leishmania-dc interaction remain largely undefined and could be critical for this process. in contrast, several macrophage receptors have been identified which mediate binding and subsequent uptake of leishmania promastigotes (blackwell, ; russell and talamas-rohana, ) . in this regard, lipophosphoglycan (lpg) and the metalloproteinase gp bind to complement receptor type (cr ), mannose-fucose, and fibronectin receptors on macrophages (blackwell, ; da silva et al., ; guy and belosevic, ; mosser, ; talamas-rohana et al., ; wilson and pearson, ) . however, the receptors implicated in amastigote uptake by dc and macrophages are poorly characterized, mainly due to the fact that isolation or in vitro culture of this intracellular form is difficult. the availability of axenic cultures has now opened the possibility of addressing the identification of leishmania amastigotes receptors on macrophages and dcs (armson et al., ; bates et al., ; debrabant et al., ; doyle et al., ; gupta et al., ; hodgkinson et al., ) . macrophages and dcs express a wide variety of pathogen-associated molecular pattern receptors, including numerous c-type lectin and lectin-like receptors (engering et al., ; figdor et al., ; mcgreal et al., ) . since leishmania spp. display an abundance of mannose-rich glycoconjugates on their surface that are important for parasite virulence (ilgoutz and mcconville, ; garami and ilg, ) , a reasonable hypothesis is that lectin-oligosaccharide interactions are involved in parasite recognition by mononuclear phagocytes. dc-specific icam- -grabbing nonintegrin (dc-sign, cd ) is a type ii transmembrane c-type lectin expressed on dcs and macrophages, and was initially described as involved in cell-cell interactions through its capacity to bind icam- and icam- (geijtenbeek et al., a, b; van kooyk and geijtenbeek, ) . the dc-sign extracellular domain comprises eight -residue tandem repeats and a cterminal carbohydrate-recognition domain (mitchell et al., ) . dc-sign is now known to be a receptor for hiv (geijtenbeek et al., c; pohlmann et al., ) , ebola virus (alvarez et al., ) , schistosoma mansoni , sindbis virus (klimstra et al., ) candida albicans (cambi et al., ) mycobacterium tuberculosis , hepatitis c (wang et al., ) , helicobacter pylori (bergman et al., ) and the fungal pathogen aspergillus fumigatus . most leishmania amastigote-dc studies have been carried out with tissue-derived opsonized parasites, which might be bound via fc and complement receptors, thus precluding the identification of opsonization-independent binding mechanisms. to analyze the participation of the receptor dc-sign in binding and internalization of leishmania, we used axenic amastigotes (armson et al., ; pan and mcmahon-pratt, ) , which are devoid of opsonizing antibodies. we first analyzed the interaction of l. pifanoi (that causes cl) axenic amastigote with k -dc-sign transfectants and demonstrated that they are specifically recognized by dc-sign (colmenares et al., ) . subsequently, l. pifanoi amastigotes were found to bind dc-sign on the surface of immature monocytederived dendritic cells (imddcs), an interaction that was dramatically reduced in the presence of anti-dc-sign blocking antibodies (colmenares et al., ) . this set of results suggested an important role for dc-sign in the early stages of infection of dcs by leishmania. since infection of immature dermal dcs is a common step shared by all leishmania species, we next analyzed the capacity of imddcs to bind other leishmania life cycle forms and species. our results underscored the relevance of the dc-sign-leishmania interaction in both vl (l. infantum) and cl (l. pifanoi), as amastigotes and promastigotes from both species exhibited dc-sign-interaction capacity. since the membrane composition of the parasite changes throughout its life cycle (bahr et al., ; wright and el amin, ) , we tested the ability of dc-sign to bind the three main life cycle forms of the parasite (amastigotes, procyclic promastigotes and metacyclic promastigotes). amastigotes and metacyclic promastigotes showed the strongest dc-sign-dependent interaction with imddc ( fig. ) . moreover, the avidity for dc-sign increased in the transition from procyclic (non-infective) to metacyclic (infective) promastigotes (i.e., procyclic and metacyclic promastigotes, respectively) (fig. ) . hence, the avidity of the different forms of the parasite for dc-sign appears to correlate with their virulence. on the other hand, a much lower ability for dc-sign recognition was observed in leishmania major promastigotes (fig. ) . lpg is one of the most abundant glycoconjugates exposed on the cell surface of promastigotes, but scarcely expressed on amastigotes, and plays a pleiotropic role through the life cycle of leishmania (aebischer et al., ; cunningham, ; kamhawi et al., ; naderer et al., ; sacks et al., ; turco and descoteaux, ; turco et al., ) . this molecule is characterized by a high mannose content, and has been proposed to mediate promastigote interaction with dcs via dc-sign . however, our results indicate that lpg is not an important leishmania ligand for dc-sign because: ( ) lpg is strongly down-regulated in amastigotes (ilg et al., ; ilgoutz and mcconville, ; turco and descoteaux, ) , which exhibit the highest dc-sign-binding ability; ( ) lpg was unable to block leishmania binding to dc-sign for all species and parasite developmental stages assayed; and ( ) the lpg-defective l. donovani promastigotes (r d ) bind to dc-sign-expressing cells. furthermore, since lpg-defective promastigotes bound dc-sign with higher avidity than their wild-type counterparts, lpg might in fact mask other promastigote membrane ligands with affinity for dc-sign . leishmania ability to modulate the dc maturation state: conflicting results have been obtained on the ability of various leishmania species to induce dc maturation (antoine et al., ) . it is currently unknown whether this variability is due to variations in the experimental conditions or truly reflects speciesspecific or strain-specific interactions between leishmania and dc. since leishmania spp. express highly polymorphic cell surface molecules, the dc response might vary according to the leishmania species examined (bennett et al., ; flohe et al., ; henri et al., ; konecny et al., ; mcdowell et al., ; von stebut et al., von stebut et al., , . we have evaluated the ability of l. infantum to alter the maturation state of imddc. unlike lps, l. infantum amastigotes, which bind to imddc via dc-sign, did not affect the cell surface expression of cd , cd or mhc ii, commonly considered as dc maturation parameters. therefore, our results suggest that non-opsonized l. infantum amastigotes are unable to induce imddc maturation, at least during a -hour period ( fig. a) . moreover, lps induced maturation of imddcs infected with l. infantum amastigotes ( fig. a) , indicating that the parasites do not inhibit the capacity of dcs to be matured by other pathogen-derived products. the failure of imddc to mature in response to leishmania capture and entry might represent a parasite strategy to avoid immunosurveillance and to allow their establishment and multiplication before the onset of immune responses. on the other hand, l. infantum amastigotes did not induce ccr -directed imddc migration but immature monocyte-derived dcs were prepared as previously described, left untreated or pre-treated min at c with the blocking anti-dc-sign monoclonal antibody (relloso et al., ) and incubated with cfse-labeled parasites at a : ratio for h at c. afterward, the percentage of cells with bound parasites was quantified by flow cytometry and the contribution of dc-sign (%) was calculated as: À((% mr- treated cells with bound parasites  )/% untreated cell with bound parasites). inhibited the ccr -dependent migration induced upon lps maturation (fig. b) . these data are in agreement with previous results demonstrating that cytokines abundantly produced during leishmania infection (e.g., il- ) down-regulate ccr expression (antoine et al., ) . therefore, leishmania might prevent the establishment of t cell-mediated immunity by interfering with the migratory properties of dcs. l-sign: a receptor implicated in vl: l-sign is a close homologue of dc-sign ( % amino acid sequence identity) that is expressed on human liver and lymph node sinusoidal endothelial cells (soilleux et al., ) . like dc-sign, l-sign recognizes and binds high-mannose glycans, binds to icam- (bashirova et al., ) , and recognizes carbohydrate structures on pathogens such as manlam on m. tuberculosis (koppel et al., ) and high-mannose moieties on hiv- , hcv and ebola. besides, l-sign has been described as a receptor for severe acute respiratory syndrome coronavirus (jeffers et al., ) . unlike dc-sign, l-sign does not bind to the fucose-containing lewis x antigens, suggesting that l-sign-expressing liver endothelial cells and lymph node are not involved in capture and internalization of lewis x -containing pathogens such as h. pylori and s. mansoni (van liempt et al., ) . besides, binding to l-sign is not reversible at low ph, suggesting that l-sign does not release internalized ligand in low-ph vesicles and that l-sign is degraded upon internalization (guo et al., ) . because of their similar ligand specificity but differential tissue location, we have compared the capacity of dc-sign and l-sign to bind axenic amastigotes from l. pifanoi (responsible for cl), and l. infantum (responsible for vl). binding experiments with jurkat cells stably transfected with dc-sign or l-sign indicated that l. infantum amastigotes specifically bound to both dc-sign and l-sign, whereas l. pifanoi amastigotes were unable to bind to l-sign (fig. ) . therefore, only vl-causing parasites (l. infantum) appear to be recognized by l-sign. these results suggest that l-sign recognition of the distinct leishmania species might play a role in the outcome of the parasite infection (cl vs. vl). to further evaluate the relevance of l-sign in binding of leishmania species causing vl, human hepatic sinusoidal endothelial cells (hsec) were isolated from hepatic surgery donors, using a modification (in˜igo martinez and fernando vidal-vanaclocha, unpublished information) of previously described isolation procedures (daneker et al., ; heuff et al., ) . incubation of hsec with axenic amastigotes showed that l. infantum amastigotes bound strongly to hsec, whereas no binding was observed with l. pifanoi amastigotes (fig. ) . in addition, the hsec-l. infantum (alvarez et al., ) were left untreated or pretreated with receptor-specific blocking antibodies, and then incubated at c with the indicated cfse-labeled parasites ( : cell:parasite ratio), or left uninfected (colmenares et al., ) . the percentage of cells with bound parasites was quantified by flow cytometry. three independent experiments were performed with similar results, and a representative experiment is shown. amastigote interaction was reduced in the presence of a blocking monoclonal antibody against l-sign (fig. ) . these results confirmed the ability of vl-causing amastigotes to interact with l-sign. given the mechanism described for the hepatitis c virus (cormier et al., ) , it is tempting to speculate that l-signmediated capture of l. infantum by hsec could result in transinfection of kupffer cells, which are the final targets of vl-causing leishmania parasites (el hag et al., ; murray, ) . taken together, the present work demonstrates that the c-type lectins dc-sign and l-sign are broad leishmania receptors that differentially bind the distinct infective forms and species of the parasite. a deeper knowledge of the leishmania-dc/l-sign interactions, and the subsequent immune consequences, may pave the way for the design of new therapeutic approaches against leishmaniasis. the recent description that macrophage treated with il- (alternatively activated macrophages) also express dc-sign (puig-kroger et al., ) could be of major importance, due to the fact that the expression of this cytokine correlates with the pathology of the disease, and hence increases the potential relevance of these c-type lectins in leishmaniasis. fig. . l. infantum binding to hepatic sinusoidal endothelial cells (hsec) is partially mediated by l-sign. human hsec were isolated from liver biopsies, and subjected to binding assays with l. pifanoi or l. infantum axenic amastigotes, as described (colmenares et al., ) . after incubation and washing, cells were photographed (hsec, hepatic sinusoidal endothelial cells; k, kupffer cells). two independent experiments were performed with similar results, and a representative experiment is shown. a critical role for lipophosphoglycan in proinflammatory responses of dendritic cells to leishmania mexicana c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans inhibition of cd expression in human dendritic cells during intracellular infection with leishmania donovani leishmania spp.: on the interactions they establish with antigen-presenting cells of their mammalian hosts cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cell-specific icam- -grabbing nonintegrin on dendritic cells a comparison of the effects of a benzimidazole and the dinitroanilines against leishmania infantum expression of lipophosphoglycan, highmolecular weight phosphoglycan and glycoprotein in promastigotes and amastigotes of leishmania mexicana dendritic cells and the control of immunity a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection axenic cultivation and characterization of leishmania mexicana amastigote-like forms silent infection of bone marrowderived dendritic cells by leishmania mexicana amastigotes /t helper cell balance through phase-variable interaction between lipopolysaccharide and dc-sign receptors and recognition mechanisms of leishmania species parasitism of epidermal langerhans cells in experimental cutaneous leishmaniasis with leishmania major dendritic cells in leishmania infection the c-type lectin dc-sign (cd ) is an antigen-uptake receptor for candida albicans on dendritic cells unique gene expression profiles of human macrophages and dendritic cells to phylogenetically distinct parasites dendritic cell (dc)-specific intercellular adhesion molecule (icam- )-grabbing nonintegrin (dc-sign, cd ), a c-type surface lectin in human dcs, is a receptor for leishmania amastigotes the dendritic cell receptor dc-sign discriminates among species and life cycle forms of leishmania l-sign (cd l) and dc-sign (cd ) mediate transinfection of liver cells by hepatitis c virus parasitic adaptive mechanisms in infection by leishmania cr , the c b receptor, mediates binding of infective leishmania major metacyclic promastigotes to human macrophages culture and characterization of sinusoidal endothelial cells isolated from human liver generation of leishmania donovani axenic amastigotes: their growth and biological characteristics leishmania donovani: long-term culture of axenic amastigotes at degrees c subversion of a young phagosome: the survival strategies of intracellular pathogens liver morphology and function in visceral leishmaniasis (kala-azar) immune escape through c-type lectins on dendritic cells c-type lectin receptors on dendritic cells and langerhans cells antigenpulsed epidermal langerhans cells protect susceptible mice from infection with the intracellular parasite leishmania major disruption of mannose activation in leishmania mexicana: gdp-mannose pyrophosphorylase is required for virulence, but not for viability dc-sign-icam- interaction mediates dendritic cell trafficking identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-sign, a dendritic cellspecific hiv- -binding protein that enhances trans-infection of t cells mycobacteria target dc-sign to suppress dendritic cell function interaction of leishmania parasites with dendritic cells and its functional consequences structural basis for distinct ligand-binding and targeting properties of the receptors dc-sign and dc-signr in vitro cultivation and characterization of axenic amastigotes of leishmania comparison of receptors required for entry of leishmania major amastigotes into macrophages hierarchy of susceptibility of dendritic cell subsets to infection by leishmania major: inverse relationship to interleukin- production isolation of rat and human kupffer cells by a modified enzymatic assay leishmania amazonensis: cultivation and characterization of axenic amastigote-like organisms proteophosphoglycans from leishmania promastigotes and amastigotes function and assembly of the leishmania surface coat leishmania major promastigotes inhibit dendritic cell motility in vitro cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus the vectorial competence of phlebotomus sergenti is specific for leishmania tropica and is controlled by species-specific, lipophosphoglycan-mediated midgut attachment dc-sign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses murine dendritic cells internalize leishmania major promastigotes, produce il- p and stimulate primary t cell proliferation in vitro identification of the mycobacterial carbohydrate structure that binds the c-type lectins dc-sign, l-sign and signr il- p production by leishmania majorharboring human dendritic cells is a cd /cd liganddependent process leishmania priming of human dendritic cells for cd ligand-induced interleukin- p secretion is strain and species dependent divergent roles for c-type lectins expressed by cells of the innate immune system dendritic cells: specialized and regulated antigen processing machines a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr. subunit organization and binding to multivalent ligands experimental cutaneous leishmaniasis: langerhans cells internalize leishmania major and induce an antigen-specific t-cell response the role of dendritic cells at the early stages of leishmania infection dendritic cells induce immunity to cutaneous leishmaniasis in mice receptors on phagocytic cells involved in microbial recognition tissue granuloma structure-function in experimental visceral leishmaniasis surface determinants of leishmania parasites and their role in infectivity in the mammalian host monoclonal antibodies specific for the amastigote stage of leishmania pifanoi. i. characterization of antigens associated with stage-and species-specific determinants dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans leishmania major lipophosphoglycan modulates the phenotype and inhibits migration of murine langerhans cells icam- )-grabbing nonintegrin in thp- human leukemic cells, monocytes, and macrophages leishmania amazonensisdendritic cell interactions in vitro and the priming of parasite-specific cd (+) t cells in vivo dc-sign (cd ) expression is il- dependent and is negatively regulated by ifn, tgf-beta, and anti-inflammatory agents leishmania and the macrophage: a marriage of inconvenience evasion of innate immunity by parasitic protozoa the role of phosphoglycans in leishmania-sand fly interactions dendritic cells and immunity to leishmaniasis and toxoplasmosis dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin mediates binding and internalization of aspergillus fumigatus conidia by dendritic cells and macrophages dc-sign; a related gene, dc-signr; and cd form a cluster on p lipophosphoglycan from leishmania mexicana promastigotes binds to members of the cr , p , and lfa- family of leukocyte integrins the lipophosphoglycan of leishmania parasites is lipophosphoglycan a virulence factor? a surprising diversity between leishmania species skin dendritic cells in murine cutaneous leishmaniasis the dendritic cell-specific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x a novel adhesion pathway that regulates dendritic cell trafficking and t cell interactions molecular basis of the differences in binding properties of the highly related c-type lectins dc-sign and l-sign to lewis x trisaccharide and schistosoma mansoni egg antigens uptake of leishmania major amastigotes results in activation and interleukin release from murine skin-derived dendritic cells: implications for the initiation of anti-leishmania immunity leishmania major-infected murine langerhans cell-like dendritic cells from susceptible mice release il- after infection and vaccinate against experimental cutaneous leishmaniasis dc-sign: binding receptors for hepatitis c virus roles of cr and mannose receptors in the attachment and ingestion of leishmania donovani by human mononuclear phagocytes leishmania infection: surfaces and immunity this work was supported by the ministerio de educacio´n y ciencia (grants saf - -c - , gen - -c - /nac and agl - -ali) and fundacio´n para la investigacio´n y prevencio´n del sida en espan˜a (fipse / ) to alc. mc was supported by the ministerio de educacio´n y ciencia (ramo´n y cajal programme). ec was supported by a fellowship fpi from the ministerio de educacio´n y ciencia. key: cord- -eot authors: wang, hongliang; rao, shuan; jiang, chengyu title: molecular pathogenesis of severe acute respiratory syndrome date: - - journal: microbes infect doi: . /j.micinf. . . sha: doc_id: cord_uid: eot the global outbreak in – of severe acute respiratory syndrome (sars) posed a serious threat to public health and had a significant impact on socioeconomic stability. although the global outbreak of sars has been contained, there are serious concerns over its re-emergence and bioterrorism potential, and up to date, no specific treatment exists for this disease. here we review the progress of studies on the pathogenesis of the disease, in particular, studies on the molecular level. during the winter of e , a new 'plague' emerged in guangdong province, china, and quickly spread to other countries. patients were characterized by fever, dry cough, dyspnea, headache, and hypoxemia; and death could be a result of progressive respiratory failure due to alveolar damage. the syndrome was designated 'severe acute respiratory syndrome' (sars). the identification of the etiologic agent, a novel coronavirus, as sars-associated coronavirus (sars-cov) was quickly made by international collaboration [ , ] . by july , when the pandemic terminated, people in countries had been diagnosed with probable sars, of whom died [http://www.who.int/csr/sars/country/ table _ _ /en/index.html]. in the winter of e , sporadic cases were reported, including four cases in guangdong province, and laboratory acquired sars from singapore, taiwan and beijing [http://www.who.int/csr/don/ archive/disease/severe_acute_respiratory_syndrome/en/index. html]. although the death rate is low in comparison with fatalities during previous pandemics, the rapidity of spread due to air travel, the coverage in the media and the enormous economic and social impacts, the fear of renewed outbreaks as well as the potential misuse of the virus as a biological weapon all contribute to the far more pronounced impact of sars-cov. since the identification of the etiological agent, rapid progress has been made towards understanding this newly emerged pathogen. most previously published reviews focused on the epidemiology, clinical presentation and potential treatment of sars-cov infection; this review focuses on the molecular mechanisms of sars pathogenesis. the newly identified virus is a new member of the family coronaviridae, genus coronavirus. it is a family of large, enveloped, positive-sense single-stranded rna viruses that replicate in the cytoplasm of animal host cells. the genome of sars-cov is about kb with e open reading frames (orfs). downstream of orf a and b is orfs that encode the four main structural proteins, namely, spike (s), envelope (e), membrane (m), and nucleocapsid (n) protein [ , ] . the spike protein of sars-cov, which is involved in virus binding, fusion, and entry, is a typical class i viral fusion protein, similar to the transmembrane glycoproteins of many enveloped viruses [ ] . the amino-terminal s and carboxylterminal s subunits of the sars-cov s protein can be identified through their homology with the s and s subunits of other coronaviruses. coronaviruses are usually subdivided into three phylogenetic groups. sars-cov is different from the known coronaviruses; depending on the method of sequence analysis used, sars-cov either constitutes a new phylogenetic group [ e ] or a subgroup of group [ ] . studies using pseudotyped lentiviruses carrying the s, m and e glycoproteins of sars-cov demonstrated that the spike protein is both necessary and sufficient for virus attachment to susceptible cells [ ] . the cellular receptors were detected by taking advantage of this tag. in an in vitro study, li et al. demonstrated that the angiotensin-converting enzyme (ace ) is a functional cellular receptor of sars-cov, by using coimmunoprecipitation of the virus glycoprotein (s ) with lysates from cells that are susceptible to virus infection (vero e cells) followed by mass spectrometry analysis [ ] . later, our group proved that ace is crucial for sars-cov infection in vivo employing an ace knockout mouse [ ] . later, the structure of sars coronavirus spike receptor-binding domain (rbd) complexed with ace was determined [ ] . all these findings will influence the ongoing vaccine development and biological analysis. immunostaining techniques identified ace on the surface of type and type alveolar epithelial cells, enterocytes of the small intestine and the brush border of the proximal tubular cells of the kidney [ ] . these localizations explain the documented tissue tropism of sars-cov for the lung and gastrointestinal tract. however, it should be noted that colonic enterocytes as well as the liver tissue were largely negative for ace protein expression, while sars-cov replication does occur therein. in contrast, whereas ace is strongly expressed on the endothelial cells of arteries and veins of all tissues and the smooth muscle cells of the intestinal tract, there is no evidence of virus infection at any of these sites [ ] . these observations suggest that another cellular factor is required for successful virus infection. pseudotyped virus containing the spike protein has also been shown to bind to dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign) [ ] . dc-sign is a type ii transmembrane adhesion molecule found on dendritic cells. it consists of tandem repeats of a highly conserved -amino acid sequence and a c-type lectin domain that recognizes carbohydrate residues on a variety of pathogens. unlike the ace receptor on pneumocytes and enterocytes, dc-sign does not permit sars-cov infection of the dendritic cells but can transfer the virus to susceptible target cells through a synapse-like structure (trans infection), and this cell-mediated transfer can be blocked by the mouse anti-sars-cov spike protein antiserum [ ] . besides dc-sign, there is also a report showing that cells expressing a molecule which is % identical to dc-signe l-sign (cd l) can enhance sars-cov infection [ ] . another study proved that l-sign is a receptor for sars-cov with the expression cloning method [ ] . this study also indicated that cells expressing l-sign became susceptible to sars-cov infection, but l-sign was a less efficient receptor when compared with ace [ ] . l-sign also is a type ii transmembrane glycoprotein in the c-type lectin family. it has a structure similar to that of dc-sign, except that l-sign has considerable polymorphism in the tandem repeat domain. since l-sign has much higher polymorphism, there must exist different combinations in the population. chan et al. found that homozygous expression of polymorphic variants of l-sign plays a protective role in sars-cov infection, because cells homozygous for l-sign show higher binding capacity for sars-cov, higher proteasomedependent viral degradation and a low capacity for trans infection [ ] . all these findings indicate that lectins may play a role in the pathogenehost interaction. whether there are other molecules involved in the sars-covehost interaction remains open. after the virus attaches to the host cells, the virus enters the cell either through ph-dependent receptor-mediated endocytosis or through direct membrane fusion. currently, there is no consensus about this question in the sars-cov case. some experiments demonstrated that sars-cov might gain cell entry via ph-dependent endocytosis, but there are also studies showing that the sars coronavirus entered the cells through direct membrane fusion. the n-terminal half of the s protein (s ) contains the receptor-binding domain, whereas the c-terminal half (s ) is the membrane-anchored membrane-fusion subunit, which contains two heptad repeat regions (hr and hr ). in the native state, spike proteins on the virus surface could be in oligomeric form and form the stems of the spikes on sars-cov; hr regions may be in random coil conformation covered by the s domain. after binding to ace on the target cells, the s domain changes conformation by forming a-helices, extending and inserting its inert fusion peptide into the target cell membrane, and exposing hr and hr regions. then the hr and hr regions form a six-helix oligomeric complex, with the hr trimer as a core. this fusion-active core structure brings the viral and target cell membranes into close proximity, resulting in fusion between the membranes and formation of fusion pores, which allows the virus genome to enter the target cell [ ] . apart from direct membrane fusion at the target cell surface, sars-cov might gain cell entry via ph-dependent endocytosis, which is also mediated by the s protein. the spike glycoproteins of most of the enveloped rna viruses can mediate the attachment, fusion and entry of the virus. however, an activating trigger, which can result in a conformational change, is usually required to make the spike glycoproteins fulfill their functions. for example, binding to specific receptors/coreceptors is the activating trigger for mlv and hiv, while the influenza virus requires only an acidic milieu. however, this is different in the case of sars-cov, as some groups have found that a special endosomal protease, cathepsin l, was crucial in the activation of viral infectivity; inhibitors of cathepsin l can prevent sars-cov entry [ ] . so sars-cov may first bind to ace on the cell surface and be taken up into a vesicle (endocytosis). later, cleavage of spike protein or ace by cathepsin l facilitates fusion of the viral membrane and the vesicle membrane. these results are consistent with previous findings showing that the inhibitors of vacuolar acidification could block infection by s-bearing pseudotypes in a dose-dependent manner [ ] , as cathepsin l is a ph-dependent cysteine protease with its maximal activity in an acidic milieu and may lose its activity with increasing ph. therefore, the spike protein mediated entry of sars-cov might be through direct membrane fusion or in a ph-dependent endocytosis fashion, and certain factors might influence this process. the mean incubation period for this disease is estimated to be . days ( % ci . e . ). the mean time from onset of clinical symptoms to hospital admission varied from to days [ ] . the most common symptoms included fever, chills, rigors, and myalgia. cough and headache were also reported in more than % of the patients. other common findings were lymphopenia, thrombocytopenia, and elevated lactate dehydrogenase and creatine kinase levels [ ] watery diarrhea has also been reported [ ] . results from autopsied organs showed that the predominant damage occurred in lungs. the pathological characteristics of the lung include acute pulmonary exudative and hemorrhagic inflammation in lung tissue, greatly increased permeability of capillaries leading to a leakage of proteins such as cellulose, or even erythrocytes, into the alveolar wall and space, and accumulation of exudates and edema fluids in most of the alveoli spaces, and even hyaline membrane formation in some alveoli spaces [ ] . airspace opacity distributed peripherally in the lower lung zone is the most commonly encountered radiographic image in patients with sars at presentation. radiographic progression to multifocal or bilateral lung involvement followed by radiographic improvement occurs during treatment in about % of patients with sars. no cavitation, lymphadenopathy, or pleural effusion was demonstrated [ ] . typically, sars follows a three-phase clinical course [ ] . phase is a viral replication phase that involves an initial presentation of high fever and myalgia of a few days' duration, which generally improve after a few days. the increasing viral load during this phase suggests that the symptoms are largely related to the effect of viral replication and cytolysis; however, this may also be due to antiviral and immunomodulatory therapies. in phase , which begins about days after onset of fever, patients frequently had recurrence of fever, onset of diarrhea, and oxygen desaturation. the timing of igg seroconversion, which starts on day , seems to correlate with falls in viral load, which occurs between days and . severe clinical worsening also occurs at this time. therefore, the lung damage at this phase is most likely related to immunopathological damage as a result of host response, rather than uncontrolled viral replication itself [ ] . most cases improve after steroid treatment and enter a third phase of rehabilitation, while about % deteriorate with evidence of severe lung injury characterized by acute respiratory distress syndrome (ards) necessitating ventilation [ ] . epidemiological analysis of the patient population shows that some factors may influence the final outcome of the disease. firstly, age: in patients older than years, the mortality rate exceeds % [ ] , while sars in children, especially those under years, is generally associated with an uneventful course and a good outcome. secondly, coexisting illnesses, especially diabetes mellitus and heart disease, are consistently found to be independent prognostic factors for poor outcome, which is defined as death, need for mechanical ventilation, or admission to an intensive care unit [ , ] . thirdly, in some studies, an increased lactate dehydrogenase level and elevated neutrophil count at the time of admission, as well as low cd and cd lymphocyte counts, were associated with a poor prognosis [ ] . although much has been learned about sars in the three years since its discovery, aspects of the pathogenesis of the disease are still not fully understood. but it needs to be emphasized that since there is no specific drug or vaccine available, research on molecular mechanisms is crucial to identify potential treatment targets. and those with immunosuppressive conditions such as hiv infection or treatment with immunosuppressors is less severe, also indicating that immunopathology plays an important role in phase . the use of steroids for sars in this phase seems to be beneficial, which is also evidence that the inflammation and immune response play a role in this phase. in general, during a viral infection, most cell types in the body respond by secreting high levels of type interferons. this is not the case in the sars-cov infection. the immune-related genes that were overexpressed after the onset of sars are usually associated with the innate immune response against bacterial infection and not against a viral infection. for example, the expression of lactoferrin is upregulated [ ] . in addition, other innate immune defenses, such as the collectins, which can bind the glycosylated sars-cov s protein, may play an important role in host defense. this suggests that the response of sars affected patients is mainly an innate inflammatory response rather than a specific immune response against a viral infection [ ] . besides the function of limiting virus spread, the immunological response against viral infection can also cause pathological damage to the host tissues. this is especially a concern in the case of proinflammatory cytokines. nicholls and colleagues suggested that pro-inflammatory cytokines released by activated macrophages in alveoli could have a prominent role in the pathogenesis of sars [ ] . t cells are essential for adaptive immunity against viral infections in vivo. antiviral cd þ helper t cells help in the production of virus-specific antibodies by b cells, while cd þ ctls can kill virus-infected host cells. in the case of sars-cov infection, there is no conclusion as to the degree that t cells can influence the progress of the disease, since lymphopenia with a rapid decrease in both cd and cd t cells is common during the acute phase of sars [ ] . neutralizing antibodies (igg) have been detected in sars patients, suggesting that humoral immunity plays an important role in the elimination of the virus. this is further evidenced by the activity of an anti-s human monoclonal antibody, r, in neutralizing sars-cov infection and inhibiting syncytia formation [ ] . virus-induced apoptosis is a common phenomenon in viral infection, especially rna virus infections, including some coronaviruses. apoptosis can be used by the host cells to clear off the virus in the infected cells and also can assist virus dissemination by the release of viral particles, thereby facilitating its survival in vivo. therefore, apoptosis can have two opposite roles on the pathogenicity of viral infection, enhancing or suppressing the viral infection. in the case of sars, apoptosis was observed in patients' lung epithelial cells; thus, sars-cov induced apoptosis would certainly have a deleterious pathogenic role, leading to severe tissue damage [ ] . this might explain the severe respiratory system damage in sars patients. apoptosis may also play an important role in the hematological changes besides the direct injury in the lung. as mentioned above, hematological changes in patients with sars are common and include lymphopenia, thrombocytopenia and occasionally leukopenia. the mechanism underlying this phenomenon remains unclear, but there are studies indicating that apoptosis as well as the immune response are involved [ ] . several studies have focused on the mechanism of sars-cov induced apoptosis, for example, proapoptotic components of the virus genes and apoptosis pathways. sars-cov infection in vero e cells can lead to apoptosis. further studies showed that a variety of signaling pathways were phosphorylated or dephosphorylated when the cells were infected with sars-cov. specifically, p mitogen-activated protein kinase (mapk) is thought to be involved in induction of apoptosis, since mizutani et al. found that the p mapk and its downstream targets, mapkapk- and hsp- were activated during viral replication [ ] . a p inhibitor was able to partially prevent cytopathic effects induced by sars-cov infection. the signal transducer and activator of transcription (stat)- , which is usually constitutively phosphorylated at a tyrosine residue ( ), is dephosphorylated by sars-cov-induced activation of p [ ] . although akt, an inhibitor of apoptosis, was also partially activated, this weak activation cannot prevent sars-cov infection-induced apoptosis in vero e cells. and recently, research on the kda ribosomal s kinases (p rsks), an important substrate of the erk, showed that the specific serine residue ( ) of p rsks, that has been reported to be involved in autophosphorylation by activation of the c-terminal kinase domain, was phosphorylated in confluent sars-cov infected cells, and this phosphorylation can be inhibited by an inhibitor of p mapk [ ] . all these results suggest that sars-cov induced apoptosis in vero e cells is related to p mapk. besides the whole virus, certain components of the virus can also induce apoptosis. it has been reported that sars-cov proteins a, b and a can induce apoptosis; the sars coronavirus nucleocapsid protein can induce apoptosis in cos- cells in the absence of growth factors; the c-terminal domain of sars-cov spike protein is sufficient to induce apoptosis in vero e cells. a recent study showed that the induction of apoptosis by the a protein also is related to its ability to activate p mapk [ ] . in adult sars patients, respiratory distress is the principal cause of mortality. the histological change associated with ards is called diffuse alveolar damage (dad), which is characterized by structureless non-cellular exudates filling the bronchioles. dad is associated with a high mortality rate, and apart from supportive clinical care, there are few specific therapeutic options of proven benefit. recent studies have shown that the renin-angiotensin system plays an important role in the sars-cov caused acute lung failure. acid aspiration or sepsis in wild-type mice, which mimics human acute lung injury, resulted in impairment of lung function as assessed by lung elasticity, blood oxygenation and pulmonary edema. the mice developed edema, alveolar wall thickening, bleeding, inflammatory cell infiltration and hyaline membrane formation. but in the ace knockout mice, lung injury was much more severe than in the wildtype mice when assessed similarly. a rescue experiment using the recombinant human ace protein showed a decreased degree of acute lung injury in both the knockout and wildtype mice. all these results demonstrated that loss of ace is essential for lung injury [ ] . both ace and ace are key enzymes in the renin-angiotensin system. ace cleaves the decapeptide angiotensini (angi) into octapeptide angiotensinii (angii). ace cleaves a single residue from angi to generate ang e , and a residue from angii to generate ang e . in this way, ace counterbalances the function of ace and negatively regulates the angii level. thus loss of ace expression will lead to a high level of angii, which will then, through receptor at a, have a causative role in acute lung failure (fig. ). we speculated that the acute lung failure caused by sars-cov was mainly due to the function of ace . first we found that experimental infections of sars-cov in wild-type mice resulted in considerably reduced ace expression in the lungs, while ace expression was not changed. to further test our hypothesis, we established a defined model system using recombinant sars-cov spike protein. this model system allowed us to avoid possible secondary effects resulting from viral replication or infections in vivo and to directly test whether sars-cov spike protein might adversely affect acute lung injury through modulation of ace . with this system we found that binding of spike to endogenous ace in vero e cells also resulted in downregulation of ace surface expression. further studies showed that treatment with spike protein worsened the lung function in wild-type mice. moreover, spike treatment of acid-challenged wildtype mice augmented the pathological changes in the lung parenchyma and increased lung edema, while in vivo spike protein administration did not affect the severity of lung failure in ace knockout mice, indicating that the effect of spike protein on acute lung injury is ace specific. thus we hypothesize that infection with sars-cov can result in ace downregulation through binding of sars-cov spike protein to ace . given that ace is a key negative regulatory factor for severity of lung edema and acute lung failure, sars-cov spike protein-mediated ace downregulation then contributes to the severity of lung pathologies [ ] (fig. ) . about one million people suffer from ards due to different disposing factors, and the death rate can reach %. as the recombinant ace and angiotensin ii receptor inhibitors are already in clinical use for control of blood pressure, this finding points to a possible therapy for the otherwise incurable disease (fig. ) . besides ards that contributes significantly to sars-cov caused death, other sars-cov associated diseases, such as hypertension, glucose increase, and heart dysfunctions cannot be ignored [ , ] . the mechanisms underlying the pathogenesis are still unclear and need to be understood. currently, there is no antiviral therapy of proven value for sars. clinically, treatment of sars includes anti-sars-cov therapy and anti-inflammatory treatment to limit viral pneumonitis and subsequent pulmonary fibrosis. with insights into the field of sars pathogenesis and sars-cov genome structure, novel potential therapeutic targets for antiviral therapy were evaluated. for example, inhibitors of each step of the virus life cycle, like binding inhibitors, fusion inhibitors, rna transcription (replication) inhibitors as well as protease inhibitors, were designed and evaluated. only a few potential anti-sars agents have been tested in animal models, and their efficacy in humans is still unknown. inconsistent results between different groups investigating the same compound may be related to testing methodology, in particular, differences between in vitro and in vivo antiviral mechanisms. new technologies such as sirna may be important. however, this new technology is still riddled with practical difficulties, like efficacious delivery systems and safety concerns, limiting its application to patient treatment. using sera from people convalescing from sars to treat sars patients was proved to be effective, implying that neutralizing antibody can serve as a therapeutic strategy. a retrospective study in a limited number of patients using human sars convalescent plasma suggested that passive immunization had no obvious adverse effects [ ] . however, the use of convalescent sera is not a practical therapy, especially when there is an outbreak worldwide. there are still no effective antiviral therapies that can be used immediately on patients. the most promising method to prevent the disease is a protective vaccine. immediately after the sars outbreak, researchers began to investigate whether inactivated vaccine could be used to prevent sars. in china, a collaborative group from the chinese academy of medical sciences, china's cdc, and the sinovac biotech company passed the inactivated sars-cov strain pumc vaccine clinical trial phase from the sfda in the summer of . although the inactivated sars vaccine seems useful and reasonable in sars prevention, the safety of the inactivated sars-cov vaccine is a serious concern. vaccines targeting structural genes, especially the recombinant spike protein, seem the safest strategy. a dna vaccine encoding s protein can elicit neutralizing antibodies and induce t cell response, which can protect mice from sars infection [ ] . varieties of vectors were used to express the spike protein and then used to immunize mice, african green monkeys, and hamsters. neutralizing antibodies were detected which protected them from infection. the dna sars vaccine invented by scientists at the national institute of allergy and infectious diseases and produced by vical inc. of san diego entered clinical trial phase under us fda guidelines at the end of . recently, a group proved that rbd could elicit much higher titers of neutralizing antibodies than did the full-length s protein [ ] , suggesting that rbd may be a potential vaccine candidate. however, the discovery that spike rbd alone could worsen acute lung injury in mice indicated the potential safety issue of spike as protein vaccine. the harm of spike rbd to the lung was thought to be caused by its binding to sars receptor ace [ ] . it is reported that s proteins from the e sars outbreak and from the much less severe e outbreak have different binding affinities to the receptor ace . the latter has lower affinity to human ace , mainly due to an alteration of amino acid residues at sites and [ ] . therefore, in order to obtain safer recombinant spike protein vaccine, site-directed mutagenesis at these sites might be necessary to eliminate the binding affinity of spike to ace . in summary, the global outbreak of sars has led to the formation of a successful network of laboratories, and much has been learned about sars in the three years since its discovery. however, some aspects of the molecular pathogenesis of the disease are still not fully understood. further investigations of various aspects, including the life cycle of the virus, the molecular mechanisms of the disease, and the factors that can influence the progress of the disease, will result in a more thorough understanding of this new pathogen. results from further research will certainly suggest more promising treatment strategies and may lead to prevention of this disease. fig. . schematic representation of the role of ras in the sars-cov induced acute lung injury and the potential therapeutic drugs. ace can cleave angiotensin i to produce angiotensin ii, which can then either bind to at ar leading to lung injury, or bind to at r reducing the severity of lung injury. ace , on the contrary, can counteract ace by converting the angiotensin ii to a less damaging molecule. sars-cov infection or spike protein treatment can down-regulate the expression of ace , and thus aggravate lung injury. based on these findings, ace inhibitor (such as lisinopri, captopri), at ar inhibitor (such as losartan, valsartan) as well as recombinant human ace (rhuace ), are all potential drugs for this kind of acute lung injury. identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome nabel, ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign unique and conserved features of genome and proteome of sars-coronavirus, an early split-off from the coronavirus group lineage angiotensin-converting enzyme is a functional receptor for the sars coronavirus sars coronavirus-induced lung injury structure of sars coronavirus spike receptor-binding domain complexed with receptor tissue distribution of ace protein, the functional receptor for sars coronavirus. a first step in understanding sars pathogenesis dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus homozygous l-sign (clec m) plays a protective role in sars coronavirus infection interaction between heptad repeat and regions in spike protein of sars-associated coronavirus: implications for virus fusogenic mechanism and identification of fusion inhibitors sars coronavirus, but not human coronavirus nl , utilizes cathepsin l to infect ace -expressing cells epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong a cluster of cases of severe acute respiratory syndrome in hong kong clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study pathological study on severe acute respiratory syndrome severe acute respiratory syndrome: radiographic appearances and pattern of progression in patients clinical features and short-term outcomes of patients with sars in the greater toronto area haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis expression profile of immune response genes in patients with severe acute respiratory syndrome lung pathology of fatal severe acute respiratory syndrome evaluation of human monoclonal antibody r for immunoprophylaxis of severe acute respiratory syndrome by an animal study, epitope mapping, and analysis of spike variants sars coronavirus induces apoptosis in vero e cells hematological findings in sars patients and possible mechanisms tyrosine dephosphorylation of stat in sars coronavirus-infected vero e cells regulation of p rsk phosphorylation by sars-cov infection in vero e cells palese, a protein of severe acute respiratory syndrome coronavirus inhibits cellular protein synthesis and activates p mitogen-activated protein kinase angiotensinconverting enzyme protects from severe acute lung failure retrospective comparison of convalescent plasma with continuing high-dose methylprednisolone reatment in sars patients a dna vaccine induces sars coronavirus neutralization and protective immunity in mice severe acute respiratory syndrome: vaccine on the way receptor and viral determinants of sars-coronavirus adaptation to human ace the authors thank dr. wenhui li of harvard medical school for reading this manuscript and for his useful comments and suggestions. key: cord- -rdhuc n authors: anderson, nancy l. title: pet rodents date: - - journal: saunders manual of small animal practice doi: . /b - - - / - sha: doc_id: cord_uid: rdhuc n nan many small rodents are commonly kept for companionship and enjoyment. this chapter provides information needed to diagnose and treat the most frequently encountered problems of mice, rats, gerbils, hamsters, guinea pigs, and chinchillas. • cages should be made of stainless steel, hard plastic, or glass. these materials are cleaned and sanitized easily and are resistant to gnawing or corrosion from urine and fecal matter. minimum floor space and height requirements are listed for each species in • guinea pigs can be housed in open-topped enclosures with walls higher than inches. ensure that dogs, cats, wild animals, and small children do not have unsupervised access to these cages. • clean cages as needed, usually to times per week for most rodents. a scrub brush, dish soap, and water work well. if cages are not kept clean, ammonia, other irritants, moisture, and bacteria concentrations rise to harmful levels, predisposing animals to disease. • disinfect the cage twice a month with part sodium hypochlorite (household bleach) mixed in parts water. let the bleach solution stand for at least minutes. rinse the cage well afterward. • all solid-floored cages need to be covered in bedding. shredded paper, non-resinous wood shavings, wood wool, and corn cobs are all acceptable. provide at least inches of bedding. most rodents enjoy burrowing in deeper bedding when it is provided in one corner of a cage. do not, however, fill the entire cage with deeper bedding. this usually leads to poor sanitation as a result of owners' failure to recognize buildup of hidden wastes such as moisture from leaking water bottles, cached foods, urine, and feces. • wire mesh floors can be used successfully only if the dimension of the mesh is correct. size the openings to be just large enough for an adult to retract a tarsal joint back through the mesh. larger holes make it difficult for the animals to walk and cause pressure sores. smaller openings may cause injuries such as tibial fractures and self-mutilation while struggling to free trapped appendages. bedding above the wire keeps waste from dropping through the wire and therefore is not recommended. wire bottom cages do not work well for breeding animals because neonatal rodents must be surrounded by nesting material to maintain moisture in the nest and prevent dehydration. young rodents often cannot walk correctly on mesh sized for adult feet. • all pet rodents require visual security. tubes, jars, or cans made of nontoxic, nonabrasive substances work well for this purpose. also provide objects for gnawing. rodents possess open-rooted teeth, and constant wear is necessary to maintain normal dentition. mice, rats, gerbils, and hamsters enjoy and benefit from exercise wheels. • a good room temperature range for most pocket pets is °f to °f. keep rodents with disease at °f to °f unless hyperthermia is of concern (some chinchillas). • provide to hours of darkness to to hours of light. this light cycle is essential if breeding is desired. • hamsters, guinea pigs, and chinchillas that are exposed to temperatures below °f may hibernate for a few days or until the ambient temperature rises. heart rates may be less than bpm during hibernation. m key point feed pet rodents laboratory animal chow appropriate for their species (table - ). seed diets are deficient in protein and contain excessive fat. • seeds, as well as vegetables and other foods, may be fed as treats but not to provide more than % of calories. intermittent exposure to vegetables and seeds causes mild, transient diarrhea. • supplementation of vitamin c is recommended for all guinea pigs. • adult chinchillas that are not obese should be fed high quality, fresh grass hay ad libitum. obese animals may need to have the hay rationed. chinchillas require / to / -cup of fresh pellets per animal each day. feeding pellets free choice leads to obesity, and the high protein and calcium levels in these diets may predispose animals to urinary tract disease. most pellets also do not provide sufficient fiber to maintain normal gastrointestinal (gi) motility. • store food in tightly sealed containers at less than °f; keep food refrigerated if possible. • feed all diets within days of milling to ensure the highest nutritional value. encourage owners to check dates on packages and ask pet store managers about providing dates on bulk items. • if possible, feed pet rodents except guinea pigs from overhead racks. these devices reduce wastage and eliminate fecal contamination of food. covered hoppers, heavy crocks, or stainless steel bowls that are attached to the side of the cage to eliminate spillage are acceptable and recommended for guinea pigs. • feed breeding females and their litters from the floor of their cages until the young are large enough to reach overhead feeders or crawl in and out of crocks. • cannibalism of neonates commonly occurs as the result of stress associated with cage cleaning. to minimize cannibalism, clean the cage and provide a to -day food supply to days before parturition. m key point provide fresh water in clean containers daily. • do not provide water in open crocks. these are contaminated or spilled easily and are a common cause of dehydration and poor sanitation. • sipper tubes and water bottles work well. clean with dish soap and water daily and disinfect them weekly. guinea pigs expel food from their mouths into their sipper tubes, so more frequent cleaning is needed. • some water bottles have special valves to minimize backflow. supplement guinea pigs' water daily with mg vitamin c/l. if the water is not dechlorinated, it will inactivate the vitamin c. quarantine all newly acquired animals in a different room from current pets for a minimum of days. feed and handle quarantined animals last. recommend that caretakers wash their hands and change clothes before handling current pets. avoid the introduction of adult animals because this frequently results in fighting. instead, place animals together while young and allow them to mature together. avoid keeping more than one male per cage because this also usually leads to fighting. a systematic history and physical examination are mandatory. many disease syndromes are caused by poor husbandry. pets that have been kept isolated from other rodents or acquired from a private breeder are less likely to harbor infectious disease than animals obtained from a pet store, laboratory, or wholesaler. see table - for normal physiologic data. obtain the following information: • observe the pet in its cage for mentation, activity, locomotion, dyspnea, head posture, haircoat, and any grossly observable abnormalities. • note respiratory and heart rates before restraint when possible. observe the condition of cagemates. m key point if dyspnea or severe depression is detected, warn the owner that the animal is critically ill and could die of stress brought on by an examination. • handle such animals as little as possible. initially, treat severely ill animals symptomatically, then • note the type of diet and bedding as well as the level of sanitation and compare these with what was described in the history. • observe quantity and quality of feces and urine. diarrhea, soft stools, absence of stools, copious urine, and discolored urine all can be signs of illness. • coprophagy is a normal behavior in rodents. • check the diet and water supply for freshness, quantity, source, and accessibility. • evaluate the presence and suitability of cage furniture. adequate visual security and the ability to exercise and gnaw are extremely important. • an accurate weight in grams is extremely important for evaluating an animal's body condition, calculating drug dosages, and monitoring treatment. the easiest method of weighing a pet rodent is to place it in a box and then subtract the weight of the container. • restraint of pet rodents is easy with experience. pets that have been handled frequently and gently by the owners require only minimal restraint. gentle pressure directs the animal as needed. grasp less cooperative patients (except chinchillas and guinea pigs) by the scruff over the back of the neck with thumb and forefinger ( fig. - ) . take care to pinch enough skin to prevent the animal from turning around, yet leave enough slack for respiration. on smaller specimens, hold the base of the tail, if present, between the fourth and fifth fingers to provide additional restraint. • hold docile guinea pigs with the palm of one hand supporting the chest while the other hand supports the hind quarters ( fig. - ). place the thumb and forefinger of the first hand in the axillas for additional control. • take care to minimize damage to the fur when handling chinchillas because they lose hair easily. grasp the animal by the tail and scoop it up into the palm of the same hand ( fig. - ). if necessary, grasp the thorax just behind the axillas. • calm uncooperative rodents by placing an appropriately sized towel over the head. complete the physical examination by wrapping the patient in a towel and exposing only needed areas. even oral, ophthalmic, and aural examinations can be performed with minimal effort if the animal is given the chance to relax in the towel "burrow." • remove particularly aggressive patients from their cages by scooping them up in a can or bucket; then slide them out onto a slick surface and pick them up or transfer them to a holding area or scale. m key point lift the hind quarters of mice and rats by the base of their tails to facilitate scruffing. never use the tip of the tail for restraint, or the skin of the tail may slough. once the animal is restrained properly, examine the head. assess the cranial nerves. check the nose for presence and character of discharge. examine the mouth for ptyalism, swelling, overgrown incisors, or discharges. to inspect the oral cavity, place an avian speculum across the mouth just caudal to the incisors. use a light source and a pair of hemostats as retractors to improve access. alternatively, use an otoscope with a pediatric head to examine the premolars and molars of guinea pigs and chinchillas for overgrowth. examine the cheek pouches of hamsters for swelling, impaction, or discharge. an ophthalmic examination, including a fundic examination, is important. • use a slit lamp to identify superficial pathology, especially corneal ulcers or foreign bodies. • if indicated, perform fluorescein stain and conjunctival scrapings or cultures. • note the presence of conditions such as discharge, asymmetry, and exophthalmos. m key point gerbils, rats, and mice produce red tears (chromodacryorrhea) with stress or disease. do not confuse them with hemorrhage. • guinea pigs suffering from hypovitaminosis c often produce dry, white tears. • check ears for discharge, foreign bodies, and mites. bluish discoloration of the ears is a sign of cyanosis. bright red injected coloration is associated with septicemia. sores behind the ears and on the neck are often a sequela of aural disease. • evaluate submandibular, axillary, inguinal, and popliteal lymph nodes for size and consistency. enlargements usually indicate infectious or neoplastic disease. • reevaluate respirations and heart rate after the stress of handling and compare them with the resting rate noted when the animal was in the cage. note dyspnea or respiratory sounds. auscultate animals weighing more than g. counting every third or fourth beat and multiplying by the appropriate factor allows recording of heart rates up to bpm. • palpate the abdomen. pay special attention to differentiating pregnancy from the bladder, kidneys, abdominal masses, enlarged cecum, and fecal balls in the colon. while palpating the abdomen, examine the mammary chain of all female rodents for signs of mastitis, lactation, or neoplasia. also check male mice and rats for mammary neoplasia. mammary tissue extends from the base of the neck to the base of the tail. gerbils typically have an elliptical sebaceous gland on their ventral midline. do not confuse this with neoplasia or infection. check the rectum and perineal area for signs of diarrhea, prolapse, irritation, parasites, and bite wounds. note that coprophagy is normal in rodents. • evaluate the urogenital tract for signs of inflammation, foreign bodies, urine scalding, and vaginal discharge. locate and palpate the testicles in males. the easiest method of determining sex in pet rodents is to compare the anogenital distance, which is twice as long in males as in females. other characteristics that allow the determination of sex are as follows: • visualization or palpation of testicles or extrusion of the penis from the prepuce indicates a male. • the presence of two external openings (i.e., anus and urethra) indicates a male. • the presence of three openings (i.e., anus, vagina, and urethra) indicates a female. • examine the skin and fur for conditions such as crusts, alopecia, masses, herniations, and wounds. • check tail and feet for swellings, coloration, sores, length of toenails, and condition of footpads. • evaluate the extremities for trauma or other abnormalities. apply cellophane tape to crusted areas of the skin and view under a microscope as an aid in diagnosing ectoparasites such as lice, mites, and fleas. skin scrapings are beneficial in detecting mites and dermatophytes. dermatophytes are diagnosed best through culture of broken hairs or crust on dermatophyte test medium. use small, cotton-tipped swabs to obtain ear swabs from animals weighing more than g. mix debris with mineral oil and view under low magnification to test for ear mites, or roll onto a glass slide and gram stain to look for bacterial or yeast infections. collect urine by placing the rodent in a clean meshbottomed cage with a plastic liner. after enough urine has been produced, collect it off the bottom of the cage with hematocrit tubes or a syringe and a -gauge needle. perform cystocentesis on non-pregnant animals weighing more than g with a -to -gauge needle. collect feces over several hours to provide a volume sufficient for fecal flotation. flotation allows the detection of nematodes and some trematodes and cestodes. cellophane tape applied to the perineal area and then viewed under a microscope often reveals oxyurid eggs. use a fresh saline smear or fecal sedimentation to diagnose protozoal parasites. fecal cultures are useful in diagnosing bacterial diarrhea. radiology is an extremely useful tool. machines capable of exposures as low as kvp and to mas effectively image mice. most radiograph machines are capable of generating diagnostic radiographs of guinea pigs, chinchillas, and mature rats at settings used for kittens. positioning is accomplished with masking tape or velcro straps. sedate unruly animals. techniques used in cats for contrast studies of both urinary and gi systems are modified easily for use in pocket pets. • use lateral or medial saphenous veins to obtain samples in animals heavier than g. liberally clip the area to allow exposure of the vessel before attempting venipuncture. place a -to -gauge needle in the vein and collect blood into microtainers or hematocrit tubes as it drips from the hub of the needle (fig. - ) . take extreme care not to col-lapse and lacerate the vein with overzealous aspiration if a syringe is attached to the needle. • it is also possible to use the cephalic vein in guinea pigs. • jugular veins are good alternatives in thin individuals under sedation. • an alternative technique that is useful in smaller animals is to coat the skin over the vein with a thin layer of petroleum jelly and then to puncture the vessel. blood is collected with a hematocrit tube as it exits the wound. samples up to % of the animal's weight are considered safe, even in stressed animals. m key point attempt tail bleeding only as a last resort in mice, rats, gerbils, and hamsters. these techniques often are not acceptable to owners. to bleed the tail, warm the tail with water or compresses to dilate the tail vessels. in large rats, perform venipuncture with a needle and obtain blood in the usual fashion. in smaller animals, lacerate the tip of the tail. blood from the wound is collected as described previously. see tables - and - for hematology and chemistry values. incorporate oral medications into a treat, or administer them in liquid form. if the medication is palatable, administer it by placing the tip of a dosing syringe into the diastema. m key point take care not to place the tip into the contralateral cheek pouch, or the patient may store the medication and expel it later. administer medication in small amounts. ensure that the animal swallows the medication in its mouth before more is administered. this technique is useful for force-feeding pellet gruels to anorexic pets if the caregiver is patient. medication or food that is administered too quickly will be spit out or aspirated. for rodents that are intractable or for administration of unpalatable substances, pass a stomach tube per os. • metal feeding needles, red rubber urinary catheters, or infant feeding tubes work well. selection is based on the size of the animal and individual preference. metal feeding needles with ball tips frequently are used in patients weighing less than g . the metal provides the necessary stiffness to pass a tube of small diameter. the ball at the end of the needle makes it difficult (but not impossible) to pass the tube into the trachea. these tubes have the potential to create esophageal tears with improper restraint or when excessive force is applied. • measure the length from the tip of the nose to the last rib. ventroflex the head slightly, and place the tip of the tube through the diastema and over the tongue. if the tube does not pass easily down the esophagus to the premeasured distance, check the tube size and/or reposition the tube before attempting further advancement. the needle is easily palpable percutaneously if it is placed correctly. it is usually safe to administer up to ml/ g body weight. • a flexible catheter is ideal for use as a stomach tube in larger rodents ( fig. - ). use a speculum to prevent chewing on the tube. an otoscope head, avian speculum, or piece of wood or plastic with a hole drilled in the center works well. measure and mark the tube for the distance from the tip of the nose to the last rib. place the speculum in the mouth and over the tongue. pass the tube while holding the speculum in place and slightly ventroflexing the head. resistance is encountered if the tube is malpositioned or is an inappropriate size. the tube must pass over the tongue before it can be advanced down the esophagus. this is difficult in some animals. palpate the throat to confirm the presence of the feeding tube in the esophagus. m key point because the placement of a stomach tube is a blind procedure, administer a small volume of sterile saline into the tube before administering the medication to ensure that the tube is not in the trachea. misplaced medications are fatal. • this method is also useful for administration of nutrition to anorexic patients. place a pharyngostomy tube if repeated dosing is necessary, using the technique for cats. flush pharyngostomy tubes with water at least every to hours. nasogastric tubes are not recommended because they are difficult to place and maintain patency because of their small size. securely suture all tubes to the skin. place a tube collar made of radiographic film or use rear leg hobbles to prevent removal of tubes. • nutritional support is critical in rodents because of their high metabolic rate. provide supplements in animals that are anorexic for longer than hours. if the gi tract is capable of digestion, use a slurry of pellets mixed with a high-calorie supplement. if the tube diameter is too small for this mixture, use avian hand-feeding formula or a mixture of vegetable and cereal baby foods in place of the pellets. if the ability of the gi tract to tolerate enteral feeding is questionable, first try isotonic electrolyte or dextrose solutions. parenteral nutrition is used successfully in research animals and may be feasible in select pet cases. administer sc injectable medications or fluids over the shoulder blades or in folds of skin on the flank. • avoid irritating substances in rats and mice because their mammary tissue extends into these areas. the resulting inflammatory response is thought to increase the occurrence of mammary neoplasia. m key point in general, avoid streptomycin and the carrier procaine in all pet rodents because of a high incidence of toxicity and hypersensitivity reaction. give im injections in the semimembranous and semitendinous muscles. inject only small volumes of nonirritating substances, or tissue damage with resulting self-mutilation may occur. use the epaxial or triceps muscles if repeated injections are necessary. m key point use intraperitoneal (ip) injections only as a last resort for large volumes of fluids or for irritating injections that cannot be administered via an iv or io route. express the bladder and aseptically prepare the abdomen. restrain the rodent with its head down to move the abdominal organs cranially. give the injection . to cm lateral to the midline in the caudal abdomen. aspirate before injecting to ensure that the injection is not being given into the bladder or bowel. never use this technique in pregnant animals. give iv injections into any of the veins as previously described. in addition, the penile vein may be used in hamsters and guinea pigs. placement of iv catheters is possible in animals heavier than g. for small rodents, give a bolus of fluids every - hours, followed by a diluted heparin flush. a pediatric iv pump is used for continuous infusion of fluids to mark distance from nose to last rib larger animals. maintenance of catheters in active animals is extremely difficult. for io injections, place a spinal needle into the proximal tibia or femur following the technique used for placing an intramedullary pin. once the needle is seated, remove the stylet. aspirate and check the hub of the needle for bone marrow. the tip of the needle should be in the bone marrow cavity that directly drains into the central venous system in normal bones (i.e., the cortex must be intact). administer drugs, blood, or fluids at a rate similar to that used for iv catheters. • in chinchillas and guinea pigs, withhold food for hours before anesthesia. withhold food from smaller, mature rodents for hours. withhold food from immature animals for up to / hour depending on age and condition. • use heat lamps and heating pads to prevent hypothermia. have a prewarmed incubator available for recovery. preoperative or intra-operative warmed sc or iv fluids are strongly recommended. place iv or io catheters whenever possible. • administer atropine preoperatively to reduce airway secretions. acepromazine, diazepam, or midazolam work well as premedications for other anesthetics. avoid acepromazine in gerbils because it potentiates seizures. see table - for anesthetic drugs and dosages. surgical anesthesia is reached when toe, tail, and ear pinch fail to generate a withdrawal reaction. depth of anesthesia is best monitored by pulse and respiratory rate and character. pulses drop to within normal ranges after induction. further reduction, especially to less than % of the original stabilized value, is an indication to lighten the plane of anesthesia. monitor the electrocardiogram (ecg) of small patients by clamping the alligator clips onto the hubs of all-metal -gauge needles or steel sutures placed through the skin at the usual sites. tape cables to the table to maintain placement. doppler units taped over the chest also provide accurate heart rates. pulse oximeters are easier to use, more sensitive, and more expensive than the instruments mentioned previously. these instruments are easily taped to the patient's ear, foot, or tail and provide heart rates as well as information regarding oxygenation. respirations are often shallow and rapid during induction. they become deep and regular as a surgical plane of anesthesia is reached. the corneal reflex varies markedly between individuals and anesthetic agents. if the animal has a corneal reflex after induction and then loses it, reduce the anesthetic. induce gas anesthesia using small face masks purchased from laboratory supply houses or make them from syringe cases and latex gloves ( fig. - ) . induction in an anesthetic chamber is also possible. all rodents induced and maintained on gas anesthesia require some form of non-rebreathing system. usual induction is achieved between % and % for isoflurane and % and % for halothane. maintenance for isoflurane and halothane varies from . % to %. there is marked individual variation in the amount of anesthetic required for induction and maintenance. use of % nitrous oxide in oxygen reduces anesthetic concentration requirements for other gases. m key point some chinchillas and guinea pigs hold their breath while being induced with gas anesthetics and then take deep rapid breaths. if the concentration of anesthetic gases is high enough, this behavior results in death. the risk of this behavior is reduced by premedication with tranquilizers, initial induction with nitrous oxide with later addition of primary anesthetic gas after relaxation, and low induction settings. changes in respirations, especially erratic or apneustic patterns and decreased respiratory rates, indicate deepening anesthesia. most pet rodents are not intubated for anesthesia because of their small size. when necessary, as in prolonged oral and other procedures, endotracheal intubation is accomplished with the animal in dorsal or ventral recumbency, depending on the clinician's preference. small non-cuffed or cole endotracheal tubes work well. a stylet usually is required to provide enough stiffness for the tube to pass the larynx. extend the animal's head and neck. grasp the tongue with forceps and use gentle traction. the tip of the tube then is advanced above the tongue and just past its base. the hard palate is used to deflect the tip of the tube ventrally into the glottis. this is a blind procedure that is difficult to master. use of a laryngoscope is helpful in larger rodents. another technique is to place an over-the-needle catheter in the trachea and move it up retrograde through the larynx to act as a guide. the catheter is removed after the endotracheal tube is in place. it is extremely important that the tube be checked for patency. rodents produce copious respiratory secretions, which frequently clog endotracheal tubes. the small diameter allows these tubes to collapse or kink, resulting in asphyxiation of the patient. check patency at least every minutes by applying positive pressure ventilation at to cm water and watching for excursion of the chest wall. if extending the head and neck does not result in air flow, suction the tube. if this is either not successful or impossible, remove the tube and continue anesthesia with a mask or reintubate the animal with a new tube. because of the small diameter of the trachea, endotracheal tube-induced tracheitis and subsequent swelling of the trachea may become a life-threatening situation. doses and routes for injectable anesthetics are listed in table - . needed doses for injectable anesthetics are tremendously variable among species and individuals. most injectable anesthetics provide safe sedation for minor procedures, but very few induce a safe surgical plane of anesthesia on a consistent basis. • ketamine in combination with diazepam is easily obtainable, is given intramuscularly, and has a wide margin of safety, but it does not provide good analgesia. • intraperitoneal injections of barbiturates provide surgical anesthesia but have a low margin of safety and a significant mortality rate. barbiturate anesthesia can result in fatal ileus. euthanasia is performed easily by induction of inhalant anesthetic through a mask or chamber followed by an overdose of barbiturates given intraperitoneally, iv, or intracranially. euthanasia by ip injection of barbiturates alone causes pain in some animals. • surgical techniques for pet rodents are similar to those used in cats and birds. • hemostasis is critical because of small blood volumes. • electrosurgery for incisions and cautery is highly recommended. • if necessary, give fresh blood transfusions drawn from a donor of the same species and mixed with sodium heparin ( iu/ml) at a rate of . ml/ ml of blood directly into an iv or io line. • the lack of a filter creates a potential for thrombosis. • transfusion reactions are possible. administer postoperative analgesics to all rodents undergoing surgical or dental procedures. common analgesics include buprenorphine, butorphanol, ketoprofen, carprofen and meloxicam. see table - for dosages. the most common surgeries are laceration repair and removal of dermal or sc masses. • most rodents will not gnaw on skin sutures. • if this occurs, use steel sutures, subcuticular sutures, or tissue glue. • if an animal still chews at its suture line, physical restraint, such as a tube or an elizabethan collar, is required. castration is a common procedure in guinea pigs. this usually is performed when owners want to house more than one male together or do not wish to breed their female any longer. common abdominal surgeries include cystotomy for urolith removal in guinea pigs and rats, and cesarean section (c-section) in guinea pigs and chinchillas because of dystocia. use a technique similar to those described for dogs and cats. preplaced stay sutures are recommended to define incision edges for closure. use - polyglactin or polydioxanone (pds) on a taper needle and suture in a simple continuous pattern to close the body wall. close the skin with a subcuticular suture (absorbable) or interrupted skin monofilament, nonabsorbable suture. fracture fixation is accomplished best with intramedullary pinning or kirschner apparatus. rodents gnaw on bandages until they remove them. if they are unable to remove a splint, self-mutilation often results in self-amputation. if a cast or splint is necessary, physical restraint often is required. healing usually takes to weeks. incisors can be trimmed with nail trimmers, but this technique often fractures the tooth, causing abscesses of the root. instead, use a high-speed dental burr or a flat cutting disk on a dremel hand tool. trim molars with a high-speed drill or pediatric rongeurs. a mouth speculum that deflects the tongue and other soft tissues is essential to prevent lacerations and provide working space ( fig. - ) . intubate the trachea to prevent aspiration pneumonia when working on molars. if a tooth is abscessed, extract both it and the occlusal tooth. • if necessary, approach cheek teeth via an incision through the cheek. • use a fine dental elevator to loosen the teeth. • patience and firm but gentle pressure are needed, or the root or surrounding bone may fracture. • the roots of the maxillary incisors curve dramatically back into the head. take care to follow the curve of the tooth. • packing an infected tooth socket with a calcium hydroxide paste may decrease the occurrence of persistent infection. remove the paste in to days. • administer meloxicam, carprofen or ketoprofen postoperatively to control pain. see table - for dosages. in chinchillas with dental malocclusion, the roots of the molars can become impacted, causing swelling of the mandible or exophthalmos and epiphora. these teeth are extremely difficult to extract without causing extensive bony and soft-tissue damage. discourage breeding of animals with malocclusion, unless it was acquired as a result of trauma or infection, because this trait is hereditary. most pet and laboratory mice are derived from mus musculus, which is the common house mouse. mice sold in the pet trade are randomly bred and less likely to suffer from the genetic problems associated with inbred laboratory rodents. mice possess brown fat tissues between their scapulae that also are known as hibernating glands; these are thought to provide an energy store. the spleen in male mice is % larger than that of females. ectoparasites usually are found in new acquisitions. • alopecia and pruritus, especially on the back of the head and dorsal midline, usually are associated with lice (polyplax serrata), mites, or fleas. • mite infestation (e.g., mycoptes musculinus, myobia musculini, radfordia affinis) often causes a greasy haircoat and folliculitis. transmission of lice and mites occurs via direct contact. fleas are transmitted by other household pets, such as cats and dogs. • diagnosis is based on clinical signs, history, visualization of parasite, skin scrape, and cellophane tape test. • treatment of fleas and lice is with pyrethrin powder. ivermectin is recommended for treatment of mites (see table - ). • change the bedding and thoroughly clean the cage between treatments to prevent reinfestation. occasionally, the surrounding environment needs to be treated with a premises spray used for killing fleas. • alopecia also may be the result of dermatophytes (see chapter ). lesions are often hyperkeratotic. • diagnosis is made by skin scrape or isolation on culture. most dermatophytes found in pet rodents do not fluoresce under a wood's lamp. • treatment is with lime-sulfur dip or griseofulvin (table - ). • ulcerative dermatitis is a common syndrome caused by staphylococcus aureus characterized by pododermatitis, mastitis, and abscesses in other areas. • administer antibiotics based on culture and sensitivity tests. chloramphenicol is recommended pending culture results (see table - ). the application of hot packs, local drainage, and topical medications are also beneficial in selected cases. • mastitis also may be caused by escheria coli or pasteurella, klebsiella, pseudomonas, or streptococcus species. mastitis usually is caused by poor sanitation, abrasive bedding, or overly aggressive young. • preputial gland abscesses are fairly common in males and are usually caused by e. coli or s. aureus. local flushing and topical treatment are usually adequate. • sc abscesses can be the result of the aforementioned bacteria or actinobacillus spp. or corynebacterium kutscheri. corynebacteria is associated with widespread abscesses, septic arthritis, gangrene, and ulcerated draining tracts. diagnosis is based on finding grampositive pleomorphic rods on gram stain and isolation on culture. • the bacteria are usually sensitive to ampicillin, chloramphenicol, and tetracycline (see table - ). • lymphoma and mammary neoplasia are common causes of sc masses. mammary neoplasia is usually malignant in mice, and metastasis to the lungs is common. (mice have five pairs of mammary glandsthree thoracic and two abdominal.) • obtain thoracic radiographs before surgery. • give a guarded prognosis for long-term survival. • other possibilities for sc masses are fungal granulomas, nodules from the psorergates simplex mite, hematoma, hernia, non-neoplastic lymphadenopathy, or emphysema. • otitis externa usually is caused by ear mites, although bacteria or fungi also may cause primary or secondary otitis. • clinical signs include erythema, pruritus, waxy debris, and excoriations behind the ears. • mites may be diagnosed by identification on otoscopic examination or microscopic examination of ear swabs (see "techniques"). • treatment requires cleaning debris from ears with a commercial ear cleanser followed by administration of three doses of ivermectin at -week intervals or topical acaricides used daily for to weeks (see table - ). • bacterial and fungal otitis is diagnosed by identification of organisms or gram-stained specimens and isolation on culture. • treatment is similar to that used in cats. • otitis media/interna usually are caused by hematogenous spread or local invasion of bacteria from a primary abscess. • clinical signs include head tilt, circling, facial nerve paralysis, and otitis externa. • rule out mouse hepatitis virus as the cause of the head tilt (see "gastroenterology"). • if treatment of the primary disease is successful, the otitis media usually resolves, although a residual head tilt may persist. • if a cluster of pseudomonas infections occurs in a population, evaluate the water source and produce for contamination. use sodium hypochlorite in the drinking water at ppm to control an outbreak while water quality is being restored. • damage to the pinnae can be associated with trauma, dermatitis, pox virus, hypersensitivity reactions, and vasculitis. dry gangrene is a common sequela and is usually self-limiting. i have observed a steroid-responsive pruritus in pet mice. the pruritus is severe enough to result in significant self-mutilation. this condition has been nonresponsive to treatment with ivermectin, lime-sulfur dips, griseofulvin, multiple antibiotics, oral prednisolone, and antihistamines. attempts at bacterial and fungal culture have failed to identify a pathogen. an inflammatory response is observed on histologic examination. mice with this condition respond to repository methylprednisolone injections every to weeks ( . mg/kg im). most owners have not elected to continue injections for longer than a few months. once the injections are stopped, the pruritus returns, often requiring euthanasia of the affected mouse. • bilateral alopecia found around the muzzle associated with no other abnormalities usually is caused by friction from overhead feeders. • alopecia occurring in smaller, weaker individuals is often the result of barbering. removal of the mice in best condition from the cage results in normal appearance of barbered mice in to weeks. • tailhead alopecia and scabbing are usually the result of aggression. separate affected animals, or additional trauma may occur. • other rare causes of alopecia are endocrinopathies, leprosy, and hereditary alopecia in nude mice. • epiphora is a common condition of pet mice. the most common causes in pets owned for longer than months are ammonia fumes and overgrown incisors. • ammonia causing contact irritation is diagnosed by examining an uncleaned cage and checking for odor. • treatment is improved sanitation. • overgrown incisors are diagnosed easily by oral examination. treat by trimming the affected teeth and providing opportunities for gnawing. • foreign bodies and the resultant corneal ulcers can cause epiphora. an eye examination, including fluorescein stain, is indicated. treat by removing the foreign body and administering an ophthalmic antibiotic (gentamicin, tetracycline, or chloramphenicol in affected eye, q h-q h). • in newly acquired pets, epiphora is often the first clinical sign of an upper respiratory infection. pasteurella pneumotropica is the most common pathogen, although salmonella spp., mycoplasma, sendai virus, lymphocytic choriomeningitis, and mouse pox also may cause epiphoria. the ocular discharge later appears mucopurulent (see "respiratory"). retinal degeneration can be either hereditary or (in albino mice) may be caused by exposure to highintensity lighting. the resulting blindness often goes undetected because patients adapt well and behave normally in their cages. • clinical signs include dyspnea (often described as chattering), mucopurulent oculonasal discharge, hunched posture, and anorexia. animals with a chronic history of this disease are often cachexic. • radiology aids in determination of the extent and severity of the pneumonia and the absence or presence of distant foci of infection. • treatment with tylosin is successful in controlling the disease if it is not too advanced. tetracycline, enrofloxacin, and chloramphenicol also have been used (see table - ). • many patients need nutritional support. • mice with pyometra or other abscesses require surgical debridement. • recovered animals are carriers and stress elicits clinical signs. strictly quarantine these animals. a common cause of pneumonia in newly acquired mice is sendai virus. acute fatalities are seen in suckling or weanling mice. • transmission is by aerosol or direct contact. • clinical signs in adults are caused by secondary bacterial infections and are similar to those in mrm. • diagnosis is based on clinical signs and serologic testing. • treat with antibiotics to control the secondary bacterial infection and provide supportive care as needed. • prohibit breeding for to weeks. • a killed vaccine is available. • recovered animals are resistant to new infection. common primary or secondary pathogens causing respiratory signs in mice are streptococcus pneumoniae, corynebacterium kutscheri, pasteurella pneumontropica, pseudomonas aeruginosa, and klebsiella pneumoniae. treatment is empiric or based on culture and sensitivity of a tracheal swab sample. dyspnea often is caused by metastasis to the lungs from mammary adenocarcinomas. primary lung tumors, especially pulmonary adenomas, also occur frequently. although not frequently diagnosed, cardiac disease can result in signs of respiratory disease. diagnosis is based on radiographic evidence of cardiomegaly and pulmonary edema. • tapeworms usually do not cause clinical signs. occasionally, heavy burdens may cause diarrhea or weight loss. the chief concern is the zoonotic potential of one species, hymenolepis nana, which is directly transmissible to humans. • diagnosis is made from visualization of eggs in the feces. treat with praziquantel or niclosamide (see table - ). improve sanitation, and remove indirect hosts (e.g., fleas, beetles, roaches). • pinworms (syphacia obvelata, aspiculuris tetraptera) may cause anal pruritus and, in severe cases, rectal prolapse. • diagnosis is based on clinical signs and observation of eggs on cellophane tape after application to the perineal region. treat with piperazine or mebendazole every to days for three treatments or with fenbendazole once daily for days (see table - ) and provide improved sanitation. • the protozoal parasite spironucleus muris causes diarrhea and slow growth associated with a pot-bellied appearance in young mice. • diagnosis is by fecal wet mount, although falsenegative findings are common. • treat with oxytetracycline (see table - ). supportive care to combat dehydration and hypothermia is extremely important. • control is achieved with improved sanitation. • giardia spp. and, rarely, eimeria falciformis show signs similar to spironucleus. giardiasis is zoonotic. treat with metronidazole. treat eimeria with sulfadiazine/trimethoprim (see table - ). most other protozoa are considered nonpathogenic. • cysticercus fasciolarus causes nonpathologic cysts of the liver. these cysts are the infective form of taenia taeniaformis in carnivores. viral diseases • diagnosis is based on clinical presentation, serology, and presence of syncytial giant cells in the epithelium of the small intestine. • treat supportively, and quarantine affected individuals. the prognosis is grave. • although less commonly seen in pet mice, reovirus occurs in older suckling mice. it is characterized by an oily diarrhea, which results in a greasy haircoat. other signs are conjunctivitis, stunted growth, and tremors. transmission is by ingestion. • diagnosis is based on clinical signs, histology, and serology. • treat supportively. the long-term prognosis is grave. initial survivors are weak and jaundiced, suffer from alopecia, and eventually die. • transmissible murine colonic hyperplasia (mch) caused by citrobacter freundii is characterized by diarrhea followed by rectal prolapse and stunted growth. transmission is feco-oral. • diagnosis is made by clinical signs and fecal culture. • treat with neomycin, tetracycline, or sulfamethazine until sensitivity results are available (see table - ). severe thickening of the distal half of the colon is observed at necropsy. • salmonellosis, also known as mouse typhoid, is transmitted by latent carriers or contaminated feed or bedding. incubation lasts for to days. • clinical signs are lethargy, anorexia, purulent conjunctivitis, arthritis, and diarrhea. • diagnosis is based on clinical signs and fecal culture. treat supportively. use of antibiotics is controversial. • quarantine survivors. • sanitation is extremely important because salmonella spp. are zoonotic. on gross postmortem examination, erythema of distal ileum and congestion of the spleen and liver are seen. with more chronic infections, necrotic foci are seen in the liver, spleen, and lymph nodes. prevent infection by feeding a fresh laboratory chow from a reputable source. thoroughly wash all produce and then dip it in a diluted bleach solution. rinse completely before feeding. • tyzzer disease is caused by bacillus piliformis. transmission is feco-oral. • clinical signs are precipitated by stress and consist of anorexia, diarrhea, and high mortality in weanlings. • diagnosis is made by clinical signs or isolation on culture. enteritis and multiple gray-yellow necrotic foci in the liver are seen on gross postmortem examination. • administer tetracycline for to days (see table - ) and reduce stress to control the disease. breeding systems vary; from one to six females may be placed with one male. all animals are housed together, and the young are removed after weaning. females in proestrus have swollen vulvas. vaginal plugs are present post-copulation. female mice that have been bred within days abort if a new male is placed in the cage. inappropriate light cycle, inappropriate age, crowding, and poor nutrition are the most common causes of infertility in pet mice. pyometra due to pasteurella pneumontropica, mycoplasma spp., or other bacteria is also common. desertion of litters is usually a result of stress, lack of nesting materials, agalactia, or mastitis. • urethral obstruction from proteinaceous plugs of inspissated ejaculum may develop in aged male mice. pseudomonas, e. coli, or proteus are the most frequently cultured pathogens. before complete obstruction, chronic hematuria may be noticed by the owner. • antibiotics, which are chosen based on the results of urine culture, are often curative with early presentation. complete obstruction requires surgical removal. • glomerulonephritis is very common in geriatric mice. it frequently is secondary to chronic viral infection. clinical signs are anorexia, lethargy, dehydration, and cachexia. urinalysis demonstrates proteinuria. as the disease progresses, the urine becomes isosthenuric, the blood urea nitrogen (bun) and creatinine levels rise, and other electrolyte abnormalities typical of chronic renal failure occur. treat supportively. prognosis for long-term survival is grave. • coccidia (e.g., klosseilla muris) occasionally is found in the urine. the clinical significance of its presence is unknown. • mice can be asymptomatic carriers of leptospirosis; however, this is rarely seen in pet mice. • diagnosis is based on darkfield microscopy of urine, serology, or histopathology. euthanasia of carriers is recommended. • infectious polyarthritis or mouse rheumatism is caused by streptobacillus moniliformis. in humans, it is known as rat bite or havernill fever. transmission is by direct contact. clinical signs are cachexia, keratitis, edema and ulceration of the appendages, and ankylosing arthritis. • diagnosis is based on the bacterial culture findings or the presence of caseous pericarditis and arthritis on necropsy. • treat with antibiotics chosen through the results of culture and sensitivity tests. use penicillin while awaiting results. supportive care is important. animals that recover remain arthritic. control is achieved through quarantine and sanitation. • the most frequently diagnosed neurologic disease in pet mice is head tilt resulting from bacterial otitis media (see "otitis"). the second most common cause of neurologic signs is trauma. • diagnosis is based on history and clinical signs. consider neoplasia in aged mice with slowly progressive signs. • lymphocytic choriomeningitis is a zoonotic arenavirus. transmission is airborne, transplacental, or by direct contact, fomites, or insect vectors. acute signs usually occur in mice that are to weeks old. approximately % of infected individuals show acute clinical signs, which include lethargy and photophobia followed by convulsions and paralysis. in animals that are latently infected, glomerulonephritis develops later. mice infected after weaning and before year in age lose weight, appear arthritic, and show signs of conjunctivitis and photophobia. the virus runs its course in several weeks. animals that recover show no residual signs. • diagnosis is based on clinical signs and the presence of immunofluorescent antibody (ifa). pleural effusion, splenomegaly, and hepatic lipidosis are found on necropsy. treat supportively. house survivors separately. • prevent the disease by improving sanitation, providing pest control, and cleaning produce. consider euthanasia because of the zoonotic potential of the virus. • mouse poliomyelitis/encephalomyelitis, also known as theiler disease, causes clinical signs in in , infected mice. two-thirds of healthy mice are carriers. transmission is by oral or respiratory routes. mice younger than weeks of age show signs of encephalitis. animals that are to weeks old are weak in the rear legs and progress to paralysis. the tail may remain mobile. affected mice continue to eat and be alert. albino mice are predisposed to show clinical signs. • diagnosis is based on clinical signs, serology, or histopathology that shows necrosis of the ganglionic cells of the anterior horn of the spinal cord. • treat supportively. consider euthanasia because of poor prognosis. • seizures in mice commonly result from otitis media, trauma, liver or kidney failure, toxin, bacterial meningitis, neoplasia, or viral encephalitis. • leukemia in mice is usually viral in origin. transmission is trans-mammary or trans-placental. • clinical signs are anemia, dyspnea (with thymic involvement), and those signs that are compatible with chronic disease. • diagnosis is based on complete blood count (cbc), bone marrow aspirate, or histopathology. prognosis is grave. • eperythrozoon coccoides is a rickettsial red blood cell (rbc) parasite of mice. affected mice are usually asymptomatic. occasionally, fever, anemia, and splenomegaly develop in infected animals. transmission is through the louse polyplax serrata. control is by extermination of the louse. • treat with tetracyclines. pet rats are derived from the norwegian or brown rat (rattus norvegicus), which did not originate from norway, but from asia. breeds of rats are called strains when they are inbred extensively and stocks when strains are hybridized. rats have brown fat, as discussed in the section on mice. they do not possess a gallbladder. their mandibular symphysis is articulated normally. rats are neophobic; therefore, make gradual changes in food or environment when possible. • fleas, mites (e.g., radfordia ensifera, ornithonyssus bacoti), lice (i.e., polyplax spinulosa), ear mites (i.e., notoedres muris), and dermatophytes cause similar signs in both mice and rats. treatment also is similar (see "mouse"). • sc masses in rats are similar to mice. pasteurella pneumotropica is a very common pathogen in mastitis and sc abscesses. • treat with chloramphenicol until culture results are available (see table - ). • mammary cancer develops in % to % of adult female rats and in approximately % of male rats. always submit biopsy specimens for histologic examination. most, but not all, of these tumors are fibroadenomas, which are benign. prognosis for longterm survival after surgical removal is good. other common neoplasms include interstitial cell tumors of the testes, which cause sc swellings in the inguinal region, and squamous cell carcinomas of the zymbals gland of the external ear canal. • ulcerative dermatitis occurs in rats as well as mice. staphylococcus aureus is the causative agent. c. kutscheri follows a similar course in rats and mice (see "mouse"). • ringtail is the formation of constrictive bands of fibrous tissue around the tail in nestling rats. these bands result in gangrene of the distal tail. this disease occurs when environmental humidity is less than %. • treat by making a longitudinal incision of the ring to release the stricture and apply topical dimethyl sulfoxide (dmso), steroid, and antibiotic solution ( ml dmso, ml mg/ml amikacin, ml mg/ml dexamethasone) four times daily. • to prevent ringtail, keep humidity above %, use solid-bottom cages and provide ample nesting material. prognosis for life is excellent. prognosis for retention of the distal tail is guarded. • epiphora and blepharospasm are caused mostly by ammonia fumes, overgrown incisors, or foreign bodies (see "mouse"). • sialodacryoadenitis virus is a coronavirus that is endemic in many rat populations. • clinical signs vary from mild keratoconjunctivitis to blepharospasm, chromodacryorrhea, severe uveitis, hyphema, buphthalmos, periorbital swelling, and pneumonia. the clinical course of the disease lasts to days. rats maintain normal activity levels and appetite. • treatment is not necessary for mild infections. place rats showing marked ocular disease or discomfort on the appropriate ophthalmic ointments (e.g., atropine, antibiotic, steroid) based on presentation. administer parenteral antibiotics to animals that show signs of respiratory problems. recovery is usually complete unless the eye ruptures or selfmutilation occurs. • control is achieved by not introducing new animals for weeks. • in contrast to mice, sendai virus rarely causes clinical signs in rats. • mucopurulent ocular discharge also may be caused by infection with mycoplasmosis, streptococcus pneumoniae, pseudomonas spp., and other less common bacterial or viral agents that cause pneumonia. • cataracts are primary hereditary defects or occur secondary to severe uveitis or diabetes mellitus. retinal dystrophy and colobomas are also inheritable traits in rats. retinal degeneration occurs in rats housed under intense lighting. • mrm is extremely common in pet rats. its presentation is similar to the disease in mice (see "mouse"). • streptococcus pneumoniae is normal flora for rats. however, during stressful situations, bacteremia may occur, resulting in pneumonia. clinical signs are similar to mrm. differentiation is based on culture and the presence of extensive fibrinopurulent pleural effusion on necropsy. • ampicillin controls clinical signs if treated early in the course of disease (see table - ). prevent the condition by minimizing stress. • corynebacterium kutscheri and pasteurella pneumotropica cause signs similar to mrm (see "mouse"). there is a serologic test for c. kutscheri. see the mouse section for a discussion of pseudomonas aeruginosa. • pneumocystosis carinii is an uncommon protozoa that infects the lung. cysts and trophozoites live in the alveoli. clinical signs occur only in immunocompromised or geriatric individuals. signs are cachexia, cyanosis, and dyspnea. • diagnosis is based on clinical signs, tracheal wash, response to therapy, or histologic examination. • treat with sulfadiazine/pyrinrethamine (see table - ). • myocardial degeneration and subsequent congestive heart failure are fairly common in geriatric rats. diagnosis is based on radiographs of the thorax and clinical signs. treat supportively, and use furosemide and digitalis at cat dosages to alleviate pulmonary edema. • polyarteritis nodosa is an idiopathic condition of geriatric rats that results in thickening and tortuosity of arteries, especially in the mesentery, pancreas, and testicles. affected areas are predisposed to clot formation and aneurysms. • nematode (syphacia muris), cestode, and intestinal protozoal parasite infestations are similar to those in mice. • capillaria hepatica has no clinical significance. yellow streaks on the liver are an incidental finding at necropsy. the causes and treatment of malocclusion are similar to those for mice. • epizootic diarrhea of suckling rats is a viral disorder found in rats to days old. the infection causes a mild diarrhea. most animals recover. occasionally, stunting occurs. treat supportively. • salmonellosis in rats is similar to that in mice. • if breeding is desired, take females showing signs of estrus (e.g., lordosis, hyperactivity, quivering ears, and swollen vulva) to a male rat's cage for hours, or keep one male in a cage with up to six females. check females for a post-copulatory plug to confirm breeding. remove females just before parturition, and house females individually while raising the young. a vaginal discharge is seen . to hours before labor. parturition is accompanied by stretching and extension of the rear legs. all neonates usually are delivered within to hours. • two extremely common conditions in geriatric rats are nephrocalcinosis and chronic progressive nephropathy. clinical signs are compatible with those of chronic renal failure. enlarged or small irregular kidneys may be found on physical or radiographic examination. isosthenuria and marked proteinuria are found in urinalysis. • definitive diagnosis is based on renal biopsy. • treat supportively. prognosis for long-term survival is grave. • trichasomoides crassicauda is an uncommon parasite of the urogenital tract. the adult worms usually reside in the kidney, but they occasionally may wander into the genital tract. the ova are passed in the urine. • clinical signs are hematuria and stranguria. proliferative mucosa of the bladder occasionally may be palpated as an abdominal mass. • treatment is somewhat successful with methyridene (see table - ). sanitation is critical in control of this disease. • klossiella muris is an incidental coccidia of the urinary tract. • many geriatric pet rats have chronic progressive radiculoneuropathy. • clinical signs are compatible with cauda equina syndrome, including posterior paresis progressing to paralysis, urine retention, and incontinence. prognosis is grave. • treat supportively or euthanize. • streptobacillus moniliformis, a normal bacteria found in the oral, nasal, and pharyngeal cavities of rodents, is isolated from % of middle ear infections and % of chronic pneumonias in rats. the bacteria is nonpathogenic for gerbils and guinea pigs. • clinical signs vary with the site of infection. head tilt and circling, septic arthritis, and respiratory disease commonly are seen. • diagnosis is based on isolation on culture. the clinical signs mimic many other diseases, especially mrm and pseudomonas infection (see "mouse"). • head tilt in rats also may be the result of trauma or neoplasia, especially pituitary adenomas. • hemobartonella muris is an rbc parasite of rats that is nonpathogenic unless the rat is immunocompromised or splenectomized. transmission is through the louse polyplax spinulosa. • clinical signs result from hemolytic anemia and hemoglobinuria. • treat with tetracyclines (see table - ). mesocricetus auratus, better known as the golden or syrian hamster, is a primarily nocturnal rodent that originated in the middle east. almost all hamsters in the united states are the offspring of three siblings imported in the s. many color variations are available. long-haired hamsters are called "teddy bear" hamsters. the stomach has two compartments, a non-glandular forestomach, which functions like a rumen, and a glandular stomach. hamsters are very territorial. they possess flank glands, which are larger in males, that are rubbed against objects to mark their territory. females are larger than males. except during estrus, they use this size advantage to attack males. do not allow groups to estivate together or recently awakened animals may cannibalize sleeping hamsters. m key point hamsters are extremely sensitive to antibiotics. penicillins, clindamycin, lincomycin, streptomycin, tylosin, erythromycin, and cephalosporins eliminate the normal intestinal flora, allowing overgrowth of pathogenic bacteria, particularly clostridium difficile. diarrhea, which is almost always fatal within to days, subsequently occurs. even antibiotics considered to be safe can have this effect. treat by discontinuing antibiotics, providing a lactobacillus supplement, and giving supportive therapy. • hamsters are susceptible to demodex criceti and d. aurati mites. d. criceti is limited to skin folds. d. aurati causes hyperpigmentation, alopecia, and seborrhea sicca affecting the dorsal midline. demodex is carried by many normal-appearing hamsters. • clinical signs occur in immunosuppressed animals, as would occur with stress, chronic infection, pregnancy, or malnutrition. • diagnosis is based on clinical signs and deep-skin scrapings. • treat with amitraz every weeks for two treatments past two consecutive negative skin scrapings. use the manufacturer's recommended dilution for dogs. • sarcoptes mites infrequently cause facial alopecia. diagnosis is based on skin scraping. treat with ivermectin (see table - ). do not confuse this condition with alopecia caused by contact with feeders or barbering. • notoedres mites affect only the external ear canal in female hamsters but may affect the ears, feet, geni-talia, and tail in males. diagnosis is made by observation of mites on samples from ear swabs, skin scrapings, or both. treat with ivermectin (see table - ). • other less common causes of alopecia in hamsters are dermatophytosis, endocrinopathies, and genetic defects. • dermal sc masses are usually abscesses caused by pasteurella pneumotropica, s. aureus, or streptococcus spp. treatment is based on results of culture and susceptibility testing. use chloramphenicol until culture results are available. other frequent causes of sc swellings are distended cheek pouches and testicles, mastitis, hernias, neoplasia, and lymphadenopathy. • epiphora and conjunctivitis are caused most frequently by increased environmental ammonia concentrations, incisor overgrowth, foreign body, or lymphocytic choriomeningitis (see "rat"; "mouse"). • mucopurulent discharge is caused by secondary infection by pasteurella or streptococcus spp. • hamsters are predisposed to rupture of the eye following trauma or infection. surgical enucleation is advised. electrosurgery is extremely helpful in controlling bleeding but do not apply heat to the stump of the optic nerve or vessels, or thermal injury to the brain may result. place gelfoam in the socket to enhance clot formation. • hamsters are susceptible to viral respiratory infections of humans. • clinical signs include nasal discharge, sneezing, otitis media, fever, and pneumonia. uncomplicated cases last to days. complications are usually the result of secondary bacterial infections. • treat supportively. use of antibiotics is indicated if copious nasal discharge, dyspnea, anorexia, or marked lethargy is observed. overuse of antibiotics may cause diarrhea-related death in hamsters that might have recovered uneventfully if left untreated. • most dyspnea in hamsters is caused by blunt thoracic trauma. hamsters often bite when startled. reflex actions on the part of humans, especially children, cause hamsters to be flung against hard objects. • diagnosis is by history and presence of fresh epistaxis. • treat supportively. emergency shock therapy, consisting of supplemental heat, oxygen administration, parenteral fluids, and glucocorticoids, frequently is required. • sendai virus can cause death in suckling hamsters housed with mice. adults show no clinical signs (see "mouse"). • primary bacterial pneumonia most frequently is caused by yersinia pseudotuberculosis, pasteurella pneumotropica, or streptococcus. clinical signs are compati-ble with those of pneumonia seen in other species, as well as weight loss and conjunctivitis. all three agents have a tendency to form distant abscesses, especially in the uterus. • diagnosis is based on clinical signs and isolation on culture. • treat with chloramphenicol until antibiotic susceptibility results are available. abscesses require surgical debridement; however, anesthesia in affected animals is very risky. recovered hamsters are carriers and must be quarantined from other rodents. prognosis is guarded. • cardiac thrombosis is seen in % of geriatric hamsters. most thromboses occur in the left atrium and are secondary to degenerative cardiomyopathy, cardiac amyloidosis, sepsis, or calcification of the great vessels. congenital myocardial necrosis also occurs. • clinical signs include cyanosis, dyspnea, and acute death. enlargement of the cardiac silhouette and pulmonary edema sometimes can be seen on thoracic radiographs. • furosemide and digitalis (using standard cat doses) may temporarily alleviate clinical signs. • hamsters can carry the zoonotic tapeworm h. nana (see "mouse"). • treat with niclosamide or praziquantel (see table - ) and provide improved sanitation. • pinworms (aspicularis tetraptera, syphacia muris, s. obvelata) occur in hamsters as well as in mice. • treat with fenbendazole (see table - ). • hamsters are predisposed to dental caries. a large percentage of affected teeth become abscessed, causing facial swelling, ptyalism, and anorexia. • diagnosis is based on clinical signs, oral examination, skull radiographs, and isolation on culture. • extract the tooth and administer antibiotic therapy based on results of susceptibility testing. prognosis is variable depending on the condition of the animal, tooth affected, and extent of the abscess (see "mouse"). • overgrown incisors also occur, as in mice. • the cheek pouches are very distensible. impaction of the pouches occurs on occasion. • clinical signs vary from ptyalism to swelling from abscess. in simple cases, removal of the material from the pouch with fine forceps is sufficient. sedation usually is not required. • if a fungal or bacterial infection of the pouch is present, remove the exudate, submit samples for gram staining and bacterial or fungal culture, and flush the pouch with diluted iodine solution. if cellulitis is present, administer systemic antibiotics as well. fistulas often heal spontaneously. • proliferative ileitis (i.e., wet-tail disease) is thought to be caused by a campylobacter-like organism with or without concurrent bacterial or viral infections. more than % of animals with clinical signs die. the highest morbidity and mortality rates occur in hamsters to weeks of age. teddy bear hamsters may be more susceptible to infection than shorter-haired varieties. transmission is feco-oral. • clinical signs include diarrhea, which mats on the ventrum and perineum, and results in anorexia, dehydration, and a hunched posture. the abdomen frequently seems painful on palpation. bowel loops often are distended on palpation because of ileal obstruction or intussusception. rectal prolapse usually occurs. • administer neomycin, gentamicin, metronidazole, or tetracycline (see table - ). supportive care is critical. prognosis is grave, even with treatment. gross postmortem findings include gas and yellow diarrhea in the distal intestinal tract, mucosal thickening in the ileum and distal jejunum, peritonitis, and liver abscesses. • other common causes of bacterial diarrhea include e. coli, tyzzer disease, or salmonella spp. (see "mouse"). in hamsters older than year of age, liver cysts that are derived from the biliary duct often develop. less frequently, similar cysts arise from the pancreas, epididymis, and seminal vesicles. this syndrome is called polycystic disease. no clinical signs are associated with cysts in these structures, which are an incidental finding on abdominal palpation. no treatment is recommended. • timing is critical to prevent injury to the male when breeding hamsters. transfer the female to the male's cage in the early evening days after a creamy, viscous vaginal discharge is noticed. monitor the pair carefully. remove the male immediately if the female is aggressive. remove the male after mating or after to hours even if mating has not occurred. two days after successful copulation, a gray malodorous vaginal discharge is observed. pregnancy is highly likely if there is no translucent vaginal discharge to days post-breeding. pseudopregnancies last to days. normal gestation is to days. before par-turition, a hemorrhagic vaginal discharge appears, and the female may pant. hamsters rarely suffer from pregnancy toxemia (see "guinea pig"). • infertility may be caused by pyometra (see p. pneumotropica and lymphocytic choriomeningitis). • cannibalism is most frequently a result of stress or mastitis. • in almost % of geriatric hamsters, renal amyloidosis develops. the disease tends to develop more rapidly in females. • clinical signs include edema and ascites due to protein loss in the urine, as well as the typical signs of chronic renal failure. • treat supportively. prognosis for long-term survival is grave. • head tilt is usually secondary to otitis media. also consider lymphocytic choriomeningitis or neoplasia as differential diagnosis (see "rat"). • in hamsters fed all-seed diets and deprived of exercise, cage paralysis syndrome often develops. usually pets are presented for acute posterior paresis which, in reality, was slowly progressive. the distinction is important in ruling out trauma. in mild cases, the hamster is able to move its hind limbs but unable to support weight. vitamin d and e supplementation, along with nutritional improvement and providing exercise, is curative in to weeks. in severe cases, recovery is negligible or incomplete. lymphoma and lymphosarcoma may be viral in origin. diagnosis is made by biopsy or fine-needle aspiration of affected lymph nodes. rule out lymphadenopathy caused by lymphadenitis from infection with streptobacillus moniliformis (see "rat"). although many hamsters initially respond well to chemotherapy protocols established for cats and dogs, prognosis for long-term survival is grave. • rarely, demodex spp. mites cause alopecia in gerbils. • diagnosis is based on skin scraping. • treat with rotenone ointment or amitraz dips every weeks for three to six treatments. use manufacturer's recommended dilution for dogs. • acute moist dermatitis usually is caused by s. aureus infection. infection on the face often begins with the harderian glands. the gland secretion is viscous and causes matting, with secondary staphylococcal infection occurring under the mats. attempts at grooming spread the infection to the feet and abdomen. • diagnosis is based on clinical signs and isolation on culture. • administer enrofloxacin, tetracycline, or chloramphenicol and apply warm, moist compresses to remove dried debris. remove possible irritants from cage (e.g., pine or cedar shavings, ammonia). occasionally, surgical removal of a chronically infected or inflamed gland is needed. • alopecia of the facial area, especially when it is symmetric, is usually the result of self-trauma from feeders, cage bars, or overzealous burrowing. • treat by changing cage construction or providing better visual security. • gerbils that catch their tails in crevices or are restrained inappropriately by their tails often are presented for avulsion of the skin from their tails. • treat initially by controlling hemorrhage and hypovolemic shock. • amputate the tail after patient stabilization to prevent ascending infection. in some animals, the infection is localized to the distal tail, which is sloughed in approximately to weeks. • generalized alopecia is normal in some weanling gerbils. the hair grows in as the animals mature. • melanomas are found most frequently on the ears, feet, or base of the tail. • diagnosis is based on biopsy. • treat by surgical removal. • sebaceous gland disease is usually the result of bacterial infections or neoplasia. • diagnosis is based on cytologic examination, culture, histologic examination, and response to antibiotic therapy. • treat bacterial infections with parenteral or topical antibiotics based on the severity of signs. • sebaceous gland adenomas, basal cell tumors, and squamous cell carcinomas are the most frequently encountered neoplasms. • take a radiograph of the thorax to diagnose metastases. prognosis for long-term survival is based on tumor type, stage, and character. • treat by surgical excision. chromodacryorrhea and epiphora occur as in mice. tapeworms (i.e., h. nana and h. diminuta) and pinworms (i.e., syphacia obvelata, dentostomella translucida, and aspicularis tetraptera) occur as in mice. incisor overgrowth occurs as in mice. • salmonella spp. cause transient diarrheas in gerbils. the source of infection is usually unwashed greens, contaminated feed, or carrier rodents of another species. most recover. animals that die have a fibrinosuppurative peritonitis. • treat supportively. use antibiotics in severe cases based on results of culture and susceptibility testing. • tyzzer disease, caused by bacillus piliformis, is seen most often in weanlings at to weeks of age and in post-partum females. • clinical signs include anorexia, lethargy, rough haircoat, and sometimes diarrhea. gross postmortem findings include yellow-gray nodules in the liver and hemorrhage at the ileocecal junction. • diagnosis is based on postmortem examination or response to therapy. • treat with oxytetracycline (see table - ) and supportive care. hepatic lipidosis and gallstones are frequent sequela to lipemia in gerbils fed diets with excessive fat. • breeding is most successful if animals are paired at weaning and kept in these pairs. male gerbils aid in raising the young. pairing older animals causes fighting. an average of % of neonates fail to survive to weaning. this is usually the result of agalactia and crushing. • chronic hemorrhagic discharge from the vulva is usually the result of cystic ovaries or ovarian tumors. most tumors occur in animals older than years of age and consist of granulosa cell tumors or theca cell tumors. leiomyomas of the uterus also cause similar clinical signs. • rule out urinary tract disease by performing a urinalysis via cystocentesis. large masses may be visualized on abdominal ultrasound. definitive diagnosis is based on vaginal cytology followed by exploratory laparotomy. • ovariohysterectomy is curative for cystic ovaries and tumors if they have not metastasized. • chronic renal failure develops in most gerbils older than . years of age. • clinical signs are polyuria, polydipsia, weight loss, and anorexia. urinalysis demonstrates proteinuria, hematuria, casts, and an increase in white and red blood cells. • treat supportively. prognosis for long-term survival is grave. • up to % of gerbils in certain family lines suffer spontaneous epileptiform seizures. the seizures are induced by stress and are self-limiting. seizures usually start as the gerbil reaches months of age. • treatment is unnecessary. chinchilla laniger and c. brevicaudata are nocturnal rodents from the andes mountains in south america. most animals kept in the united states are the descendants of animals. aside from pets, chinchillas are raised commercially for their pelts. the most common coat color is gray; the most valuable coat color is black. m key point chinchillas are sensitive to antibiotics (see "hamster"); therefore, avoid use of penicillins, lincomycin, erythromycin, and cephalosporins. house chinchillas in a cool environment because they are prone to overheating. if heat stroke occurs, treat with tepid water baths and supportive therapy. • chinchillas require dust baths to keep their skin in condition. use commercially available chinchilla dust only. sand substitutions do not condition the coat and occasionally cause conjunctivitis. offer dust at least once a week. • dermatophytosis occurs as in guinea pigs. • fur chewing is a serious problem in chinchillas that are farmed for pelts and often is seen in pet chinchillas that are recent culls from a ranch. the etiology of fur chewing is unknown. some cases seem to be related to chronic disease, malnutrition, poor caging, or stress. theories for undiagnosed cases include genetic abnormality; undiagnosed dermatophytosis; or adrenal, pituitary, or thyroid gland abnormalities. • diagnostics such as skin scraping, fungal culture, fecal, cbc, history profile, and biopsy are recommended. in general, if changes in diet and husbandry do not elicit a response or an underlying treatable disease condition is not discovered, prognosis for cure is grave. • one source advocates plucking all remaining underfur in chewed areas in an attempt to stimulate new hair growth. place collars after this procedure until the fur has grown in completely. • cystic sc masses may be caused by the intermediate stage of multiceps serialis. transmission is by ingestion of feed contaminated with canine feces. • diagnosis is made by histopathologic or cytologic examination of tissue samples. treat by surgical removal of the masses. • otitis caused by pseudomonas spp. occurs as in rats. • conjunctivitis occurs as in mice. • cataracts are congenital or developmental. • asteroid hyalosis occurs as a degenerative change. pneumonia occurs as in guinea pigs. parasites tapeworms (i.e., h. nana) occur as in mice. malocclusion of incisors and cheek teeth occurs as in guinea pigs. • diarrhea is caused most often by coccidia or giardia spp. or a bacterium. • clinical signs range from soft stools and weight loss to fluid diarrhea, dehydration, bloating, septicemia, and sudden death. • the protozoal parasites are best diagnosed on fresh saline smear or necropsy. • bacterial diarrhea is most often caused by contaminated feed and is diagnosed by isolation on culture. clostridium spp., pseudomonas aeruginosa, e. coli, salmonella enteritidis, and pasteurella spp. are the most common isolates. • treat supportively and use appropriate antiprotozoal or antibiotic drugs. • pasteurella pseudotuberculosis causes acute death from septicemia or a chronic weight loss with intermittent diarrhea. enlarged mesenteric lymph nodes are a hallmark of this disease. • diagnosis is based on clinical signs, histopathologic examination of tissue samples, and isolation on culture. • treat with sulfa drugs until sensitivity results are available. prognosis for recovery is poor. gross postmortem examination reveals yellow to white necrotic foci in the liver. • check male chinchillas four times per year for penile hair rings. roll back the prepuce and expose the penis. roll hair rings off the penis after application of a water-soluble lubricant. treat ulcerations topically or systemically as needed. • dystocia is fairly common in chinchillas (see "guinea pig"). • metritis is suspected when post-partum vaginal discharge, failure to return to a normal estrus cycle, anorexia, weight loss, polydipsia, polyuria, and chewing at flank and abdomen are present. • diagnosis is based on history, physical examination, abdominal radiographs, culture, ultrasound, and cbc. it usually is caused by bacteria introduced by the male or spread from an internal abscess. retained placentas, macerated fetuses, and dystocia are predisposing factors toward metritis. • treat with ovariohysterectomy after stabilization. females used only for breeding purposes may be treated with antibiotics alone, but the prognosis is poor. • female chinchillas are aggressive toward male chinchillas when not in estrus. breeding operations usually have separate cages for females and an interconnecting run for the male. females are kept out of the male's run by their larger size or collars. the young are precocious and do not need a nest. chinchillas only produce two litters per year. • clinical signs include hot, swollen mammary glands. suspect mastitis if previously healthy neonates become restless, then lethargic. • perform bacterial cultures on milk samples, and treat with antibiotics based on susceptibility testing. administer sulfa drugs until susceptibility results are available. local hot packing is also beneficial. occasionally, surgical drainage is required. foster neonates to another female if possible, or use puppy or kitten milk replacers to hand-raise babies. • chinchillas seem to be particularly sensitive to listeria monocytogenes. clinical signs can mimic p. pseudotuberculosis and include anorexia, lethargy, abortion, generalized central nervous system (cns) signs, hepatitis, mild enteritis, and mild emphysematous pneumonia. necropsy shows yellow foci in the liver. • diagnosis is based on isolation on culture. • treat with sulfa drugs (see table - ) until sensitivity results are available. the prognosis is poor. • other less common causes of neurologic disease in chinchillas include lymphocytic choriomeningitis, streptococcus spp., balisascaris procyonis (i.e., aberrant migration of raccoon roundworm), lead poisoning, and thiamine deficiency. guinea pigs (caviae porcellus) are nocturnal rodents that originated in the andes mountains. they are known for their dietary need for vitamin c. they are used as a food source in their native lands. there are three basic types: english, which have short hair; abyssinian, which have short, cowlicked hair; and peruvian, which have long hair. male guinea pigs are known as boars and the females as sows. guinea pigs become neophobic as they mature. offer a variety of foods early in life and make changes in diet or environment gradually. guinea pigs stampede when excited. square cages and strategically placed barriers on external walls prevent the trampling of small or weak animals. the smooth muscle of the bronchial tree is quite developed in guinea pigs. this places them at high risk for asthmatic-type anaphylactic reactions. both male and female guinea pigs have one pair of inguinal mammary glands; however, only the female's are well developed. m key point antibiotic toxicity (see "hamster"): guinea pigs also may be sensitive to tetracyclines. • fleas occur as in mice. • lice (i.e., gliricola porcelli, gynopus ovalis) usually cause no clinical signs except occasional alopecia, seborrhea, and trauma secondary to pruritus. • diagnosis is made by observation of lice on skin scraping. • treat with ivermectin, % malathion dust, or pyrethrin shampoo (see table - ). • the mite trixacarus caviae causes severe pruritus and is zoonotic. it mainly affects the dorsal midline and is difficult to find on skin scraping. it occurs most frequently in recently post-partum females, in which alopecia is the predominant clinical sign. treat with excellent sanitation and ivermectin (see table - ). • chirodiscoides caviae lives on the hair shaft of the perineal regions. it does not cause clinical signs. • treat with % carbaryl or lime-sulfur dip ( : ) (see table - ). sanitation is critical in preventing reinfestation. • about % to % of guinea pigs are carriers of trichophyton mentagrophytes. • clinical signs are alopecia and seborrhea sicca, usually starting on the face and spreading along the dorsum. • treat with lime-sulfur dips or griseofulvin (see table - ) combined with topical povidone iodine or chlorhexadine shampoos. • other causes for alopecia are barbering, alopecia of the flanks in late-gestation females, and generalized alopecia of young at weaning. subclinical hypovitaminosis c causes a poor hair coat and seborrhea sicca, as well as anorexia and large, malodorous stools. • "lumps" is the lay terminology for cervical lymphadenitis, which is characterized by lymphadenopathy in the ventral neck region. • pododermatitis and sore hocks are very common in guinea pigs. predisposing factors are untrimmed toe nails, poor sanitation, and wire flooring. s. aureus is the most commonly cultured pathogen. • clinical signs range from small ulcers on the soles of the feet to abscesses and gangrene. radiography is essential in determining whether bony involvement is present. untreated pododermatitis usually develops into osteomyelitis, which is very difficult to cure. • treat mild cases by improving sanitation and grooming. place affected individuals in solidfloored cages with paper bedding. use sulfa drugs (see table - ) until results of susceptibility testing are available. surgically remove or curette abscesses, and apply topical therapy and hot packing. amputation may be necessary when severe osteomyelitis exists. • conjunctivitis and epiphora occur as in mice. • inclusion body conjunctivitis is caused by chlamydia psittaci and is self-limiting in to weeks. • perform a conjunctival scraping to differentiate inflammatory conjunctivitis secondary to infection from allergy. i have observed an idiopathic, topical steroid-responsive lymphoplasmacytic conjunctivitis in guinea pigs. • white, dry ocular discharge is an early sign of hypovitaminosis c. • "pea-eyes" is the lay terminology for subscleral fatty deposits or protrusion of the lacrimal gland through the lower conjunctiva. the condition is thought to be hereditary. treatment is not required. • cataracts occur and are either congenital or developmental. • diabetes mellitus in guinea pigs also may cause cataracts. usually, no other clinical signs are present and urine glucose is greater than mg/dl whereas blood glucose remains within normal limits. • corneal or scleral calcification is usually an incidental finding. a thorough workup, including serum chemistry profile and radiographs, is recommended to ensure that generalized metastatic calcification is not present. • pneumonia in guinea pigs usually is caused by infection with s. pneumoniae, s. zooepidemicus, or bordetella bronchiseptica. s. aureus, p. aeruginosa, klebsiella pneumoniae, and pasteurella multocida also are cultured frequently. transmission is by direct contact, fomites, or aerosol. hypovitaminosis c and stress often predisposes guinea pigs to bacterial respiratory infections. weanlings are particularly susceptible. clinical signs and diagnosis are similar to other small mammals (see "mouse"). • take radiographs to rule out abscesses, pleural effusion, or pericardial effusion in refractory cases. • treat with chloramphenicol, sulfa drugs, or enrofloxacin (see table - ) and vitamin c (table - ) until results of culture and susceptibility testing are available. cats, dogs, rabbits, and rats are reservoirs for bordetella spp. as in other rodents, respiratory infections may lead to otitis interna/media. bordetella spp. also cause pyometra and abortions. • nasal discharge is most frequently a sign of upper respiratory tract infection but also may be associated with allergies or volatile irritants. • the diagnosis of allergic rhinitis is made by exclusion and through response to antihistamines or environmental changes. • bronchogenic papillary adenoma develops in approximately % of guinea pigs older than years of age. • diagnosis is often an incidental finding when thoracic radiography is performed for another problem. • occasionally, clinical signs are seen as a result of pressure on the heart or great vessels. • dyspnea most frequently is caused by heat stress or trauma. other causes are pregnancy toxemia, gastric bloat, volatile irritants, pleural effusion, pneumonia, or pulmonary edema. rhabdomyomatosis is a common necropsy finding. gross lesions appear as pale foci located on the endomyocardium and valves. histologic examination reveals myocardial cells that have stored excessive glycogen. do not confuse these areas with thrombi, abscesses, or neoplasia. their clinical significance is unknown. • paraspidodera ucinata is the cecal pinworm of guinea pigs. they are generally asymptomatic, but heavy infestations can cause diarrhea and weight loss. • diagnosis is based on fecal examination or cellophane tape test. • treat with piperazine or fenbendazole (see table - ). • coccidiosis caused by eimeria caviae is a fairly common cause of diarrhea in guinea pigs recently purchased from pet stores. • clinical signs are tenesmus, diarrhea, dehydration, and death. • diagnosis is based on fecal examination. on gross postmortem examination, petechiation and thickening of the colon are seen. • treat supportively and administer sulfa drugs (see table - ). • cryptosporidium wrairi and giardia spp. are found rarely. they cause a chronic enteritis. balantidium spp. are thought to be nonpathogenic. • malocclusion in guinea pigs is diagnosed on oral examination. • clinical signs are ptyalism and anorexia. the premolars are the most commonly affected teeth. • long-standing hypovitaminosis c predisposes guinea pigs to malocclusion. • treat malocclusion as in other rodents (see "dental procedures"). • hypovitaminosis c (i.e., scurvy) is associated with soft, malodorous feces. degeneration of the epithelium of the intestinal tract adversely affects digestion and absorption and allows secondary bacterial infections. • diagnosis of scurvy is based on clinical signs, the exclusion of other causes of diarrhea, and response to vitamin c therapy (see table - ). • salmonellosis usually is contracted through contaminated feed. • clinical signs range from sudden death to diarrhea and anorexia. the diarrhea is frequently light colored. sepsis is common and may cause conjunctivitis, shock, pneumonia, abortion, and neurologic symptoms. • diagnosis is based on isolation on culture of feces or other appropriate tissue samples. • treatment is controversial because recovered individuals remain carriers. use sulfa antibiotics or enrofloxacin (see table - ) until sensitivity testing results are available. supportive care is essential. • e. coli, arizona, and clostridium are other commonly cultured diarrhea-causing organisms. clostridium are diagnosed most easily by finding large numbers of spores on a gram stain fecal specimen. treat with metronidazole (see table - ). • yersinia pseudotuberculosis either causes an acutely fatal diarrhea or localizes into regional lymph nodes. • diagnosis is based on culture. • treat by surgical removal or drainage of abscessed lymph nodes. mesenteric lymph node involvement necessitates abdominal surgery. treat with sulfa drugs or enrofloxacin until susceptibility testing results are available (see table - ). • one male usually is housed with four to six females for breeding purposes. signs of estrus are vulvar swelling, lordosis, and opening of the vaginal closure membrane. fetuses are palpable at to weeks of gestation. parturition occurs within hours after the pubic symphysis has reached mm. neonates weighing less than g have a grave prognosis for survival even with intensive care. neonates normally do not nurse for the first to hours. litters with five or more fetuses usually result in abortion. • dystocia commonly occurs in females bred after the age of to months. after this age, the symphysis fuses and is unable to open the to cm required to allow passage of a fetus. dystocia in younger guinea pigs may be caused by obesity, large fetal size, fetal malpresentation, subclinical ketosis, or uterine inertia. on presentation, check the pelvic symphysis. if active contractions are present and the symphyseal gap is less than cm, perform a c-section. normal parturition is very rapid, with a rest of only to minutes between fetuses. • perform a c-section if active straining does not produce a fetus within to minutes. radiograph sows with a history of weak contractions to determine the stage of pregnancy and evaluate the size of the fetuses. if well-developed skeletons of appropriate size are seen and the pubis has not yet fused, give oxytocin and calcium (see table - ). if no fetuses are produced within to minutes, perform a c-section. • if poorly developed fetuses are seen radiographically, consider fetal death, ketosis, or a nonreproductive disorder as possible causes of dystocia. m key point pregnancy toxemia usually is seen in obese sows with large litters in late pregnancy. other risk factors include systemic disease or diet change causing anorexia, genetics, stress, and first litter. • clinical signs are tachypnea, depression, malodorous breath, seizures, and icterus. a urine ph of less than with marked proteinuria is compatible with pregnancy toxemia. a marked hyperkalemia and elevation of liver enzymes often occurs. thrombocytopenia may be present. • treat with iv or io saline, dextrose, glucocorticoids, and calcium. surgical abortion of the fetuses may be attempted, but the anesthesia risk is quite high. prognosis for survival is grave. do not rebreed affected females. do not breed sows heavier than g. • large litters can cause a hemorrhagic syndrome. compression of the portal vein and liver causes hepatic dysfunction, which results in vitamin k and clotting factor deficiency. • treat with vitamin k supplementation (see table - ). response is poor in severely compromised patients. affected individuals are at risk of ketosis developing. prognosis is guarded. • vaginitis in guinea pigs frequently is caused by foreign bodies, usually bedding. • diagnosis is made on vaginal examination. • treat by flushing the vagina to remove the foreign material. • vaginal discharge also can be caused by pyometra, uterine torsion, urinary tract infection, or urogenital neoplasia. • diagnosis is based on findings on abdominal palpation, vaginal cytology and culture, urinalysis, abdominal radiographs, ultrasound, and exploratory. • treatment varies with the condition and is similar to that used in cats. • ovarian teratomas and uterine tumors occasionally are diagnosed and usually resolve with ovariohysterectomy. • a symmetric alopecia with concurrent abdominal enlargement may be seen in female guinea pigs with cystic ovaries. • diagnosis is based on abdominal palpation, cytology, and ultrasound. • treat by performing an ovariohysterectomy. if the guinea pig is not a good candidate for surgery, human chorionic gonadotropin (hcg, usp units im, repeat in week) may temporarily resolve clinical signs. • male guinea pigs are prone to preputial foreign bodies. a preputial discharge is the usual presenting complaint. • diagnosis is based on physical examination. • treat by removing foreign bodies and performing local flushing. chronic problems require a change in bedding. • male guinea pigs produce sebaceous secretions in the folds around their perineal area. clean these areas with soap and water semiannually to prevent localized pyoderma. • if pyoderma occurs, treat with topical therapy and oral antibiotics. bacterial cystitis and urolithiasis are relatively common in guinea pigs. diagnosis is based on a history of stranguria, hematuria, painful abdomen, and anorexia, in addition to abdominal palpation, urinalysis, urine culture, abdominal radiographs, and ultrasonography. treatment consists of antibiotics based on results of culture and susceptibility testing and surgical removal of calculi, if present. prevention of recurrence is difficult if the calculi are not caused by a bacterial infection. addition of vitamin c to the drinking water as well as changing the brand of diet are sometimes helpful in preventing recurrence of metabolic stones. klossiella cobayae is a coccidia that lives in the renal tubules. it has no clinical significance. the most common orthopedic problem seen in guinea pigs is overgrown toenails. this leads to pododermatitis and sore hocks as well as to degenerative joint disease and a predisposition to tibial fractures. tibial fractures are the most common fracture seen in guinea pigs. they most frequently occur after foot entanglement. internal fixation with an im pin or application of a kirschner apparatus is the repair of choice. m key point signs of hypovitaminosis c or scurvy start to develop in guinea pigs as early as - days if they are placed on diets % deficient in vitamin c. early signs are soft, malodorous stools, weight loss, poor hair coat, and anorexia. later, petechia, gingivitis, cutaneous and oral sores, swollen costochondral junctions, joint pain and hemorrhage resulting in lameness, and conjunctivitis become apparent. treat supportively and administer parenteral vitamin c ( mg/day). • lymphocytic choriomeningitis occurs as in mice. • guinea pig paralysis syndrome starts with mild pyrexia and urinary incontinence, followed by weight loss and posterior paresis that progresses to paralysis. currently, the etiology is unknown, but it does not appear to be contagious. • treat with supportive care. prognosis for long-term survival is grave. • head tilt is usually the result of otitis or trauma (see "mouse"). cavian leukemia has a viral etiology. the liver, spleen, and lymph nodes are the primary organs involved. there is no current treatment. quarantine exposed individuals. death usually occurs within days after discovery of lymphoblasts in the peripheral blood. neutrophils normally have red granules. kurloff bodies are normally occurring eosinophilic intracytoplasmic inclusion bodies that are found in mononuclear cells. they are seen most frequently in females and appear to correspond positively with estrogen levels. metastatic calcification occurs in most guinea pigs older than year of age. it is more severe in females than in males. the stomach is one of the first organs affected. dysfunction in motility causes obstruction. the tendency appears to be exacerbated by high calcium and low phosphorus diets. exotic animal formulary the biology and medicine of rabbits and rodents veterinary clinics of north america key: cord- - mezubh authors: plazolles, n.; humbert, j.‐m.; vachot, l.; verrier, b.; hocke, c.; halary, f. title: pivotal advance: the promotion of soluble dc‐sign release by inflammatory signals and its enhancement of cytomegalovirus‐mediated cis‐infection of myeloid dendritic cells date: - - journal: j leukoc biol doi: . /jlb. sha: doc_id: cord_uid: mezubh dc‐sign is a member of the c‐type lectin family. mainly expressed by myeloid dcs, it is involved in the capture and internalization of pathogens, including human cmv. several transcripts have been identified, some of which code for putative soluble proteins. however, little is known about the regulation and the functional properties of such putative sdc‐sign variants. to better understand how sdc‐sign could be involved in cmv infection, we set out to characterize biochemical and functional properties of rdc‐sign as well as naturally occurring sdc‐sign. we first developed a specific, quantitative elisa and then used it to detect the presence sdc‐sign in in vitro‐generated dc culture supernatants as cell‐free secreted tetramers. next, in correlation with their inflammatory status, we demonstrated the presence of sdc‐sign in several human body fluids, including serum, joint fluids, and bals. cmv infection of human tissues was also shown to promote sdc‐sign release. based on the analysis of the cytokine/chemokine content of sdc‐sign culture supernatants, we identified ifn‐γ and cxcl /il‐ as inducers of sdc‐sign production by modc. finally, we demonstrated that sdc‐sign was able to interact with cmv gb under native conditions, leading to a significant increase in modc cmv infection. overall, our results confirm that sdc‐sign, like its well‐known, counterpart mdc‐sign, may play a pivotal role in cmv‐mediated pathogenesis. dc-sign is a tm type ii protein, which belongs to a family of calcium-dependent lectins diversely used by human apcs, such as tissue-residing myeloid dcs, alveolar and ln macrophages, and endothelial cells from liver sinusoids [ ] [ ] [ ] [ ] . dc-sign contains a crd that is highly conserved in lectins and a neck region consisting of ig-like domain repetitions, which mediate a ph-dependent oligomerization of dc-sign monomers and thus, increase affinity with its ligands [ ] [ ] [ ] [ ] . during monocyte differentiation toward modcs, dc-sign expression has also been reported to be induced by il- and to be negatively regulated by ifns, tgf-␤, and dexamethasone [ ] . dc-sign is a receptor for self-glycoproteins, such as icam- / [ , ] , and is also able to recognize high mannose-containing structures and fucosylated lewis blood ags (le x/y/b/a ) [ , ] , expressed by several pathogenic microorganisms including viruses, bacteria, yeasts, and parasites (for review, see ref. [ ] ). interactions between dc-sign and pathogenic-derived sugar moieties have been shown to play a prominent role in vivo in favoring the capture and internalization of microbes [ ] , with or without the help of other ag-capture receptors, such as the macrophage mannose receptor (cd ), dec- (cd ) [ ] , or the mac-rophage galactose-type c-type lectin [ ] . in contrast to other c-type lectins, several transcripts have been described for dc-sign, most likely originating from alternative splicing and potentially leading to the expression of sdc-sign proteins [ ] . as described already for the dc-sign homologue, dc-signr (or cd ligand) [ ] , such cdnas have been reported by others to encode nonsecreted sdc-sign molecules [ ] . in addition, the same study showed that sdc-sign displayed no functional activity in terms of icam- -dependent cosignaling compared with mdc-sign. some years ago, we demonstrated that cmv, like hiv, is able to bind to dcs via dc-sign and enter them more easily than the free viral particles [ ] . consequently, the dc-sign-mediated increase in virus entry resulted in a strong cmv infection of modc. the study in question shed new light on the molecular interactions that favor dc infection by cmv and especially the pivotal role played by dc-sign as a docking and internalizing receptor. cmv is a widespread herpesvirus that infects - % of the populations worldwide. it induces lifelong viral persistence and may cause severe disease in immunocompromised individuals, such as those with hiv or transplant patients. the major cmv host entry sites are myeloid dc-containing peripheral tissues, especially oropharyngeal and genital mucosa. in the present study, we investigated expression regulation and functional properties of naturally occurring as well as rsdc-sign variants. we demonstrated that such variants can promote cmv infection of modcs, as already shown for their counterparts, mdc-sign. these data suggest that sdc-sign variants may play a crucial role in the cmv-mediated pathogenesis. after signing an informed consent, peripheral or cord blood and bal samples, as well as vaginal mucosa explants, resulting from a partial or complete hysterectomy (vaginal tumor resection or prolapsus), were collected and used in our experiments, in accordance with protocols approved by the local ethical committee. bal samples were isolated from hospitalizedinfected (cmv primary infection, influenza, coronavirus, pneumococcus, or hemofilus influenza) or polytrauma patients. bal were classified as "inflammatory" or "noninflammatory" according to the crp serum concentration (respectively, [crp] serum Ͼ mg/ml, and [crp] serum Ͻ mg/ml). two endotheliotropic human cmv strains, tb /e and vhl/e, were kindly provided by dr. christian sinzger (institute for medical virology and epidemiology, university of tubingen, tubingen, germany) [ ] . tbgfp was a kind gift of dr. martin messerle (department of virology, hannover medical school, hannover, germany). all viruses were propagated, purified, and titered as described [ ] . for gel filtration analyses, the rcrd of dc-sign was kindly provided by dr. franck fieschi (institute for structural biology, grenoble, france). this molecule was produced in escherichia coli. anti-dc-sign antibodies were obtained from dr. bernard verrier (fre cnrs-biomérieux, lyon, france; mab clone e a , purified and biotinylated) from the french agency for aids research program (mab clone b ; ac . [ ] ) or purchased from bd biosciences (san jose, ca, usa; mab clone dcn ) or santa cruz biotechnology (santa cruz, ca, usa; pab h- ). mouse mab against specific human surface ags were used in a direct immunostaining assay: pe-cyanin -conjugated anti-cd a (clone bl ), pe-conjugated anti-cd / / (clones mab , hb a, and ha . b ), and conjugated isotypic control mab (beckman coulter, fullerton, ca, usa); and pe-conjugated anti-hla dr (clone l ) and fitc-conjugated anti-dc-sign/cd (#dcn ; bd biosciences). neutralizing polyclonal antibodies directed against human cxcl- /il- , cxcl- /ip- , ifn-␥, or il- were purchased from r&d systems (minneapolis, mn, usa). hek t clones were cultured in % fcs dmem supplemented with mm glutamine. hek cells were used for transient recombinant protein expression assays. dcs were generated in vitro from adult blood monocytes according to a modified version of the protocol described previously by sallusto and lanzavecchia [ ] . briefly, cd ϩ monocytes were highly enriched from pbmcs of healthy donors by a negative magnetic depletion using hapten-conjugated cd , cd , cd , cd , cd b, and glycophorin a antibodies (stemsep™ human monocyte enrichment kit, stem cell technologies, vancouver, canada). routinely, two enrichment steps resulted in Ͼ % pure cd ϩ cells. purified cd ϩ monocytes were cultured in six-well plates (nunc, thermo scientific, rochester, ny, usa) for days with rpmi- medium, supplemented with % fcs (biowhittaker, cambrex, charles city, ia, usa) and ng/ml human ril- and ng/ml human rgm-csf (peprotech ec ltd., london, uk). every days, one-half of the medium was replaced by fresh il- /gm-csf-supplemented medium. at day , virtually all cells displayed the typical phenotype cd a ϩ , cd -, hla-dr -, cd low , cd low , cd -, dc-sign ϩ of immature modcs, as assessed by flow cytometry. alternatively, dcs were generated from cd ϩ cbps, as described already elsewhere [ ] . immunomagnetically purified cbps were cultured in medium supplemented with ng/ml stem cell factor and ng/ml gm-csf (r&d systems). tnf-␣ ( ng/ml) was added on day , and on day of culture, il- ( ng/ml) was added to induce dc-sign synthesis for additional days. total rna was isolated from modc using the trizol™ reagent isolation method according to the manufacturer's instructions (invitrogen corp., carlsbad, ca, usa). total rna was then reverse-transcribed with the m-mlv rt and polydt oligonucleotide (promega, madison, wi, usa). dc-sign-encoding cdnas were amplified by pcr using a high-fidelity taq polymerase (roche diagnostics, meylan, france) with the following oligonucleotides: forward Ј-aagaattcgactacaaggatgacgatga-caagggaatgagtgactccaaggaa- Ј, allowing to insert a flag tagencoding sequence, and reverse Ј-tattatgcatctacgcag-gaggggaattctt - Ј. pcr products were directly cloned at the ecori sites of the pet a prokaryotic expression vector (novagen, vwr international s.a.s, fontenay sous bois, france) or alternatively, of the pcdna . plasmid (invitrogen corp.). tm-missing cdna were screened using two internal primer pairs to amplify, respectively, all and tm-containing cdna: forward/reverse dc-sign_sm/dc-sign_as (tmϭ . °c, Ј-ctc-caaggaaccaagactgc- Ј/tmϭ . °c, Ј-ttgttgggctctcctct-gtt- Ј) and forward/reverse dc-sign_ss/dc-sign_as (tmϭ . °c, Ј-aactcctctccttcacgc- Ј). retained, tm-missing cdnas were then sequenced by the dideoxynucleotide termination method (genomexpress, meylan, france) and submitted to a basic local alignment search tool request (http://blast.ncbi.nlm.nih.gov/blast.cgi). for clarity reasons, our constructions consisting of the sdc-sign a type i isoform encoding cdna cloned into pet a and pcdna . vectors and were renamed pet -flag-sdcsign at and pcdna . -flag-sdcsign at , respectively. the sdc-sign a type iii-encoding sequence was also cloned into the pet a plasmid and produced similarly as described below for sdcsign at . for protein production, pet a-flag-sdcsign at and pet a-flag-sdcsign at recombinant plasmids were transferred to rosetta™ (de ) plyss-competent cells (novagen, a merck company, germany). this modified bacterial strain is usually used to synthesize native recombinant eukaryotic protein, even in the absence of a signal peptide. the recombinant protein expression was induced for - h by the addition of mm iptg to a growing culture at °c, according to the manufacturer's instructions, and in the presence of g/ml chloramphenicol (c , sigma-aldrich, st. louis, mo, usa). iptg-induced cells were harvested and lysed directly with the bacterial-protein extraction reagents (thermo scientific). both sdc-sign isoforms were then purified by a two-step affinity chromatography consisting of one passage on mannan-conjugated agarose (elution was done with g/ml mannan in tbs), followed by a second passage on a m affinity resin-loaded column at low pressure (sigma-aldrich). then, rsdc-sign elution was obtained with a -g/ml flag peptide solution (in tbs), according to sigma-aldrich protocols. after the second round of purification, each eluted fraction was concentrated onto centricon- filtration devices (millipore, bedford, ma, usa) to allow the contaminating flag peptide to be removed. each treated fraction was then analyzed by western blot and silver nitrate staining to estimate purity ( - % estimated purity was usually achieved). positive fractions were then pooled and stored in glycerol ( % v/v) at - °c until used. sdc-sign concentration was measured by elisa for every flag-sdc-sign batch production. when required, samples were submitted to gel filtration chromatography to allow mw determination of native multimer or monomer proteins. briefly, . - ml dc-sign-containing samples were loaded onto a hiprep / sephacryl s- hr column (ge healthcare, waukesha, wi, usa) and separated according to their mw. fractions ( ml) were collected and analyzed by western blot or elisa to document the presence or absence of dc-sign. mean elution volumes were then determined for each tested sample, thus enabling the calculation of dc-sign mw using mw standards (bio-rad france, marnes-la-coquette, france). a specific sdc-sign elisa was developed in our laboratory. a polyclonal anti-dc-sign antibody (clone h- , santa cruz biotechnology) was coated onto presaturated reactibind -well plates, according to the manufacturer's instructions (pierce biotechnology, rockford, il, usa). after washing plates, samples (sera, bal, synovial fluid, cell culture supernatants, etc.) were diluted in tbs or left undiluted, mixed with an equal volume of tbst containing the biotin-conjugated e anti-dc-sign mab ( . g/ ml), and incubated further for h at room temperature. a final incubation step with a hrp-conjugated neutravidin™ solution (pierce biotechnology) allowed for the detection of sdc-sign with tetramethylbenzidine, a specific chromogenic hrp substrate. absorbance was read at nm and nm (background substraction) on a labsystems multiskan microplate reader (labsystem multiskan ms, finland) and analyzed with the biolise™ software (version . , labtech international, uk). quantitative sdc-sign concentration determination was achieved when ods were compared with a standard curve, obtained with flag-sdc-sign at protein, produced in e. coli. validation tests of our homemade elisa sdc-sign are presented in table . exosomes were prepared from the supernatant of -day-old il- /gm-csfdifferentiated modcs cultured in complete medium using a simplified version of a protocol reported previously by raposo et al. [ ] . three successive centrifugations at g ( min), g ( min), and , g ( min) were performed to eliminate cells and debris. supernatants were ultracentrifuged at , g for h onto a d / % sucrose gradient-density cushion (dϭ . ). the exosome-enriched pellet was resuspended in pbs and kept frozen for further experiments, and clarified supernatant was concentrated with an amicon centrifugal filter device ( kda cut-off, millipore). total protein contents of supernatants and exosome pellets were quantified using the bca assay (pierce biotechnology). cytokines and chemokines were quantified into cell/tissue culture supernatants or biological fluids by the cba flex set (bd biosciences). when needed, cervical or vaginal epithelium biopsies were infected with tb /e cmv strain ( ϫ percentages of gfp ϩ cells directly reflected the infectious rate, as assessed already in our former work [ ] . total proteins were obtained from concentrated cell culture supernatants or cell lysates. when required, hek t cells were transiently transfected with the pcdna . -flag-sdcsign at eukaryotic expression vector using the exgen transfection reagent according to the manufacturer's instructions (euromedex, france). after days, cells were harvested, washed three times with pbs, and resuspended into a lysis buffer ( mm tris, phϭ . , mm nacl, . % triton x- ), supplemented with a protease inhibitor cocktail (p ; sigma-aldrich) and cleared from nuclei by centrifugation at , g. for each kind of experiment, indicated total protein amounts were loaded and separated onto % acrylamide sds-page gels before being transferred to nitrocellulose membranes. saturated membranes ( h, room temperature in . % tbst, % nonfat milk) were then hybridized with specific primary antibodies against dc-sign ( e a or b clones) or actin (rabbit polyclonal antibody; sigma-aldrich) and secondary hrp-conjugated goat anti-mouse or rabbit mab (amersham pharmacia biotech, sweden). proteins of interest were finally detected using an ecl detection kit (pierce biotechnology). alternatively, protein lysates were diluted in tbs and spotted onto appropriate nitrocellulose membranes using a minifold-i dot-blot system (schleicher & schuell, germany). then, membranes were saturated and hybridized as described in the previous paragraph. human cervical or vaginal mucosa explants were collected by prorfessor c. hocke (department of gynecological surgery, general hospital of bordeaux, france), in accordance with international ethical rules. explants were extracted as circular pieces of ϳ cm and submitted or not to cmv infection (vhl/e; ϫ pfu/cm ). oct (sakura finetek usa, inc., torrance, ca, usa)-embedded frozen tissue sections were air-dried for min, washed in pbs (ph . ), and fixed/permeabilized in : vol:vol cold acetone/methanol at °c for min. the fixed sections were saturated using pbs containing . % bsa and % normal goat serum (sigma-aldrich) at room temperature for h. subsequently, the sections were incubated with the anti-dc-sign dcn mab or the anti-ie/e cmv ag antibody (argene sa, varilhes, france), followed by incubation with an alexa -conjugated goat anti-mouse mab (molecular probes, invitrogen corp.). concomitantly, nuclei were stained with dapi. slides were then washed twice in pbs for min and rinsed in distilled water before being air-dried. stained sections were mounted with dako fluorescent mounting medium (dako, carpinteria, ca, usa) and analyzed on a sp confocal microscope (leica microsystems, germany). statistics were generated with the graphpad prism . software (graphpad software inc., la jolla, ca, usa). unpaired sample comparisons were performed using a mann-whitney nonparametric rank test. a previous study by mummidi and colleagues [ ] reported the cloning of cdna, potentially coding for sdc-sign. to determine whether corresponding proteins exist, we first isolated dc-sign-encoding cdnas from monocyte-or cd ϩ cord blood cell-derived dcs by pcr. the primer used in this pcr allowed for the insertion of a flag tagencoding sequence in the Ј position (fig. a) . only selected cdna clones were submitted to a second round of pcr to retain the sole tm region lacking cdnas, thought to probably code for sdc-sign (fig. b) . four of the clones were not amplified by the tm-specific internal pcr (clones , , , and ; fig. a and b). these cdna clones lacking the tm exon were sequenced and blasted against known cdna libraries. then, we confirmed that three out of these four isolated cdnas were completely identical to a sequence reported previously by mummidi et al. [ ] (i.e., sdc-sign a type i isoform-encoding sequence; genbank accession number nm_ ) and that the last one encoded a spliced variant, the sdc-sign a type iii (gen-bank accession number ay ). we then subcloned sdc-sign a type i-and iii-encoding cdnas (called thereafter flag-sdc-sign at and t ) into prokaryotic and eukaryotic expression plasmids as required. an elisa was produced simultaneously to measure sdc-sign isoform concentrations in cell culture supernatants or in any other biological fluids. the flag-sdc-sign at protein of prokaryotic origin was used to obtain a standard curve and set up our homemade elisa (fig. c ). although our sdc-sign elisa was moderately sensitive with a typical concentration range of - . ng/ml, we were able to validate its specificity. in fact, by means of selective ip experiments on sdc-sign standard solutions, we could demonstrate that b mab or h- pab (the coating pab), directed against dc-sign, abrogated sdc-sign detection, whereas an irrelevant mab had no effect, i.e., flag-sdc-sign at was still detected by elisa (table ) . interestingly, a homotypic elisa, i.e., the same antibody (clone e ) used as the primary (capture) and secondary antibody, led to low signal detection. this observation evoked the possibility that the flag-sdc-sign at protein was expressed as multimers. this issue is addressed at a later stage in this paper. previous studies have reported that tm-lacking encoding dc-sign sequences are transcribed as soluble cytoplasmic pro-teins but not secreted by the sdc-sign-expressing transfectant cells or immature modcs [ ] . it was our hypothesis that the lack of secreted proteins in culture supernatants may be a result of a secretion defect or the lack of a signal peptide [ ] . of note, certain proteins, such as il- ␣, fgf , or galectin- / , are secreted, despite being devoid of a signal peptide [ - ] . as a result, we surmised that the same could be true for sdc-sign. thus, using our quantitative and specific elisa, we observed the presence of potential sdc-sign isoforms in ϫ concentrated cell culture supernatants of mdc-sign-expressing dcs. in vitro dcs were generated from adult monocytes or cd ϩ cbps, according to already well-known protocols [ , , ] . somewhat surprisingly, increasing quantities of sdc-sign were found in both dc-derived culture supernatants in relation to postdifferentiation time ( fig. a and b) . sdc-sign appearance in culture supernatants was correlated with the addition of ril- . this observation was consistent with previously published observations showing that upon differentiation, il- was required to induce mdc-sign expression [ ] . to know whether sdc-sign and mdc-sign expressions could be differentially regulated during differentiation, modcs were generated with several cytokine cocktails (il- /gm-csf, ifn-␣/gm-csf, and il- /gm-csf) or il- /il- alone. all combinations, except the one using ifn-␣, were able to induce sdc-sign and mdc-sign to the same extent, as assessed by western blot (fig. c and d) . taken together, these results provided strong evidence that sdc-sign may be secreted by in vitro-generated dcs and that sdc-sign and mdc-sign were regulated in a similar manner in the course of dc differentiation. we next tried to purify sdc-sign from modc culture supernatants to definitely prove that this molecule is derived from an alternative splicing event, as expected in previous studies [ ] . however, we failed to purify sufficient amounts of highpurity sdc-sign for n-terminal sequencing. to circumvent these difficulties, we alternatively treated modcs with a broadspectrum inhibitor of mmps (i.e., a disintegrin and metalloprotease/tace family), called marimastat (british biotech, uk), which was supposed to have no effect on sdc-sign release. differentiating dcs were incubated with increasing doses of marimastat. at day , mdc-sign expression was analyzed by flow cytometry, and sdc-sign was quantified in culture supernatants by elisa (fig. a , respectively, black and open bars). despite the use of high concentrations of marimastat ( m), no modification of sdc-sign versus mdc-sign expression patterns could be observed, thus weighing in favor of a sliced, sequence-derived product and not a shedding of mdc-sign by mmps. like many cell types, dcs are able to secrete - nm membrane vesicles, called exosomes. to know whether sdc-sign isoforms could be secreted as exosome-borne proteins, we separated the exosome-enriched fraction from cleared il- /gm-csf-derived modc culture supernatant, according to the modified protocol published by raposo et al. [ ] , and titered sdc-sign in all samples by elisa, i.e., medium alone, nonultracentrifuged supernatant, ultracentrifuged supernatant, and exosome-enriched pellet. the results are described in fig. b . sdc-sign was retained almost completely in the supernatant cleared from exosomes by ultracentrifugation, as it was shown to be absent from the exosome-enriched fraction (p). we concluded that sdc-sign was not expressed as exosomeassociated proteins. several groups have provided strong evidence that mdc-sign or its homologue dc-signr is expressed as homotetramers to increase affinity for their ligands [ , , ] . tetramerization is dependent on the neck length consisting of various ig-like domain repeats [ , ] and also on the extracellular ph [ ] . the question we posed ourselves was whether this was the case for sdc-sign using gel filtration. here again, we were unable to purify sufficient sdc-sign from modc cultures to perform our analysis. as a result, we produced rflag-sdc-sign at and t in e. coli and separated them according to their apparent mw by gel filtration. for each sample, fractions were collected and analyzed by elisa. the results are shown in fig. c . sdc-sign quantities were plotted against elution volumes, and elution volume of the maximum sdc-sign quantities for each sample was determined on the plot. column precalibration allowed us to calculate each apparent mw meas of the most abundant sdc-sign isoform found in each sample. thus, the flag-sdc-sign at -associated peak was eluted with ml, corresponding to an apparent mw of kd. an approximate, mw theor of kd was calculated on specialized websites (mw theor ; http://www.expasy.org/) for flag-sdc-sign at . as mw meas was about four times larger than mw theor (ratioϭ . ), we concluded that flag-sdc-sign at could be expressed as tetramers in nondenaturing and nonreducing conditions. applying the same procedure for the flag-sdc-sign at , it was estimated that unlike sdc-sign at , sdc-sign at could probably be found in culture medium as a dimer (mw meas ϭ kd; mw theor ϭ kd; ratioϭ . ). purified crd (ϳ kd) was used as a control here, as it was unable to multimerize. peak-associated fractions of the flag-sdc-sign at gel filtration run were pooled and concentrated ten-to -fold onto centrifugal filter units ( kda cutoff) and finally analyzed by western blotting in denaturing and reducing conditions. the results are shown in fig. d . in this setting, we only observed flag-sdc-sign at as a -to -kda pro- tein, suggesting that flag-sdc-sign at is expressed as a homotetramer in native conditions. all of our data indicate that sdc-sign proteins are secreted in a mmp-independent manner as exosome-free homotetramers, at least for fulllength sdc-sign variants. we demonstrated above that sdc-sign is secreted by human blood-borne, precursor-derived dc-sign ϩ dcs in vitro. thus, we assumed that it could be secreted by dcs in vivo. our study first analyzed human sera from healthy donors by western blot (nϭ ; fig. a ). much to our surprise, sdc-sign was detected in of the sera with a heterogeneity of expression between individuals. following this, we tested a broader panel of human serum from healthy donors (nϭ ) by elisa. consistently with the western blot analysis, almost all sera contained detectable amounts of sdc-sign ranging from to Ͼ ng/ml with a mean value of . ng/ml (minϭ ng/ml; maxϭ ng/ml; medianϭ ng/ml; th percentileϭ . ng/ml; th percentileϭ . ng/ml; fig. b ). the serum sdc-sign concentrations were comparable with the lower amounts of mbl variants, which are found in human serum in a broad range of concentrations, depending on their genotype [ ] . further gel-filtration experiments demonstrated that serum sdc-sign was more probably expressed as tetramers, which was consistent with our previous results obtained with rflag-sdc-sign at (supplemental fig. ). mdc-sign has previously been associated with th responses, at least in vitro [ , ] . however, certain experimental findings came to our attention, showing that dc-sign may be up-regulated in inflammatory diseases such as crohn's disease [ , ] . we hypothesized that sdc-sign may be found in inflammatory body fluids. for this purpose, bals were harvested from patients suffering from diverse lung diseases, including primary cmv infections, and tested for the presence of sdc-sign. when segregated according to the inflammatory status of samples (based on diagnoses, i.e., viral infections, etc.), the quantity of sdc-sign was clearly higher within inflammatory (meanϭ . ng/ml) compared with noninflammatory bal (meanϭ . ng/ml; pϭ . ; fig. c ). to confirm these observations, we compared sera and joint fluids from ra versus osteoarthritissuffering patients. joint fluids from ra patients were indeed prototypical inflammatory fluids, whereas joint fluids from os- teoarthritis were considered as mechanical fluids marked by the absence of inflammatory cytokines and chemokines (supplemental fig. ). as shown in fig. d (left panel) , there was a highly significant difference between sdc-sign amounts in ra versus control joint fluids (pϭ . ). when analyzing the related sdc-sign concentrations in the sera of the same patients (fig. d, right panel; pϭ . ) , a similar difference could be observed, thus promoting the conclusion that sdc-sign expression was closely correlated to the inflammatory status of these human biological fluids (fig. e) . taken together, these results largely confirmed the fact that sdc-sign is produced in distinct biological fluids, and its expression ap-pears to be up-regulated by inflammatory disorders, as exemplified here in the case of ra. on the basis of previous results of this study showing that higher amounts of sdc-sign are generally found in inflammatory, virally infected bal (fig. c) , we next endeavored to ascertain whether such inflammation was dependent on viral infection. we therefore infected freshly isolated mucosal explants of human origin (vagina or cervix biopsies) with a high viral load and further incubated them for days before freezing. frozen tissue sections were stained to reveal nuclear ie/e cmv ag in infected cells or dc-sign. as shown in fig. a , dc-sign ϩ cells were located exclusively in the lamina propria (fig. a, d and e) of freshly isolated explants (fig. a, a) or of -day noninfected explants (fig. a, b) , whereas after a -day infection period, dc-sign ϩ cells were distributed equally among the mucosal epithelium and the lamina propria (fig. a, f) , as shown by a sharp nuclear staining of ie/e cmv agpositive cells (fig. a, c) . in the course of these experiments, explant culture supernatants were collected and analyzed by elisa to quantify secreted dc-sign. the results are described in fig. b . upon infection, sdc-sign release was ϳ . times higher in explants when compared with the basal sdc-sign release after days in noninfected explant supernatants, as well as in freshly isolated explant supernatants. we then confirmed that cmv infection contributed to the establishment of an inflammatory environment by showing that typical cytokines (il- and ifn-␥) or chemokines (cxcl- /il- and cxcl- /ip- ) were overproduced in explant supernatants upon infection (fig. c ). lps stimulation for h was used as a positive control of cytokine/chemokine release. together, these results clearly demonstrate that sdc-sign expression could be up-regulated by a viral infection of freshly isolated peripheral tissues. we next sought to identify proinflammatory cytokines or chemokines that might be responsible for the induction of sdc-sign up-regulation. focusing on molecules overexpressed in explant culture supernatants upon cmv infection or lps activation, experiments were performed using fully differentiated immature modc, subcultured with various doses of human rcxcl- /il- , rcxcl- /ip- , rifn-␥, and ril- , alone or in combination. to minimize the effect of exogenous il- , which is necessary to differentiate modc but also able to induce sdc-sign, potentially leading to misleading conclusions, cells were first starved of il- . in that setting, sdc-sign release was up-regulated significantly by ifn-␥ and to a lesser extent, by cxcl- /il- in a dosedependent manner when compared with the spontaneous level of sdc-sign secretion by il- -starved cells (i.e., "no il- " experimental conditions; respectively, pϭ . and pϭ . ), whereas no significant effect could be shown after adding exogenous cxcl- /ip- or il- ( fig. ) . it should be noted that cxcl- /il- and ifn-␥ act in an additive but not synergistic manner to induce sdc-sign production by il- -starved immature modcs. the stimulation level in that case is equivalent to the "il- alone" experimental condition. il- unresponsiveness is most likely a result of the absence of a functional receptor on the cell surface, as no significant staining for gp , i.e., the signaltransducing chain of il- r, could be observed by flow cytometry (data not shown). in accordance with our previous data, we hereby demonstrated that sdc-sign is re-in-duced by exogenous ifn-␥ and/or cxcl- /il- in fully differentiated immature modc cultures. we demonstrated above that sdc-sign secretion is promoted by an inflammatory setting and even upon cmv infection of human tissue explants. thus, it remained to be determined whether sdc-sign has a protective or facilitating effect on cmv infection. first, we tested the functionality of flag-sdc-sign at through its ability to interact directly with the cmv envelope gb, which we identified previously as a ligand for mdc-sign [ ] . lysates of transiently transfected hek cells were spotted on a nitrocellulose membrane and incubated further with flag-sdc-sign at or specific mab as a detection control to document interactions between gb and sdc-sign under native conditions (fig. a) . when incubated with flag-sdc-sign at , gb ϩ cell lysates could be revealed by a hrp-conjugated anti-flag antibody, whereas the anti-flag alone did not provide any signal. flag-sdc-sign at ϩ hek cell lysates were used as positive controls for dc-sign and flag detection. an additional actin detection was used as a loading sample control. these results suggested that the flag-sdc-sign at was functional. however, the functional role of sdc-sign during the cmv infection remained unclear. on the basis of the mbl-mediated inhibition of hiv susceptibility reported in the literature, we first hypothesized that sdc-sign might neutralize the cmv infection of modc. in this setting, we were unable to block modc infection, even using higher concentrations of flag-sdc-sign at (data not shown). in addition, as mdc-sign expression was reported to be responsible for modc cmv cis-infection, we assumed that flag-sdc-sign at may function as a promoter of the infection. for this purpose, modc were infected with tbgfp cmv (moiϭ ) for - h in the presence of decreasing amounts of flag-sdc-sign at (from to . ng/ml). the gfp ϩ modc, indicating the percentage of early infected cells, was estimated by flow cytometry. infected modc frequencies were approximately twice as high ( . Ϯ %) as the control infection ( . Ϯ . %) when incubated with ng/ml flag-sdc-sign at -supplemented culture medium (pϭ . ; fig. b ). a -ng/ml concentration was not sufficient to mediate a significant infection enhancement when compared with control infection. surprisingly, it was observed that flag-sdc-sign at concentrations higher than ng/ml and lower than ng/ml did not modify the infectious rate of modc. to confirm that the deleterious effect that we could observe was specific to sdc-sign, we conducted similar experiments but only using an optimal amount of flag-sdc-sign at ( ng/ml), which was first captured or not onto a mannan-conjugated resin (fig. c, upper panels) . here again, the addition of flag-sdc-sign at doubled the gfp ϩ modc percentage ( . % vs. . %). this enhanced infection was readily a result of flag-sdc-sign, as it was abrogated by a preincubation step of the recombinant lectin on mannan-conjugated agarose. as a negative control, we also performed the same assay with fsf, which we reported previously as being prone to cmv infection in a dc-sign-independent manner ( [ ] ; fig. c lower panels). all of these results led us to conclude that flag-sdc-sign at is functional and is able to facilitate cmv infection of modcs. we previously reported the crucial role of mdc-sign as a docking and internalizing receptor for cmv on modc [ ] . simultaneously, other groups reported the existence of potentially sdc-sign variants at the cdna level, generated by alternative splicing of the tm-encoding exon [ ] . soluble but nonsecreted and nonfunctional dc-sign proteins were described by others [ ] . to better delineate the role of an extended dc-sign repertoire on cmv infection pathogenesis, the present study appraised the biochemical properties, the regulation, and the role of recombinant as well as naturally occurring sdc-sign variants in the cmv cis-infection of modc. martinez et al. [ ] had previously shown that sdc-sign was produced by transfected cells. they reported that sdc-sign failed to be secreted in the cell culture supernatant but instead, was retained in the cytoplasm. strangely, they sought to test its ability to promote the transmission of cosignals to t cells in comparison with mdc-sign but to no avail. in the present study, we developed and used a specific and quantitative elisa to demonstrate the secretion of sdc-sign in concentrated modc culture supernatants. the fact that martinez et al. [ ] failed to detect sdc-sign in culture supernatant may be a result of the lack of sensitivity of their own elisa and the absence of a culture supernatant concentration step before measuring sdc-sign. what is more, in keeping with the work of mummidi et al. [ ] , we cloned cdnas lacking the tm-encoding exon and coding for putative sdc-sign from distinct, in vitro-generated dc populations. in our study, a broad-spectrum inhibitor of mmps was used to avoid a possible shedding of mdc-sign at the plasma membrane. nevertheless, modc and tissue-residing dcs expressed two or three other membrane-associated mmps that might not be targeted by mmp inhibitors [ ] . however, mmp inhibition did not result in a difference of sdc-sign and mdc-sign expression by modcs. these results all confirmed previous observations reporting sdc-sign expression as a soluble, full-length molecule. another crucial issue was to check whether sdc-sign could be expressed as an exosomeassociated protein, as suggested in a recent report [ ] . briefly, exosomes derived from dc-sign ϩ murine bone marrow-derived dcs were shown to express dc-sign on the basis of cytometric analyses. on the other hand, as a result of very slight dc-sign staining of exosomes, these results were not convincing and had to be interpreted with caution. in this case, based on the measurement of sdc-sign concentrations with our elisa, we could undoubtedly argue in favor of the expression of sdc-sign as exosome-free molecules. the final proof of the secretion of a full-length sdc-sign would be to sequence the naturally occurring sdc-sign protein, but we failed to purify sufficient amounts of it to obtain any kind of irrefutable proof. feinberg et al. [ ] previously demonstrated that mdc-sign was expressed as tetramers at the plasma membrane of transfected cell lines. the mdc-sign tetramerization was dependent on the presence of at least five ig-like domain repeats in the neck region. another study also provided sound evidence that immature modcs express mdc-sign tetramers, which are supposed to physically interact with the coreceptor cd [ ] . here, by gel filtration analyses under native conditions (i.e., neutral ph and isotonic buffer), we proved that not only recombinant but also blood-borne sdc-sign consisted of tetramers, thus confirming results published previously by others [ , ] . we next demonstrated that il- and il- were potent inducers of sdc-sign and mdc-sign. these results were in line with those published by relloso and colleagues [ ] in a previous report. in addition, we did not test ifn-␥ during monocyte differentiation in dcs, but we tested ifn-␣, which was sufficient to prevent dc-sign expression when added at the very beginning of modc differentiation. in contrast, it had no effect when added after days postdifferentiation (data not shown), strongly suggesting that dc-sign expression was irreversible. this idea was borne out by a former study reporting the il- -dependent and irreversible up-regulation of pu. , a specific transcription factor of the myeloid lineage, mainly responsible for the induction of dc-sign expression in modcs and monocyte-derived macrophages during their in vitro differentiation [ , ] . in this study, besides il- or il- , we used fully differentiated immature modcs as a cellular model to document what inflammatory soluble factors could be involved in the sdc-sign secretion that we detected in several human body fluids. surprisingly, ifn-␣ and most probably, type i ifns, in general (although this remains to be experimentally ascertained), were shown to impair dc-sign during cell differentiation, whereas ifn-␥ was identified as a good inducer of sdc-sign by fully differentiated immature modc. cxcl- /il- was also demonstrated to promote sdc-sign expression but to a lesser extent. when added together as exogenous cytokines on il- -starved modcs, we observed additive effects on sdc-sign secretion, suggesting that they may be both required to induce optimal sdc-sign secretion, albeit weakly. even at higher doses of ifn-␥ and/or cxcl- /il- , we were unable to attain the sdc-sign secretion level obtained with il- or il- in vitro. to explain why, in such a case, ifn-␥ induced and did not repress sdc-sign secretion by modcs, one could argue once again that fully differentiated immature modcs have a distinct transcription program coordinated by distinct transcription factors (i.e., pu. , etc.) compared with monocytes during their differentiation. in genital human mucosa, after days of ex vivo cmv infection, we noted a significant increase of sdc-sign secretion by residing dcs. the analysis of the cytokine content of infected biopsy supernatants confirmed the presence of ifn-␥ and cxcl- /il- in sufficient amounts to imagine their direct involvement in the induction of sdc-sign. in these experiments, no difference of mdc-sign expression by dermal dcs could be observed by immunostaining. based on our own results, we expected that sdc-sign induction could be correlated with the same up-regulation of membrane isoforms. however, it is our belief that the explanation for this discrepancy can be found in a recent study reported by liu et al. [ ] , indicating that the vast majority of dc-signr, a closely related dc-sign gene product found in hiv mucosal inoculation sites, i.e., anal and genital mucosa, was encoded by tm lacking spliced cdna sequences [ ] . these data pointed to a differential expression of sdc-sign versus mdc-signr isoforms in virally infected peripheral tissues. despite being unable to document any differential expression of dc-sign in vitro, we cannot rule out this possibility in vivo. to better understand if this were the case, discriminating antibodies are currently being generated in our laboratory. interestingly, former studies provided strong evidence that ra synovia contained high levels of cxcl- /il- , which may be a result of viral infection caused by cmv [ ] . here, these molecules were detected in high amounts in infected bal or ra joint fluids, suggesting that they could also be sdc-sign inducers in vivo. moreover, cxcl- /il- was up-regulated in the female genital tract in the course of coinfection with cmv and hiv- [ ] . this observation substantially supported our sdc-sign induction data in genital mucosa explants. cxcl- /il- was already known as an efficient neutrophil and macrophage chemoattractant, and a link had been established between high serum concentrations and the cmv disease occurrence in patients after liver allotransplantation [ ] . the analysis of the sdc-sign content in sera from healthy volunteers showed a high heterogeneity of concentrations. on the basis of previous reports in the literature, we endeavored to forge a link between sdc-sign concentrations and a th cytokine environment, marked by high amounts of il- / in sera and bal from atopic patients. unfortunately, we were unable to show significant correlation (data not shown). in contrast, we demonstrated that high amounts of sdc-sign could be correlated to high amounts of cytokines and chemokines, usually overexpressed upon inflammation in diverse body fluids, such as serum, bal, or joint fluids, from patients suffering from autoimmune, inflammatory, or infectious diseases. these results match the data reporting an increased number of mdc-sign-expressing macrophages in bal from mycobacterium tuberculosis-infected patients [ ] , as well as in ra synovia [ ] . several studies have already reported a link between high frequencies of mdc-sign ϩ -infiltrating cells and the proinflammatory environment found in crohn's disease lesions [ ] . one can easily imagine that these mdc-sign ϩ cells may also be able to secrete sdc-sign upon inflammation and/or infection. thus, on the basis of these unexpected results, we suggest that dc-sign, whether sdc-sign or mdc-sign, may be considered as a new marker of tissue inflammation. our final investigation focused on sdc-sign functions. several studies have already documented the fact that rsdc-sign was able to neutralize hiv and dengue virus infection of transfectants or modc at similar mean concentrations (i.e., from . to g/ml [ , ] ). although navarro-sanchez et al. [ ] reported convincing data, they used truncated rsdc-sign, only consisting of the dc-sign crd domain, which was shown to be expressed as monomers in solution by others [ ] . crd monomers had a limited affinity for their ligands in comparison with tetramers [ ] . here, we provided strong evidence that flag-sdc-sign at was capable of interacting with the cmv gb, already known to be a ligand of mdc-sign [ ] . using our tetrameric rsdc-sign, we were unable to inhibit cmv infection of fsf, even when sizeable sdc-sign amounts were used (up to m; data not shown). we then hypothesized that sdc-sign could function as an enhancer of cmv infection. as expected, low flag-sdc-sign at concentrations (varying from to ng/ml) allowed a significant increase in the percentage of ie/e cmv ag ϩ immature modc from three distinct blood donors, whereas sdc-sign concentrations below and above this range had no potentializing effect. to explain this bell-curve effect, we proposed a hypothetical model at low but constant viral input. according to this model, at high sdc-sign concentrations, free virions are rapidly complexed, leading to an impaired binding and internalization of cmv into modc. between and ng/ml, sdc-sign may serve as an opsonin with cmv virions with an optimal stoichiometry leading to the capture and internalization of a maximum number of sdc-sign-immobilized virions. when using very low sdc-sign concentrations (below ng/ ml), the majority of virus is free, and the infection efficacy decreases to levels of infection without sdc-sign, as we postulated that sdc-sign-opsonized cmv was captured more efficiently by modc than free virions. this suggests the existence of one or several yet-unknown opsonic receptors for sdc-sign, which may not be able to interact with previously reported, truncated rsdc-sign [ , ] . a novel study is under progress in our lab to fulfil the identification of such a receptor. however, based on the literature, we discuss putative candidates. first, icam- and - , but not icam- , are ligands for mdc-sign [ , ] . however, only icam- is expressed by modc [ ] . although we cannot rule out, so far, the possibility for sdc-sign to interact with icam- , the involvement of icam- / in the sdc-sign-mediated infection enhancement in modc is likely to be inconceivable. second, mdc-sign complexes have the propensity to form multimers (di-, tri-, and tetramers, depending on the neck length [ ] ). as a consequence, one can imagine that sdc-sign, whether bound to cmv virions or not, may be able to dock on mdc-sign to form aggregates with pre-existing lectin homodimers or trimers. new experiments are needed to confirm this hypothesis. we finally considered a last potential candidate receptor enabling the sdc-sign-mediated cmv infection enhancement, the cd b molecule, also known as the ␣m integrin, a macrophage antigen- or complement receptor component when associated with the ␣ integrin. cd b is highly expressed by immature modc [ ] . interestingly, it has been involved recently in the uptake of mannosylated liposomes by macrophages in cooperation with a murine homologue of mdc-sign [ ] . furthermore, cd b has also been described to function as a facilitating agent for hiv opsonization by immature modc in a mdc-sign-dependent manner [ ] . it is thus highly tempting to speculate on the fact that sdc-sign-opsonized cmv particles may interact directly with cd b, leading to their internalization and thus, promoting modc infection. further experiments are obviously needed to confirm this hypothesis. in summary, our work has strengthened the notion that dc-sign, whether sdc-sign or mdc-sign, should be considered as an inflammatory rather than a regulatory marker. our results also raised the question of the existence of a cell surface receptor for sdc-sign-opsonized cmv virions on modc. as such, our findings shed new light on the diversity of the dc-sign repertoire and extend our knowledge of the use of mdc-sign as well as sdc-sign by cmv to divert the human innate immune response to its own benefit. f.h. designed the research and was responsible for the project and manuscript preparation; and p.n., l.v., and b.v. participated in the data analyses. all authors reviewed the paper and had access to raw data. dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells expression of dc-sign and dc-signr on human sinusoidal endothelium: a role for capturing hepatitis c virus particles dc-sign induction in alveolar macrophages defines privileged target host cells for mycobacteria in patients with tuberculosis expression of the dendritic cell-associated c-type lectin dc-sign by inflammatory matrix metalloproteinase-producing macrophages in rheumatoid arthritis synovium and interaction with intercellular adhesion molecule -positive t cells. arthritis rheum extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr binding-site geometry and flexibility in dc-sign demonstrated with surface force measurements a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr. subunit organization and binding to multivalent ligands dc-sign neck domain is a ph-sensor controlling oligomerization: saxs and hydrodynamic studies of extracellular domain dc-sign (cd ) expression is il- dependent and is negatively regulated by ifn, tgf-␤, and anti-inflammatory agents dc-sign-icam- interaction mediates dendritic cell trafficking identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses cutting edge: carbohydrate profiling identifies new pathogens that interact with dendritic cellspecific icam- -grabbing nonintegrin on dendritic cells pathogen recognition by dc-sign shapes adaptive immunity the c-type lectin dc-sign (cd ) is an antigen-uptake receptor for candida albicans on dendritic cells how c-type lectins detect pathogens the macrophage c-type lectin specific for galactose/n-acetylgalactosamine is an endocytic receptor expressed on monocyte-derived immature dendritic cells extensive repertoire of membrane-bound and soluble dendritic cell-specific icam- -grabbing nonintegrin (dc-sign ) and dc-sign isoforms. inter-individual variation in expression of dc-sign transcripts most dc-signr transcripts at mucosal hiv transmission sites are alternatively spliced isoforms dc-sign, but not sdc-sign, can modulate il- production from pmaand anti-cd -stimulated primary human cd t cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection human cytomegalovirus labeled with green fluorescent protein for live analysis of intracellular particle movements human cytomegalovirus impairs dendritic cell function: a novel mechanism of human cytomegalovirus immune escape efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/ macrophage colony-stimulating factor plus interleukin and downregulated by tumor necrosis factor ␣ b lymphocytes secrete antigenpresenting vesicles sequence and expression of a membrane-associated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp determinants in the n-terminal domains of galectin- for secretion by a novel pathway circumventing the endoplasmic reticulum-golgi complex a novel secretory pathway for interleukin- ␤, a protein lacking a signal sequence unconventional secretion of fibroblast growth factor and galectin- does not require shedding of plasma membrane-derived vesicles regulation of expression and secretion of galectin- in human monocyte-derived dendritic cells cd ϩ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to gm-csfϩtnf ␣ the structure of dc-signr with a portion of its repeat domain lends insights to modeling of the receptor tetramer structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam -grabbing non-integrin (cd ) on the cell surface interplay between promoter and structural gene variants control basal serum level of mannan-binding protein helicobacter pylori modulates the t helper cell /t helper cell balance through phase-variable interaction between lipopolysaccharide and dc-sign dc-sign association with the th environment of lepromatous lesions: cause or effect? unique cd intestinal macrophages contribute to the pathogenesis of crohn disease via il- /ifn-␥ axis increased expression of dc-signϩil- ϩil- ϩ and cd ϩil- -il- -dendritic cell populations in the colonic mucosa of patients with crohn's disease expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases define the migratory characteristics of human monocyte-derived dendritic cells mature dendritic cells pulsed with exosomes stimulate efficient cytotoxic t-lymphocyte responses and antitumour immunity proteomic analysis of dc-sign on dendritic cells detects tetramers required for ligand binding but no association with cd the transcription factor pu. controls dendritic cell development and flt cytokine receptor expression in a dose-dependent manner pu. regulates the tissue-specific expression of dendritic cell-specific intercellular adhesion molecule (icam)- -grabbing nonintegrin enhancement human cytomegalovirus replication in a human lung fibroblast cell line by interleukin- hiv type and cytomegalovirus coinfection in the female genital tract interleukin- serum concentrations after liver transplantation dc-sign-mediated internalization of hiv is required for transenhancement of t cell infection dendritic-cellspecific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses immature monocyte-derived dendritic cells are productively infected with herpes simplex virus type opsonization of hiv with complement enhances infection of dendritic cells and viral transfer to cd t cells in a cr and dc-sign-dependent manner cooperation of specific icam- grabbing nonintegrinrelated (signr ) and complement receptor type (cr ) in the uptake of oligomannose-coated liposomes by macrophages key words: opsonin ⅐ c-type lectin ⅐ inflammation this work was supported by an institutional grant from in-serm avenir (national french health institute), the aquitaine, and the pays de loire regions (j-m.h.'s salary). we thank olivier neyrolles for his critical reading of the manuscript, the etablissement français du sang d'aquitaine for blood samples, and the bordeaux imaging center (bic; confocal imaging). all authors certify that they have no conflicts of interest or competing financial interests. key: cord- - lgup yj authors: robbins, r.c.; almond, g.; byers, e. title: swine diseases and disorders date: - - journal: encyclopedia of agriculture and food systems doi: . /b - - - - . - sha: doc_id: cord_uid: lgup yj swine diseases and disorders that are significant in modern, commercial swine production systems are organized by body system; the reader will need to know basic anatomy and physiology. the industry significance, etiology, epidemiology, pathogenesis, clinical signs, postmortem and histpathologic lesions, diagnostic testing, and generic treatment, control, and prevention are described. diseases of a particular system are summarized in a differential diagnosis table. r elsevier inc. all rights reserved. autogenous vaccines vaccine made from microorganisms isolated from the animal it will be used on; in the united states these are killed and, by permit, are extended for use in a farm, production system, or region. farrow process of parturition; location where a pig is born and stays till weaning, usually - weeks of age. grow-finish phase of production that follows the nursery period where pigs reach slaughter weight; used to describe pigs from to weeks of age. histopathology; -ic the science and microscopic examination of formalin-fixed and paraffin-embedded sections of diseased tissues. lesion visible (microscopic or macroscopic) deviation from normal. national animal health monitoring service a program administered by united states department of agriculture-animal and plant health inspection service. nursery phase of production that begins after weaning, used to transition pigs from the farrowing house to finishing; used to describe pigs from to weeks of age. oie world organization for animal health created on january . pathognomonic characteristic of a specific disease or disorder that, when present, is sufficient to make a diagnosis. veterinary diagnostic laboratory location where samples are submitted and tests are run to determine the cause of disease. in an approach to investigating any suspected disease or disorder in swine production, a history should be gathered first. important history to understand from caretakers includes: age of pigs affected, duration of clinical signs, morbidity rate, mortality rate, treatments administered, response to treatments, and any other important information regarding previous diagnoses or disease in the affected group of animals. this is also the time to examine any production records that have been kept on the affected group of swine as well as previous groups for comparison. records include but are not limited to: where the animals originated from; number in the herd; age; daily mortality; number treated; name of treatment, route of delivery and dose; feed and water usage; high-low temperatures; and vaccinations received or administered. after examining the production records and obtaining a history, proceed with a visual examination of the herd. typically, it is a biosecurity custom to observe youngest groups first; however, in cases of suspected infectious diseases, it may be best to begin with the healthiest group advancing in order of increasing severity or prevalence. often, a definitive diagnosis is not achieved without an extensive clinical and pathological investigation. a postmortem examination, or necropsy, of affected pigs should occur last. any pigs recently deceased of natural causes should be examined to establish trends, with the understanding that submission of tissues from these animals may not yield valuable diagnostic results. tissues for diagnostic evaluation should be collected from clinically affected pigs that are euthanized immediately before necropsy. sampling of five or more pigs may be required to obtain a valuable diagnosis. when investigating signs referable to the central nervous system (cns), it is important to preserve brain and spinal cord tissue for microscopic evaluation in cases of neurological disease; therefore, blunt force trauma and brain penetration by captive bolt are not preferred methods of euthanasia. at minimum, fresh and formalin-fixed tissue samples should include: brain, tonsil, heart, lung, lymph nodes, spleen, kidney, liver, and intestine. additional samples that may be beneficial for diagnosis include: premortem whole blood and ethylenediaminetetraacetic acid-chelated blood (for serum chemistry and complete blood count), spinal cord, intact stifle and hock joints (remove the leg at the hip), intact eyeball with optic nerve attachment, urine, feed, and water. consult a diagnostic lab regarding any additional samples that may be required in determining an etiologic diagnosis. the etiologic diagnosis should be based on consistent history, signs, and pathology derived from a list of differential diagnoses that are most common or most likely to occur in that herd or production system. a treatment, control, or prevention program should be formulated simultaneously. before using any chemical, pharmaceutical, or biologic in swine intended for food, know the domestic use guidelines, importer requirements or producer-packer agreements regarding withdrawal times, residue and tolerance limits, prescribing guidelines, and prohibited substances. this section will focus on a practical approach to investigating signs of neurological disease in swine summarized in table . it is important to determine if clinical signs are consistent with cns or peripheral nervous system lesions (pns). common cns signs in pigs include behavioral abnormalities (most commonly stupor), ataxia, loss of righting, seizures or seizure-like activity (paddling), nystagmus, and blindness. musculoskeletal disorders may clinically confuse or complicate perceived pns signs and must be differentiated from each other. streptococcus suis is a gram-positive cocci with reported serotypes. observational studies implicate sows as carriers and piglets are colonized as they pass through the birth canal (amass et al., ) . disease occurs most frequently during the suckling and postweaning period. commingling pigs from different herds, concurrent infection with porcine reproductive and respiratory syndrome (prrs), and other stress factors may increase the risk of developing s. suis meningitis (villani, ; thanawongnuwech et al., ) . variable morbidity and mortality: mortality depends on early recognition and treatment. clinical signs of s. suis meningitis include paddling, recumbency, nystagmus, and seizure. isolation of s. suis from the lung, nasal secretions, or tonsil from normal pigs is clinically insignificant. in contrast, s. suis isolation from cerebrospinal fluid (csf), meninges, joints, endocardium, or serosal surfaces with or without lesions is relevant (pijoan, ) . few to no gross lesions may be observed during necropsy. early recognition of clinical signs followed by injection with an antimicrobial that s. suis is susceptible is the most effective means of treatment. administering an antimicrobial that s. suis is susceptible to in the drinking water has been proposed to control morbidity (villani, ) . antimicrobial susceptibility patterns for s. suis isolates from regional diagnostic laboratories can be used to assist in selection of an appropriate antimicrobial while diagnostic tests are pending; ceftiofur is effective . commercial and autogenous vaccines are available but due to s. suis serologic diversity may not be effective . haemophilus parasuis (hps), also called glässer's disease, causes bacterial meningitis, arthritis, and polyserositis similar to s. suis. infections are not clinically or grossly distinguishable from s. suis. definitive diagnosis is by bacterial isolation. however, hps is a fastidious gram-negative rod and culture media must be supplemented with v factor for successful isolation. owing to the difficulty in isolating hps, polymerase chain reaction (pcr) tests are a suitable alternative (oliveira et al., ) . like s. suis, isolation from the airways has little significance unless lesions are present (hoefling, ) . antimicrobial susceptibility testing identifies ceftiofur or florfenicol that are typically effective first choice therapeutics (oliveira, b) . prevention may be achieved with medicated early weaning. edema disease results when a fimbrial (f or f ) and shiga-like toxin (stx- e) positive strain of escherichia coli successfully attaches to brush border receptors releasing toxin that damages blood vessels including those of the blood-brain barrier causing edema and encephalomalacia. edema disease most commonly affects rapidly growing pigs, weeks postweaning. morbidity is moderate to high and mortality is high. acute death of robust pigs, ataxia, eyelid swelling, and diarrhea are typical clinical signs (rademacher, ) . at necropsy, edema may be observed in the mesentery between the loops of the spiral colon and in the cardiac region of the gastric mucosa. stomachs are usually full of feed. bacteriologic isolation of a β-hemolytic strain of e. coli from affected pigs with meningoencephalitis is not sufficient for a diagnosis. genotyping is necessary to confirm that the e. coli isolated was f or f and stx- e positive and thus capable to induce such lesions. there is no effective treatment. vaccination using an avirulent live culture of e. coli postweaning, thorough cleaning and disinfection between groups, and use of genetically resistance breeds that lack the fimbrial receptor are preventatives (fairbrother and gyles, ) . pseudorabies (prv), also known as aujezsky's disease, is caused by a herpesvirus. prv was eradicated from the us commercial swine herd in (usda aphis, ) . feral swine are potential reservoirs. cattle, sheep, dogs, and cats can also be infected with prv. high morbidity is due to large quantities of virus shed in saliva and nasal secretions for several weeks following infection. mortality is inversely related to age approaching % in neonates. clinical signs are also age dependent. neonates may die without signs. suckling and recently weaned pigs are those that commonly exhibit ataxia, tremors, excess salivation, and seizures. at necropsy, the brain appears congested and hemorrhagic. necrotic foci occur in the spleen, liver, lung, lymph node, and specifically tonsils. histopathologic lesions are characterized by nonsuppurative meningitis and intranuclear inclusion bodies. pcr, virus isolation (vi), immunohistochemistry (ihc), or fluorescent antibody can be used to confirm the diagnosis. no specific treatment is available. vaccination and eradication are effective for control (usda aphis, ) . in areas free of prv, suspicion of the disease should be reported to state and federal agencies as required. congenital tremors result when hypomyelination or demyelination of the brain and spinal cord. clinical signs are clonic muscle contractions that cause a general tremor of the entire body. pigs are affected at birth but severity subsides with note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). age (dewey, ) . mortality is variable and is the sequela of malnutrition because piglets are unable to nurse. there is no known treatment or prevention. hypoglycemia occurs when piglets fail to nurse the sow. this condition is observed within h afterbirth. there is a low morbidity but high mortality. pigs may appear disoriented, ataxic, recumbent, or dead. on necropsy, affected piglets will have empty stomachs. assigning an employee to attend farrowing to ensure piglets nurse will reduce incidence. water deprivation, also referred to as salt poisoning, is an idiopathic disease resulting from a period of inadequate water intake (carson, ) . high morbidity with variable mortality occurs. the disease is suspected when there is a history of power outage or poor management (thacker, ) . fighting over water access is the first clinical sign and occurs within hours. dog-sitting, opisthotonous, convulsions, and fighting over water follow and develop after h without water. removal of the brain from an affected animal reveals edema and eosinophilic meningoencephalitis with perivascular cuffing and this is pathognomonic (gudmundson and meagher, ) . serum or csf with a sodium level above meq l À may also be used for supporting evidence (osweiler and hurd, ) . treatment of swine showing signs with an anti-inflammatory is variably effective. when water is restored, limit intake to short, - min intervals until all animals have had a chance to drink and fighting has ceased after which water can be provided ad libitum. prevention is daily observation to ensure each animal can access water, adequate water delivery system, and equipping the facility with a generator or alternative method to deliver water during power outages. gastrointestinal diseases and disorders can occur in all ages of swine as summarized in table . most digestive diseases are referable to the gastrointestinal tract and result in diarrhea and occasional vomiting. diarrhea is the result of an intestinal dysfunction caused by malabsorption, excessive secretion, or effusion. unfortunately, this is not an exclusive characterization of diarrhea and overlap occurs (moeser and blikslager, ) . rather, differentials for diarrhea should be referable to age at onset and site of infection. gastric ulcers are noninfectious and result when glandular mucosa specifically the pars esophagea is traumatized by gastric acid. gastric ulcers have a wide variety of causes but are most commonly associated with small feed particle size (ayles et al., ) and interruption of feed intake whether caused by disease or poor management. it is common to see signs consistent with gastric ulceration increase following an acute prrs or influenza outbreak. morbidity and mortality vary with the scope of the underlying cause. clinical signs include regurgitation, vomiting, pallor or jaundice, and acute death. an acutely dead pig with blood in its stomach is indicative of an active ulcer and is sufficient evidence for a diagnosis. in chronic cases, ulceration causes hyperplasia resulting in stricture of the pars esophagea and regurgitation. feeding a coarse ground diet for weeks significantly decreases severity (ayles et al., ) but is impractical in modern production facilities. rotavirus is a nonenveloped rna virus with a doublelayered capsid allowing it to remain stable and infective in the environment for months and intrinsically resistant to some disinfectants. four serogroups infect swine: a, b, c, and e (the latter only reported from the united kingdom). in addition, infections with particular serogroups vary by age: type c mostly in suckling piglets and type a predominately in nursery pigs (stephenson et al., ) . type a is the most prevalent serogroup. severity of disease decreases with age and is self-limiting. the virus infects and destroys villous enterocytes resulting in villous atrophy. in response, crypt cells fill in the gaps but, because they are incapable of absorption, suckling piglets quickly loose body condition and have a gaunt or wasted appearance. neither clinical signs nor gross lesions are pathognomonic although loops of small intestine appear thinwalled with moderate to large amounts of watery contents. a histopathologic report of blunted villi and crypt hyperplasia is suggestive of rotavirus infection. infection with rotavirus can be confirmed by pcr or electron microscopy (em). enzymelinked immunosorbent assay (elisa) is also available but limited to detection of serogroup a. ihc and em detect rotavirus and confirm its role in pathology, but both tests lack table common gastrointestinal diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). sensitivity. treatment is supportive by administration of oral rehydration solutions. acidifiers and antibiotics are sometimes administered to control secondary bacterial infections. treatment success is variable and depends on the degree of malnourishment. prevention among neonatal piglets is through ingestion of lactogenic virus neutralizing antibody from the sow, which is stimulated by administering feedback of rotavirus positive piglet feces or intestines (arruda et al., ) or a modified-live commercial type a vaccine no less than weeks before farrowing. a modified-live commercial type a vaccine is also available for pigs. it does not induce cross-protection for other serogroups and may be cost-prohibitive. tge is caused by tgev, a coronavirus that is heat labile at temperatures above c, prone to dessication and photosensitization (bay et al., ) . the epidemic form causes acute disease in all age groups within as little as h of infection. morbidity and mortality is high, approaching %, in an epizootic outbreak. the severity of disease is age dependent but all ages will develop diarrhea (moeser and blikslager, ) . postweaning infections result in high morbidity but low mortality; the most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. necropsy reveals that the small intestine and colon are fluid filled, the small intestinal wall is thin almost translucent, and lacteals are empty. necrosis and atrophy are observed throughout the length of the villus. the colon and cecum are spared. the endemic form occurs when susceptible animals are introduced to the herd or after maternal antibody wanes. prior exposure to porcine respiratory coronavirus (prcv) may cause false-positive antibody test results. a tgev/prcv differential elisa is available. in an outbreak, sows are fed tissue of diarrheic pigs to stimulate herd immunity and new introductions of animals are stopped. after the exposure and a subsequent cool-down period of - months or after clinical signs cease, sentinels can be introduced and monitored for seroconversion (saif and sestak, ) . absence of seroconversion indicates successful elimination of tgev. commercial vaccines are available but should be used with caution and only when elimination is not an option. ped is caused by pedv, a coronavirus that causes signs and histolopathologic lesions indistinguishable from tgev. unlike tgev, pedv is more environmentally resistant making elimination more difficult. the disease has been described in europe, asia, and, as of , the united states. prevalence of the enzootic form is approximately % (chae et al., ) . morbidity approaches % and mortality is % or more in a naïve sow herd resulting in - weeks of production losses. clinical signs appear within h; piglets develop a watery, fetid diarrhea leading to dehydration, metabolic acidosis, and death before caretakers are able to humanely euthanize them. vomiting also occurs. the severity of disease is age dependent but all ages will develop diarrhea. postweaning infections result in a high morbidity but low mortality; most significant economic losses at this time are caused by reduced average daily gain, market weights, and overall system efficiency. viral shedding occurs up to days postinfection. reproductive failure and inefficiency is a sequela of an outbreak (olanratmanee et al., ) . tgev/pedv differential pcr is available to confirm a presumptive diagnosis of ped. serum can be submitted for elisa or immunofluorescent antibody but collected no sooner than weeks after diarrhea was o bserved. immunoprophylaxis using egg antibody or hyperimmune serum and supportive care including electrolyte administration have been used for treatment. in an outbreak, sows are fed tissue and feces from diarrheic pigs to stimulate herd immunity (olanratmanee et al., ) . hygiene is the key to reducing environmental contamination. preventing introduction of virus into a herd with biosecurity alone may not be sufficient because the virus has been found in aerosol up to miles from a positive farm (goede et al., ) . porcine coccidiosis is most often caused by isospora suis. farm hygiene, specifically farrowing rooms, and sow infestation influence the persistence of disease; however, age at infection rather than infectious dose has the greatest impact on severity (worliczek et al., ) . the prepatent period is approximately days. morbidity is variable and mortality is low. pasty diarrhea, unthrifty to potbellied appearance of - day old pigs, and below average wean weight is suspicious for coccidiosis. on necropsy, the small intestine often is thickened and the mucosal surface is necrotic and has an adherent pseudomembrane. histopathologic examination of the affected portion of the intestine reveals larvae in the lamina propria. sensitivity of fecal flotation is moderate. there is no effective treatment. prevention is by oral administration of an anticoccidial (maes et al., ) . heat treatment (flaming) of flooring may reduce environmental contamination. concrete, rubber coated and plastic flooring in the farrowing crates are difficult to clean and disinfect so removal may be the only option. swine dysentery (sd) is a spirochete of the genus brachyspira that is an oxygen-tolerant anaerobe giving it the ability to survive for long periods of time in manure, pits, and lagoons (schwartz et al., ) . rodents, particularly mice, are known vectors and can serve as reservoirs. brachyspira hyodysenteriae is the species known for causing sd. other species of brachyspira have been recently described in dysentery-like disease (burrough, ) . incubation is - days but disease occurs in a - week cycle. administration of tiamulin or lincomycin in the feed or water may alter the time to onset of signs after exposure. morbidity is high and mortality is low to moderate characteristically causing disease in only the finisher and mature groups. economic significance is mostly lost performance due to reduced daily gain and feed conversion. the specific mechanism of pathogenesis is not well understood but the spirochete does not invade the lamina propria. clinical signs are the presence of mucohemorrhagic diarrhea containing flakes of frank blood or appearing as a generalized brick red to rust color. lesions are mostly observed in the spiral colon where epithelial sloughing and mucosal invasion cause necrosis resulting in the formation of a pseudomembrane. the colonic walls may be thickened due to vascular congestion and mucosal hyperplasia. bacterial culture produces strong β-hemolysis. pcr test for confirmation and speciation is recommended for any isolate with characteristic growth. introduction of infected pigs and contaminated equipment or facilities are the source of infection. pleuromutilins, like tiamulin, and the lincosamide, lincomycin, are effective for treatment. however, if the environment remains contaminated, clinical signs will recur. depopulation has resulted in successful eradication (harms, ) . medicated elimination that combines thorough pulse medication with tiamulin, cleaning and disinfection, and employment of an aggressive rodent control program is also effective (burrough and sexton, ) . escherichia coli is a gram-negative rod that infects all ages of swine but must express virulence factors to cause diarrhea. escherichia coli colonize the small intestine by fimbria that binds receptors on the villous surface of enterocytes. enterotoxigenic e. coli (etec) then produce toxin(s) that increase osmolality leading to diarrhea (moeser and blikslager, ) . etec is subdivided by fimbria, toxin, and age of pig affected. neonatal diarrhea (nd) is most common in pigs - days of age. the onset of postweaning diarrhea (pwd) caused by f is delayed, occurring - days postweaning, compared to that caused by f and its severity is indirectly related to wean age. clinical signs are profuse diarrhea, rapid dehydration leading to emaciation, or death due to metabolic acidosis. fluid filled and hyperemic sections of jejunum and ileum may be present at necropsy but few consistent gross lesions occur. intestinal contents have a distinctly alkaline ph. isolation of large numbers of e. coli and with dense layers of rod-shaped bacteria covering villi seen on histopathology in samples from pigs with diarrhea is sufficient for diagnosis of e. coli but not etec. genotyping is necessary to determine fimbria and toxin types, which are essential to confirm diagnosis of etec. treatment of affected pigs/litters/groups includes administration of antibiotics and oral rehydration solution or electrolytes to correct hyperkalemia (kiers et al., ) . control and prevention of nd is by passively derived lactogenic immunoglobins from vaccinated females (kohler, ) . prevention of pwd include selection of genetically resistant breeds lacking k and f receptors, administration of an oral avirulent live culture to stimulate active immunity or competitively exclude field strains (genovese et al., ) , feeding ppm zinc oxide postweaning and probiotics. immunity and exclusion is unique to each fimbria; vaccines should include the prevalent genotype(s) causing the diarrhea. clostridium perfringens type a (cpa) is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of neonatal infection. frequency of cpa diarrhea is on the rise in the usa. in uncomplicated cases, mortality is low whereas morbidity is high and below average weaning weights result. cases of cpa diarrhea are associated with the expression of α and β toxin. cpa is cultured from the stomach and upper third of small intestine but does not bind intestinal epithelium causing few to no histologic lesions. because of its ubiquitous nature and prevalence among health pigs, cpa may be an opportunist and its role as a primary cause of neonatal enteritis is not definitive. large numbers ( þ or þ ) of gram-positive bacilli cultured from feces or intestinal contents of diarrheic pigs is suggestive of cpa. genotyping by pcr to confirm presence of cpb gene in cpa isolates and rule out of other causes of nd are supportive to the diagnosis (bueschel et al., ). treatment has variable success rates and is limited to administration of empirically selected antibiotics and oral rehydration solutions to affected piglets. control of cpa enteritis is best accomplished by preventing other causes of nd. following a thorough cleaning, sporicidal disinfectant should be applied to farrowing crates and equipment between litters and be allowed to dry before reloading. feeding of bacitracin to sows has resulted in significant increases in weaning weights (schultz, ) . a commercial cpa toxoid vaccine is available (hammer et al., ) . autogenous whole cell vaccines are also in use. if vaccine is unavailable, feedback might be considered but should be pursued with caution (robbins and byers, a) . cpc is a gram-positive bacillus and inefficient sporulator. sows are regarded as the source of infection. pathogenesis of type c is due to expression of β toxin leading to necrosis of intestinal epithelium resulting in hemorrhagic diarrhea or acute death of piglets less than days of age. gross necropsy reveals hemorrhagic and blood-filled loops of small intestine. a pseudomembrane may form on the luminal surface, and intestinal mucosa is edematous. gross and histopathologic lesions in the presence of large numbers ( þ or þ ) of grampositive bacilli cultured from feces or intestinal contents warrant a presumptive diagnosis. genotyping by pcr to confirm presence of the cpb gene is confirmatory (songer and uzal, ) . treatment of affected piglets is unrewarding due to the rapid and debilitating course of this disease. prevention is accomplished by vaccination of gestating females with a commercial toxoid and ensuring piglets consume sufficient colostral antibodies to result in protection. clostridium difficile is a gram-positive bacillus that easily sporulates making it environmentally resistant to many disinfectants. clostridium difficile associated diarrhea leads to a - % reduction in wean weights (songer and uzal, ) . although more than a third of piglet diarrhea involves c. difficile, it is the better known to cause healthcare-associated infections among humans. the pathogenesis of c. difficile infections is in response to the expression of toxins a and b. a watery diarrhea occurs in - day old piglets. mesocolonic edema may be observed at necropsy. clostridium difficile is difficult to culture and can be isolated from healthy piglets. therefore, volcano lesions on histologic exam and confirmation of toxins in fecal contents by antigen elisa are diagnostic. treatment is ill-defined but is likely similar to that for cpa enteritis, because it is likely to be initiated based on clinical signs, which are similar. autogenous vaccines are used to aid in prevention but efficacy is unclear. ppe, commonly referred to as ileitis, is the general categorization of infections caused by lawsonia intracellularis, an obligate intracellular bacterium. because the bacteria cause lesions in the ileum, ppe is also referred to as ileitis. seroprevalence in grow-finish herds can reach %. ppe can further be divided into four clinical forms (kroll et al., ) . porcine intestinal adenomatosis (pia) is most common in - week pigs and causes little mortality. porcine hemorrhagic enteritis (phe) affecting pigs weeks of age and older including breeding swine and can be associated with increased mortality and dark, bloody stools. necrotic enteritis (ne) and subclinical ileitis, the most common form, occur among postweaning pigs. in all forms, transmission is by the fecaloral route. crypt enterocytes infected with l. intracellularis become hyperplastic. the altered ratio of villous and crypt enterocytes leads to malabsorption and subsequent increases in feed conversion and time to reach market weights. pia results in variable degrees of thickened ileum that can be found at necropsy. the ileal lumen may contain a blood clot in phe or pseudomembrane in ne. when diarrhea ranging in color from normal (pia, ne, and subclinical) to dark-red or black (phe) is observed, ppe should be considered as a possible cause. subclinical ileitis usually causes no clinical signs (gebhart, ) . histopathologic lesions containing intracellular s-shaped organisms are suggestive of lawsonia infection but ihc should be used to confirm diagnosis. pcr is helpful to detect infection and is highly specific but moderately sensitive. cross-sectional or longitudinal serologic profiling using a widely available elisa is the best tool for determining timing of exposure. treatment is with effective antibiotics, such as tylosin, administered by injection or in the feed or water. control is by administration of a commercially available modified-live oral vaccine before infection or feeding antibiotics when infection is known to occur. vaccination should take place at least weeks before seroconversion (walter et al., ) . salmonellosis causing gastrointestinal disease in swine is most commonly associated with the species typhimurium. salmonella typhimurium is commonly isolated from swine. isolation of multidrug resistance strains of s. typhimurium from swine at slaughter have garnered attention from public health and food safety professionals and it is this that make this infection significant to the pork industry (foley et al., ) . some european union member states have implemented meat-juice serologic monitoring at slaughter to assess on-farm salmonella control programs. pathogenesis of s. typhimurium is similar to salmonella cholerasuis by invading enterocytes and subsequently macrophages leading to an infectious carrier state. initial infection causes inflammation and cytokine release that result in watery, yellow diarrhea containing feed particles. button ulcers may be visible on the mucosal surfaces of the colon and cecum on gross necropsy examination and, on histopathology, can be found to extend into the lamina propria. bacterial isolation without using enrichment media and the presence of histopathologic lesions is consistent with a diagnosis of salmonella enteritis. treatment is with antibiotics administered symptomatically to diarrheic pigs. antibiotic susceptibility of the isolate should be considered before initiating treatment. rearing pigs on slatted floors, decreasing stocking density, and acidification of digesta are effective in reducing the prevalence of salmonella infections in swine (funk and gebreyes, ; boyen et al., ) . cross-protection with s. cholerasuis vaccine has been reported and reduces carcass colonization (husa et al., ) . whipworm infestations of swine are the result of trichuris suis infection. pigs kept on pasture, in outdoor lots, or facilities with a history of t. suis diagnosis are at greatest risk for disease (pittman et al., a) . the prepatent period is - weeks. the egg is not immediately infective, which requires - weeks in the environment. the infectious larva hatches from the egg and invades enterocytes in the small intestine and cecum. the entire life cycle of t. suis is completed in the intestine. ulcerations in the mucosa and damage to capillary blood supply of intestinal epithelium lead to hemorrhage, anemia, and hypoalbuminemia. clinical signs are depressed weight gain, increased feed conversion, bloody diarrhea, ill thrift, and death. adult worms imbedded in the ileum, cecum, or proximal colon are sufficient for diagnosis of whipworms. eggs are intermittently shed and thus not a reliable method of diagnosis (pittman et al., a) . treatment and control are synonymous and require administration of an effective anthelmintic like fenbendazole. prevention is by steam sanitation and drying; however, eggs are resistant to common disinfectants and remain infective for years. the porcine integument or skin, like that of other domestic species, serves as a protective barrier between fragile internal tissues and harsh external hazards. skin is comprised of layers (from external to internal): epidermis, dermis (superficial and deep), and subcutis. blood vessels, hair follicles, sebaceous glands, and muscles are found in the dermis. notably, the pig's skin does not contain sweat glands; therefore, modern swine facilities are outfitted with evaporative cooling systems for thermal regulation in hot climates. skin diseases and disorders can be the result of viral or bacterial infections, parasite infestations, immunologic reactions, and idiopathic or iatrogenic causes that are summarized in table by their various macroscopic and microscopic lesions. greasy pig is a skin disease of swine caused by a toxin produced by staphylococcus hyicus. a break in the skin is the typical sequela. gilt litters reportedly have a higher incidence of this disease, presumably due to deficient maternal immunity. all ages of pigs may be affected but suckling and nursery pigs are most likely to develop disease. affected pigs develop focal crusts on the face, neck, and axillary region, and the crusts may coalesce as the disease progresses. affected areas are greasy to touch and may appear black due to dirt adhering to it. if pigs are untreated or fail to respond to treatment, the trunk and extremities may become involved. pyrexia and lethargy can be observed in severe cases and are followed by growth reduction. gross appearance of affected skin is rarely confused with other skin conditions of swine. submission of formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis for histopathologic examination is needed for a diagnosis. the pathognomonic histologic lesion is exudative epidermitis. table common integument diseases and disorders of pigs greasy pig x x x erysipelas x x porcine dermatopathy and nephropathy syndrome (pdns) x x x sarcoptic mange x x the first column provides the diseases. the remaining columns represent the type of lesion that occurs. the s. hyicus can be cultured from the surface of clinically normal skin sections. treatment includes topical application of antimicrobials or disinfectants. unaffected pigs with direct contact with affected pigs should also be treated to control spread. in cases where pigs exhibit systemic signs, administration of an injectable antimicrobial and anti-inflammatory is warranted. in the united states, no antimicrobials are labeled for the treatment, control or prevention of s. hyicus so all antimicrobial therapy is extra-label. prevention should focus on facility hygiene and include a soap degreaser and disinfectant regimen to reduce contamination. in addition, scarification of the skin of breeding age females with the farmspecific s. hyicus strain can reduce disease incidence in suckling pigs (murray and rademacher, ) . erysipelas or diamond skin disease is caused by a soilborne gram-positive bacterium, erysipelothrix rhusiopathiae. this zoonotic pathogen is transmitted by migratory fowl, turkeys, and pigs. humans may become sickened when direct contact with blood from affected animals contaminates an open wound (brooke and riley, ) . the finding of lesions at slaughter results in partial or complete carcass condemnation (bender et al., ) . the disease is most common in growing, finishing, and breeding age swine. bacterial emboli lodge in blood vessels causing vasculitis, thrombosis, and ischemia leading to lameness, abortions in gestating females, and raised, red to purple rhomboid skin lesions for which erysipelas is best known. skin biopsies from the affected area should include epidermis and dermis, but histologic lesions are only supportive. bacteriologic isolation or pcr identifying e. rhusiopathiae confirms the diagnosis. treatment with β-lactam antibiotics including penicillin is effective. commercial bacterins and avirulent live cultures are available for prevention (wood, ) or in the face of outbreaks to prevent the chronic form. pdns has been associated with porcine circovirus type (pcv ) infection, but any disease process resulting in ischemia could cause result in pdns. the condition is characterized by red to purple discoloration of skin that begins on the caudal surface of the hind limbs and the ventral surface of the abdomen resulting from ischemia. on necropsy, gross examination of the kidney cortex may be speckled with pinpoint, white foci caused by infarcted blood vessels. pig of any age can be affected with pdns, but it is more commonly observed during growing and finishing stages. submission of fresh and formalin-fixed skin sections that include the junction of affected and unaffected layers of epidermis and dermis is required. there is no specific treatment or prevention; rather, diagnose the underlying cause to determine appropriate therapy ( figure ) . sarcoptic mange is the result of an allergic reaction to the saliva of ectoparasites, sarcoptes scabiei. mange may also be caused by demodex phylloides. mortality is low and morbidity is moderate. economic losses are the result of reproductive inefficiency, growth reduction, and carcass condemnation. infestation and subsequent clinical signs in the breeding herd, most notably an incessant scratching, develop following the purchase of infested genetic replacements. in addition, growing pigs placed in facilities that previously housed infected swine or facilities that reuse straw bedding or have solid wood partitions may also become infested. the mite is rare in modern, high-health swine operations. the burrowing mite causes red pustules and flaking skin. individual pigs may develop signs in as few as weeks but a herd may not show signs for several months. in the chronic stage, thick crusts develop at the corners of and inside the ears. examination of a scraping from the crusts will reveal the mite (averbeck and stromberg, ). an elisa test is used to determine prior exposure and determine success of eradication programs. treatment can be applied topically using an antiparasitic, such as amitraz, to temporarily alleviate clinical signs. control and eradication programs utilize feeding or injection of ivermectins (mohr, ) . the musculoskeletal system is comprised of tendons, ligaments, muscles, and bones. disorders and disease of this system are typically characterized by lameness. lameness is any deviation in normal locomotion including favoring a limb or failure to bear weight on the limb. neurologic conditions, which also cause changes in locomotion, may be ruled out by postmortem examination of articular surfaces and diagnostic testing. investigation of musculoskeletal diseases and disorders should always start with the claws that are easily traumatized causing pain resulting in lameness. flooring and genetics also influence the incidence of lameness. common musculoskeletal diseases and disorders of swine can be divided into osteopathies and myopathies and summarized in table . mycoplasma hyosynoviae colonizes upper airways and tonsils resulting in a carrier state. transmission is vertical from sow to pigs and lateral between pigs (ross and spear, ) . m. hyosynoviae is most often diagnosed during the grow-finish phase. morbidity is variable but mortality is low. clinical signs are a stiff gait and difficulty in standing, most often the stifle or elbow and less frequently the hock, hip, and shoulder. signs often occur - weeks after a stressful event; lesions begin to resolve weeks postinfection. the affected joint contains yellow or blood-tinged effusion with moderate villous proliferation but is not always observed despite lameness and does not necessarily correlate with presence of histopathologic lesions. aseptic collection of synovial fluid by needle aspiration or sterile swab or submission of the affected joint intact is recommended for diagnosis. pcr is the most sensitive test; culture requires special media and lacks sensitivity (gomes neto et al., ) . histopathologic examination of formalin-fixed synovium reveals nonsuppurative fibrinous polyarthritis and lymphoplasmacytic perivascular synovitis. elisa is also available. lincomycin has historically been an effective therapeutic choice (burch and godwin, ) . treatment should be initiated when lameness is first observed; however, spontaneous resolution is common. no commercial vaccines are available. mycoplasma hyorhinis is a ubiquitous bacterium that is an early colonizer of upper airways. transmission is vertical from sow to pigs and then between pigs postweaning (rovira, ) . infection can progress to polyarthritis, polyserositis, and otitis in the pre-or postweaning phases; arthritis develops postweaning. clinical signs include lameness, arthritis, and fever that develop - days after septicemia occurs and persists for - days (gomes neto et al., ) . disease may become chronic resulting in ill thrift, reduced growth, and death. articular surfaces may be eroded. in cases of lameness, synovial fluid and formalin-fixed synovium can be submitted. alternatively, the entire affected leg can be submitted; disarticulate above the infected joint keeping the affected joint intact. submission of fibrin or fibrin covered tissue(s) should be included for pcr testing to differentiate m. hyorhinis from other bacteria that form fibrin on serosal surfaces like hps and s. suis (rovira, ). histopathology reveals fibrinosuppurative inflammation in affected tissues. treatment is empirical. erysipelas is the result of a chronic e. rhusiopathiae infection causing arthritis and endocarditis that follows the initial septicemia. lameness and joint swelling is mostly noticeable in hock and carpal joints. lameness may also occur in stifle and elbow but swelling cannot be appreciated. synovial fluid appears serosanguinous and can be submitted for testing by bacterial culture or pcr. alternatively, the entire affected leg can be removed to prevent contamination; disarticulate the leg above the infected joint. histopathologic examination of formalin-fixed synovium reveals a proliferative synovitis. other lesions that occur are nonsuppurative fibrinous polyarthritis and erosion of cartilage that can progress to pannus and ankylosis. treatment with β-lactamase antibiotics including penicillin is effective. an anti-inflammatory is added to a treatment program for pain management. commercial bacterins or avirulent live cultures are available for control and prevention. oc is the result of a delay in ossification of articular cartilage, and represents the most common lesion among culled sows. morbidity is most often reported in adult and breeding age pigs (dewey et al., ) . mortality is variable and is the result of humane euthanasia because the animal becomes nonambulatory. oc causes lameness, pain, and joint swelling. a noninfectious lameness most often affects the distal part of the humerus or femur. lesions are typically bilateral and symmetrical. diagnosis is made by ruling out other causes of lameness. ricketts occurs as a result of phosphorus deficiency, vitamin d deficiency, or secondary to iron toxicity but is not caused by dietary calcium deficiency. the condition should be suspected when there is an increase in nonambulatory pigs and broken bones during the finishing stage particularly at and immediately before marketing. occasionally, joint swelling in the nursery stage is observed. rachitic rosary (enlargement of costochondral junctions) and soft bones are observed on necropsy. if rickets are present, a bone ash analysis of the second rib will be below normal. feed analysis can identify low levels of vitamin d or phosphorus. low levels of vitamin d or phosphorous serum chemistry also will occur (madson et al., ) . supplementation of vitamin d is the only reported treatment and response that may be considered diagnostic. prevention includes proper diet formulation for the stage of production. mhd is a noninfectious disease of muscle caused by deficiency of vitamin e or selenium. it can occur if pigs are fed grain grown in selenium deficient soils (dewey, ) . clinical signs are limited to acute death of large, robust pigs. on necropsy, the heart muscle has a mottled appearance. feed analysis, response to vitamin e supplementation, and ruling out other causes support diagnosis of vitamin e/selenium deficiencies like mhd (hooser, ) . supplementation with selenium is impractical in the united states because of environmental regulations, and overzealous supplementation may cause toxicosis. splayleg is a noninfectious, congenital condition resulting from delayed myofibril development with no known cause. splayleg has low morbidity and mortality as long as it is identified and corrected before it leads to starvation or being crushed by the sow. treatment includes the use of nonslip note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). flooring in farrowing crates and application of harness or tape that holds the rear legs under the pig until it is strong enough to walk on its own. reproductive failure occurs when insemination fails to result in pregnancy or pregnancy fails to produce viable pigs due to infectious and noninfectious causes summarized in table . reproductive failure should be considered when a low conception or farrowing rate, irregular returns to estrus, abortions, stillbirths, or mummies persist at an abnormal rate. infertility occurs when fewer than four embryos are present at the time of maternal recognition of pregnancy resulting in a regular return to estrus and reduced conception rate for that breeding group. irregular returns to estrus result from embryonic death or early term abortion after implantation but before calcification of the fetuses. embryonic death of some or all of the embryos will result in low total born or irregular return to estrus, respectively. early term abortion also will reduce farrowing rates. mummies and stillborns can occur any time after calcification of the fetuses. the normal rates for mummies and stillborns are o . and o pig per litter, respectively. late-term abortions are classified as those occurring after days of gestation. total abortion rate should remain o % of a breeding group. these are general guidelines; thus, familiarity with the herd's normal reproductive performance is the most sensitive means to identify a reproductive problem. prrs is, at this time, known only to occur among swine. the estimated cost of prrs to the us pork industry is us$ million annually (holtkamp et al., ) . prrs usually results when susceptible swine are infected with either the leylystad or north american strains of prrs virus (prrsv), a member of the arteriviridae family. viremia lasts up to days, but shedding of infectious virus can last much longer (murtaugh and genzow, ) . prrsv is most commonly transmitted by introduction of infected swine or contaminated fomites, use of contaminated semen, and aerosol. the pathogenesis of the reproductive form is believed to be arteritis of fetal umbilical cords during gestation (lager and halbur, ) . swine may show no signs when reinfected with a homologous strain. conversely, infection with a heterologous strain will reproduce lesions and disease but is usually less severe than that of naïve swine (murtaugh and genzow, ) . clinical signs of prrs in a breeding herd start with an epidemic of abortions followed by an increase in low viable piglets, stillbirths, and mummies. abortions result due to fetal death or pyrexia of the gestating female. sows and gilts may be anorexic, pyrexic, or lethargic. periparturient females may become agalactic. in severe outbreaks of prrs, sow mortality also increases. in utero infection of feti can result in persistently infected piglets (rossow, ) . prewean mortality commonly increases and may remain above the herd average for weeks. diagnosis can be made by submitting lung, spleen, and lymph node from fetuses or low viable piglets. whole fetuses can also be submitted but should be refrigerated to prevent autolysis. lesions are not pathognomonic so confirmatory testing such as pcr, ihc, or vi should be conducted. tissues and thoracic fluid from stillbirths, aborted, or mummified feti can be submitted but may result in false negatives. serum collected from aborted sows or low viable piglets and tested for prrsv by pcr is another option for diagnosis. prrsv elisa indicates previous exposure but is not useful in a previously exposed herd. treatment of prrs is supportive. anti-inflammatories to reduce fever and antibiotics for control and treatment of secondary bacterial pneumonia may be necessary. the most common methods for control include depopulationrepopulation and herd closure and rollover, also called loadclose-homogenize, using commercial vaccine or herd-specific live virus exposure (corzo et al., ) . periods of closure vary based on facility capacity but a minimum of days is recommended. commercial modified-live and killed vaccines are available but do not prevent infection and should be used in accordance with label and domestic guidelines. ppv is sometimes described by the acronym smedi (stillborns, mummies, embryonic death, and infertility). ppv is an enzootic infection of swine breeding herds in the united states. the virus is ubiquitous and is transmitted through ingestion of infected feces, afterbirth, or fetal tissue. the disease most commonly affects gilts and younger parity sows (christianson, ) . the pathogenesis is through damage to table common reproductive diseases and disorders of pigs note: the first column provides the diseases. the remaining columns represent the clinical signs. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). the placental epithelium resulting in fetal death. clinical signs of ppv range from low total born, mummies of various sizes, irregular returns to estrus, and females diagnosed pregnant but fail to farrow. diagnosis is based on vaccination history, clinical signs, and pcr testing of mummified fetuses. ppv elisa may provide diagnostic value if acute and convalescent serum samples are used. there is no effective treatment for ppv; however, commercial killed vaccines are available and very effective. exposure of unbred females to tissue or cull sows from a seropositive herd has been used for immunization when vaccine is unavailable. leptospirosis is caused by infection by spirochete bacteria. leptospira species may be zoonotic (leptospira canicola, l. icterohemorrhagiae), swine-adapted (l. pomona and l. bratislava), or incidentally infect swine (l. grippotyphosa and l. hardjo). infection has been associated with exposure of swine to contaminated soil or untreated surface water, and exposure to urine from infected vectors, such as rodents. infected swine can become carriers resulting in chronic disease. the pathogenesis is due to bacteremia resulting in transplacental infection followed by fetal death. clinical signs include pyrexia, low conception rate, abortion, stillbirths, and low viability pigs resulting in increased prewean mortality. diagnosis is made using dark field microscopy or ihc performed on tissues, particularly kidney, of aborted feti or stillbirths. paired or matched serology for hemagglutination inhibition (hi) testing may be useful if suspected. treatment with antibiotics, such as chlortetracycline, may be pursued (henry et al., ) . commercial killed bacterins are available to aid in prevention and should be given at least semiannually to breeding stock (christianson, ) but may not be available in all countries. for example, federal regulations prohibit the use of these bacterins in france and the netherlands (figure ) . pcv is a ubiquitous virus in swine facilities. pigs become infected with pcv through ingestion (oral nasal contact). in addition, breeding females can become infected via insemination with contaminated semen (madson et al., a) . gilts and low parity sows are affected most often, whereas boars show no clinical signs. pcv -associated reproductive failure may occur in conjunction with ppv. infection results in variable lengths of viremia. pcv -reproductive failure is due to transplacental infection of fetuses. clinical signs depend on the stage of gestation when the infection occurs. embryonic death, early term abortions, stillbirths, mummies, low total born, or low viable pigs can result from infection. mummies may vary in size, like ppv, and measuring crown to rump length is useful to determine the time when that fetus was infected. pcv -reproductive failure is diagnosed by the presence of viral antigen confirmed by ihc or deoxyribonucleic acid confirmed by pcr along with the presence of lesions in fetal tissue notably myocardial mineralization. pcr testing of fetal thoracic fluid is sufficient to diagnose in utero infection of piglets (madson and opriessnig, ) . commerical killed baculovirus vectored vaccines are available and effective for prevention of disease but not infection or viremia (madson et al., b) . prv or aujezsky's disease virus was eradicated from the us commercial swine herd in ; a comprehensive review is available (usda aphis, ). prv is a member of the herpesviridae family and, like other herpesviruses, infection can result in a carrier state or latency within nervous tissue with the potential for recrudescence. the pathogenesis of prv results from viremia, and then replication and necrosis of epithelial tissue including the placenta (christianson, ) . the period of viremia gives prv time to cross the placenta and cause fetal death. clinical signs following acute infection include embryonic death, abortion, mummies, and stillborns. necrotic foci can be found in fetal spleen, liver, lung, and lymph node. histopathology is not definitive; ihc is required to confirm presence of antigen. diagnosis may also be made through serology; a commercial elisa test is available and can differentiate between exposure to the gene-deleted vaccine and wild-type virus used extensively in the us eradication. commercial prv vaccines are available but only should be used in accordance with federal guidelines. brucellosis is a zoonotic infection caused by the bacteria, brucella suis biovars and . brucella suis is transmitted through direct contact with susceptible swine, ingestion of infected tissue, or fluids including milk and contaminated semen. pathogenesis of b. suis is initiated when the mucosal epithelium is penetrated, thereby resulting in bacteremia that commonly persists for weeks and results in placentitis among other lesions. clinical signs of infection in gilts and sows include abortion with or without vaginal discharge, whereas boars have reduced libido and fertility. bacteriologic isolation of b. suis from vaginal discharge or tissue confirms diagnosis. serology reflects prior exposure (or vaccination) to b. suis but not for diagnosis of acute disease. the us commercial swine herd is brucellosis free. swine erysipelas (se) is a zoonotic, gram-positive bacterium, which is ubiquitous among swine. erysipelothrix rhusiopathiae is the sole causative species. carrier swine shed the bacteria in saliva, nasal discharge, and feces. infection may result from direct contact with carriers, exposure to infected facilities or soil (wood, ) . a bacteremia lasting several days precedes lesions. reproductive failure is most often due to abortion but infertility and low total born following high fevers or endometritis at the time of breeding is also possible. clinical signs including rhomboid skin lesions, high fevers, lethargy, inappetence, withdrawal, and response to treatment with penicillin of affected sows and gilts are suggestive of acute and subacute se. serology is available; availability is by veterinary diagnostic laboratory (vdl) and value is limited when vaccine is in use. bacterial culture and histopathologic examination of fetal tissue is unrewarding for diagnosis of se but is helpful to rule out other causes of abortion. in chronic se, culture of e. rhusiopathiae from vulvar discharges was successful (gertenbach and bilkei, ) . treatment involves injections of antibiotics and anti-inflammatories. commercial vaccines are available and effective. co poisoning induces hypoxia resulting in an increased number of stillborns (hooser, ) . concentrations of ppm are toxic. malfunctioning heating units or poorly ventilated farrowing rooms are the cause. diagnosis is done by ruling out infectious causes of stillbirths. fetal blood or thoracic fluid can also be measured for co concentrations. zearalenone is a luteotropic mycotoxin produced by fusarium rosea. it binds estrogen receptors resulting in irregular returns to estrus, signs of estrus in prepubertal gilts, and reduced litter size (hooser, ) . diagnosis is by detection of elevated levels in feed samples. however, definitive diagnosis is rarely possible because the contaminated feed has long been consumed by the time reproductive failure occurs. aas and seasonal infertility is a noninfectious cause of reproductive failure. the declining photoperiod and temperature fluctuations during the fall months result in declining progesterone levels. high-ambient temperature experienced during lactation and the postweaning period are suspected but not confirmed as a cause. diagnosis is done by ruling out infectious causes and careful assessment of management, facilities, and reproduction records (rueff, ) . modern facilities that utilize gestation crates and evaporative cooling systems may improve but not prevent infertility during the fall months (leman, ) . the respiratory system can be simply divided into upper and lower portions. the upper portion includes the nasal cavity and sinuses, throat, trachea, and bronchi for air conduction. the lower portion is the lung comprised of bronchioles and alveoli responsible for air exchange. the respiratory system is commonly involved in numerous infectious diseases of swine summarized in table . the most notable infectious agents are the viral pathogens, prrs and pcv , which cause primary pathologic lesions to both the respiratory and the immune system. this damage to the immune system often leads to respiratory or systemic disease incited by secondary infectious agents. such mixed respiratory infections can occur at any age, and, when they occur in growing and finishing pigs, are termed porcine respiratory disease complex (prdc). multifactorial respiratory disease can obscure histopathologic lesions complicating the diagnostic process. app is a host-adapted, fastidious, and gram-negative encapsulated rod that is transmitted vertically from sow to piglet. morbidity and mortality are strain-specific; virulence varies with expression of apxi and apxii toxins. inhalation of strains of app expressing apx toxins results in lung lesions within - h. the disease is economically significant because mortality occurs during the later part of the finishing phase, usually just before slaughter. clinical signs of fever, lethargy, dyspnea, and acute death are common. pigs found dead may have a frothy, blood-tinged discharge from the nose and mouth. focal hemorrhage occurs in the diaphragmatic lung lobe, which is firm, and its appearance is likened to that of a bull's eye. fibrinous, necrotizing bronchopneumonia containing streaming leukocytes is a key histopathologic feature. bacterial culture is difficult and requires nicotinamide adenine dinucleotide (nad)-supplemented media so diagnosis is traditionally made on finding characteristic postmortem and histopathologic lesions. a pcr test is also available. in an outbreak, the entire population should receive antimicrobial therapy parentally. unlike many other gram-negative bacteria, app is sensitive to a variety of antimicrobials; at iowa state university vdl % of isolates were sensitive to ceftiofur, enrofloxacin, florfenicol, tiamulin, tilmicosin, and tulathromycin. prevention is aimed at eliminating carrier swine through depopulation or by pulse medication (marsteller and fenwick, ) . actinobacillus suis causes a hemorrhagic, necrotizing pneumonia during nursery and grow-finish phases. actinobacillus suis infection has similar clinical signs and pathologic appearance to app. affected pigs are frequently observed in a dog-sitting position with elbows abducted. unlike app, lung lesions are random in their distribution and petechial table common respiratory diseases and disorders of pigs postweaning nursery postweaning grow-finish adult note: the first column provides the diseases. the remaining columns represent the respective phases of production. the frequency of the occurrence is þ (occasional), þþ (common), and þþ þ (routine). hemorrhages may be seen in other organs due to the septicemia that follows a. suis infection (yaeger, ) . a pcr test is available to help differentiate disease from app and hps (oliveira, a) . like app, outbreaks should be treated by parental delivery of an antimicrobial; however, treatment of only those individual pigs with clinical signs is usually sufficient. autogenous vaccines can be used for control but response is variable because most pigs are already seropositive at the time of vaccination. ascaris suum, the swine roundworm, is the most common parasitic infection of swine. the reduced growth performance and liver condemnations are responsible for economic losses (stewart and hoyt, ) . the prepatent period is - days. adult roundworms are present in the manure but it is the migration of larvae through the lungs, occurring - days after ingestion of an infective egg that causes respiratory signs. a persistent cough and dyspnea result due to verminous pneumonia. the liver develops whitish spots, called 'milk spots' that are the cause of condemnations but resolve within days (stewart and hoyt, ) . the presence of eosinophils is suggestive of a parasite infestation. treatment and control is accomplished using anthelmintics: dichlorvos, fenbendazole, levamisole eliminate adults and larvae; piperazine kills only adults. proper cleaning and disinfection particularly removing fecal material between groups reduces potential for exposure but it is virtually impossible to get rid of a. suum once a premise is infested (pittman et al., b) . it is necessary to prevent access to contaminated soil. ar is described in two forms: progressive (par) and nonprogressive (npar). par is caused by toxigenic strains of pasteurella multocida, whereas npar is the result of toxigenic strains of bordetella bronchiseptica. in both forms, the bacteria attach to cilia in the nasal passages and the cytotoxin production causes hypoplasia of nasal turbinates. clinical signs include sneezing, deviated snouts, and, in cases of par, bloody nasal discharge occurring in a large number of grow-finish pigs. mortality is low but the reduced growth that results due to ar makes it economically important. beacause the cytotoxins are responsible for ar, isolation of either bacterium from nasal passages is not sufficient for diagnosis. in addition, b. bronchiseptica and p. multocida colonize the lung leading to bronchopneumonia causing cough and dyspnea in pigs postweaning, often part of prdc (hansen et al., ) . therefore, examination of nasal turbinates at slaughter is the recommended method for diagnosis of ar (gatlin et al., ) . transmission is vertical; therefore, prefarrow vaccination of sows can protect piglets up to weeks of age. if vaccination does not prevent ar, depopulation of the herd may be necessary. hps is also called glässer's disease. there are serovars identified and prefer to colonize the nose (macinnes et al., ) . hps may not actually result in pneumonia but does cause signs of respiratory disease including nasal discharge and dyspnea. in addition, fever, lethargy, and acute death are observed. on necropsy, one or all of the pleural, pericardial, epicardial, and peritoneal serosal surfaces become covered in fibrin. effusion commonly occurs. histopathologic lesions are described as fibrinopurulent. definitive diagnosis is by bacterial isolation on culture media supplemented with v factor. owing to the difficulty in isolating hps, pcr testing is now available (oliveira et al., ) . isolation from the airways in the absence of lesions has little significance (hoefling, ) . ceftiofur, enrofloxacin, or tulathromycin delivered parenterally to affected animals are effective therapeutic drugs. use of water-soluble antimicrobials is for control. maternal immunity, medicated early weaning, and controlling infections with prrs, pcv , and influenza postpone or prevent disease onset (rapp-gabrielson et al., ) . commercial and autogenous vaccines are available but may experience limited efficacy due to serologic diversity; controlled exposure to low dose, live virulent culture is another option (oliveira et al., ) (figure ) . mh is known to infect pigs in production systems worldwide causing reduced growth performance and mortality. the disease is classified as enzootic pneumonia or a component of prdc. both manifestations of mh cause paralysis of the mucociliary escalator resulting in a severe cough and dyspnea known as thumping. vertical and lateral transmission can occur; but, owing to its slow rate of transmission between pigs, the disease primarily occurs in grow-finish pigs (meyns et al., ) . in addition, time of colonization with mh and disease severity are directly related (fano et al., ) . on necropsy, well-demarcated (red to purple lobular consolidation occurs in the apical) diaphragmatic, and accessory lung lobes is visible. histopathologic lesions characteristic of mh is bronchopneumonia with lymphocytic perivascular, peribronchial, and peribronchiolar cuffing. because mh is difficult to isolate, pcr is the most sensitive method of detection. elisa is available and is helpful in establishing herd status but must be interpreted in the context of vaccination as tests do not distinguish between antibodies produced subsequent to vaccination or field infection. treatment of affected pigs with parental antimicrobials like enrofloxacin, tulathromycin, or lincomycin or administration of water-soluble lincomycin, tiamulin, or tetracyclines to affected groups is effective in outbreaks. control can be achieved through pulse-medication in feed of chlortetracycline (thacker et al., ) beginning figure epicarditis, heart, nursery pig. fibrin gives surface a granular appearance, caused by hps infection. note the enlarged (draining) mediastinal lymph nodes located cranial to the base of the heart and the excess thoracic fluid (reddish-brown) indicative of septicemia. courtesy dr. glen almond. week before the historical onset of disease . commercial vaccines are whole cell bacterins marketed to reduce lesions but do not prevent disease or slow transmission rate. simultaneous infection with prrsv reduces efficacy of mh vaccination thacker, ) . eradication from the herd is preventative but practically difficult to accomplish. pcvad is any disease process where pcv infection results in lesions and includes pmws (ellis et al., ) and pdns. infection with pcv is widespread. morbidity and mortality is variable, often dependent on the occurrence of secondary infections and their virulence. survivors of pcvad remain stunted, owing to the economic significance of this collection of diseases. clinical signs include wasting, dyspnea, depression, ill thrift, and diarrhea. lungs are wet, heavy, and fail to collapse; pulmonary edema and lymphadenopathy also can be found at necropsy. histopathology results include presence of interstitial pneumonia, lymphoid depletion, enteritis, nephritis, and dermatitis. for a diagnosis of pcvad the following must occur: pcv antigen within characteristic lesions and lymph nodes are depleted (sorden, ) . ihc is used to confirm presence of pcv antigen within the histopathologic lesion. pcr has little value in diagnosing pcvad unless the herd is considered free. commercial vaccines are very effective and available with flex labels for administration to sows and pigs and as or doses (chae, ) . nonvaccinated, subclinically infected pigs have poorer weight gain compared to their vaccinated counterparts (kristensen et al., ) ; therefore, it is part of most vaccination protocols by us pork producers ( figure ) . prrs is the result of infection with the leylystad or north american strain of prrsv. the estimated cost of prrs to the us pork industry is us$ million annually (holtkamp et al., ) . prrsv is the most commonly diagnosed viral respiratory pathogen at vdls (gauger, ) . infection is observed to increase susceptibility to other infections, particularly opportunistic bacteria. this apparent increased susceptibility to secondary and opportunistic infections is the result of the pathologic process in which prrsv recruits and replicates in pulmonary alveolar macrophages, and then disseminates systemically (rossow, ) . clinical signs are nonspecific including fever, lethargy, and dypsnea but not cough. signs also depend on the type of secondary infection(s) present. lungs fail to collapse and appear heavy, wet, and gray on postmortem examinations. lymphadenopathy is caused by hyperplasia of germinal centers. interstitial pneumonia, alveoli are lined with hyperplastic type ii pneumocytes and contain necrotic debris, whereas the lining of bronchi and bronchioles is normal (rossow, ) . vasculitis also occurs. pcr is the most sensitive method for confirming infection. owing to the genetic diversity of prrsv, sequencing of the orf region is a common adjunct to pcr testing. sequences are then used to create dendrograms for use by production systems pursuing prrs control and epidemiologic investigations (murtaugh, ) . prrsv elisa is helpful for establishing herd status; national animal health monitoring service reports that a large percentage of us herds are seropositive. treatment is limited to maintaining pig comfort, minimizing stress, and controlling secondary infections. commercial modified-live vaccines (mlv) are available and administration during the nursery phase significantly reduces mortality and improves growth performance during the grow-finish phase of production (robbins et al., b) . mlv vaccines do replicate and should not be used in negative populations. salmonella cholerasuis is the swine-adapted salmonella from the c serogroup and, unlike s. typhimurium, is not a foodborne pathogen. ingestion or inhalation of the bacteria causes a septicemia resulting in low to moderate morbidity with high mortality within - days of infection that occurs postweaning, predominately during the grow-finish phase (baskerville and dow, ) . signs include high fevers ( c), lethargy, dyspnea, acute death, and cyanotic extremities and abdomen. the latter makes it impossible to differentiate clinically from classical swine fever virus (csfv). pleuropneumonia, interlobular edema, mediastinal, and tracheobronchial lymphoadenopathy, and occasionally white foci in the liver are apparent postmortem (turk et al., ) . acute histopathologic lesions that form in the lung are purulent bronchitis, lobular necrosis, and abscessation, whereas paratyphoid nodules are observed in the liver. isolation is best achieved from the draining lymph nodes, lung, or liver using selective culture media. serogrouping and typing is necessary for speciation and diagnostic confirmation. owing to the rapid onset of disease, parental treatment is recommended. salmonella cholerasuis isolates are commonly susceptible to ceftiofur. increased hygiene particularly eliminating access to waste and vaccination is preventive (husa et al., ) . siv is more accurately described as influenza, to encompass the infections occurring in swine, avian, and human species. influenza virus is classified by its hemagglutinin and neuraminidase proteins; the three predominant strains in pigs are h n , h n , and h n . rapid transmission and onset are characteristic; in the experimental inoculation of one nonvaccinated nursery age pig resulted in . more becoming infected (romagosa et al., ) . virus is shed for - days and uncomplicated lesions resolve days postinfection (gramer, ) . nasal discharge, fever, and lethargy occur but resolve quickly. cough and dyspnea can last up to figure pulmonary edema, lung, grow-finish pig. interlobular edema associated with pcvad, ventral portion of apical and diaphragmatic lung lobes is consolidated (purple) due to secondary bacterial infection. courtesy dr. glen almond. weeks postinfection. pcr and vi detect virus for diagnosis of clinical cases. elisa and hi detect antibodies; elisa is helpful in establishing herd status, whereas hi is best for vaccination timing and measuring postvaccination titers (allerson et al., ) . necrotizing bronchitis, bronchiolitis, and alveolitis as lesion resolves affected areas appear vacuolated. pigs recover quickly so treatment should focus on maintaining pig comfort, minimizing stress, and controlling secondary infections. all licensed vaccines are killed; commercial and autogenous products are in use in the united states. vaccination reduces lung lesions and rate of transmission, but does not prevent infection and is complicated by antigenic shift and drift. in the united states, it is typical to vaccinate the sows rather than pigs to control disease and infection (allerson et al., ) . trade diseases are those listed by the oie. when one of these diseases is suspected or confirmed, it results in closure of international market access, which would be economically devastating to import-export businesses. the primary method for managing diseases that affect trade is to prevent their introduction. foot-and-mouth disease (fmd) is caused by a picornavirus, fmdv, which causes mucosal lesions exclusively in cloven hoofed species. clinical signs are excessive salivation, anorexia, and lameness causing high morbidity but low mortality. gross lesions are vesicles at cutaneous junctions, on the snout, or in the oral cavity. similar lesions can be caused by seneca valley virus, vesicular stomatitis, swine vesicular disease, and vesicular exanthema of swine; therefore, any blister in swine warrants diagnostic investigation. fmdv is highly transmissible within and between species. african swine fever (asf) is caused by asfv, currently classified as an iridovirus. soft ticks can act as reservoirs or vectors. current outbreaks are reported throughout eastern europe and russia that have been associated with improper garbage feeding. the virus damages blood vessels resulting in clinical signs and gross lesions consistent with septicemia; including red to purple skin discoloration and enlarged spleen, liver, and lymph nodes. excess blood and fluid in body cavities may occur. classical swine fever, historically referred to as hog cholera, is caused by csfv, a pestivirus, eradicated from the united states in . transmission is associated with infected feeding, uncooked or undercooked garbage containing pork or pork by-products to swine. the virus remains infectious for months when refrigerated and years when frozen. clinical signs are nonspecific and are easily confused with s. cholerasuis. the virus replicates rapidly in tonsils, which makes it the ideal tissue to collect for diagnosis of csfv. see also: animal health: ectoparasites. animal health: foot-and-mouth disease. animal health: global antibiotic issues. slum livestock agriculture. vaccines and vaccination practices: key to sustainable animal production. zoonotic helminths of livestock the impact of maternally derived immunity on influenza a virus transmission in neonatal pig populations maternally derived antibody transfer to piglets following siv vaccination streptococcus suis colonization of piglets during parturition model for characterizing oral controlled exposure in a field setting sarcoptic mange in swine effect of dietary particle size on gastric ulcers, assessed by endoscopic examination, and relationship between ulcer severity and growth performance of individually fed pigs pathology of experimental pneumonia in pigs produced by salmonella cholera-suis some properties of the causative agent of transmissible gastroenteritis in swine erysipelothrix spp. genotypes, serotypes, and surface protective antigen types associated with abattoir condemnations non-typhoidal salmonella infections in pigs: a closer look at epidemiology, pathogenesis and control erysipelothrix rhusiopathiae: bacteriology, epidemiology and clinical manifestations of an occupational pathogen prevalence of cpb , encoding beta toxin, in clostridium perfringens field isolates: correlation of genotype with phenotype use of tiamulin in a herd of pigs seriously affected with mycoplasma hyosynoviae arthritis brachyspira-associated colitis − update and review swine dysentery: diagnostic criteria and elimination strategies toxic minerals, chemicals, plants, and gases commercial porcine circovirus type vaccines: efficacy and clinical application prevalence of porcine epidemic diarrhea virus and transmissible gastroenteritis virus infection in korean pigs stillbirths, mummies, abortions, and early embryonic death control and elimination of porocine reproductive and respiratory syndrome virus diseases of the nervous and locomotor systems clinical and postmortem examination of sows culled for lameness isolation of circovirus from lesions of pigs with postweaning multisystemic wasting syndrome postweaning escherichia coli diarrhea and edema disease effect of mycoplasma hyopneumoniae colonization at weaning of disease severity in growing pigs salmonella challenges: prevalence in swine and poultry and potential pathogenicity of such isolates risk factors associated with salmonella prevalence on swine farms the quantitation of turbinate atrophy in pigs to measure the severity of induced atrophic rhinitis porcine respiratory disease complex trends and diagnostics for practitioners tests to diagnose subclinical ileitis competitive exclusion treatment reduces the mortality and fecal shedding associated with enterotoxigenic escherichia coli infection in nursery-raised neonatal pigs erysipelas: potential involvement in urogenital disease of the sow detection of porcine epidemic diarrhea virus in air samples at varying distances to epidemic farms in oklahoma mycoplasma-associated arthritis: critical points for diagnosis siv: an update on circulating strains, advances in diagnostic tests and interpretation of test results sodium salt poisoning of swine efficacy of antimicrobial treatments and vaccination regimens for control of porcine reproductive and respiratory syndrome virus and streptococcus suis coinfection of nursery pigs serological evaluation of a clostridium perfringens type a toxoid in a commercial swine herd an investigation of the pathology and pathogens associated with porcine respiratory disease complex in denmark practitioner experiences with swine dystentery leptospira pomona: a case report in growing swine and breeding stock the various forms of haemophilus parasuis assessment of the economic impact of porcine reproductive and respiratory syndrome virus on united states pork producers swine toxicosis. swine health production a comparison of the safety, cross-protection, and serologic response associated with two commercial oral salmonella vaccines in swine effect of osmolality on net fluid absorption in non-infected and etec-infected piglet small intestinal segments protection of pigs against neonatal enteric colibacillosis with colostrums and milk from orally vaccinated sows a meta-analysis comparing the effect of pcv vaccines on average daily weight gain and mortality rate in pigs from weaning to slaughter proliferative enteropathy: a global enteric disease of pigs caused by lawsonia intracellularis gross and microscopic lesions in porcine fetuses infected with porcine reproductive and respiratory syndrome virus optimizing farrowing rate and litter size and minimizing nonproductive sow days prevalence of actinobacillus pleuropneumoniae, actinobacillus suis, haemophilus parasuis, pasteurella multocida, and streptococcus suis in representative ontario swine herds effect of porcine circovirus type (pcv ) on reproduction: disease, vertical transmission, diagnostics and vaccination rickets: case series and diagnostic review of hypovitaminosis d in swine reproductive failure experimentally induced in sows via artificial insemination with semen spiked with porcine circovirus type effect of porcine circovirus type (pcv ) vaccination of the dam on pcv replication in utero control of mycoplasma hyopneumoniae infections in pigs effects of toltrazuril on the growth of piglets in herds without clinical isosporosis actinobacillus pleuropneumoniae disease and serology quantification of the spread of mycoplasma pneumoniae in nursery pigs using transmission experiments mechanisms of porcine diarrheal disease sarcoptic mange control/eradication and their impact on pig performance: a literature review and comparison of different protocols and monitoring programs successfully implemented worldwide with ivomec s products for swine system wide sick pig management immunological solutions for treatment and prevention of porcine reproductive and respiratory syndrome (prrs) use and interpretation of sequencing in prrsv control programs impact of porcine epidemic diarrhea virus infection at different periods of pregnancy on subsequent reproductive performance in gilts and sows update on actinobacillus suis diagnosis, epidemiology, and control: on the path from good to great development of a pcr test to diagnose haemophilus parasuis infections evaluation of haemophilus parasuis control in the nursery using vaccination and controlled exposure determination of sodium content in serum and cerebrospinal fluid as an adjunct to diagnosis of water deprivation in swine diagnosis of streptococcus suis infections trichuris suis in finishing pigs: case report and review prevalence of internal parasites in a production system: part ii-finishing pigs diagnostic approaches to swine central nervous system disorders haemophilus parasuis what do we really know about feedback to gestating dams? prrsv control in finisher pigs, a large scale barn study in a high dense area in usa vaccination of influenza a virus decreases transmission rate in pigs role of the sow as a reservoir of infection for mycoplasma hyosynoviae porcine reproductive and respiratory syndrome review of mycoplasma hyorhinis diagnostic approaches to reproductive failure in pigs. swine health production transmissable gastroenteritis and porcine respiratory coronavirus effect of bmd s in sow gestation/lactation diets on clostridial infection, piglet pre-weaning performance, and sow body condition effect of waste environment on survival of brachyspira hyodysenteriae clostridial enteric infections in pigs update on porcine circovirus and postweaning multisystemic wasting syndrome (pmws). swine health production rotavirus and undifferentiated diarrhea in suckling piglets: what's new and diagnostics criteria internal parasites efficacy of a chlortetracycline feed additive in reducing pneumonia and clinical signs induced by experimental mycoplasma hyopneumoniae challenge effect of vaccination on the potentiation of porcine reproductive and respiratory syndrome virus (prrsv)-induced pneumonia by mycoplasma hyopneumoniae water in swine nutrition pathogenesis of porcine reproductive and respiratory syndrome virus-induced increase in susceptibility to streptococcus suis infection pleuropneumonia in missouri swine pseudorabies (aujeszky's disease) and its eradication: a review of the u.s. experience a retrospective evaluation of actions taken to control streptococcus suis infection serologic profiling and vaccination timing for lawsonia intracellularis swine erysipelas − a review of prevalence and research age, not infection dose, determines the outcome of isospora suis infections in suckling pigs actinobacillus suis septicemia: an emerging disease in highhealth herds key: cord- -yvdpj authors: thomson, maurine title: the cat with lameness date: - - journal: problem-based feline medicine doi: . /b - - - - . - sha: doc_id: cord_uid: yvdpj nan lameness can occur in any limb, and is defined as interference in the normal locomotion of the cat. it is usually pain related, and is secondary to tissue injury which has caused structural alteration, edema and inflammation. the severity of the lameness will vary with the severity and type of the injury or insult, varying from a mild to a non-weight-bearing lameness. generally periosteum has the densest nerve supply of the deep tissues, and has the lowest pain threshold, followed by the joint capsule, tendon, fascia and muscle. mechanical lameness can result from an abnormal limb conformation such as shortening, angulation or a rotational abnormality. cat with an abnormal gait part • localized tetanus (p ) occasionally causes paresis or paralysis of a solitary limb in the initial stages, but usually advances to generalized tetanus. • viral arthritis (p ) can occur secondary to calici virus exposure or vaccination; also coronavirus. • fungal infections (p ) histoplasmosis, coccidiomycosis, sporotrichosis usually cause non-healing lesions, draining sinuses or osteomyelitis. • toxoplasmosis (p ) more often causes cns signs such as ataxia, and paresis but can cause stiff gait, shifting lameness and muscle and joint pain. • neospora caninum (p ) causes encephalomyelitis with ataxia and paresis and polymyositis. • feline progressive polyarthritis (erosive) (p ) rare disease; proliferative subtype occurs as acute onset in - -year-old male cats, deforming subtype occurs as chronic onset in older cats. usually several joints affected, most commonly carpus and tarsus. proliferative subtype often has systemic signs of illness. deforming subtype may be felv positive. • immune-mediated polyarthritis (non-erosive) (p ) rare disease, due to deposition of immune complexes in the synovial membrane. usually carpus and tarsus affected. may be idiopathic or associated with sle or chronic infection. • fractures, luxation, muscular contusions*** (p ) very common cause of presenting lameness. • cruciate rupture (p ) occurs much less frequently in the cat than the dog, and usually less symptomatic. occurs only rarely in the cat, predisposition in devon rex cats. continued referred pain is pain felt in a part of the body other than that where the cause of pain is located. signs of weakness can be confused with lameness. see the cat with generalized weakness (page ). paresis and ataxia can also be confused with lameness. the weak and ataxic or paralyzed cat see (page ). the origin of the lameness may be from bone, muscle, joint or neurological. diagnosis requires a good physical examination and orthopedic examination to localize the lameness in the affected limb or limbs. the most common causes of lameness in the cat are cellulitis or an abscess from a cat bite injury, and motor vehicle trauma resulting in fracture or luxation. the site of the injury is generally localized by palpation of pain and swelling at the affected site. cellulitis is often associated with pyrexia and lethargy. motor vehicle trauma is often associated with other signs of external injury such as grazes or scuffed nails, with or without dyspnea or internal injuries. • acute lameness of affected limb. • pain and soft tissue swelling at site of injury. • often associated with pyrexia, anorexia, lethargy. • wounds in close proximity to a joint can cause septic arthritis. occurs secondary to a penetrating bite wound, and is one of the most common causes of lameness in the cat. many bacteria are present in the oral cavity of cats, but pasteurella, bacteroides and fusobacterium are the most common organisms responsible for abscess formation, though a mixture of anaerobes and aerobes may also be present. infection may take the form of a discrete abscess or a diffuse cellulitis. occasionally l-forms of bacteria which lack bacterial walls are involved. acute lameness. soft tissue swelling and pain at the affected site. often a small scab and evidence of a bite wound can identify the area of penetration. associated with variable signs of systemic illness, including depression, pyrexia and anorexia. l-forms produce a syndrome of fever and persistently draining, spreading cellulitis and synovitis that often involve extremities. lesion is unresponsive to most antibiotics except tetracycline group. clinical signs of penetrating injury. abscess formation can be demonstrated by aspiration of purulent material. l-form bacteria produce a discharge containing predominantly macrophages and neutrophils, but organisms cannot usually be detected cytologically or grown on culture. response of syndrome only to tetracycline group antibiotics suggests diagnosis. soft tissue injury caused by another form of external injury. foreign body reaction. osteomyelitis often causes swelling and sinus formation. rare in the cat and usually associated with an open fracture or bite wound. abscess formation generally requires lancing, and flush with weak solution of chlorhexidine, followed by course of antibiotics sensitive to pasteurella, i.e. amoxycillin and clavulonic acid mg/kg for days. rarely there may be extensive regions of necrosis, which may require debridement and delayed closure. cellulitis treated by broad-spectrum antibiotics, i.e. amoxycillin and clavulonic acid, or cephalosporins. osteomyelitis may be a rare secondary complication resulting in sinus formation after initial healing of the abscess. l-form bacteria respond rapidly to tetracycline ( mg/kg tid po) or doxycycline ( mg/kg bid po). normally excellent, and resolves after initial treatment. with extensive injury the prognosis is still excellent, although it may require more prolonged treatment. with septic arthritis some degree of degenerative joint disease will occur, although cats tolerate this with less clinical symptoms than dogs. • acute, usually non-weight-bearing lameness of affected limb or limbs. • soft tissue swelling at the site of injury. • abnormal movement or angulation of the affected bone or joint. usually due to external trauma from a motor vehicle accident. can also occur from other external trauma such as dog attack, malicious attack or fall. hindlimb lameness in young cats less than months of age is usually due to disruption of the growth plate to the femoral head rather than hip luxation. type and location of the fracture or luxation will depend on the type of forces that have occurred at the time the injury was sustained. acute lameness of the affected limb. pain and swelling localized by physical examination. most common sites of fractures are femur, humerus, pelvis, tibia and radius. hip luxation is the most common form of joint disruption in the mature cat. pain localized to the hip in young cats is more likely due to separation of the femoral head. fracture/luxation injuries may be open or closed. open shearing injuries of the carpus and tarsus often occur due to the minimal degree of soft tissues covering over these regions. usually history of trauma. localization by physical examination. radiography is normally required to demonstrate exact type and location of the fracture or luxation. cellulitis can cause a similar degree of soft tissue swelling and lameness. usually differentiated by evidence of penetrating wound and pyrexia. neoplasia of a bone or joint can present suddenly if associated with a pathological fracture. very rare in the cat, and usually differentiated on radiographs. nutritional hyperparathyroidism in the young cat can present suddenly due to spontaneous fractures. radiographically will see generalized osteopenia, with history of poorly balanced diet. will depend on the location and extent of the injury. simple fractures in young animals may be amenable to external coaptation, i.e. splints or casts. more extensive fractures generally require some form of internal fixation. see relevant textbooks dealing with orthopedic diseases and management. joint sprains or other soft tissue injuries usually respond well to brief periods, i.e. weeks or less, of immobilization, or simple rest. depends on the location and degree of severity of the injury. with appropriate treatment most injuries have a very good prognosis for return to normal function. excision arthroplasty in the cat for treatment of femoral head separation or fracture has a very good to excellent prognosis following surgery. cats are generally less symptomatic for osteoarthritis following joint trauma than are dogs. • chronic, insidious lameness of the affected limb. • lameness may be worse after sleeping, or when first rising. • may have thickening of the affected joint, with decreased range of movement. • much less common in the cat than the dog. generally secondary to joint trauma such as open fracture, or penetrating bite wound. can occur secondary to joint conformational abnormality such as hip dysplasia in persian cats, patellar luxation or cruciate rupture. may be part of an immune mediated polyarthritis, e.g. systemic lupus erythematosus. can occur secondary to septic arthritis from hematogenous spread or direct contamination. occasionally occurs in association with acromegaly from excess growth hormone. insidious lameness which may be exacerbated by strenuous activity. usually some degree of joint thickening and reduced range of motion on physical exam. often does not cause any clinical signs of lameness and may be incidental finding. history of insidious or vague intermittent lameness. radiographs demonstrate signs of osteoarthritis such as osteophytes, periosteal proliferation and periarticular joint thickening. joint neoplasia, which is very rare in the cat. usually expect more extensive swelling and consistent lameness. most cats are not symptomatic for osteoarthritis, or if associated with a sudden exacerbation of the joint normally respond well to conservative management. this involves rest and short-term non-steroidal antiinflammatory drugs, i.e. aspirin mg/kg every hours or corticosteroids, i.e. prednisolone . mg/kg daily, or other non-steroidal anti-inflammatory drugs, e.g. carprofen, ketofen or meloxicam depending on availability and registration for use in cats. encourage weight loss in fat cats. in severe cases of osteoarthritis associated with erosive arthritis or joint instability, arthrodesis may be warranted. with severe polyarthritis long-term prognosis is poor, with most cases relapsing after therapy. • much rarer in the cat than the dog. • usually results from traumatic herniation of the disc. • clinical signs vary with the location of the disc herniation. • signs may be acute or progressive in onset. usually results from traumatic herniation of the disc, resulting in acute spinal cord compression. location of the site of herniation dictates type of clinical signs seen. usually associated with paresis of the hindlimbs. see the weak and ataxic or paralyzed cat (page ). lesion identified by neurological examination. confirmation of disc herniation by radiographs, myelography or ct imaging. • acute onset of hindlimb lameness. • may have thickening of the affected joint due to synovitis, with pain evident on palpation of the stifle. • positive cranial drawer sign or tibial thrust. • much less common in the cat than the dog. usually due to external trauma such as a fall, hyperextension injury of the stifle, or excessive internal rotation of the tibia. generally causes rupture of the anterior cruciate, rarely is the caudal cruciate involved, unless there has been major stifle disruption. acute lameness of the affected hindlimb. generally there will be stifle swelling and pain present on joint palpation. a positive cranial drawer sign or tibial thrust is generally evident on palpation, though pain may prevent an adequate examination of the joint. chronic cases may show a mild intermittent lameness as opposed to the acute injury. by identification of a positive cranial drawer sign and pain in the stifle. traumatic disruption of all stifle ligaments due to excessive trauma. usually palpation will reveal laxity in a mediolateral direction also if the collaterals have been disrupted. degenerative joint disease will usually occur, but this does not normally cause clinical signs of lameness. in refractory cases the stifle can be stabilized with an extra-articular surgical technique. see relevant texts on surgical management of cruciate disease. generally the prognosis is very good to excellent with both conservative and surgical management. • can be congenital or acquired secondary to trauma. • much rarer in the cat than the dog, though a predisposition occurs in purebred cats such as the devon rex. • lameness may be worse after sleeping, or when first rising. • may have thickening of the affected joint, with decreased range of movement. the congenital form occurs secondary to malalignment of the quadriceps mechanism, generally resulting in a medial luxation of the patella. varying degrees of deformity of the trochlear groove and tibial tuberosity can occur depending on the severity of the luxation. the acquired form can occur secondary to external joint trauma from a fall or motor vehicle accident. the congenital form is characterized by varying degrees of lameness depending on the severity of the condition. the lameness if often insidious and intermittent. usually occurs in young, purebred cats. the acquired form can occur at any age, usually presenting with an acute lameness. palpation of the stifle will demonstrate luxation of the patella. palpation of the stifle demonstrates patellar luxation, usually in a medial direction. lameness present in the affected limb, which varies with the severity of the underlying disease. in cats with clinical disease surgery is generally warranted. type of surgery varies with the severity of the disease, ranging from lateral imbrication, deepening of the trochlear groove and transposition of the tibial tuberosity. generally excellent, despite the progression of some degree of degenerative joint disease. • lameness may be of a single limb or multiple. • if all limbs involved will be reluctant to ambulate at all. • can cause erosive or non-erosive signs radiographically. • considered rare in the cat as a cause of lameness. • generally from a blood-borne infection that arises from a septic foci elsewhere in the body. • can occur from direct bite wound or open joint injury. • rarer in cats than dogs. • can occur in kittens secondary to umbilical vein infections. • pasteurella species are most commonly identified. • can be due to bacterial l forms, which are mutant bacteria, which have lost their cell wall. • calicivirus polyarthritis can occur uncommonly in young kittens following natural exposure or after live attenuated vaccination. • lameness usually occurs - days after mild respiratory tract signs, though can occur in the absence of respiratory signs. • coronavirus has also been associated with polyarthritis. • the effusive form of feline infectious peritonitis (fip) virus occasionally causes lameness, with leakage of fluid into joint spaces. • the granulomatous form of fip may cause paresis or paralysis as a result of spinal cord inflammation. • m. gatae is linked to arthritis and tenosynovitis in older cats. • mycoplasmal infection may incite an antigenic response resulting in the formation of immunemediated disease such as rheumatoid-like (deforming) arthritis. acute onset of lameness in one or several limbs. usually associated with systemic signs such as pyrexia, lethargy. may appear weak rather than lame if all four limbs involved. usually demonstrate joint pain on manipulation of limbs. radiographic signs may demonstrate changes of erosion and periosteal proliferation in advanced cases, but may show no abnormalities initially. arthrocentesis generally required for cytology and culture and sensitivity. weak or paretic cat can appear similarly. see the weak and ataxic or paralyzed cat (page ). immune-mediated polyarthritis. antibiotics for bacterial infection depending on results of culture and sensitivity. l form infections respond well to - -day course of tetracycline or doxycycline. calicivirus is often self-limiting, and cats recover well. • more commonly causes cns signs such as ataxia and paresis. • can cause a stiff gait, shifting leg lameness, hyperaesthesia on muscle palpation and joint pain. with signs of acute small bowel diarrhea) and page (the cat with generalized weakness). cns signs seen most frequently, see the paretic cat (page ). may cause shifting limb lameness. can cause arthritis and myositis. high igm titers suggest recent infection, but do not confirm that t. gondii is responsible for the clinical signs in a given cat. the organism can sometimes be seen in a muscle biopsy or very rarely in csf. • cats less commonly affected than dogs. • lameness due to fungal osteomyelitis. • can be a single bone involved, or more commonly multiple sites affected. • spores usually inhaled from the soil. occurrence tends to be location dependent: • coccidioides: south-western usa, mexico, central and south america. • blastomyces: small area of distribution mainly in north america and near fresh water. • histoplasma: widespread in temperate and subtropical regions of the world, not australia. • cryptococcus: worldwide distribution. • sporotrichosis: worldwide distribution. result in non-healing tumor-like lesion of the distal limbs and draining sinus tracts (sporothrix) or lameness due to fungal osteomyelitis. radiographically, see tumor-like lesions present in the bone with bone bony destruction and proliferation seen. organism is often evident in cytological preparation of aspirates or discharge from lesion or in a biopsy section. positive fungal titer may aid diagnosis. • neurological signs such as ataxia and paresis. • polymyositis frequently present. polymyositis and encepalomyelitis with ataxia and paresis are seen most frequently. serum titers and muscle biopsy results are the most common methods of diagnosis. • retrograde axonal transport of the toxin from an infected focus. • can cause paralysis of single limb. • usually progresses to generalized tetanus. with an abnormal third eyelid). occurs following penetrating trauma and tissue devitilization. can see paresis or paralysis of a solitary limb in the initial stages. generally advances to generalized tetanus. history of recent wound and clinical signs. emg findings characteristic with persistent electrical motor unit discharges following needle insertion or tapping of muscles. serum antibody titer to tetanus toxin compared with control cats may aid diagnosis. • are considered rare diseases in the cat. • can be acute or chronic onset. • often associated with other systemic signs such as lethargy, pyrexia and anorexia. • can cause erosive or non-erosive signs radiographically. • chronic progressive polyarthritis has proliferative and deforming subtypes. • proliferative form occurs in male cats aged - years. • deforming type occurs more rarely in older cats. • severe subchondral bone destruction may occur, leading to joint instability and deformity. • radiographic signs are similar to those of rheumatoid arthritis in other species. • often associated with feline leukemia viruspositive cats. • mycoplasmal products have been postulated to chronically stimulate immune-mediated diseases such as deforming arthritis. • can be due to systemic lupus erythematosus, chronic infection elsewhere, or idiopathic. • due to deposition of immune complexes in the synovial membrane which incites an inflammatory response. • the inciting agent is usually not identified, but if the cat is on medication this may be a potential source and should be discontinued. deforming type of progressive polyarthritis has a chronic onset. all other forms tend to be acute onset of lameness and stiffness. often affects carpus and tarsus more severely than other joints. radiographic signs in erosive forms show marked destruction and deformity of the distal joints often resulting in luxation or subluxation but with minimal periosteal proliferation. the proliferative form is characterized by periarticular soft tissue swelling in the early stages, then progressing to extensive periarticular proliferation. this is most frequently seen in the carpi, tarsi and smaller distal joints. arthrocentesis preferably done on two or more joints. joint fluid is generally slightly increased in amount, slightly turbid, and with reduced viscosity. cytology typically reveals a non-septic purulent inflammation, with cell counts in the range of - nucleated cells/μl, and predominantly non-degenerative neutrophils. a variable number of small and large mononuclear cells may also be present. mycoplasma. rheumatoid factor, ana and le cell tests are consistently negative. hematological parameters are generally unremarkable. infectious arthritides, such as viral infections. causes of weakness and paresis in the cat. see the weak and ataxic or paralyzed cat (page ). remove the cause if secondary to drug administration or chronic infection. treatment consists of immunosuppressive doses of corticosteroids for several months, or in combination with chlorambucil at . - . mg/cat once daily, and prednisolone - mg twice daily until resolution of clinical signs, then slowly taper the dose of prednisolone over - months. continue the chlorambucil at the same dose every other day. combination of prednisolone and gold salts (aurothioglucose mg/kg by injection once a week) has also been used. good initial response to drugs, but greater than % of cases are likely to relapse, becoming more refractory to treatment. classical signs • rarer in the cat than dog. • may be acute or insidious onset of lameness. • usually associated with swelling at site of neoplasia. primary bone tumors are rare in the cat. osteosarcoma is the most commonly identified tumor. generally occurs in cats older than years. primary bone tumors are much less metastatic in the cat than the dog. metastatic spread from carcinomas to bone is very rare in the cat, compared to the dog. can get carcinomas of the digits secondary to primary lung tumors. joint neoplasias are very rare in the cat, and are due to synovial cell sarcomas or other soft tissue sarcomas. multiple cartilaginous exostoses or feline osteochondromatosis can transform to a malignant disease in the cat. lameness can also occur from nerve sheath tumors of the plexus or spinal cord, but these are very rare in the cat. can be acute or insidious onset of lameness. rarely can be associated with pathological fracture. usually thickening identified at site of disease, which may or may not be painful. radiographically, will see bone lysis in the metaphyseal region of long bones, or joint thickening and proliferation. histopathology is required for definitive diagnosis. thoracic radiographs should be taken to demonstrate primary lung tumor if multiple digits are swollen, or prior to amputation to rule out metastatic disease. osteomyelitis. septic arthritis. if no evidence of metastatic disease, amputation is the treatment of choice. if a solitary tumor of a digit is present, often this can be removed and the limb saved. if no evidence of metastatic disease, then amputation is often curative, with median survival times greater than years. primary bone tumors in the cat are a lot less aggressive than in the dog and generally not followed with chemotherapy. • usually occurs in persian and long-haired cats. • usually asymptomatic in cats compared to dogs. • generally requires only conservative management. congenital disorder of persian and other long-haired cats. radiographically, see luxation of the coxofemoral joint, with varying degrees of osteoarthritis present. usually no clinical signs of lameness are seen. pain may be exhibited on flexion and extension of the hip joints. occasionally may show signs of a stiff gait or lameness, which is exacerbated by excessive activity. pain on manipulation of the hip joints in a long-haired cat. radiographic evidence of hip laxity and osteoarthritis. usually conservative management consisting of rest, weight loss and intermittent anti-inflammatory drugs is all that is required in symptomatic cats. if lameness persists femoral head and neck arthroplasty results in an excellent prognosis. • onset of forelimb lameness in the cat less than year of age. very rare in the cat. has been reported as cause of shoulder lameness. occurs due to a defect in osteochondral ossification. insidious forelimb lameness in cats less than year of age. pain identified on full extension and flexion of the shoulder joint. radiographically a thickened flap of cartilage or joint mouse can be identified in the shoulder joint. surgical removal of the cartilaginous flap. excellent. • onset of hindlimb lameness in the cat less than year of age. uncommon condition. reported in male cats less than years of age. idiopathic necrosis of femoral neck. vague, progressive hindlimb lameness. may be bilateral. radiographically, see radiolucency of the femoral neck and proximal femoral metaphysis. traumatic fracture of the femoral neck. femoral head and neck osteotomy. excellent. • rare inherited disorder in siamese and siamese cross cats. • causes a stiff gait and paraparesis. • cats also have a broad maxilla, corneal clouding, pectus excavatum and neurological abnormalities. rare inherited disorder that occurs as a result of a lysosomal enzyme deficiency in siamese and siamese cross cats. usually present at less than months of age. typically have dysmorphic facial features (a broad maxilla), plump paws (from thickened skin) and corneal clouding. generally exhibit a stiff gait and paraparesis with diffuse neurological signs. also present with a chronic mucoid ocular discharge and chronic respiratory tract infections. constellation of clinical signs suggests the diagnosis. radiographs show dysplastic femoral heads and necks and subluxated hip joints. excessive granulation of neutrophils, and vacuolation of lymphocytes are suggestive. positive urine toluidine test for sulfated beta glycosaminoglycans is consistent with diagnosis. definitive diagnosis is via enzyme assays of leukocytes and skin fibroblasts to demonstrate deficient beta glucuronidase activity. • a defect in osteogenesis results in multiple spontaneous fractures. • present with multiple sites of bony pain due to fractures, or unwilling to ambulate. • lameness generally of young cats. a heritable disease with failure of osteoprogenitor cells to develop into mature osteoblasts. results in bones that are very brittle and fracture spontaneously. spontaneous fractures can occur in any bones, although the long bones are more commonly affected. radiographically the cortical bone is very osteopenic, with multiple fractures present. • disease affecting cats with at least one fold-eared parent. • lameness generally occurs at - months of age. inherited disorder of fold-ear cats. get shortening of the coccygeal vertebrae, metacarpal and metatarsal bones and phalanges. they may develop periarticular exostoses and joint ankylosis. • a congenital disorder characterized by dorso-ventral flattening of the thorax. • kittens and cats show variations in respiratory and cardiovascular abnormalities. • also associated with lateral limb deviation. a deformity of the sternal and costal cartilages resulting in narrowing of the thorax. occurs as a congenital abnormality of cats, with the specific cause unknown. can be associated with the so-called "wimmer's"syndrome, in which there is lateral deviation of the limbs and impaired ambulation. kittens and young mature cats < year of age may show respiratory and cardiovascular abnormalities associated with flattening of the thorax. based on clinical signs and radiographic findings of dorso-ventral flattening of thorax and lateral deviation of limbs. improper closure of the neural tube during embryogenesis occurs, with varying severity of neurological signs seen affecting hindlimb function and urinary and fecal control. in the manx cat it is often associated with sacral dysgenesis. based on clinical signs, radiographic findings. • due to excessive intake of vitamin a, usually from an all-liver diet. • lameness occurs due to bony exostoses around the cervical vertebrae and ankylosis of joints. due to excessive vitamin a intake over several months, usually from an all-liver diet. causes bony exostoses of spine and osseous hyperplasia and proliferation at joint margins, particularly the shoulder and elbow joints. excess vitamin a causes increased reactivity of the periosteum, resulting in bony proliferation at sites of joint capsule and soft tissue attachments to bone. cats are often reluctant to move around, and are very painful. they often display an abnormal posture with a stiff neck and gait. radiographically there is ankylosis of cervical and cranial thoracic vertebra, and new bone proliferation around shoulder and elbow joints. history of diet almost exclusively of liver. correction of the diet, ideally on a balanced commercial diet. may require short-term analgesics. correction of the diet halts the progression of disease, and some remodeling of existing bone may occur. however, bony changes are often permanent and pain may persist. • lameness present in multiple sites in young kittens. • due to a diet deficient in calcium, such as an all-meat diet. due to increased parathyroid hormone from persistent hypocalcemia, either from an absolute dietary calcium deficiency or secondary to excessive phosphorus intake relative to calcium. causes generalized osteopenia. most commonly seen in kittens on an all-meat diet. occurs with an all-meat diet even if the kittens have access to milk. osteopenia can result in spontaneous fracture. history of inadequate diet. radiographs demonstrate diffuse osteopenia, with or without folding fractures. correct the diet by adding calcium, or more appropriately, change to a balanced commercial diet formulated for kittens. confine if multiple folding fractures are evident. very good if fractures have not resulted in malunion. residual deformity of the spine and pelvis may result in chronic partial bowel obstruction with constipation. • lameness present in multiple sites in young kittens. • due to a diet deficient in vitamin d or insufficient mineral content, such as an allmeat diet. diet deficient in vitamin d or with insufficient mineral content causes the cartilaginous matrix of the growth plate not to calcify. results in osteopenia and widening of the growth plates. osteopenia can result in spontaneous fracture. abnormalities of the growth plate with rickets can cause angular limb deformities. diffuse osteopenia, and may see pathological fractures. with rickets see widened growth plates and osteopenia. correct the diet by feeding a balanced commercial diet, and adequate exposure to light. may require confinement if multiple fractures have occurred. usually excellent prognosis if there is not abnormal limb angulation. correction of the diet corrects the underlying osteopenia and growth plate defect. disease mechanisms in small animal surgery slatter d. textbook of small animal surgery key: cord- -lylt qld authors: van breedam, wander; pöhlmann, stefan; favoreel, herman w.; de groot, raoul j.; nauwynck, hans j. title: bitter‐sweet symphony: glycan–lectin interactions in virus biology date: - - journal: fems microbiol rev doi: . / - . sha: doc_id: cord_uid: lylt qld glycans are carbohydrate modifications typically found on proteins or lipids, and can act as ligands for glycan‐binding proteins called lectins. glycans and lectins play crucial roles in the function of cells and organs, and in the immune system of animals and humans. viral pathogens use glycans and lectins that are encoded by their own or the host genome for their replication and spread. recent advances in glycobiological research indicate that glycans and lectins mediate key interactions at the virus‐host interface, controlling viral spread and/or activation of the immune system. this review reflects on glycan–lectin interactions in the context of viral infection and antiviral immunity. a short introduction illustrates the nature of glycans and lectins, and conveys the basic principles of their interactions. subsequently, examples are discussed highlighting specific glycan–lectin interactions and how they affect the progress of viral infections, either benefiting the host or the virus. moreover, glycan and lectin variability and their potential biological consequences are discussed. finally, the review outlines how recent advances in the glycan–lectin field might be transformed into promising new approaches to antiviral therapy. many emerging and re-emerging viral diseases in animals and humans pose significant global health problems for which novel antiviral measures are in urgent demand. in general, rational design of new prophylactic and curative antiviral strategies requires a detailed knowledge of the viral infection mechanism and the host's innate and adaptive immune defense. historically, cell biological and microbiological research was mainly focussed on the nucleic acid and protein level. however, over the last few decades it has become clear that also glycans account to a great extent for the structural and functional diversity displayed by animal and human cells and their pathogens. at this moment, glycobiology is one of the most rapidly expanding disciplines in biology. a rapidly evolving array of powerful, novel techniques for the analysis of glycan structure and function (glycomics) has prompted many scientists to apply these tools to the field of virology, and this glycovirological approach has yielded a wealth of information on the various glycobiological aspects of viral infection and antiviral immunity. particularly fascinating in this context are the many distinct glycan-lectin interactions that may occur during viral infection of a host. virion-associated glycans often serve as ligands for specific host lectins. conversely, the glycan portions of host glycoconjugates function as receptors for various viruses that employ viral lectins for host cell entry. this review reflects on glycan-lectin interactions in the context of viral infection and antiviral immunity. following a general introduction on glycan and lectin biology, specific glycan-lectin interactions are highlighted and discussed within the larger framework of viral infection and immunity. distinction is made between interactions that benefit the host and interactions that benefit the virus. in addition, different factors that contribute to glycan and lectin variationand that consequently affect glycan-lectin interactionsare explored and various approaches to modulate specific glycan-lectin interactions in antiviral therapies are briefly discussed. glycans like nucleic acids, proteins and lipids, glycans are essential components of the animal cell and organism. the term 'glycan' refers to the carbohydrate portion of glycoproteins and glycolipids typically found at cell surfaces, in extracellular matrices and in cell secretions. unlike nucleic acid and protein synthesis, the biosynthesis of glycans is not a template-driven process. instead, glycosylation depends on the concerted action of different glycosyltransferase, glycosidase, and other enzymes. several important glycan types are exclusively assembled by the enzyme sets present in the endoplasmic reticulum (er) and the golgi network: glycan/glycoconjugate synthesis is typically initiated in the er or early golgi and gradual processing and diversificationor 'maturation'occurs as these molecules move further through the different enzyme-equipped compartments of the secretory pathway. the variability inherent to glycan synthesis and maturation forms the basis of the considerable diversity and complexity of the glycan repertoires found on animal glycoconjugates (varki et al., ; taylor & drickamer, ) . according to the basic glycan 'core' structure, the type of molecule the glycans are linked with and the type of the linkage, glycans and glycoconjugates can be categorized in different classes. two major classes of proteinlinked glycosylation are the n-and o-linked glycans. n-linked glycans are covalently linked to the nitrogen atoms of specific amino acid (aa) residues (typically asparagine) via an n-glycosidic bond. n-acetylglucosamine-to-asparagine (glcnacb-asn) type glycans represent the most common form of n-linked protein glycosylation (weerapana & imperiali, ; varki et al., ; larkin & imperiali, ; schwarz & aebi, ; taylor & drickamer, ) . o-linked glycans are covalently attached to the oxygen atoms of specific amino acid residues (typically serine or threonine) via an o-glycosidic bond. a common o-glycan typeand potentially the one most studiedis the n-acetylgalactosamine-to-serine/threonine (galnaca-ser/thr) type or mucin-type o-glycan. other (nonmucin) o-glycan types include a-linked o-mannose, a-linked o-fucose, b-linked o-xylose, b-linked o-glcnac (n-acetylglucosamine), a-/b-linked o-galactose, and a-/b-linked o-glucose glycans (van den steen et al., ; peter-katalinic, ; varki et al., ; jensen et al., ; gill et al., ; taylor & drickamer, ) . interestingly, various proteins are also modified with glycosaminoglycans (gags), linear polysaccharide chains composed of repeated disaccharide subunits consisting of a uronic acid/galactose residue and an amino sugar. glycosaminoglycan-carrying glycoproteins are generally referred to as proteoglycans (prydz & dalen, ; varki et al., ; taylor & drickamer, ) . figure illustrates the general structure and classification of some common types of protein glycosylation. similarly as for protein-linked glycosylation, different types of lipid-linked glycosylation can be discerned: the glycosphingolipids (varki et al., ; taylor & drickamer, ; yu et al., ) and the glycophospholipid anchors or glycosylphosphatidylinositol (gpi) anchors (paulick & bertozzi, ; varki et al., fig. . classification and basic structure of common types of protein-linked glycosylation. (a) glcnacb-asn type n-linked glycans are covalently attached to the amide nitrogen atoms of asn side chains and are almost exclusively found on asn residues within the sequence asn-x-ser/thr, in which x can be any amino acid except pro. the nature of the glycan structures that decorate the common glycan corethe glycan part shown in a dashed boxdictates classification of n-linked glycans as high-mannose type, hybrid type or complex type glycans, examples of which are shown in the panel. (b) galnaca-ser/thr type o-linked glycans have a galnac residue a-linked to the oxygen atom of the hydroxyl group of ser or thr residues. unlike for glcnacb-asn type n-linked protein glycosylation, there are no clear amino acid motifs that mark these o-linked glycosylation sites. a single galnac residue linked to the ser/thr is termed the 'tn antigen'. depending on the basic structure of the glycan core, more complex (extended) o-linked glycans are categorized into different 'core types'. cores - are the most common core structures, but also other core types exist. the tn antigen and examples of extended core , , , and o-glycans are shown in the panel. the distinct glycan cores are shown in dashed boxes. (c) glycosaminoglycans (gags) are linear polysaccharide chains composed of repeated disaccharide subunits of a uronic acid/galactose residue and an amino sugar. glycosaminoglycans are classified as hyaluronan (ha), heparan sulfate/heparin (hs), chondroitin sulfate (cs), dermatan sulfate (ds), or keratan sulfate (ks), depending on the structure of their basic disaccharide subunits (shown in square brackets) and further modification (e.g. sulfation at different positions) of the glycan chain. with exception of hyaluronan, all major glycosaminoglycan types are sulfated and occur covalently linked to proteins. hs, cs, and ds are found on ser-linked xylose residues. although no unambiguous consensus sequence for xylosylation exists, the ser attachment site is consistently flanked by a gly residue at its carboxyterminal side. as depicted in the figure, heparan sulfate and heparin have the same basic structure. although they share a common biosynthesis, heparin generally undergoes more extensive sulfation and epimerization of uronic acid to iduronic acid. moreover, heparin is synthesized only in connective tissue mast cells as part of serglycin proteoglycans, whereas heparan sulfate is synthesized in virtually all mammalian cells. ks is found on asn-linked n-glycan core structures (ks i) or ser/thr-linked o-glycan core structures (ks ii). capping or further modification of the glycosaminoglycan chainssulfation exceptedis not depicted (adapted from varki et al., ). ; taylor & drickamer, ) represent important glycolipid classes. their basic structure is introduced in fig. . despite their different core structures and linkages to carrier molecules, distinct glycan types can still share conserved structural characteristics as they often follow partially overlapping biosynthetic pathways. although some glycan features may be exclusively found in one specific glycan class, many (sub)terminal glycan modifications can be found in different glycan classes. common (sub)terminal modifications include poly-n-acetyllactosamine chains, abh and lewis histo-blood group antigens (hbga), and sialic acids in different linkages (varki et al., ; taylor & drickamer, ) . consequently, the glycan moieties of glycoproteins and glycolipids often have more in common than one would expect based on their core structure. apart from the different glycan types introduced here, several other forms of glycosylation exist. however, an in-depth discussion of these is beyond the scope of this review. for more detailed information on glycan structure and biosynthesis, readers may refer to recent reference works on this topic (varki et al., ; taylor & drickamer, ) . not only the animal and human hosts, but also their pathogens can benefit from the fine-tuned cellular biosynthetic pathways that govern glycosylation. this is most obvious in the case of obligatory intracellular pathogens such as viruses. glycans form an important part of the surface of many viruses. as the glycosylation of viral components is facilitated by the cellular glycosylation machinery, viral glycosylation is similar to that of the host. however, important differences have been noted: viral glycoproteins are often more heavily glycosylated than their cellular counterparts and the composition of individual glycan chains can diverge greatly between virus and host. the biological basis of this variability is further classification and basic structure of major types of lipid-linked glycosylation. (a) glycosphingolipids consist of a hydrophilic glycan moiety linked to a hydrophobic sphingolipid. in higher animals, a ceramide lipid molecule is initially modified with a b-linked glucose or galactose residue, after which further extension and modification of the glycan moiety can occur. extension to larger glycan chains is common on ceramide-linked glucose residues, whereas further glycan extension on ceramide-linked galactose residues is more rare. depending on their glycan core structure, glycosphingolipids are classified in 'series'. the figure depicts a number of glycosphingolipid core structures. the key features that characterize each series are shown in dashed boxes. core structures can be further modified with sialic acids or sulfate groups, which allows subclassification of glycosphingolipids as neutral (lacking charged carbohydrates or ionic groups), sialylated or sulfated. (b) glycosylphosphatidylinositol (gpi) anchors are found in association with certain membrane proteins and serve as linkers between the protein and the lipid membrane. glycosylphosphatidylinositol anchors have a common core structure comprising ethanolamine-po - mana - mana - mana - glcna - myo-inositol- -po -lipid. differential derivatization of this common core structure through lipid remodeling and modification of the glycan moiety can cause significant glycosylphosphatidylinositol anchor heterogeneity. the protein is linked to the glycosylphosphatidylinositol anchor via an amide linkage between the c-terminal carboxyl group of the protein and the amino group of phosphatidylethanolamine (adapted from varki et al., ). situated later in this review (see 'glycan and lectin variation at the virus level'). in line with the similarity between viral and host glycosylation, many of the basic functions covered by glycans in normal animal and human physiology and in viral infection biologywhich is intrinsically linked to the biology of the hostare essentially the same. glycans play important structural roles and are for instance implicated in protein folding and solubility, protease resistance, and masking of highly immunogenic protein stretches ('glycan shielding') (varki et al., ; taylor & drickamer, ) . alternatively, glycans can also have nonstructural roles and take part in specific recognition events, in which they usually interact with complementary glycan-binding proteins called lectins (varki et al., ; taylor & drickamer, ) . lectins may simply be defined as carbohydrate-binding proteins, although some definitions are more restrictive and exclude mono-/oligo-saccharide transport proteins, enzymes, glycan-specific antibodies, and even glycosaminoglycan-binding proteins (elgavish & shaanan, ; weis, ; loris, ; varki et al., ; gabius et al., ) . according to the more strict definitions, glycosaminoglycan-binding proteins and lectins are distinguished based on different factors, including their ligand range, the structural basis of their glycan recognition, and their conservation (varki et al., ) . in general, glycosaminoglycan-binding proteins interact with negatively charged glycosaminoglycans via clusters of positively charged aa residues andwith exception of hyaluronan-binding proteins, which seem to share an evolutionarily conserved fold do not appear to be evolutionarily related to each other (varki et al., ) . in contrast, most strict sense lectins belong to protein families with defined 'carbohydrate recognition domains' (crd). the crds within a lectin family share structural and functional properties and selectively recognize specific portions of n-glycans, o-glycans, or glycolipids (sometimes also glycosaminoglycans) (varki et al., ) . although some crds can efficiently bind monosaccharides, other crds show no apparent affinity for monosaccharides and favor oligosaccharide ligands. the latter crd type often has a preference for ligands with specific linkages between the monosaccharide subunits, as these linkages determine the d structure of the glycan ligand and therefore the portions of the glycan that are available for interaction with the crd. interactions between a single crd and a single mono-/ oligo-saccharide (affinity) are often weak, and strong interactions are usually the result of multivalent binding, i.e. the interaction of multiple crds with multiple ligands (avidity). whereas some lectins contain multiple crds that can participate in ligand binding, others contain only a single crd and rely on clustering of individual lectin molecules for high avidity binding. clustering of crds does not only allow stronger interactions with ligands, but also contributes to the specificity/selectivity of interactions at the multivalent level. the relative spacing of the crds allows highly avid, multivalent binding to specific saccharide ligands in a certain density and particular presentation. ultimately, the avidity of lectins for specific glycoconjugates depends on the structure, multivalency, and density of glycans on these molecules (elgavish & shaanan, ; weis, ; loris, ; varki et al., ; gabius et al., ) . animal lectins are typically expressed in a cell-and/or tissue-specific manner. they are involved in many different biological processes, including glycoprotein trafficking, cell adhesion and signaling, and their expression is usually tightly regulated (varki et al., ) . particularly striking is the great number of membrane-associated and soluble lectins that are linked with host immunity. immune system lectins are involved in intercellular communication, positive and/or negative regulation of activation, regulation of inflammation, disposal of damaged and apoptotic cells, etc. several of these lectins have also been identified as 'pattern recognition receptors' (prrs), which act as molecular sensors for pathogens and endogenous stress signals and often trigger specific immune reactions/mechanisms in response to their detection (gordon, ; janeway & medzhitov, ; cambi & figdor, ; mcgreal et al., ; cambi et al., ; mcgreal et al., ; crocker et al., ; crocker & redelinghuys, ; van kooyk & rabinovich, ; garcia-vallejo & van kooyk, ; geijtenbeek & gringhuis, ; sato et al., ; bottazzi et al., ; dam & brewer, ; kumagai & akira, ; svajger et al., ; davicino et al., ; osorio & reis e sousa, ; sancho & reis e sousa, ) . the capacity of many immune system lectins to couple glycan recognition events with specific signaling and/or effector functions gives them a key regulatory position in the immune system. figure gives a schematic overview of different types of animal lectins that are considered in this review. lectins play a pivotal role in different aspects of the physiology, including the immune defense against (viral) pathogens. however, it has become apparent that several viruses exploit host lectins to promote their spread. in addition, many viruses encode lectins for the recognition of and infectious entry into target cells. the following section explores how glycan-lectin interactions shape the virus-host interplay, mainly focussing on glycan-lectin interactions directly involving infectious virions. glycan-lectin interactions that benefit the host lectins cover essential roles in the animal host's immune defense. importantly, several membrane-associated host (immune system) lectins act as pathogen recognition molecules: they can bind pathogens and activate signaling mechanisms or capture pathogens for subsequent degradation and presentation to cells of the adaptive immune system (e.g. mhcii-restricted presentation of antigens to t cells), resulting in the induction of a pathogen-specific adaptive immune response. alternatively, pathogens attached to such cell surface lectins may also be directly presented to neighboring immune cells in trans, a process that seems especially significant at sites with a high density of immune cells (e.g. the lymph nodes). hence, binding of a viral pathogen to membrane-associated (immune system) lectins can lead to its clearance and degradation. a prominent example is the interaction between the human immunodeficiency virus type (hiv- ) and human langerin, a c-type lectin mainly expressed on langerhans cells (de witte et al., ; . de witte et al. ( ) reported that hiv- interacts with langerin via high-mannose glycans on the gp envelope protein and is subsequently internalized into birbeck granules, leading to virus degradation (de witte et al., ) . also dc-sign, a mannose-binding c-type lectin mainly expressed on dendritic cells (dcs), can bind and internalize hiv- virions for degradation and promotes mhcii-restricted as well as exogenous mhci-restricted presentation of hiv- antigens (moris et al., (moris et al., , . similarly, various other membrane-associated host lectins can aid as prrs in the defense against viral pathogens. however, growing evidence illustrates that many of these lectins, including dc-sign, are also abused by viruses to gain access to their target cells and facilitate viral spread, as is discussed further below. in contrast to the dual role played by different membrane-associated host lectins, soluble host lectins have mainly been associated with protection against viral infection. several soluble host lectins have been reported to aid in neutralization and clearance of various viral pathogens. table gives an overview of membrane-associated and soluble host lectins that are linked with the host's defense against different viruses. current experimental data strongly implicate these host lectins in the defense against the listed viral pathogens and do not attribute explicit proviral effects to the host lectinunlike for the lectin-virus pairs listed further in table . the basic principles of soluble lectin-mediated antiviral protection are most easily conveyed using some specific, wellcharacterized examples. for instance, various studies point out an important role of surfactant protein a (sp-a), surfactant protein d (sp-d), and mannose-binding lectin (mbl) in the defense against influenza a virus (iav) infection. sp-a, sp-d, and mbl are all soluble c-type lectins and share a similar basic structure: lectin monomersconsisting of an n-terminal cysteine-rich domain, a collagen-like domain, a coiled coil neck domain, and a c-terminal crdassemble into trimers which, under physiologic conditions, further multimerize via their n-termini to form typical cruciform-(sp-d) or bouquet-(sp-a and fig. . schematic overview of different types of membrane-associated (a) and soluble (b) animal lectins that are considered in this review. the lectin domains are highlighted and listed in the key. c-type lectin/c-type lectin domain: lectins are classified as c-type lectins based on their ca + -dependency and shared primary structure. in the c-type crd, a ca + ion is directly involved in carbohydrate binding by making coordination bonds to both the crd surface and key hydroxyl groups of the carbohydrate. the c-type lectin family contains both membraneassociated (a. ) and soluble (b. ) lectins. the collectins are soluble c-type lectins characterized by the presence of collagen-like domains. r-type lectin domain: this term refers to a crd that is structurally similar to the crd in ricin, a toxin found in the plant ricinus communis. i-type lectin/ i-type lectin domain: i-type lectins are glycan-binding proteins that belong to the ig superfamily, but are not antibodies or t-cell receptors. the 'sialic acid-binding ig-like lectin (siglec)' family of membrane-associated lectins is currently the only well-characterized group of i-type lectins (a. ). ficolin: ficolins (b. ) are soluble lectins characterized by the presence of collagen-like domains and fibrinogen-like globular domains with a lectin activity. galectin/s-type lectin (domain): galectins (b. ) are soluble lectins that typically bind b-galactose-containing glycoconjugates and show primary structural homology in their crds. galectins were initially referred to as s-type lectins to reflect their sulfhydryl dependency, the presence of cysteine residues and their solubility; however, at present, not all identified galectins fit this initial description anymore. pentraxin/pentraxin domain: pentraxins (b. ) are characterized by the presence of pentraxin domains, which contain an eight amino acid long conserved 'pentraxin signature' (hxcxs/twxs, where x is any amino acid) and display an l-type (legume-type) lectin fold. sap is a soluble lectin that requires ca + ions for carbohydrate ligand binding (adapted from fujita, ; varki et al., ; bottazzi et al., ) . (van de wetering et al., ; veldhuizen et al., ) . despite their similar structure, these lectins show distinct glycan ligand specificities (veldhuizen et al., ) and also their interaction with iav and their effects on infection and spread appear to differ. studies using distinct iav isolates indicate that sp-d and mbl bind mannose-rich glycans on the viral surface glycoproteins hemagglutinin and neuraminidase (a viral lectin and a viral glycosidase, involved in iav entry and release; see discussion on viral lectins) through their crds (malhotra et al., ; reading et al., ; kase et al., ; hartshorn et al., ; hillaire et al., ) . although sp-a may interact with some iav isolates in a similar manner (malhotra et al., ) , binding of this molecule to most of the iav variants tested to date appears not to involve the lectin activity of sp-a; in contrast, virus binding depends on the interaction of the viral hemagglutinin with a sialylated n-glycan on the sp-a crd (hartshorn et al., ; benne et al., benne et al., , hartshorn et al., ; van eijk et al., ; mikerov et al., ) . in other words: sp-d and mbl binding depend on viral glycosylation, whereas sp-a binding mainly depends on the specificity of the hemagglutinin, and this is mirrored in the spectrum of iav variants these lectins can effectively bind (hartshorn et al., ; malhotra et al., ; benne et al., benne et al., , hartshorn et al., ; reading et al., ; kase et al., ; hartshorn et al., ; van eijk et al., ; mikerov et al., ; hillaire et al., ) . interestingly, the porcine variant of sp-d appears to combine the above-mentioned iav-binding functionalities: this molecule may not only bind iav virions through interaction of its crd with high mannose glycans on the virion surface (similar to other sp-d molecules), but also through interaction of a sialylated glycan on the lateral surface of its crd with the iav hemagglutinin (similar to sp-a) and can therefore bind to a broader array of iav variants (van eijk et al., (van eijk et al., , (van eijk et al., , hillaire et al., ) . in vitro assays show that sp-a and sp-d can directly neutralize iav infectivity (benne et al., ; reading et al., ; hartshorn et al., ; van eijk et al., ; hawgood et al., ; hillaire et al., ) . both sp-a and sp-d inhibit the viral hemagglutinating activity that is required for iav attachment to target cells (hartshorn et al., ; malhotra et al., ; benne et al., ; hartshorn et al., hartshorn et al., , hartshorn et al., , van eijk et al., van eijk et al., , van eijk et al., , mikerov et al., ; hillaire et al., ) and sp-d was reported to inhibit the viral neuraminidase (reading et al., ; hillaire et al., ) . interestingly, both lectins also induce viral aggregation (hartshorn et al., (hartshorn et al., , (hartshorn et al., , van eijk et al., ) and function as potent opsonins. sp-a for instance was identified as an opsonin for iav phagocytosis by alveolar macrophages (benne et al., ) . moreover, sp-a and sp-d were shown to enhance iav binding to neutrophils (hartshorn et al., (hartshorn et al., , (hartshorn et al., , van eijk et al., ) and sp-d-iav complexes were found to internalize upon attachment to neutrophils (hartshorn et al., ; van eijk et al., ) . pre-incubation of iav with sp-a or sp-d also enhances the virus-induced h o responses in neutrophils (hartshorn et al., (hartshorn et al., , (hartshorn et al., , van eijk et al., ) and pre-incubation of the virus with sp-d can protect neutrophils from iav-induced deactivation (hartshorn et al., (hartshorn et al., , (hartshorn et al., , . mbl can counteract iav by roughly the same mechanisms as sp-d (hartshorn et al., ; anders et al., ; malhotra et al., ; hartshorn et al., hartshorn et al., , reading et al., ; kase et al., ) , although its ability to activate the complement cascade expands its capabilities (anders et al., ; reading et al., ; chang et al., ) . ligand binding by mbl (or alternatively ficolins) can lead to activation of mbl-associated serine proteases (masps) and initiate the complement cascade via the so-called lectin pathway (blue et al., ; bottazzi et al., ) . complement deposition on a virus may interfere directly with crucial steps in the viral infection process (e.g. receptor binding), but can also trigger complement receptor-mediated uptake of the pathogen into immune cells. in addition, for enveloped viruses, complement activation may result in membrane attack complex (mac) formation on the viral envelope and subsequent virolysis (blue et al., ; bottazzi et al., ) . in line with the available in vitro data, recent work with sp-a-, sp-d-, and mbl-knockout mice also confirmed the antiviral potential of these soluble lectins in vivo (levine et al., (levine et al., , zhang et al., ; li et al., ; hawgood et al., ; levine et al., ; kingma et al., ; chang et al., ) . noteworthy caveats regarding these in vivo studies are, however, that direct antiviral effects of these lectins can be hard to uncouple from othere.g. immune-regulatoryeffects and that mice do not represent natural hosts for iav. another interesting example of antiviral activity mediated by soluble host lectins was recently reported for nipah virus (niv): the physiologic, homodimeric form of the soluble lectin galectin- can inhibit niv envelope protein-mediated membrane fusion (levroney et al., ; garner et al., ) . niv encodes two viral membrane although capture of a virus by these lectins may have certain antiviral effects or promote the specific immunity against this pathogen, current experimental data suggest that the listed viruses may also employ these lectins to promote viral infection, spread or persistence. references in table are listed in supporting information, data s . dimt: discussed in main text. glycoproteinsthe attachment protein niv-g and the fusion protein niv-fthat mediate viral entry and direct the endothelial cell syncytia formation typically associated with niv infection (levroney et al., ; garner et al., ; lee & ataman, ) . binding of niv-g to cell surface receptors induces a conformational change in niv-f, thereby activating its fusogenic activity (levroney et al., ; garner et al., ; lee & ataman, ) . galectin- associates with glycans on the niv envelope proteins and interferes with the membrane fusion process in multiple ways (levroney et al., ; garner et al., ) . not only does galectin- binding directly inhibit the crucial conformational change in niv-f associated with membrane fusion, it also reduces the lateral mobility of niv-f and niv-g in the lipid membrane and consequently counteracts the physical separation of niv-f and niv-g that is essential for this conformation change (garner et al., ) . moreover, galectin- binding impedes endocytosis and maturation of the niv-f precursor niv-f in infected cells (garner et al., ) , further illustrating the capacity of this lectin to block different stages of the viral infection/ replication process. in sum, soluble host lectins can counteract viral infection in various ways: they may directly neutralize virus by destabilizing or aggregating virions, interfere with crucial steps in the viral infection process (e.g. entry), and/or opsonize virus to facilitate uptake and degradation. upon virus binding, some soluble lectins can trigger complement deposition on the virus, which may inhibit viral infection, enhance viral uptake via complement receptors and subsequent degradation in immune cells, and/or cause virolysis. considering the above, it is clear that lectins contribute significantly to the host's antiviral defense. host lectins are involved in neutralization and clearance of free virus, immune regulation, andalthough not extensively discussed in this overviewthe detection and clearance of virus-infected cells. figure illustrates how membraneassociated and soluble host lectins can aid in antiviral defense. like their animal hosts, also viruses can benefit from interactions between glycans and glycan-binding proteins. for instance, many viruses, including hiv- , herpes simplex virus- , and dengue virus, have been shown to interact with glycosaminoglycan molecules present on target cells (patel et al., ; chen et al., ; krusat & streckert, ; summerford & samulski, ; dechecchi et al., dechecchi et al., , vanderheijden et al., ; delputte et al., ; trybala et al., ) . viral association with glycosaminoglycans is usually attributed to charge-based attractions between clusters of positively charged aa residues on the virion surface and the negatively charged glycosaminoglycan chains. the interaction with glycosaminoglycans often constitutes the first contact between a virus and its target cell and typically increases infection efficiency. however, it has been documented for several viruses that the ability to interact with glycosaminoglycans can result from adaptation to growth in cell culture (sa-carvalho et al., ; klimstra et al., ; hulst et al., ; mandl et al., ) . clearly, use of primary virus isolates is of crucial importance when assessing the occurrence and relevance of such interactions in vivo. aside from potential glycosaminoglycan-binding capacity, several viruses are endowed with a true lectin activity: they carry virally encoded lectins on their surface and use these as keys to gain entry into their target cells (fig. a ). similar to animal lectins, such virally encoded lectins often possess characteristic glycan binding regions ('glycan binding pockets') and recognize specific portions of protein-and lipid-linked glycans. the hemagglutinin protein of iav is generally regarded as the prototype of a viral lectin. hemagglutinin is a membrane glycoprotein that forms noncovalently linked fig. . schematic overview of how membrane-associated (a) and soluble (b) host lectins are implicated in antiviral defense. (a. ) binding of virion-associated glycans with membrane-associated host lectins can lead to virus uptake, degradation, and presentation of viral antigens to cells of the adaptive immune system. binding may trigger specific signaling that promotes an effective antiviral immunity. (a. ) binding of virionassociated glycans with membrane-associated host lectins may promote direct presentation of the virus to immune cells in trans. binding may trigger specific signaling that promotes an effective antiviral immunity. (b. ) binding of soluble host lectins to virion-associated glycans may interfere directly with viral infection by destabilizing virions, blocking interaction of the virus with its receptors or interfering with other crucial steps in the infection process (e.g. membrane fusion). soluble host lectins may also aggregate virions, which often negatively impacts viral infectivity (not depicted). (b. ) soluble host lectins can act as opsonins: lectin binding to virion-associated glycans may facilitate viral uptake in immune cells via lectin receptors, leading to viral degradation and potential presentation of viral antigens to cells of the adaptive immune system. lectin binding may also trigger complement deposition on the virus (through the lectin pathway) and facilitate viral uptake via complement receptors. (b. ) detection of virion-associated glycans by soluble host lectins may trigger complement deposition on the virus (through the lectin pathway), which may directly inhibit viral infection and/or elicit lysis of the (enveloped) virus. homotrimers in the (viral) membrane and is responsible for both virus attachment and penetration (skehel & wiley, ; harrison, ; gamblin & skehel, ) . the mature hemagglutinin protein consists of two disulfide-linked subunits, termed ha and ha (skehel & wiley, ; harrison, ; gamblin & skehel, ) . the ha subunit forms the globular 'head' region of hemagglutinin that covers the lectin function of this molecule: sialic acid-binding pockets in the membrane distal part of ha allow interaction with sialic acidcontaining receptors on target cells (skehel & wiley, ; harrison, ; gamblin & skehel, ) . variation in the ha subunit determines the affinity and specificity (e.g. a - vs. a - -linked sialic acids) of this molecule (skehel & wiley, ; gamblin & skehel, ) . as seen for animal lectins, also hemagglutinin binds with its glycan counterparts with a relatively low affinity and efficient virus attachment and entry depends on the interaction of multiple hemagglutinin molecules with multiple sialic acid-containing receptors (skehel & wiley, ; gamblin & skehel, ) . the stalk-like ha subunit of hemagglutinin mediates the ph-dependent fusion process upon internalization of the iav virion in the endosomal compartment of the target cell (skehel & wiley, ; harrison, ; gamblin & skehel, ) . similar to iav, various other enveloped [e.g. influenza b and c viruses (nakada et al., ; herrler et al., a; rogers et al., ; vlasak et al., ; herrler et al., ; herrler & klenk, ; rosenthal et al., ; lamb & krug, ; suzuki & nei, ; wang et al., b; wang et al., a) , mumps virus (bowden et al., ; harrison et al., ; chang & dutch, ) ] as well as nonenveloped [e.g. murine norovirus (taube et al., (taube et al., , , feline calicivirus (stuart & brown, ) , and rhesus rotavirus (dormitzer et al., ) , among others (taube et al., ) ] viruses employ sialic acid-binding viral lectins to infect target cells. importantly however, viral lectins with a different glycan specificity have also been identified. for instance, many virusesincluding human noroviruses (estes et al., ; le pendu et al., ; cao et al., ; bu et al., ; choi et al., ; donaldson et al., ; shirato, ) , rabbit hemorrhagic disease viruses (ruvoen-clouet et al., ; rademacher et al., ; guillon et al., ; nystrom et al., ) , and the rhesus monkey tulane virus (farkas et al., ) were found to interact with specific hbga types. although a broad spectrum of viruses has evolved to use viral lectins to secure efficient target cell infection, the use of viral lectins for cellular attachment comes with a price. the glycan receptors for viral lectins are not necessarily target cell-specific and, whereas low affinity/avidity interactions may be reversible, high affinity/avidity binding of viral lectins to nontarget cell-associated glycoconjugates ('decoy receptors') can prevent the virus from efficiently targeting susceptible host cells. in line with this, it was shown for iav that interaction of the viral lectin hemagglutinin with soluble, sialylated host glycoproteins e.g. sp-a (cfr. supra) or a -macroglobulincan interfere with the viral hemagglutinating activity that is crucial for receptor binding (rogers et al., ; pritchett & paulson, ; ryan-poirier & kawaoka, ; matrosovich et al., ; ryan-poirier & kawaoka, ; hartshorn et al., ; malhotra et al., ; benne et al., ; gimsa et al., ; benne et al., ; hartshorn et al., ; matrosovich et al., ; van eijk et al., ; mikerov et al., ; chen et al., ; cwach et al., ) . although the presence of 'high avidity' glycan decoys invariably puts a strain on viral infection efficiency, this burden may be lighter on viruses that are equipped with a receptor destroying enzyme (rde) that matches the specificity of the viral lectin. intriguingly, the best-known examples in this context are again influenza viruses. as situated above, both influenza a and b viruses display hemagglutinin proteins on their surface, which bind to sialic acids displayed on the host cell surface and mediate ph-dependent fusion of the viral membrane with the host cell membrane (skehel & wiley, ; lamb & krug, ; wang et al., b; wang et al., a; harrison, ; gamblin & skehel, ). an rde activity was mapped to another viral membrane glycoprotein, designated neuraminidase (gottschalk, ; colman, ; fig. . (a) illustrates how viral lectins promote target cell infection. (b) shows how many viruses that employ viral lectins also benefit from a matching receptor-destroying enzyme (rde) activity, which provides a counterweight against (high avidity) lectin activity. (a) interaction of viral lectins with glycosylated receptors on a target cell promotes viral entry and infection (attachment/internalization/fusion, depending on specific virus biology). (b) although they clearly benefit the virus, the use of (high avidity) viral lectins comes with a price. for instance, viral lectin activity can cause virions to aggregate (b. ) and can impair efficient release of newly formed virions from (glycosylated) infected cells (b. ). moreover, binding of viral lectins to nontarget cell-associated glycoconjugates (decoy receptors) can prevent the virus from efficiently targeting susceptible host cells (b. ). intriguingly, several lectin-carrying viruses are also equipped with an rde that matches the specificity of the viral lectin and provides a counterweight against lectin-mediated glycan binding. in fact, for viruses equipped with both viral lectins and rdes, a functional balance between these molecules appears to be an important determinant of the viral (replicative) fitness. lamb & krug, ; gamblin & skehel, ) . this enzyme removes sialic acid moieties from glycoproteins and glycolipids by catalyzing the hydrolysis of the a-ketosidic linkage to the subterminal sugar residue and consequently destroys potential receptors for the viral hemagglutinin (gottschalk, ; colman, ; lamb & krug, ; gamblin & skehel, ) . viral use of an enzyme that can actually destroy receptors for the virus may seem peculiar at first. importantly, however, the neuraminidase activity can prevent virions from aggregating via hemagglutinin-sialic acid interactions, promotes efficient release of newly formed virions from (sialic acidcarrying) infected cells, and provides a counterweight to the interaction of hemagglutinin molecules with nontarget cell-associated glycans: neuraminidase-mediated removal of sialic acids from decoy receptors prevents virions from establishing high-avidity interactions with these glycoconjugates and may even provide an escape route for virions after hemagglutinin-mediated binding to nontarget cellassociated glycans (colman, ; suzuki et al., ; gimsa et al., ; barrere et al., ; gamblin & skehel, ) . conceivably, a functional balance between the hemagglutinin and neuraminidase activities is an important determinant of the (replicative) fitness of iav variants in vivo. using iav as a paradigm, fig. b illustrates how viral rde activity can balance the high avidity of viral lectins where favorable and consequently improve viral infection efficiency. in contrast to influenza a and b viruses, other viruses combine both lectin and rde functions in one protein complex. for example, the viral membranes of mumps virus, newcastle disease virus, sendai virus, and human parainfluenza virus and are studded with hemagglutinin-neuraminidase proteins (bowden et al., ; harrison et al., ; chang & dutch, ) . another example is the influenza c virus, which carries the hemagglutination, rde and fusion protein functions in one single envelope protein named the hemagglutininesterase-fusion protein (nakada et al., ; herrler et al., a, b; rogers et al., ; vlasak et al., ; herrler et al., ; schauer et al., ; herrler & klenk, ; rosenthal et al., ; pekosz & lamb, ; lamb & krug, ; suzuki & nei, ) . whereas the rde of influenza a and b viruses is a neuraminidase, which cleaves off entire sialic acid residues, the rde of influenza c functions as a sialate-o-acetylesterase and cleaves off specific o-acetyl groups (herrler et al., b; vlasak et al., ; herrler et al., ; schauer et al., ; pekosz & lamb, ) . a similar situation is seen for certain coronaviruses and toroviruses that carry an accessory protein called hemagglutinin-esterase on their surface (de groot, ) . as the name implies, functional hemagglutinin-esterase proteins combine a hemagglutinating activity with a sialate-o-acetylesterase activity (de groot, ) . interestingly, for some coronaviruses, the hemagglutinin-esterase protein is not the only envelope protein endowed with a lectin activity. for example, the spike proteins of bovine coronavirus and human coronavirus oc have been shown to be potent sialic acid-binding lectins kunkel & herrler, ) . also the spike protein of the transmissible gastroenteritis coronavirus of pigs has been shown to possess such a lectin activity (schultze et al., ; krempl et al., krempl et al., , schwegmann-wessels et al., ) . however, transmissible gastroenteritis coronavirus has no hemagglutinin-esterase protein that serves as rde to counteract the hemagglutinating activity of the spike protein. viruses do not only benefit from virally encoded lectins, but can also use host lectins to their advantage. paradoxically, many of the host lectins that are exploited by viruses form part of the immune system. although capture of viral pathogens by these lectins may have certain antiviral effects or promote the specific immunity against this pathogen (cfr. supra), many viruses can also employ such interactions to promote efficient infection and spread or to facilitate persistence (table ) . virus binding to membrane-associated lectins can lead to concentration of virions at the cell surface and can facilitate infection of target cells. in many cases, host lectins appear to function as true portals for viral entry: the virus binds to the lectin, which drives subsequent internalization of the virus into specific cellular compartments from which the virus can initiate the next stage of infection. however, it has also been shown that lectins present on non-target cells may facilitate infection of target cells, a process called trans-infection. many membrane-associated (immune system) lectins also participate in specific signaling pathways and engagement of such lectins by a virus may modulate both viral infection and the immune response in favor of the pathogen. an immune system lectin that can be used as a paradigm in this context is dc-sign. this molecule is mainly expressed on dendritic cells (dcs), but expression on distinct other cell-typesincluding macrophages, b lymphocytes, platelets, and (immortalized) podocyteshas also been described (geijtenbeek et al., a, b; soilleux et al., ; granelli-piperno et al., ; gurney et al., ; chaipan et al., ; rappocciolo et al., ; mikulak et al., ; svajger et al., ) . prototypic dc-sign molecules consist of a c-terminal c-type crd, a neck region made up of and a half aa residue repeats, a transmembrane domain, and a cytoplasmic domain containing motifs involved in receptor internalization and signaling (svajger et al., ; tsegaye & pohlmann, ) . lectin monomers typically multimerize via their neck regions to form tetramers, which in turn organize in nanoclusters on the cell membrane (svajger et al., ; tsegaye & pohlmann, ; manzo et al., ) . dc-sign functions as a receptor for various ligands. the t cell-expressed molecule icam- is probably its most prominent endogenous ligand: dc-sign binds icam- on the t cell surface and thereby contributes to the transient, nonantigen-specific interaction of dc with t cells that is required for efficient screening of mhcii-peptide complexes and eventual t cell priming (geijtenbeek et al., a; svajger et al., ) . moreover, dc-sign has been implicated in various other processes, including dc differentiation, migration, and antigen capture (svajger et al., ) . over the last decade, it has become apparent that dc-sign interacts with a wide variety of viral pathogens. a textbook example of a virus that recruits dc-sign is hiv- (lekkerkerker et al., ; wu & kewalramani, ; piguet & steinman, ; tsegaye & pohlmann, ; da silva et al., ; van der vlist et al., ) . dc-sign can bind the enveloped hiv- particle and mainly recognizes mannose-rich glycans on the viral envelope glycoprotein gp (curtis et al., ; geijtenbeek et al., a, b; feinberg et al., ; hong et al., ; lin et al., ; su et al., ; hong et al., ) . the efficiency of hiv- capture by dc-sign has been linked to receptor density. experiments in t-rex cells using an inducible dc-sign expression system have shown that high surface expression levels of dc-sign correlate with optimal binding of hiv- particles, and that lowering the dc-sign expression levels can significantly reduce the efficiency of hiv- binding . these data suggest that the high dc-sign surface expression levels on certain (immature) dc subsets are compatible with optimal capture of hiv- virions, whereas lower dc-sign expression levels on b lymphocytes and especially platelets may be mirrored in a less efficient hiv- capture (baribaud et al., ; boukour et al., ; chaipan et al., ; rappocciolo et al., ) . nevertheless, studies have shown that also b lymphocytes and platelets can effectively bind hiv- virions via dc-sign (boukour et al., ; chaipan et al., ; rappocciolo et al., ) . evidently, the interaction between hiv- and dc-sign is also critically dependent on the viral glycome. recent research has shown that virion-associated gp of peripheral blood mononuclear cell (pbmc)grown virus predominantly carries oligomannose n-glycans (doores et al., ; bonomelli et al., ) , which constitute optimal ligands for dc-sign (van liempt et al., ) . nevertheless, virus originating from different host cell types can display a different glycosylation profile, and this may modulate the efficiency of dc-sign recruitment (lin et al., ) . binding of hiv- to dc-sign can entail both negative and positive effects for the virus. in dc-signexpressing antigen-presenting cells, most of the dc-sign-captured virions appear to be internalized into the endolysosomal pathway and rapidly degraded (moris et al., ; turville et al., ) . in line with this, dc-sign was found to promote mhcii-restricted presentation of hiv- antigens (moris et al., ) . intriguingly however, in cells that co-express cd and ccr / cxcr (hiv- receptor and co-receptors, respectively), dc-sign expression also facilitates hiv- fusion: dc-sign efficiently captures and concentrates viral particles at the cell surface, and binding of this lectin to the hiv- envelope protein appears to increase exposure of the cd binding site nobile et al., ; burleigh et al., ; hijazi et al., ) . although dc-sign-mediated enhancement of hiv- fusion may promote mhci-restricted presentation of hiv- antigen (proteasome and tap-dependent pathway) and activation of cytotoxic t lymphocytes (moris et al., ) , enhanced hiv fusion inevitably leads to more efficient infection. indeed, several studies confirm that dc-sign facilitates productive (cis-) infection in dc-signexpressing cells that also co-express cd and ccr / cxcr nobile et al., ; burleigh et al., ; hijazi et al., ) . dc-sign has also been implicated in hiv- transinfection. an initial study showed that dc-sign can efficiently capture hiv- particles and transfer them to adjacent target t cells, without the need for productive infection of the dc-sign-expressing cell (geijtenbeek et al., b) . subsequent studies on the subject reported dc-sign-mediated internalization of infectious hiv- virions into low ph nonlysosomal compartments, and advocated that the virus traffics in intracellular compartments towards the zone of t cell contact, where it is released into the infectious synapse (i.e. the contact zone between the virus-loaded cell and the target t cell) (kwon et al., ; mcdonald et al., ) . dc-captured hiv- virions as well as t-cellexpressed cd and ccr /cxcr were found to concentrate at the dc-t-cell interface, rendering it an ideal micro-environment for efficient infection of target t cells (mcdonald et al., ) . moreover, it was postulated that dc-sign-mediated capture of hiv- virions temporarily protects them from degradation and preserves viral infectivity (geijtenbeek et al., b; kwon et al., ) . the above data supporting dc-sign-mediated capture, uptake, intracellular transport and ultimately transfer of intact hiv- particles from dcs to target t cells were united in the 'trojan horse model' of mucosal hiv- transmission. this model posits that submucosal dcs capture and internalize hiv- virions via dc-sign and, by homing to the lymph nodes, provide a means of transport for the virus to a compartment rich in target cells. the virus-loaded dcs then interact with cd + t cells and the virions are transferred to the target t cell via the infectious synapse, ultimately resulting in efficient target cell infection (geijtenbeek et al., b; baribaud et al., ; sewell & price, ) . however, recent research is not always in line with this initial model and has challenged several of its key features. cavrois et al. ( ) reported that hiv- trans-infection does not require intracellular virus trafficking, but primarily depends on cell surface-associated virions that reach the infectious synapses via transport on the cell surface (cavrois et al., ) . in line with this, yu et al. ( ) reported that hiv- traffics towards the infectious synapse through a specialized, surface-accessible intracellular compartment (yu et al., ) . in addition, reports stating that virus capture by dc-sign mainly leads to virus internalization into the endolysosomal pathway and subsequent degradation (moris et al., ; turville et al., ; moris et al., ) and that dc-sign-mediated trans-infection can only occur within the first hours after virus attachment (turville et al., ) seem to downplay the importance of dc-sign-mediated trans-infection for efficient hiv- infection and spread. these and other data counter the theory that hiv- capture by dcs preserves viral infectivity, and suggest that the presenceand transferof infectious virus at later time points may be ascribed to productive dc infection (turville et al., ; nobile et al., ; burleigh et al., ; wang et al., a) . it is also noteworthy that, whereas initial studies identified dc-sign as the main factor involved in hiv- capture and transmission by dcs (geijtenbeek et al., b) , recent studies also implicate other lectins in this process (turville et al., ; izquierdo-useros et al., ) or even conclude that dc-sign is not involved in dc-mediated hiv- trans-infection (boggiano et al., ) . differences in virus strains, cell types, and experimental setup might in part explain these conflicting data. intriguingly, other recent work indicates that dc-sign-expressing b lymphocytes and platelets may effectively capture and transfer infectious hiv- via dc-sign (boukour et al., ; chaipan et al., ; rappocciolo et al., ) , potentially implicating these cells/cell fragments in hiv- dissemination in infected patients, although recent work suggests that platelets might negatively regulate viral spread by secretion of cxcl (auerbach et al., ; tsegaye et al., ) . clearly, further research is necessary to allow a better understanding of dc-sign-mediated hiv- trans-infection and its relevance for viral infection and spread in vivo. importantly, the role of dc-sign in hiv- infection appears not to be restricted to purely physical capture of virions for subsequent degradation or cis-or transinfection. recruitment of dc-sign by hiv- also triggers signal transduction that modulates immune responses and infection of dcs and adjacent target cells more indirectly. for example, hodges et al. ( ) reported that binding of hiv- to dc-sign compromises dc maturation and primes these cells for trans-infection: upregulation of cd and mhcii is suppressed, whereas synapse formation between dcs and cd + t cells is promoted (hodges et al., ) . moreover, hiv- binding to dc-sign was shown to activate cdc and promote formation of membrane extensions that facilitate hiv- transfer to cd + lymphocytes (nikolic et al., ) . other work by gringhuis et al. ( gringhuis et al. ( , showed that binding of hiv- to dc-sign triggers raf- dependent signaling, which modulates toll-like receptor (tlr)-elicited signals to induce synthesis of fulllength hiv transcripts as well as production of the immunosuppressive cytokine il- ( gringhuis et al., ( gringhuis et al., ( , . in general, the data discussed above suggest that dc-sign recruitment by hiv- might affect viral infection and transmission, as well as the host defense against this pathogen in several ways. over the last decade, dc-sign has become a prototype for lectin-mediated cis-and trans-infection and has been implicated in the infection process of various viruses, including hiv, dengue virus, ebola virus, and iav (see table ). importantly, however, dc-sign is not the only host lectin that is (ab)used by viruses to promote target cell infection or avoid immune recognition and clearance. various other membrane-associated host lectins seem to be exploited by virusesin ways similar to dc-signto aid cis-infection, trans-infection and/or viral persistence. analysis of recent literature suggests that membrane-associated host lectins may constitute weak links in the host's defense against viral pathogens (table ) . although generally implicated in antiviral defense, soluble host lectins may also support viral infection (table ) . for example, galectin- has been proposed to promote hiv- infection (ouellet et al., ; mercier et al., ; st-pierre et al., ; sato et al., ) . in vitro experiments pointed out that galectin- can enhance hiv- infection of different cell typesincluding human lymphoid cell lines, pbmc, cd + t lymphocytes, and monocyte-derived macrophages (ouellet et al., ; mercier et al., ) and increase hiv- infection in an ex vivo lymphoid tissue model (ouellet et al., ) . further experiments showed that galectin- accelerates virion binding to the target cell surface, probably by crosslinking viral and cellular glycans (ouellet et al., ; mercier et al., ) . a more recent study confirmed these findings and showed that galectin- binds to clusters of n-linked glycans on the viral gp envelope protein in a b-galactoside-dependent manner (st-pierre et al., ) . data from the same study identify the hiv- receptor cd as a ligand for galectin- and suggest that galectin- can cross-link gp and cd (st-pierre et al., ) . in sum, it appears that the dimeric lectin galectin- can enhance hiv- infection efficiency by cross-linking viral and host cell glycans and thereby promoting firmer adhesion of the virus to the target cell surface and facilitating virus-receptor interactions (ouellet et al., ; mercier et al., ; st-pierre et al., ; sato et al., ) . some studies have also attributed proviral effects to the collectins mbl, sp-a, and sp-d. for some viruses, it was reported thatunder certain conditionsviral recognition by collectins may enhance cis-or trans-infection (hickling et al., ; sano et al., ; gaiha et al., ; brudner et al., ; madsen et al., ) . it is conceivable that these collectins can bind the virus and subsequently associate with collectin receptors on the surface of target/ transmitting cells, thereby concentrating virions at the cell surface and facilitating infection or viral transfer. nevertheless, involvement of other mechanisms (e.g. collectinmediated cross-linking of virus-and host cell-displayed glycans, cfr. the galectin- -hiv- example described above) can currently not be excluded. further research is necessary to elucidate the biology behind the potential proviral effects of collectins and to estimate the occurrence and relevance of these events in an in vivo context. figure gives a schematic overview of how membraneassociated and soluble host lectins can be implicated in interactions that benefit the virus and facilitate viral infection and spread. as glycan-lectin interactions often represent key events in viral infection and/or antiviral immunity, variation in glycan or lectin expression and structureeither at the host or at the virus levelmay significantly shift the balance between host and pathogen. a basic insight into the nature and origin of this variability is therefore germane to a proper understanding of glycan-lectin interactions in the context of viral infection biology and immunology. glycan formation is a very complex and versatile biosynthetic process. in contrast to the primary amino acid sequences of proteins, glycan structures are not directly encoded in the host genome. instead, they are synthesized in a step-wise manner via the concerted action of various host-encoded glycosyltransferase, glycosidase, and other enzymes. the availability of these glycoenzymes, the availability of precursor molecules and the accessibility of specific glycosylation sites govern (the efficiency of) glycan addition and modification and hence determine glycan variability. the genetic make-up of the host evidently has a major impact on glycosylation, but also other host-related factors can have pronounced effects. recent research has shown that different cell types within a host can assemble radically different glycomes (roth, ; haslam et al., ) and that factors such as the activation (comelli et al., ; bax et al., ; haslam et al., ) or infection (lanteri et al., ) status of a cell can significantly influence glycosylation processes. clearly, various biological factors contribute to the high glycan heterogeneity that is seen for many animal glycoconjugates. although host glycan variation may influence virtually all viral infections in several ways, its potential impact is probably most evident for viruses that are equipped with viral lectins. a notable example in this context are the noroviruses, a major cause of nonbacterial gastroenteritis in humans. it is well known that the viral capsids of most human noroviruses display an affinity for hbgas, structurally related but highly polymorphic carbohydrate structures found on proteins and lipids of epithelial cells in the gastrointestinal and respiratory tract, on the surfaces of red blood cells and as free antigens in body fluids such as saliva, blood, and intestinal contents (bu et al., ; choi et al., ; shirato, ) . different noroviruses display distinct hbga specificities and can be categorized according to the (range of) hbga structures they preferentially bind shirato, ) . human hbga synthesis is controlled by various enzymes, including the glycosyltransferase enzymes encoded in the abo, fut , and fut gene loci. the presence of variant (functional or nonfunctional) alleles at these and other relevant gene loci is a key determinant of hbga phenotype, as it controls which abh and lewis antigens an individual can synthesize (le pendu et al., ; shirato, ) . although it is still unclear whether they function as primary receptors for noroviruses, current data indicate that hbgas are important determinants of the noroviral tissue specificity. moreover, several studies have established a link between hbga geno-/ phenotype and individual susceptibility to (clinical) infection with specific norovirus variants: hbga phenotypes matching the specificity of the viral lectin correlate with a higher risk of (clinical) infection, whereas nonmatching hbga phenotypes correlate with relative resistance (le pendu et al., ; shirato, ) . several studies have revealed significant heterogeneity relating to animal lectins. lectin expression is governed by various genetic and nongenetic (e.g. hormone balance, immune status) factors. importantly, gene polymorphisms that affect protein expression and/or functionality have been described for several animal lectins, including mbl and dc-sign. for mbl, mutations in the promoter region of the mbl gene were found to affect protein expression levels, probably by influencing binding of transcription factors (eisen & minchinton, ; dommett et al., ; heitzeneder et al., ) . moreover, specific polymorphisms in mbl exon , encoding the collagen-like domain of mbl, appear to hinder correct and stable oligomerization of mbl protein chains and impede efficient ligand binding and activation of the lectin complement pathway (eisen & minchinton, ; dommett et al., ; heitzeneder et al., ) . several studies suggest a correlation between mbl deficiency and susceptibility to hiv infection, but conflicting data have been reported and further research is clearly necessary to corroborate this link (eisen & minchinton, ; dommett et al., ; heitzeneder et al., ) . similar findings have been recorded for dc-sign. polymorphisms in the promoter region of the dc-signencoding cd gene can affect protein expression levels and have been linked with altered susceptibility to and/or altered disease progression after infection with several viral pathogens, including hiv- and dengue virus (martin et al., ; sakuntabhai et al., ; koizumi et al., ; selvaraj et al., ; wang et al., ; boily-larouche et al., ) . moreover, distinct gene polymorphisms in the dc-sign-encoding region as well as alternative splicing events give rise to different isoforms of the protein, ranging from variants containing single nucleotide polymorphisms (snps) to variants with truncated lectin domains, variable numbers of -aa-residue repeats in the neck domain, alternative cytoplasmic domains or a lacking transmembrane region (mummidi et al., ; liu et al., ; serrano-gomez et al., ; boily-larouche et al., ) . information on the expression and biological activity of most of these dc-sign variants is rather limited. recently, however, boily-larouche et al. ( ) reported that naturally occurring genetic variants of dc-sign, carrying specific snps in the neck domain-encoding exon , have an enhanced capacity to capture and transfer hiv- virions to cd + t lymphocytes. moreover, liu et al. ( ) described differ-ent neck domain length variants of dc-signcarrying variable numbers of -aa-residue repeats in the neck regionand correlated neck domain length heterozygosity with a reduced risk of hiv- infection. recent experimental data provide evidence that naturally occurring dc-sign neck domain variants can differ in multimerization competence in the cell membrane and display altered glycan binding capacity (serrano-gomez et al., ) . moreover, the presence of such neck domain variants appears to modulate multimerization of the prototypic dc-sign molecule (serrano-gomez et al., ) . the fact that neck domain variation may influence the presence, stability, and functionality of dc-sign multimers on the cell surface can provide a molecular explanation for the link between dc-sign polymorphisms and susceptibility to hiv- and other pathogens, although further research is needed to substantiate this (serrano-gomez et al., ) . although viruses rely on the host cell machinery for glycoconjugate synthesis, viral glycosylation profiles can significantly differ from the standard glycosylation profile of their host cell. for instance, it is well known that viral glycoproteins are often more heavily glycosylated than host glycoproteins, and that also the nature of their glycan modifications can significantly differ. a prototypic example in this context is the gp glycoprotein of hiv- . the hiv- envelope is studded with trimers of noncovalently associated gp /gp heterodimers . gp is one of the most heavily n-glycosylated proteins in nature: it contains more than n-linked glycosylation sites, and n-glycans account for about half of its molecular weight (zhu et al., ; wei et al., ; pantophlet & burton, ; scanlan et al., ) . intriguingly, whereas mammalian glycoproteins typically carry mainly complex type n-glycans, this is not the case for the viral gp glycoprotein. recent reasearch has shown that virion-associated gp of pbmc-grown virusas opposed to recombinantly expressed monomeric gp predominantly carries oligomannose n-glycans (doores et al., ; bonomelli et al., ) . the synthesis of this unusual glycosylation profile appears to be partially directed by the structure of the gp /gp spike itself: the presence of a dense n-glycan cluster in gp , combined with the steric consequences of gp /gp trimerization, seems to hinder further processing of (normally transient) biosynthetic glycan intermediates by er and golgi a-mannosidases, ultimately yielding hiv- virions with oligomannose-enriched gp glycoproteins (zhu et al., ; doores et al., ; eggink et al., ; bonomelli et al., ) . clearly, although viral glycosylation is critically dependent on the glycosylation machinery of the host cell, the genetic and structural background of a virus can have a decisive influence in this process. importantly, viral infection itself may also have strong repercussions on the glycosylation biology of a host cell. considering the restricted glycosylation enzyme and precursor availabilities, it is conceivable that overexpression of viral glycoproteins in an infected target cell can result in an increased glycan heterogeneity of both viral and cellular glycoconjugates. moreover, viruses may also actively modify the host and viral glycome by modulating the expression of host cell glycoenzymes (hiraiwa et al., ; cebulla et al., ; hiraiwa et al., ) or via expression of virally encoded glycoenzymes in infected cells (jackson et al., ; willer et al., ; nash et al., ; sujino et al., ; vanderplasschen et al., ; markine-goriaynoff et al., , a . an additional source of glycan variation can be discerned for viruses that can infect multiple cell types, or even different host species. for instance, it is well known that hiv can productively infect multiple cell types, and that hiv glycosylation is cell type-dependent (liedtke et al., ; willey et al., ; liedtke et al., ; lin et al., ) . cell type-dependent glycosylation differences for hiv have been shown to impact viral interaction with and trans-infection via dc-sign (lin et al., ) , as well as viral sensitivity to antibody neutralization (willey et al., ) . another, particularly fascinating example in this context is dengue virus (dv). dv is a mosquito-borne flavivirus that can replicate in mosquitos as well as in humans (navarro-sanchez et al., ; dejnirattisai et al., ) . in humans, immature skin dcs are considered the primary target cell for the virus after a mosquito bite, and dc-sign is believed to be the main dv receptor on these cells (navarro-sanchez et al., ; dejnirattisai et al., ) . in a recent study, it was shown that insect cell-derived dv can efficiently infect dcs, whereas dc-derived dv is not able to reinfect dcs (dejnirattisai et al., ) . similarly, insect cell-derived dv could efficiently bind and infect a dc-sign-expessing cell line, whereas this was not the case for dcderived dv (dejnirattisai et al., ) . finally, it was found that insect cell-derived dv predominantly contains high-/pauci-mannose-type n-glycans, whereas dcderived virus contains only complex type n-glycans (dejnirattisai et al., ) . projected against the background of dv infection, these data outline a tentative model of the first stages of dv infection in humans: during dv replication in mosquito cells, newly formed dv virions obtain mannose-rich glycans. upon viral transfer to a human host, these virions efficiently infect immature skin dcs via dc-sign. importantly, dv replication in skin dcs yields virions with complex type n-glycans, thus creating a 'glycan mismatch' with dc-sign. due to this mismatch, newly synthesized dc-derived virus will not readily reinfect dcs via dc-sign, but preferentially infect other potential host cells via other receptors (dejnirattisai et al., ) . although much more research is needed to verify this tentative model, it elegantly illustrates how cell type-dependent glycan variability may impact a viral infection process. another notable factor to be considered in the context of virus-related variability is the rapid evolution of many viral pathogens. this seems especially significant for rna viruses, as these viruses generally evolve more rapidly than dna viruses due to factors inherent to their biology and infection strategy (belshaw et al., ; holmes, ; lauring & andino, ) . the higher mutation frequency of many rna viruses directly implies a higher chance for addition or deletion of putative glycosylation sites. as has been shown for iav, acquisition or deletion of glycosylation sites may affect crucial steps in the viral infection/replication process (e.g. receptor binding, fusion, release of newly formed virions) (ohuchi et al., ; wagner et al., ; tsuchiya et al., ; kim & park, ) , alter the capacity of the virus to avoid induction of/recognition by virus-specific antibodies (glycan shielding) wei et al., ; wanzeck et al., ; kim & park, ; job et al., ; sun et al., ) , and modulate viral interaction with various immune system lectins (reading et al., ; vigerust et al., ; reading et al., ; tate et al., a, b) . clearly, mutational changes in the viral glycome may affect the virus-host interactome in various ways. ultimately, the net benefit of a specific glycome change will determine if a glycosylation variant may become dominant in the virus population. however, not only the viral glycosylation status, but also the affinity and specificity of viral lectins for specific glycoconjugates may change as a result of mutations. for instance, ample data show that amino acid changes at specific sites of the iav hemagglutinin protein can significantly alter its affinity and/or specificity for particular sialic acid-containing receptorsa factor that is crucial for the virus to infect new host species (skehel & wiley, ; wagner et al., ; suzuki, ; gamblin & skehel, ) . finally, functional alterations in the viral rde due to mutations may also have a strong impact on the interaction of viral lectins with host cell glycans. in the case of iav, balanced lectin and rde functions appear to be crucial for efficient viral replication. for iav variants that are well adapted to a certain host species, the substrate specificity and activity of the neuraminidase generally match the ligand specificity and affinity of the hemagglutinin . disruption of this balancefor instance due to reassortment or transmission to a new host speciesoften results in a decreased replicative fitness . interestingly, however, the virus may overcome this hurdle and evolve towards replicative competence by selecting for compensatory mutations in hemagglutinin and/or neuraminidase that restore the functional balance between these molecules . considering these data on iav, it is conceivable that the balance between viral lectin and rde is also an important determinant of the replicative fitness of several other viruses. on a related note, glycan-lectin interactions in virus biology are typically studied using a limited number of (prototypic) virus variants. although the information obtained in these studies can often be extrapolated to include other virus variants, there are important exceptions. for iav, for instance, it is well documented that different virus variants can carry hemagglutinin lectins with distinct glycan ligand specificities and therefore associate with distinct spectra of (decoy) receptors. moreover, iav variants can display different glycan arrays on their surface, which has been shown to modulate viral infection, glycan shielding, and recognition by various immune system lectins (cfr. supra). the fact that the specific genetic make-up of a virus determines specificity/ affinity of viral lectins, co-directs viral glycosylation, etc., and that this can be mirrored in a distinct virus-host interactome remains an important issue in glycovirology. in sum, an intricate web of glycan-lectin interactions can modulate viral infection, and host and virus inherent variability in glycans and lectins adds a further layer of complexity to this matter. considering the pivotal roles of glycan-lectin interactions in many viral infections, interfering with these interactions seems an attractive strategy in the combat against these pathogens. conversely, strategies that promote recognition of viruses by specific immune system lectinsinvolved in viral inhibition and clearancemay also prove useful in antiviral therapies. several possibilities have been and are currently being explored. perhaps the most obvious strategy to modulate glycanlectin binding is the use of molecules that can physically interfere with these interactions. glycan decoys (e.g. carbohydrate-containing drugs, sugar analogs/glycomimetics) or carbohydrate-binding agents (cbas) may be used in antiviral therapies to directly block key glycan-lectin interactions at the side of the lectin and at the side of the glycan, respectively. binding of glycan decoys with a specific lectin can inhibit binding of other ligands by this lectin. for instance, it has been shown that multivalent mannosecontaining molecules or mannose-based glycomimetics can compromise binding of hiv- gp with dc-sign (wang et al., b; luallen et al., ; martinez-avila et al., b; becer et al., ) and inhibit dc-sign-mediated trans-infection of cd + t lymphocytes (martinez-avila et al., a; sattin et al., ; berzi et al., ) . likewise, sialic acid-containing glycan decoys and sialic acid analogs are being evaluated for their capacity to block the sialic acid binding site of iav hemagglutinin to inhibit interaction of the virus with sialic acid-containing receptor molecules on the surface of target cells (landers et al., ; matrosovich & klenk, ; matsubara et al., ; papp et al., papp et al., , . alternatively, binding of cbas to glycans displayed on the virion surface can inhibit viral cis-or trans-infection via host cell lectins. this is elegantly exemplified in several recent studies, showing that mannose-as well as n-acetylglucosamine-specific cbas can effectively prevent dc-sign-mediated hiv- capture and subsequent transmission to t lymphocytes balzarini et al., a; bertaux et al., ; huskens et al., ; hoorelbeke et al., ; alexandre et al., ) . although cba binding to host cell-associated glycans may inhibit viruses that employ viral lectins, the potential of this strategy for antiviral therapy remains virtually unexplored. it is noteworthy that the antiviral activity of cbas or glycan decoys that bind to virion surfaces is not necessarily limited to direct inhibition of crucial glycan-lectin interactions, as they can mask greater portions of the virus and interfere with other crucial (including non-glycanlectin) interactions or steps in the infection process. moreover, recent research on hiv- highlights the antiviral potential of cbas from yet another angle. although the heavily glycosylated hiv- gp protein generally provides multiple ligands for mannose-and glcnac-specific cbas, prolonged cba pressure selects for hiv- variants with multiple n-glycosylation site deletions in the gp protein that are less sensitive to cba-mediated neutralization (balzarini et al., (balzarini et al., , a witvrouw et al., ; balzarini et al., ; balzarini, b, c; balzarini et al., b; huskens et al., ) . interestingly, however, deletion of n-glycosylation sites can also increase the immunogenicity of the virus and weaken the glycan shield that protects the virus from recognition by virus-specific antibodies and other (nonlectin) immune receptors (botarelli et al., ; back et al., ; reitter et al., ; bolmstedt et al., ; kang et al., ) , and may decrease the efficiency of hiv- trans-infection via immune system lectins like dc-sign (hong et al., ; liao et al., ) . in addition, it appears that accumulation of mutations under cba pressure is often paralleled by a significant reduction of the viral fitness, which is obviously advantageous in the context of an antiviral treatment (balzarini et al., a; balzarini, b, c) . for further information on cbas and their potential in antiviral therapy, readers may refer to recent expert reviews on this topic (balzarini, c; francois & balzarini, ) . although the use of glycan decoys and cbas may seem the most intuitive strategy to interfere directly with glycan-lectin interaction, other options exist as well. for instance, similar to glycan decoys and cbas, lectin-or glycoconjugate-specific immunoglobulins may be used to block specific interactions. evidently, the increasing availability of such 'direct' modulators of glycan-lectin interaction is mirrored in an increasing number of potential antiviral applications. apart from direct modulation, also strategies that influence glycan-lectin interactions more indirectly can be employed. in fact, many of the molecules used to examine glycan-lectin interactions in vitro suggest themselves as potential therapeutics. one approach to indirectly govern glycan-lectin interaction is via the use of drugs that alter the host and/or viral glycome. glycosidases and other enzymes may be used to alter the glycan portions of fully formed and matured glycoconjugates. alternatively, various drugs may be employed to directly modify glycan synthesis: glycoconjugates produced in the presence of such molecules will obtain aberrant glycosylation, which may promote or annihilate their interaction with specific lectins. promising results in glycovirological research have highlighted the antiviral potential of such compounds. for instance, ample data indicate that sialidases may be used to counteract infections where sialic acids play important roles as cellular receptors for viral lectins [e.g. iav binds to sialic acid receptors on the airway epithelium (skehel & wiley, ; malakhov et al., ; belser et al., ; harrison, ; chan et al., ; triana-baltzer et al., a , or as viral ligands for host lectins that serve as portals for viral entry [e.g. sialic acids on the porcine reproductive and respiratory syndrome virus bind the macrophage-specific entry mediator sialoadhesin (delputte & nauwynck, ; delputte et al., ; van breedam et al., a, b) ]. sialidase treatment may also enhance recognition of viral glycoconjugates by mannose-specific immune system lectins that can limit viral infection: in vitro experiments have shown that enzymatic removal of sialic acids from the hiv- virion surface can significantly enhance virus binding and neutralization by mbl (hart et al., (hart et al., , . in line with this, production of hiv- in the presence of the golgi a - -mannosidase i inhibitor -deoxymannojirimycinwhich blocks the biosynthesis of complex-type, sialylated oligosaccharidesincreased susceptibility of the virus to mbl-mediated neutralization (hart et al., ) . interestingly, -deoxymannojirimycin treatment of hiv- -infected cells was also shown to potentiate the antiviral effects of mannose-specific plant cbas towards the newly produced hiv- virions (balzarini, a) . these and other examples illustrate the potential of these molecules as antiviral drugs. considering the various structural and nonstructural roles of glycans, it is clear that glycome modulation can also have effects beyond the alteration of glycan-lectin binding events. for instance, interfering with host cell glycosylation processes using specific inhibitors may inhibit assembly of infectious virions (leavitt et al., ; katz et al., ; pizer et al., ; herrler & compans, ; montefiori et al., ; pal et al., ; mehta et al., ; dwek et al., ; wu et al., ; durantel et al., ; lazar et al., ; scanlan et al., ; durantel, ; merry & astrautsova, ) . moreover, glycome modulation may significantly alter the capacity of the virus to evade recognition by virus-specific antibodies and b-and t-cell receptors via glycan shielding (botarelli et al., ; back et al., ; willey et al., ; reitter et al., ; bolmstedt et al., ; kang et al., ; aguilar et al., ; wang et al., ; francica et al., ; kobayashi & suzuki, ) . clearly, glycome-modifying drugs can counteract viruses in various ways and constitute versatile tools in the control of viral infection. also other strategies that can indirectly influence glycan-lectin interactions are certainly worth exploring. for instance, drugs that alter host or viral lectin expression (e.g. cytokines or rnai) may prove useful in antiviral strategies (ochiel et al., ; relloso et al., ; ge et al., ; arrighi et al., ; ge et al., ; nair et al., ; yagi et al., ; raza et al., ) . furthermore, patients infected with a virus that employs both viral lectins and rdes may also benefit from treatment with rde-specific inhibitors, as these can alter the balance between viral lectin and rde activity which is often crucial for efficient viral replication and spread. notable examples in this context are the several neuraminidase inhibitors that have been used successfully for the treatment of iav infections (kim et al., ; lew et al., ; roberts, ; garman & laver, ; alymova et al., ; von itzstein, ; von itzstein & thomson, ; gamblin & skehel, ; ikematsu & kawai, ) . in sum, drugs that modulate glycan-lectin interactionseither directly or indirectlycan be powerful instruments in the combat against viral pathogens. however, the antiviral strategies suggested above can also have drawbacks. intensive use of antiviral therapeutics may elicit rapid selection of drug-resistant virus variants. another possible drawback relates to the potential off-target effects of these therapies: therapeutics aimed at influencing specific glycan-lectin interactions that play key roles in viral infection processes may also affect general host glycosylation, lectin expression or glycan-lectin interactions that are crucial for normal functioning of the host and its immune defense. it is also conceivable that such drugs, despite their antiviral effects, may benefit the virus in some ways. for instance, although a glycome-modifying drug may promote viral recognition by specific immune system lectins that aid in viral clearance, it may also promote viral interaction with host lectins that can aid in cis-or transinfection. ultimately, the potential of specific agents as antiviral drugs depends on their net (antiviral) effects in vivo. other potential disadvantages concern the pharmacokinetic properties of specific drugs. for instance, if glycan decoys or cbas used in antiviral therapy show a broad reactivity and can respectively bind with multiple (nontarget) lectins or glycans in the host, it is possible that much of the antiviral effect is lost. also, when using peptidic cbas that are not native to the host, an antibody response might be mounted against these components, leading to neutralization and/or faster clearance of the active compound. in spite of these and other potential pitfalls, it is clear that glycan decoys and cbas, as well as various indirect approaches to modulate glycan-lectin interaction, show potential for the treatment of diverse viral infections, either or not in combination with other antiviral strategies. a good understanding of relevant glycan-lectin interactions facilitates specific targeting of these binding events and can help to minimize possible off-target effects and to reduce the risk of drug resistance. glycans and lectins cover crucial roles in virus biology and their interplay often shapes the virus-host interaction. in general, the nature of the glycan, the lectin, and the specific conditions under which their interaction occurs determines the outcome of a specific binding event and directs the virus to a certain fate. based on current knowledge, it is clear that viral lectins generally facilitate viral infection and spread. on the other hand, although it may seem intuitive that host lectin prrs and other immune system lectins exclusively act in defense of the host, there is ample evidence that contradicts this. although many host lectins are involved in the induction of an efficacious immune response against viral pathogens, many viruses can also abuse these lectins to promote infection and spread. intriguingly, analysis of the many studies regarding the role of host lectins in viral infections suggests that soluble host lectins tend to be associated with antiviral activity, whereas membrane-associated host lectins seem to play a more dubious role and are often implicated in pro-as well as antiviral mechanisms (tables & ) . it is however noteworthy that the information provided in this manuscript reflects current views on glycan-lectin interactions in virus biology, and that future research may alter our understanding and interpretation of specific interactions. the fact that (aspects of) viral glycobiology may change during virus-host co-evolution even advocates periodic re-evaluation of specific glycan-lectin interactions. the biology of glycans and lectins is complex and has long been poorly accessible to virologists and other scientists outside this field. this situation is changing with the emergence of international glycomics consortia (e.g. consortium for functional glycomics), which can provide state-of-the-art techniques and expertise to analyze and interpret virologically/immunologically relevant glycanlectin interactions. still, there are specific pitfalls associated with glycovirological research. in vitro experiments need to be designed and interpreted considering key issues like glycan and lectin variability, the cell-type dependency of host and virus glycosylation and the influence of the lectin-expressing cell type on the final outcome of a glycan-lectin interaction. in addition, the results of in vitro experiments must ultimately be compared withand re-interpreted in the context ofdata obtained in animal models. in fact, our current understanding of specific glycan-lectin interactions in viral infection is mostly based on in vitro experiments, underlining the need for experimental validation of these results in the context of the infected host. in the context of viral infections, many different (lectin-dependent or -independent) interactions and processes take place simultaneously, resulting in a complex network of virus-host factor interaction, signaling, and effector mechanisms, the net effect of which may benefit the host or the virus. many of the interactions taking place are not yet well defined and probably more are still unknown. key to combating viral disease is to make these black boxes more transparent. synergisms between different branches of life sciences are essential to sustain and advance our knowledge in this important field of research. n-glycans on nipah virus fusion protein protect against neutralization but reduce membrane fusion and viral entry the lectins griffithsin, cyanovirin-n and scytovirin inhibit hiv- binding to the dc-sign receptor and transfer to cd (+) cells neuraminidase inhibitors as antiviral agents complement-dependent neutralization of influenza virus by a serum mannose-binding lectin lentivirus-mediated rna interference of dc-sign expression inhibits human immunodeficiency virus transmission from dendritic cells to t cells identification of the platelet-derived chemokine cxcl /pf- as a broad-spectrum hiv- inhibitor an n-glycan within the human immunodeficiency virus type gp v loop affects virus neutralization the alpha( , )-mannosidase i inhibitor -deoxymannojirimycin potentiates the antiviral activity of carbohydrate-binding agents against wild-type and mutant hiv- strains containing glycan deletions in gp carbohydrate-binding agents: a potential future cornerstone for the chemotherapy of enveloped viruses? targeting the glycans of glycoproteins: a novel paradigm for antiviral therapy profile of resistance of human immunodeficiency virus to mannose-specific plant lectins carbohydrate-binding agents cause deletions of highly conserved glycosylation sites in hiv gp : a new therapeutic concept to hit the achilles heel of hiv marked depletion of glycosylation sites in hiv- gp under selection pressure by the mannose-specific plant lectins of hippeastrum hybrid and galanthus nivalis mutational pathways, resistance profile, and side effects of cyanovirin relative to human immunodeficiency virus type strains with n-glycan deletions in their gp envelopes carbohydrate-binding agents efficiently prevent dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin (dc-sign)-directed hiv- transmission to t lymphocytes pradimicin a, a carbohydrate-binding nonpeptidic lead compound for treatment of infections with viruses with highly glycosylated envelopes, such as human immunodeficiency virus pradimicin s, a highly soluble nonpeptidic small-size carbohydrate-binding antibiotic, is an anti-hiv drug lead for both microbicidal and systemic use the role of dc-sign and dc-signr in hiv and siv attachment, infection, and transmission quantitative expression and virus transmission analysis of dc-sign on monocyte-derived dendritic cells a novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of mdck cells in culture dendritic cell maturation results in pronounced changes in glycan expression affecting recognition by siglecs and galectins high-affinity glycopolymer binding to human dc-sign and disruption of dc-sign interactions with hiv envelope glycoprotein das , a novel sialidase fusion protein, protects mice from lethal avian influenza h n virus infection pacing a small cage: mutation and rna viruses interactions of surfactant protein a with influenza a viruses: binding and neutralization surfactant protein a, but not surfactant protein d, is an opsonin for influenza a virus phagocytosis by rat alveolar macrophages entry of hepatitis c virus and human immunodeficiency virus is selectively inhibited by carbohydrate-binding agents but not by polyanions a glycomimetic compound inhibits dc-sign-mediated hiv infection in cellular and cervical explant models the relevance of complement to virus biology dendritic cell-mediated trans-enhancement of human immunodeficiency virus type infectivity is independent of dc-sign naturally-occurring genetic variants in human dc-sign increase hiv- capture, cell-transfer and risk of mother-to-child transmission enhanced immunogenicity of a human immunodeficiency virus type env dna vaccine by manipulating n-glycosylation signals. effects of elimination of the v n glycan the glycan shield of hiv is predominantly oligomannose independently of production system or viral clade n-glycosylation of hiv-gp may constrain recognition by t lymphocytes an integrated view of humoral innate immunity: pentraxins as a paradigm lentivirus degradation and dc-sign expression by human platelets and megakaryocytes shared paramyxoviral glycoprotein architecture is adapted for diverse attachment strategies lectin-dependent enhancement of ebola virus infection via soluble and transmembrane c-type lectin receptors structural basis for the receptor binding specificity of norwalk virus infection of dendritic cells (dcs), not dc-sign-mediated internalization of human immunodeficiency virus, is required for long-term transfer of virus to t cells dual function of c-type lectin-like receptors in the immune system how c-type lectins detect pathogens structural basis for the recognition of blood group trisaccharides by norovirus in vitro derived dendritic cells trans-infect cd t cells primarily with surface-bound hiv- virions cytomegalovirus induces sialyl lewis (x) and lewis(x) on human endothelial cells dc-sign and clec- mediate human immunodeficiency virus type capture by platelets das inhibits h n influenza virus infection of human lung tissues paramyxovirus fusion and entry: multiple paths to a common end lack of the pattern recognition molecule mannose-binding lectin increases susceptibility to influenza a virus infection dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate the essentiality of alpha- -macroglobulin in human salivary innate immunity against new h n swine origin influenza a virus atomic resolution structural characterization of recognition of histo-blood group antigens by norwalk virus influenza virus neuraminidase: structure, antibodies, and inhibitors activation of murine cd + and cd + t lymphocytes leads to dramatic remodeling of n-linked glycans siglecs as positive and negative regulators of the immune system siglecs and their roles in the immune system envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens specificity and affinity of sialic acid binding by the rhesus rotavirus vp * core celgosivir, an alpha-glucosidase i inhibitor for the potential treatment of hcv infection glucosidase inhibitors as antiviral agents for hepatitis b and c targeting glycosylation as a therapeutic approach lack of complex n-glycans on hiv- envelope glycoproteins preserves protein conformation and entry function impact of mannose-binding lectin on susceptibility to infectious diseases lectin-carbohydrate interactions: different folds, common recognition principles noroviruses everywhere: has something changed? genetic diversity and histo-blood group antigen interactions of rhesus enteric caliciviruses structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr steric shielding of surface epitopes and impaired immune recognition induced by the ebola virus glycoprotein potential of carbohydrate-binding agents as therapeutics against enveloped viruses evolution of the lectin-complement pathway and its role in innate immunity from lectin structure to functional glycomics: principles of the sugar code surfactant protein a binds to hiv and inhibits direct infection of cd + cells, but enhances dendritic cell-mediated viral transfer influenza hemagglutinin and neuraminidase membrane glycoproteins endogenous ligands for c-type lectin receptors: the true regulators of immune homeostasis controlling influenza by inhibiting the virus's neuraminidase endothelial galectin- binds to specific glycans on nipah virus fusion protein and inhibits maturation, mobility, and function to block syncytia formation rna interference of influenza virus production by directly targeting mrna for degradation and indirectly inhibiting all viral rna transcription use of sirnas to prevent and treat influenza virus infection signalling through c-type lectin receptors: shaping immune responses identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells location, location, location: new insights into o-galnac protein glycosylation two evolutionary strategies of influenza viruses to escape host non-specific inhibitors: alteration of hemagglutinin or neuraminidase specificity pattern recognition receptors: doubling up for the innate immune response neuraminidase: the specific enzyme of influenza virus and vibrio cholerae dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin/cd is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction c-type lectin dc-sign modulates toll-like receptor signaling via kinase-dependent acetylation of transcription factor nf-kappab hiv- exploits innate signaling by tlr and dc-sign for productive infection of dendritic cells association between expression of the h histo-blood group antigen, alpha , fucosyltransferases polymorphism of wild rabbits, and sensitivity to rabbit hemorrhagic disease virus binding and transfer of human immunodeficiency virus by dc-sign+ cells in human rectal mucosa viral membrane fusion paramyxovirus assembly and budding: building particles that transmit infections high mannose glycans and sialic acid on gp regulate binding of mannose-binding lectin (mbl) to hiv type glycosylation inhibitors and neuraminidase enhance human immunodeficiency virus type binding and neutralization by mannose-binding lectin human mannose-binding protein functions as an opsonin for influenza a viruses evidence for a protective role of pulmonary surfactant protein d (sp-d) against influenza a viruses neutrophil deactivation by influenza a viruses: mechanisms of protection after viral opsonization with collectins and hemagglutination-inhibiting antibodies mechanisms of anti-influenza activity of surfactant proteins a and d: comparison with serum collectins mechanism of binding of surfactant protein d to influenza a viruses: importance of binding to haemagglutinin to antiviral activity characterizing the glycome of the mammalian immune system pulmonary collectins modulate strain-specific influenza a virus infection and host responses mannan-binding lectin deficiency -good news, bad news, doesn't matter? posttranslational modification and intracellular transport of mumps virus glycoproteins structure and function of the hef glycoprotein of influenza c virus neuraminic acid is involved in the binding of influenza c virus to erythrocytes the receptor-destroying enzyme of influenza c virus is neuraminate-o-acetylesterase the glycoprotein of influenza c virus is the haemagglutinin, esterase and fusion factor -o-acetylated sialic acid, a receptor determinant for influenza c virus and coronaviruses lung surfactant protein a provides a route of entry for respiratory syncytial virus into host cells dc-sign increases the affinity of hiv- envelope glycoprotein interaction with cd assessment of the antiviral properties of recombinant porcine sp-d against various influenza a viruses in vitro human t-cell leukemia virus- encoded tax protein transactivates alpha -> fucosyltransferase fuc-t vii, which synthesizes sialyl lewis x, a selectin ligand expressed on adult t-cell leukemia cells transactivation of the fucosyltransferase vii gene by human t-cell leukemia virus type tax through a variant camp-responsive element activation of the lectin dc-sign induces an immature dendritic cell phenotype triggering rho-gtpase activity required for hiv- replication the evolution and emergence of rna viruses human immunodeficiency virus envelope (gp ) binding to dc-sign and primary dendritic cells is carbohydrate dependent but does not involve g or cyanovirin binding sites: implications for structural analyses of gp -dc-sign binding identification of the optimal dc-sign binding site on human immunodeficiency virus type gp differences in the mannose oligomer specificities of the closely related lectins from galanthus nivalis and zea mays strongly determine their eventual anti-hiv activity norovirus and histo-blood group antigens: demonstration of a wide spectrum of strain specificities and classification of two major binding groups among multiple binding patterns passage of classical swine fever virus in cultured swine kidney cells selects virus variants that bind to heparan sulfate due to a single amino acid change in envelope protein e(rns) resistance of hiv- to the broadly hiv- -neutralizing, anti-carbohydrate antibody g microvirin, a novel alpha ( , )-mannose-specific lectin isolated from microcystis aeruginosa, has anti-hiv- activity comparable with that of cyanovirin-n but a much higher safety profile laninamivir octanoate: a new long-acting neuraminidase inhibitor for the treatment of influenza siglec- is a novel dendritic cell receptor that mediates hiv- trans-infection through recognition of viral membrane gangliosides myxoma virus encodes an alpha , -sialyltransferase that enhances virulence innate immune recognition mucin-type o-glycosylation-putting the pieces together addition of glycosylation to influenza a virus hemagglutinin modulates antibody-mediated recognition of h n pandemic viruses modified hiv envelope proteins with enhanced binding to neutralizing monoclonal antibodies human mannan-binding lectin inhibits the infection of influenza a virus without complement antiviral activity of tunicamycin on herpes simplex virus n-linked glycosylation in the hemagglutinin of influenza a viruses neuraminidase inhibitors as anti-influenza virus agents correction of pulmonary abnormalities in sftpdÀ/À mice requires the collagenous domain of surfactant protein d adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor evidence for n-glycan shielding of antigenic sites during evolution of human influenza a virus hemagglutinin rantes - g delays and dc-sign - c enhances aids progression in hiv type -infected japanese hemophiliacs point mutations in the s protein connect the sialic acid binding activity with the enteropathogenicity of transmissible gastroenteritis coronavirus characterization of the sialic acid binding activity of transmissible gastroenteritis coronavirus by analysis of haemagglutination-deficient mutants heparin-dependent attachment of respiratory syncytial virus (rsv) to host cells identification and functions of pattern-recognition receptors structural and functional analysis of the surface protein of human coronavirus oc dc-sign-mediated internalization of hiv is required for trans-enhancement of t cell infection orthomyxoviridae: the viruses and their replication prevention of influenza pneumonitis by sialic acid-conjugated dendritic polymers altered t cell surface glycosylation in hiv- infection results in increased susceptibility to galectin- -induced cell death the expanding horizons of asparagine-linked glycosylation quasispecies theory and the behavior of rna viruses treatment of hepatitis b virus-infected cells with alpha-glucosidase inhibitors results in production of virions with altered molecular composition and infectivity mendelian resistance to human norovirus infections tunicamycin inhibits glycosylation and multiplication of sindbis and vesicular stomatitis viruses modes of paramyxovirus fusion: a henipavirus perspective cis expression of dc-sign allows for more efficient entry of human and simian immunodeficiency viruses via cd and a coreceptor viral piracy: hiv- targets dendritic cells for transmission surfactant protein d enhances clearance of influenza a virus from the lung in vivo absence of sp-a modulates innate and adaptive defense responses to pulmonary influenza infection surfactant protein-d enhances phagocytosis and pulmonary clearance of respiratory syncytial virus novel innate immune functions for galectin- : galectin- inhibits cell fusion by nipah virus envelope glycoproteins and augments dendritic cell secretion of proinflammatory cytokines discovery and development of gs (oseltamivir): an orally active influenza neuraminidase inhibitor surfactant protein-a-deficient mice display an exaggerated early inflammatory response to a beta-resistant strain of influenza a virus identification of the dc-sign-interactive domains on the envelope glycoprotein of hiv- crf _bc oligosaccharide profiles of hiv- external envelope glycoprotein: dependence on host cells and virus isolates host-cell-specific glycosylation of hiv- envelope glycoprotein differential n-linked glycosylation of human immunodeficiency virus and ebola virus envelope glycoproteins modulates interactions with dc-sign and dc-signr analysis of genetic polymorphisms in ccr , ccr , stromal cell-derived factor- , rantes, and dendritic cell-specific intercellular adhesion molecule- -grabbing nonintegrin in seronegative individuals repeatedly exposed to hiv- principles of structures of animal and plant lectins a yeast glycoprotein shows high-affinity binding to the broadly neutralizing human immunodeficiency virus antibody g and inhibits gp interactions with g and dc-sign surfactant protein d modulates hiv infection of both t-cells and dendritic cells sialidase fusion protein as a novel broad-spectrum inhibitor of influenza virus infection binding of human collectins (sp-a and mbp) to influenza virus adaptation of tick-borne encephalitis virus to bhk- cells results in the formation of multiple heparan sulfate binding sites in the envelope protein and attenuation in vivo the neck region of the c-type lectin dc-sign regulates its surface spatiotemporal organization and virus-binding capacity on antigen-presenting cells the core beta- , -n-acetylglucosaminyltransferase-mucin encoded by bovine herpesvirus was acquired from an ancestor of the african buffalo glycosyltransferases encoded by viruses the core beta- , -n-acetylglucosaminyltransferase-m encoded by bovine herpesvirus is not essential for virus replication despite contributing to post-translational modifications of structural proteins association of dc-sign promoter polymorphism with increased risk for parenteral, but not mucosal, acquisition of human immunodeficiency virus type infection multivalent manno-glyconanoparticles inhibit dc-sign-mediated hiv- trans-infection of human t cells gold manno-glyconanoparticles: multivalent systems to block hiv- gp binding to the lectin dc-sign natural and synthetic sialic acid-containing inhibitors of influenza virus receptor binding influenza viruses differ in recognition of -o-acetyl substitution of sialic acid receptor determinant molecular mechanisms of serum resistance of human influenza h n virus and their involvement in virus adaptation in a new host sialic acid-mimic peptides as hemagglutinin inhibitors for anti-influenza therapy recruitment of hiv and its receptors to dendritic cell-t cell junctions divergent roles for c-type lectins expressed by cells of the innate immune system ligand recognition by antigen-presenting cell c-type lectin receptors alpha-glucosidase inhibitors as potential broad based anti-viral agents galectin- promotes hiv- infectivity in macrophages through stabilization of viral adsorption alternative approaches to antiviral treatments: focusing on glycosylation as a target for antiviral therapy inhibition of hemagglutination activity of influenza a viruses by sp-a and sp-a variants expressed in cho cells dc-specific icam- -grabbing nonintegrin mediates internalization of hiv- into human podocytes role of protein n-glycosylation in pathogenesis of human immunodeficiency virus type dc-sign promotes exogenous mhc-i-restricted hiv- antigen presentation dendritic cells and hiv-specific cd + t cells: hiv antigen presentation, t-cell activation, and viral transfer extensive repertoire of membrane-bound and soluble dendritic cell-specific icam- -grabbing nonintegrin (dc-sign ) and dc-sign isoforms. inter-individual variation in expression of dc-sign transcripts rnai-directed inhibition of dc-sign by dendritic cells: prospects for hiv- therapy influenza c virus hemagglutinin: comparison with influenza a and b virus hemagglutinins post-translational modification of the myxoma-virus anti-inflammatory serpin serp- by a virally encoded sialyltransferase dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses hiv- activates cdc and induces membrane extensions in immature dendritic cells to facilitate cell-to-cell virus propagation covert human immunodeficiency virus replication in dendritic cells and in dc-sign-expressing cells promotes long-term transmission to lymphocytes histo-blood group antigens act as attachment factors of rabbit hemorrhagic disease virus infection in a virus strain-dependent manner uterine epithelial cell regulation of dc-sign expression inhibits transmitted/founder hiv- trans infection by immature dendritic cells regulation of receptor binding affinity of influenza virus hemagglutinin by its carbohydrate moiety c ( ) myeloid c-type lectin receptors in pathogen recognition and host defense galectin- acts as a soluble host factor that promotes hiv- infectivity through stabilization of virus attachment to host cells role of oligosaccharides in the processing and maturation of envelope glycoproteins of human immunodeficiency virus type gp : target for neutralizing hiv- antibodies inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold nanoparticles inhibition of influenza virus activity by multivalent glycoarchitectures with matched sizes cell-surface heparan sulfate proteoglycan mediates hiv- infection of t-cell lines the glycosylphosphatidylinositol anchor: a complex membrane-anchoring structure for proteins cell surface expression of biologically active influenza c virus hef glycoprotein expressed from cdna methods in enzymology: o-glycosylation of proteins the interaction of hiv with dendritic cells: outcomes and pathways effect of tunicamycin on herpes simplex virus glycoproteins and infectious virus production dc-sign interactions with human immunodeficiency virus type and and simian immunodeficiency virus basis for the potent inhibition of influenza virus infection by equine and guinea pig alpha -macroglobulin synthesis and sorting of proteoglycans nmr experiments reveal the molecular basis of receptor recognition by a calicivirus dc-sign on b lymphocytes is required for transmission of hiv- to t lymphocytes selection of predicted sirna as potential antiviral therapeutic agent against influenza virus a serum mannose-binding lectin mediates complement-dependent lysis of influenza virus-infected cells collectin-mediated antiviral host defense of the lung: evidence from influenza virus infection of mice glycosylation as a target for recognition of influenza viruses by the innate immune system loss of a single n-linked glycan from the hemagglutinin of influenza virus is associated with resistance to collectins and increased virulence in mice a role for carbohydrates in immune evasion in aids dc-sign (cd ) expression is il- dependent and is negatively regulated by ifn, tgf-beta, and anti-inflammatory agents anti-influenza drugs and neuraminidase inhibitors differential sensitivity of human, avian, and equine influenza a viruses to a glycoprotein inhibitor of infection: selection of receptor specific variants influenza c virus uses -o-acetyl-n-acetylneuraminic acid as a high affinity receptor determinant for attachment to cells structure of the haemagglutinin-esterase-fusion glycoprotein of influenza c virus protein glycosylation in the endoplasmic reticulum and the golgi apparatus and cell type-specificity of cell surface glycoconjugate expression: analysis by the protein a-gold and lectin-gold techniques binding of rabbit hemorrhagic disease virus to antigens of the abh histo-blood group family distinct glycoprotein inhibitors of influenza a virus in different animal sera alpha -macroglobulin is the major neutralizing inhibitor of influenza a virus in pig serum tissue culture adaptation of foot-and-mouth disease virus selects viruses that bind to heparin and are attenuated in cattle a variant in the cd promoter is associated with severity of dengue disease signaling by myeloid c-type lectin receptors in immunity and homeostasis lactoferrin and surfactant protein a exhibit distinct binding specificity to f protein and differently modulate respiratory syncytial virus infection galectins in innate immunity: dual functions of host soluble beta-galactoside-binding lectins as damage-associated molecular patterns (damps) and as receptors for pathogen-associated molecular patterns (pamps) glycans, galectins, and hiv- infection inhibition of dc-sign-mediated hiv infection by a linear trimannoside mimic in a tetravalent presentation exploiting the defensive sugars of hiv- for drug and vaccine design isolation and characterization of sialate ( )-o-acetylesterase from influenza c virus the s protein of bovine coronavirus is a hemagglutinin recognizing -o-acetylated sialic acid as a receptor determinant transmissible gastroenteritis coronavirus, but not the related porcine respiratory coronavirus, has a sialic acid (n-glycolylneuraminic acid) binding activity mechanisms and principles of n-linked protein glycosylation the sialic acid binding activity of the s protein facilitates infection by porcine transmissible gastroenteritis coronavirus cd gene polymorphisms in south indian hiv and hiv-tb patients structural requirements for multimerization of the pathogen receptor dendritic cell-specific icam -grabbing non-integrin (cd ) on the cell surface dendritic cells and transmission of hiv- norovirus and histo-blood group antigens receptor binding and membrane fusion in virus entry: the influenza hemagglutinin constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro host-soluble galectin- promotes hiv- replication through a direct interaction with glycans of viral gp and host cd alpha , -linked sialic acid acts as a receptor for feline calicivirus dc-sign binds to hiv- glycoprotein in a distinct but overlapping fashion compared with icam- and icam- a novel viral alpha , -sialyltransferase (v-st gal i): transfer of sialic acid to fucosylated acceptors membrane-associated heparan sulfate proteoglycan is a receptor for adeno-associated virus type virions n-linked glycosylation of the hemagglutinin protein influences virulence and antigenicity of the pandemic and seasonal h n influenza a viruses sialobiology of influenza: molecular mechanism of host range variation of influenza viruses origin and evolution of influenza virus hemagglutinin genes sialoglycoproteins that bind influenza a virus and resist viral neuraminidase in different animal sera c-type lectin dc-sign: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity norovirus-host interaction: implications for disease control and prevention specific sites of n-linked glycosylation on the hemagglutinin of h n subtype influenza a virus determine sensitivity to inhibitors of the innate immune system and virulence in mice glycosylation of the hemagglutinin modulates the sensitivity of h n influenza viruses to innate proteins in airway secretions and virulence in mice ganglioside-linked terminal sialic acid moieties on murine macrophages function as attachment receptors for murine noroviruses glycosphingolipids as receptors for non-enveloped viruses murine noroviruses bind glycolipid and glycoprotein attachment receptors in a strain-dependent manner introduction to glycobiology belshe rb & fang f ( a) inhibition of neuraminidase inhibitor-resistant influenza virus by das , a novel sialidase fusion protein novel pandemic influenza a(h n ) viruses are potently inhibited by das , a sialidase fusion protein das , a sialidase fusion protein, protects human airway epithelium against influenza virus infection: an in vitro pharmacodynamic analysis phenotypic and genotypic characterization of influenza virus mutants selected with the sialidase fusion protein das glycosaminoglycan-binding ability is a feature of wild-type strains of herpes simplex virus type the multiple facets of hiv attachment to dendritic cell lectins platelet activation suppresses hiv- infection of t cells effect of addition of new oligosaccharide chains to the globular head of influenza a/ h n virus haemagglutinin on the intracellular transport and biological activities of the molecule diversity of receptors binding hiv on dendritic cell subsets immunodeficiency virus uptake, turnover, and -phase transfer in human dendritic cells the m/gp( ) glycoprotein complex of porcine reproductive and respiratory syndrome virus binds the sialoadhesin receptor in a sialic acid-dependent manner concepts and principles of o-linked glycosylation langerin functions as an antiviral receptor on langerhans cells innate signaling in hiv- infection of dendritic cells porcine surfactant protein d is n-glycosylated in its carbohydrate recognition domain and is assembled into differently charged oligomers porcine pulmonary collectins show distinct interactions with influenza a viruses: role of the n-linked oligosaccharides in the carbohydrate recognition domain interactions of influenza a virus with sialic acids present on porcine surfactant protein d specificity of dc-sign for mannose-and fucose-containing glycans effects of heparin on the entry of porcine reproductive and respiratory syndrome virus into alveolar macrophages a multipotential beta- , -n-acetylglucosaminyltransferase is encoded by bovine herpesvirus type the carbohydrate recognition domain of collectins n-linked glycosylation attenuates h n influenza viruses the influenza c virus glycoprotein (he) exhibits receptor-binding (hemagglutinin) and receptor-destroying (esterase) activities the war against influenza: discovery and development of sialidase inhibitors anti-influenza drugs: the development of sialidase inhibitors interdependence of hemagglutinin glycosylation and neuraminidase as regulators of influenza virus growth: a study by reverse genetics functional balance between haemagglutinin and neuraminidase in influenza virus infections functionally distinct transmission of human immunodeficiency virus type mediated by immature and mature dendritic cells structural basis for receptor specificity of influenza b virus hemagglutinin crystal structure of unliganded influenza b virus hemagglutinin targeting the carbohydrates on hiv- : interaction of oligomannose dendrons with human monoclonal antibody g and dc-sign glycans on influenza hemagglutinin affect receptor binding and immune response a/g polymorphism is associated with dengue hemorrhagic fever and correlated to dc-sign expression and immune augmentation glycan shielding of the influenza virus hemagglutinin contributes to immunopathology in mice asparagine-linked protein glycosylation: from eukaryotic to prokaryotic systems antibody neutralization and escape by hiv- influenza viruses: role of glycans in viral evolution and vaccine design cell-surface carbohydrate recognition by animal and viral lectins molecular architectures of trimeric siv and hiv- envelope glycoproteins on intact viruses: strain-dependent variation in quaternary structure the complete genome sequence of shope (rabbit) fibroma virus differential glycosylation, virion incorporation, and sensitivity to neutralizing antibodies of human immunodeficiency virus type envelope produced from infected primary t-lymphocyte and macrophage cultures resistance of human immunodeficiency virus type to the high-mannose binding agents cyanovirin n and concanavalin a dendritic-cell interactions with hiv: infection and viral dissemination antiviral effects of an iminosugar derivative on flavivirus infections inhibition of dc-sign-mediated transmission of human immunodeficiency virus type by toll-like receptor signalling in breast milk macrophages hiv traffics through a specialized, surface-accessible intracellular compartment during trans-infection of t cells by mature dendritic cells structures, biosynthesis, and functions of gangliosides-an overview complementation of pulmonary abnormalities in sp-d(À/À) mice with an sp-d/conglutinin fusion protein mass spectrometric characterization of the glycosylation pattern of hiv-gp expressed in cho cells the authors thank leslie bosseler for critical reading of the manuscript and assistance in creating the figures. the authors apologize to all colleagues whose work has not been cited due to space limitations. key: cord- -ybncwweg authors: snyder, greg a.; colonna, marco; sun, peter d. title: the structure of dc-signr with a portion of its repeat domain lends insights to modeling of the receptor tetramer date: - - journal: journal of molecular biology doi: . /j.jmb. . . sha: doc_id: cord_uid: ybncwweg the dendritic cell-specific icam- non-integrin (dc-sign) and its close relative dc-signr recognize various glycoproteins, both pathogenic and cellular, through the receptor lectin domain-mediated carbohydrate recognition. while the carbohydrate-recognition domains (crd) exist as monomers and bind individual carbohydrates with low affinity and are permissive in nature, the full-length receptors form tetramers through their repeat domain and recognize specific ligands with high affinity. to understand the tetramer-based ligand binding avidity, we determined the crystal structure of dc-signr with its last repeat region. compared to the carbohydrate-bound crd structure, the structure revealed conformational changes in the calcium and carbohydrate coordination loops of crd, an additional disulfide bond between the n and the c termini of the crd, and a helical conformation for the last repeat. on the basis of the current crystal structure and other published structures with sequence homology to the repeat domain, we generated a tetramer model for dc-sign/r using homology modeling and propose a ligand-recognition index to identify potential receptor ligands. the structure of dc-signr with a portion of its repeat domain lends insights to modeling of the receptor tetramer greg a. snyder , , marco colonna and peter d. sun * introduction the dendritic cell-specific icam- non-integrin (dc-sign) and its close relative dc-signr are members of the c-type lectin family. originally discovered as a human immunodeficiency virus (hiv)-binding protein, dc-sign has been shown to bind carbohydrates on various pathogens, including ebola, mycobacterium tuberculosis, hepatitis c virus and cytomegalovirus. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] in the case of hiv- infection, dc-sign and dc-signr (together referred to as dc-sign/r) have been proposed to facilitate the viral infection of t-cells in trans through binding with hiv gp . , however, recent evidence suggests that dc-sign/r function as antigen capturing receptors to facilitate the presentation of hiv- antigen by dendritic cells. dc-sign/r each consist of four domains: a cytoplasmic domain with a di-leucine motif for internalization, a single-spanning transmembrane region, a region with seven and one-half amino acid residue repeats, and a carbohydrate-recognition domain (crd). dc-signr is % identical with dc-sign in amino acid sequence, and differs mainly in tissue and cellular expression patterns although recent reports indicate that it may differ in binding and processing of pathogens. [ ] [ ] [ ] it has been established that dc-sign/r recognize specifically high-mannose carbohydrates. previous structural studies have shown the molecular details of this interaction. more recently, however, dc-sign/r were shown to recognize terminal fucose and galactose-containing carbohydrates, such as blood group antigens b lewis a , and lewis x structures in addition to mannose. , while recognition between various carbohydrate substructures gives insight into how various carbohydrate model compounds are recognized by the crd of the receptor, the overall receptor binding affinity appears to depend on the e-mail address of the corresponding author: psun@nih.gov multivalent nature of the ligand. for example, while dc-sign/r bind to model carbohydrate with millimolar affinity, the receptors recognize hiv gp , which carries multiple high-mannosebased, n-linked glycosylations with nanomolar affinity. this carbohydrate valency-dependent avidity effect was shown to be the result of dc-sign/r tetramerization through its repeat region. , to understand the nature of the receptor-carbohydrate interaction, we have determined the crystal structure of dc-signr with a portion of the repeat domain. we propose a tetramer model for the intact extracellular receptor, and formulate a scheme to predict the potential ligands. the crystals of dc-signr crd with its last repeat belong to the orthorhombic space group p . the protein solution was supplemented with mm mannose and mm cacl prior to crystallization. crystals were obtained under several conditions that include polyethylene glycol with various molecular masses ( - da) and buffers with a ph between . and . . crystals contained one molecule per asymmetric unit with a solvent content of . % (v/v) (matthew's coefficient of . ). molecular replacement rotation and translation correlation coefficients were ranked and yielded a single solution well above the background. the initial phased map had clear electron density for both the main chains and the side-chains. after rebuilding of loops, the electron density was continuous throughout the structure and the final structure consists of residues - , with met modeled in two alternative conformations. the final refined crystallographic r-factors are . % and . % for r work and r free , respectively, at . Å resolution ( table ). the overall structure is a typical long-form c-type lectin, and the crd portion superimposes with a root-meansquare (r.m.s) deviation of . Å (for c a atoms) to the crd-only structure (figure (b) ). although both mannose and calcium were present in the crystallization solution, only calcium was observed bound in the canonical calcium binding site. this structure contains additional residues at both the amino and carboxyl termini including a disulfide bond linking the two termini as well as a short a-helix at the beginning of the repeat domain. additional amino acids were present in both the n and c termini compared with the crd-only structure. at the c-terminus we observed additional density for amino acids - , including a disulfide bond between cys and cys , which links both the n-and c-termini into close proximity ( figure ). as a result, the ring of the n-terminal histidine residue (his ) stacks against the ring of the c-terminal phe . the helical repeat domain has been shown to be responsible for tetramerization of the receptor. our dc-signr r construct contains the last repeat that immediately precedes the crd domain. this repeat region encompasses residues gln -cys . a portion of this repeat, ala -cys , is ordered in the structure and forms a short a-helix. although the rest of the r repeat appears disordered in our crystal, presumably due to the proximity to the n terminus of the expressed recombinant r construct, the presence of a short helix is consistent with the secondary structure prediction that the repeat domain is mainly a-helical. the n-terminal crd disulfide bond (cys -cys ) and the helical repeat conformation was observed recently in the structure of dc-signr r (crd with its last two repeats). , the r.m.s deviation between the crd domain of dc-signr r and that of r is . Å for c a atoms. however, the hinge angle between the crd and the repeat domain differs by about ( and , respectively) between the two structures, indicating a domain flexibility between the crd and the repeat domain of the receptor (figure ). the primary calcium site involved in binding where f c is the calculated and f o is the observed structure factor amplitude of reflection h for the working or % free set, respectively. carbohydrate (ca ) has a well-ordered calcium ion in this structure. the amino acid residues involved directly in coordinating the calcium ion are glu , asn , glu , and asp , and a water molecule (w ). with the exception of asn , which is rotated out of the calcium coordination, the ligand positions are well conserved between the r and crd-only structures of dc-signr (figure (a) and table ). in contrast, no attributable electron density was found near the two auxiliary calcium ions (ca and ca ) binding site and the two residues coordinating the auxiliary calcium, asn and asn , moved . Å and . Å , respectively, compared to the crd-only structure ( k j). the movement of asn and asn effectively disrupts the coordination of ca and ca , further evidence that both auxiliary calcium ions are absent from the dc-signr r structure. despite the presence of mannose in the crystallization buffer and the existence of additional electron density at the putative carbohydrate-binding site, attempts to fit mannose were not satisfactory, and instead, water molecules were built throughout the carbohydrate-binding site. the comparison between the current apo-dc-signr r and the mannose-containing crd structure showed both the primary calcium/carbohydrate-binding loop (residues - ) and the secondary calcium-binding loop (residues - ) assumed an "open" conformation in the apo state while adopting a "closed" conformation in the carbohydrate-bound state of the receptor ( figure ). in the presence of carbohydrate, the conformation of the primary carbohydrate-binding loop (residues - ), the closed conformation, is defined by the coordinating hydrogen bonds between the sidechains of asn and ser , and the bound n-acetyl-d-glucosamine (glcnac ) as well as between asn and asn , and their bound ca and ca . in the absence of carbohydrate, however, (table and figure ). interestingly, an arginine residue from a symmetry-related molecule, arg , is found near the putative ca and ca sites, forming a hydrogen bond with the secondary calciumbinding loop to neutralize, as a surrogate to the missing calcium ion, the partial negative charges of the region. despite the presence of mm cacl in the crystallization setup, both ca and ca appear to be absent, suggesting that these auxiliary calcium sites are of low affinity compared to that of the primary calcium-binding site (ca ), and that their occupancies are coupled to the binding of the carbohydrate ligand. namely, they are glycaninduced calcium-binding sites. in the absence of the bound carbohydrate, both calcium coordination loops adopt an open, conformation ejecting the auxiliary calcium ions and become less ordered. the result suggests the function of these glycaninduced auxiliary calcium is to stabilize the conformation of the glycan-binding loops synergistically to the bound glycan rather than to preconform the glycan-binding loop. [ ] [ ] [ ] modeling of the dc-sign/r tetramer a homology search was performed using sequences corresponding to various lengths of the repeat domain of dc-signr against known structures in the protein data bank (pdb). the resulting sequence identities between segments of known structures and portions of dc-signr repeats are % between residues - of the focal adhesion kinase (pdb code k ) and repeats r -r of dc-signr (figure ), % between residues - of muts (pdb code nne) and repeats r -r , % between residues - of the large ribosomal subunit from deinococcus radiodurans (pdb code nkw) and repeats r and r , % between residues - of the monomeric isocitrate dehydrogenase (pdb code itw) and repeats r -r . all homologous structures are helical in nature. both homology modeling and sequence-based secondary structure prediction resulted in similar secondary structure assignment, including the boundary of helices, turns and loops throughout the r -r repeat domain of dc-signr. additional structural information derived from gel-filtration experiments on truncated receptors showing that receptor tetramerization requires r -r repeats and analytical ultracentrifugation observations suggesting an elongated shape of the tetramer were included in the modeling of the tetramer. based on the overlapping homologous structures and the biophysical shape consideration and using the focal adhesion kinase (pdb code k ) as a template (supplementary data figure ), a polyalanine model of dc-signr tetramer was built manually using the crystallographic program o and subjected to energy minimization using cns ( figure ). the tetramer model displays a -fold symmetry, with the core tetramerization domain adopting a four-helix bundle structure similar to that of the focal adhesion kinase (see supplementary data for a more detailed description of the model). the arrangement of the r and r helices in this model agree with the recently deposited structure of dc-signr containing both r and r repeats (pdb code sl ). the dimensions of the model proposed here are w Å ! Å ! Å with individual crd separated by w Å . on the table . we carried out limited proteolysis using trypsin to explore the likelihood of the proposed model of helical repeat bundles for the tetrameric dc-signr versus a model consisting of an elongated linear concatenation of helical repeats (figure ). since identical trypsin digestion sites are found within each repeat region, a differential use of each potential trypsin site would suggest differential protection from the protease. the tight packing of the proposed model predicts a biased protease-sensitivity for the different repeats with the core tetramer packing repeats less accessible than the peripheral repeats, while the linear helical concatenation model predicts an equal proteasesensitivity for each repeat. the digestion with trypsin was carried out using a recombinant expressed and refolded full extracellular dc-signr, termed dc-signr r , that has been characterized to be a tetramer. digestion of dc-signr r with trypsin resulted in four major fragments f w kda, f w kda , f w kda, and f w kda, with the f and f appearing before f and f in time-based digestions (supplementary data figure ). no other intermediate fragment could be identified. the amino-terminal sequencing revealed fragments f (residues - ) and f (residues - ) resulting from cleavages at trypsin sites between repeats r and r (site ) and within the crd (site ). fragments f (residues - ) and f (residues - ) appeared to be derived from f and f by further digesting at site and , respectively ( figure ). these results indicate that most of the tetramerization repeats (r -r ) remain resistant to digestion by trypsin, consistent with it being a compact tetramer unit rather than an elongated linear helical tetramer in which all repeats appear equally susceptible to protease. digestion experiments with subtilisin are consistent with these results, indicating protease-sensitive sites being primarily between repeats r and r , and after the helical repeat domain at the beginning of the crd region. evaluating potential dc-sign/r ligands earlier studies of the dc-sign/r crd binding to model carbohydrate compounds suggest that the receptors prefer a high-mannose type of carbohydrate. , , more recently, the receptors were shown to recognize also sialyl-lewis-like carbohydrates. the dissociation constant (k d ) between dc-sign/r crd and the model compounds, however, are millimolar at best, while the functional ligand recognition by the receptor has better than micromolar affinity. thus, much of the receptorligand binding affinity appears to be derived from an avidity effect of the dc-sign/r tetramer. the requirement of tetramer binding for ligand recognition would, in turn, impose limitations to its ligand selection. namely, ligands carrying multiple glycosylations capable of engaging the multimeric dc-sign/r crd simultaneously would be preferred by the receptor. the surface area encompassed by the tetrameric crd in our current dc-sign/r model is approximately Å , or Å per crd molecule. this requires the potential ligands of dc-sign/r to possess a surface glycosylation level exceeding one glycan molecule per Å of its surface area. this enables us to formulate a potential ligand index i to evaluate potential ligands of dc-sign/r on the basis of their surface glycosylation density: ( ) where n is the number of predicted potential glycosylation sites and m is the molecular mass of the candidate protein. a potential dc-sign/r ligand would possess an index greater than . and proteins with the indices less than . are less likely to be ligands of the receptor. the calculation of this potential ligand index for a number of viral envelope glycoproteins as well as for some cell-surface glycoproteins is summarized in table . of the potential viral targets of dc-sign/r, hiv- , coronavirus and marburg virus are known to bind dc-sign. in addition, hrsv, influenza and human foamy viruses appear to be good candidates for dc-sign/r. among the cellular targets, in addition to the known icam- ligand, several surface glycoproteins also score favorably for dc-sign binding. dc-sign and dc-signr are part of an antigencapturing network of receptors expressed on dendritic cells. previously, the structures of a mannose-bound and a lewis x -bound form of the receptor showed critical residues involved in both calcium and carbohydrate interactions. , our current structure of dc-signr r represents an apo form of the receptor. the structure revealed that much of the crd adopts a conformation very similar to that observed in the carbohydrate-bound receptor, with the exception of two loops that are involved in the coordination of carbohydrate (residues - ) and auxiliary calcium ions (residue - ) in the bound-form. in the absence of the bound carbohydrate, both loops adopt open conformations that are likely attributed to the loss of interactions with the putative carbohydrate and calcium. the absence of two bound auxiliary calcium ions compared with the structure of the carbohydrate-bound receptor suggests that the auxiliary calcium sites are of low affinity compared to the primary calcium site, and their presence appears to be ligand-induced. the multivalent nature of dc-sign/r indicates that recognition of small carbohydrate compounds by individual crd alone is not sufficient to achieve the high-affinity interactions of dc-sign and dc-signr with pathogens like hiv- gp . the functional receptors have been shown to be tetramers. , in addition, biochemical studies with repeat domain deletion mutants have shown that a minimum of three repeats are necessary to form tetramers, with additional repeats functioning to stabilize the tetramer. on the basis of the current crystal structures and available biophysical data, a tetramer for the entire extracellular dc-signr receptor was constructed by homology modeling in which the repeat regions form helical bundles to bring together their crds in a -fold related symmetry. this helical bundle-mediated oligomerization resembles superficially the trimer of rat mannose-binding protein. while the receptor repeat domain is conserved in most species, a notable exception is that of old world rhesus monkey, whose dc-signr gene (cd l ) is missing all the repeats and dc-sign gene is missing the fourth repeat. cd l is predicted to be a monomer and has been shown to be less efficient in binding to both icam- and hiv gp . the fourth repeat in our model serves as a connecting helix between the two helical bundles. deletion of this repeat would most likely shorten this connecting helix but may not affect the formation of the helical bundles (r -r and r -r ). the results of trypsin digestion studies appear to support a model in which the helical repeats are protected from protease by forming tightly packed helical bundles rather than by forming a single elongated helical domain (figure ). using this dc-signr tetramer model and the assumption that high-affinity ligand binding requires simultaneous engagement of multiple crd of the tetrameric receptor, we formulated a prediction scheme for potential ligands of dc-sign/r based on their predicted gross glycosylation density. the results show that several viral envelope glycoproteins, including hiv- gp , marburg virus gp, coronavirus spike protein, and hrsv glycoprotein g, possess high ligand indices. among them, gp of hiv, gp of ebola, and the spike protein of coronavirus are known ligands of dc-sign. of the potential cellular targets, in addition to the known icam- ligand, mucins are notably ranked high in our scoring scheme. the low-scoring molecules, such as igg, kir dl and hla-cw did not exhibit binding to dc-sign/r (data not shown). it should be noted that the receptor-ligand binding will also depend on the geometrical constraint, including the distance between and the orientation of the crds. the distance between glycans, in general, should correlate with their surface density. situations in which local spacing variation resulting in the distance between glycans either too close or too far apart to simultaneously engage the multimeric crd would clearly affect the recognition by the receptor. nevertheless, the known flexibility of glycans and the observed variation in the hinge angle between the receptor crd and repeat domains of dc-signr illustrate the built-in flexibilities in both the receptor and ligands, and thus lend some degree of freedom to the receptor-ligand recognition. these intrinsic flexibilities would lead to greater variability in distance and orientation, and marginalize the geometric constraint. nonetheless, the most obvious reasons to use the surface area of crd instead of the distance are ( ) to enable us to derive a prediction scheme based on surface glycan density of a potential ligand, and ( ) to have the prediction less dependent on the precise conformation, thus, the degree of correctness, of the tetramer model. in addition, equation ( ) assumes a globular shape for proteins and a uniform distribution of their glycosylation. clearly, both the local distribution of glycans and the actual shape of the protein presenting the glycans influence the receptor recognition. for example, despite a low score for dengue and hemorrhagic fever, evidence suggests that these viruses are recognized by dc-sign/r. , although the dengue virus envelope protein is not heavily glycosylated, the crystal structures of both the type and type dengue envelope protein e showed that the two conserved glycosylation sites are located at the protein dimer interface, resulting in four glycans distributed symmetrically at w Å apart across the interface. , this generates four closely packed glycan residues, which enables the recognition by the tetrameric dc-sign. this equation is thus a first-order approximation that does not reflect variations in protein shape or distribution of glycosylation. in conclusion, the mechanism of receptor-carbohydrate recognition may be more complicated than previously thought. the high-affinity binding strategy employed by these receptors appears to be twofold. first, the structure of each individual crd determines the preference of the receptor for particular carbohydrate structures. secondly, and perhaps more importantly, the high-affinity interaction as well as ligand specificity rely on receptor oligomerization, which would increase the affinity of ligand binding and impose constraints on the density and distribution of carbohydrates found on target pathogens. protein expression, purification and crystallization dna encoding amino acid residues - of the human dc-signr, which includes the last repeat (r ) and the crd, referred as dc-signr r , was inserted into the pet b vector (figure (a) ). the expression of the full-length extracellular domain of dc-sign and dc-signr has been described. proteins were expressed as inclusion bodies in escherichia coli bl (de ) and reconstituted in vitro. refolded dc-signr r was loaded onto a source q column (amersham) and further purified by size-exclusion chromatography using a superdex s column (amersham). the peak fractions were then concentrated to mg/ml and characterized using sds-page, n-terminal sequencing and mass spectrometry. initial crystallization screening trials were carried out by microbatch experiments using an automated crystallization robot (douglas instruments oryx ). , repeated attempts to crystallize the entire ectodomain of either dc-sign or dc-signr did not yield any diffraction-quality crystals. in contrast, rod-like crystals of the dc-signr r construct appeared in many conditions within six hours of setup. optimization of crystal growth conditions was performed by fine-screening of ph and precipitant concentration. crystals used for x-ray data collection were grown by the hanging-drop, vapordiffusion method in a well solution of mm mgcl , mm sodium cacodylate (ph . ), % (w/v) polyethylene glycol . crystals of dc-signr r were briefly transferred into well solution supplemented with % (v/v) glycerol and flash-frozen in a liquid nitrogen stream at k. the x-ray diffraction data were collected on a x chargecoupled device detector at the structural biology center collaborative access team beamline id and processed using hkl . the crystals diffracted to . Å and were indexed to the orthorhombic space group p with cell dimensions az . Å , bz . Å , and cz . Å . molecular replacement using the coordinates for dc-signr crd (pdb accession code k j) provided phase information. diffraction data from - . Å were used for the rotation and translation functions with the program amore. after rigid body refinement using program packages amore and cns. a complete model was built with the occupancies for disordered side-chains and loops set to zero. initial refinement in cns included simulated annealing, conjugate gradient minimization and individual temperature factor refinement. further refinement using maximum likelihood methods was performed with the program refmac . the final geometry of the structure was evaluated using the program procheck. least-squares superpositions were performed using the program lsqman. modeling of the dc-sign tetramer a protein search using the program blastp for sequences corresponding to one, two, three, four and all eight repeat domains of dc-signr in various combinations was used to query the pdb. from this search we identified a representative set of structures that includes focal adhesion kinases, taq muts and dna-binding proteins, with sequence identity of - % (pdb accession codes k , p , iom, nne, nkw, ewr, itw and hp ). the homologous portions of these structures were aligned on the basis of sequence homology to each corresponding repeat subunit of dc-signr, and their secondary structure was viewed using the program o. the structure of the focal adhesion kinase (pdb code k ) was used as a template for tetramer formation, with the additional structures being used primarily to predict location of turns. the final model was refined in cns using rigid body and energy minimization. ribbon diagrams were prepared using the program molscript. evaluating potential ligands of dc-sign/r since both receptors use multiple crd domains to modulate avidity-mediated binding to various carbohydrates found on a variety of pathogens, we derived a formula to evaluate and identify potential receptor ligands. let the surface area encompassed by the tetramer of dc-signr crd be s o , the surface area of a protein of interest be s, then the binding of dc-sign/r requires the number of glycosylations n satisfying: the structure of dc-signr n=so =s o thus, an index for potential ligands can be defined as: when the likelihood index i is greater than , the protein of interest possess, on average, higher glycosylation density than is required for binding to dc-sign/r and, conversely, when i is less than , the target protein is under glycosylated for dc-sign/r binding. assuming a spherical nature for proteins, which is only a crude approximation but will nonetheless result in a correct power-dependence on the molecular mass, the surface area s of a given protein can be calculated, to the first approximation, from its molecular mass by: where n a is avogadro's constant ( . ! ), m is the molecular mass (in da), and d is the average density of a protein, which has a value of . - . g/ml. if dz . g/ml is taken and equation ( ) is substituted in equation ( ), then: where s o is in Å . coordinates have been deposited with the protein data bank under accession code xph. c-type lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans hcv and hiv binding lectin, dc-signr, is expressed at all stages of hcv induced liver disease sequence and expression of a membrane-associated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp cd l) is a liver-specific capture. receptor for hepatitis c virus dc-sign, a dendritic cell-specific hiv- -binding protein that enhances trans-infection of t cells human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection are high affinity binding receptors for hepatitis c virus glycoprotein e c-type lectins l-sign and dc-sign capture and transmit infectious hepatitis c virus pseudotype particles hepatitis c virus glycoproteins interact with dc-sign and dc-signr dc-sign and dc-signr bind ebola glycoproteins and enhance infection of macrophages and endothelial cells dc-sign: escape mechanism for pathogens dc-sign: a novel hiv receptor on dcs that mediates hiv- transmission dc-sign promotes exogenous mhc-i-restricted hiv- antigen presentation dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans dc-sign; a related gene, dc-signr; and cd form a cluster on p a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection a novel mechanism of carbohydrate recognition by the c-type lectins dc-sign and dc-signr. subunit organization and binding to multivalent ligands structural basis for selective recognition of oligosaccharides by dc-sign and dc-signr structural basis for distinct ligand-binding and targeting properties of the receptors dc-sign and dc-signr contribution to ligand binding by multiple carbohydrate-recognition domains in the macrophage mannose receptor characterization of dc-sign/r interaction with hiv- gp and icam molecules favors the receptor's role as an antigen capturing rather than adhesion receptor extended neck regions stabilize tetramers of the receptors dc-sign and dc-signr c-type lectin-like domains structure of a c-type carbohydrate recognition domain from the macrophage mannose receptor collectin structure: a review oligolysinebased oligosaccharide clusters: selective recognition and endocytosis by the mannose receptor and dendritic cell-specific intercellular adhesion molecule (icam- )-grabbing nonintegrin novel member of the cd (dc-sign) gene family in primates dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses cd ) mediates dengue virus infection of human dendritic cells variable surface epitopes in the crystal structure of dengue virus type envelope glycoprotein a ligand-binding pocket in the dengue virus envelope glycoprotein comparative studies of protein crystallization by vapour-diffusion and microbatch techniques the role of oil in macromolecular crystallization the ccp suite: programs for protein crystallography crystallography & nmr system: a new software suite for macromolecular structure determination procheck: a program to check the stereochemical quality of protein structures not your average density basic local alignment search tool improved methods for building protein models in electron density maps and the location of errors in these models molscript: a program to produce both detailed and schematic plots of protein structures on the nature of the protein interior we thank c. hammer for mass spectrometry, m. garfield for n-terminal sequencing, b. hagos for assistance with protein expression, and c. foster, a. johnson, z. lu, s. ginell, and n. duke for assistance with synchrotron data collection. we thank j. arthos for helpful discussions. we thank c. foster, s. garman and s. radaev for helpful comments with structure and manuscript. use of the argonne national laboratory structural biology center beamlines at the advanced photon source was supported by the us department of energy, office of energy research, under contract no. w- - -eng- . this work was supported by niaid intramural funding. key: cord- -f o owg authors: navarre, christine b.; pugh, d.g. title: diseases of the gastrointestinal system date: - - journal: sheep & goat medicine doi: . /b - - - / - sha: doc_id: cord_uid: f o owg nan the gastrointestinal system is, arguably, more prone to disease than any other part of the sheep or goat. gastrointestinal parasitism alone is the most significant cause of production and animal losses in much of north america. , there is no substitute for a thorough physical examination when trying to determine the affected body systems of a sick animal; this is especially true in diseases of the gastrointestinal system. a complete physical examination should include palpation for body condition, assessment of abdominal shape and rumen motility, observation of the consistency of the stool, and evaluation for the presence of bloat. however, because rectal palpation cannot be performed in sheep and goats, diagnosis of disease in a particular segment of the gastrointestinal system can be difficult. therefore, the clinician may have to perform ancillary diagnostic procedures to characterize gastrointestinal diseases properly. clinicopathologic data consisting of a complete blood count (cbc), serum biochemical evaluation (sbe), and urinalysis can be helpful in differentiating gastrointestinal diseases, developing a prognosis and plan for treatment, and monitoring treatment. a cbc rarely identifies a specific disease, but it can be helpful in evaluating the severity of dehydration, anemia, and hypoproteinemia. the clinician must take care to interpret the packed cell volume (pcv) and total protein in light of the hydration status of the animal as noted on physical examination. an anemic or dehydrated hypoproteinemic animal may have normal pcv and total protein values. both the cbc and sbe can be helpful in characterizing the presence and severity of an inflammatory disease process. changes in the total and differential white blood cell count indicate acute or chronic inflammation; increases in globulins or fibrinogen suggest a chronic inflammatory disease. low protein levels, especially albumin, can point to chronic blood loss from intestinal parasitism or infiltrative bowel disease. liver disease should be suspected if liver enzymes or bilirubin are elevated. however, liver enzymes can be normal in chronic liver disease. also, albumin levels rarely drop in ruminants with liver disease, as they do in other species. changes in electrolytes can occur with gastrointestinal disease, especially if the animals are anorexic. electrolyte measurements also are helpful in formulating a treatment plan. hypochloremia and metabolic alkalosis occasionally occur in abomasal disease. a mild hypocalcemia may be encountered in some small ruminants with gastrointestinal atony. because many animals with gastrointestinal disease are dehydrated and therefore azotemic and possibly hypoproteinemic, urinalysis is helpful to eliminate urinary disease as a cause of these pathologies. normal ranges for clinicopathologic values are included in this textbook (see appendix iii) and also have been published in several other textbooks. [ ] [ ] [ ] [ ] however, clinicians would do well to learn the normal values, especially serum biochemistry values, established by the laboratory most commonly used for analysis. analysis of rumen fluid can help differentiate diseases of the forestomachs. an appropriately sized orogastric tube can be passed through the oral cavity for fluid collection (figure - ) . the clinician must properly restrain the animal, using a mouth speculum (figure - ) to prevent tube chewing. if the tube is chewed, its roughened surface may damage the esophagus; parts of a broken tube can be swallowed. rumen fluid also can be collected using percutaneous rumenocentesis , [ ] [ ] [ ] [ ] [ ] (figure the clinician then aspirates fluid with a syringe. local anesthesia and sedation of the animal may be necessary. this technique avoids the saliva contamination that can occur during collection with an orogastric tube, and it appears to be less stressful. rumenocentesis presents a slight risk of peritonitis, but this risk can be minimized with proper restraint. percutaneous rumenocentesis should not be performed on pregnant females. after the fluid is collected, it can be analyzed for color, odor, ph, protozoal species and motility, methylene blue reduction time (mbr), gram's staining characteristics, and chloride levels. normal values are listed in table - . anorexia may cause the fluid to appear darker, the ph to increase, and the number and motility of protozoa to decrease. a gray color, low ph, and dead or no protozoa are seen in rumen acidosis from grain overload. the mbr is prolonged with any type of indigestion. large numbers of gram-positive rods (lactobacillus species) also may be seen in rumen acidosis. elevated rumen chloride indicates an abomasal or proximal small intestinal obstruction (either functional or mechanical). the most important reason for examining feces in sheep and goats is to determine the presence and relative number of nematode parasites infesting an animal or flock. the quantitative technique for determining eggs per gram of feces (epg) is shown in box - . fecal epg values of more than to indicate serious infestation and the need for intervention. fecal occult blood testing and acid-fast staining of fecal smears also can be performed. fecal occult blood tests can detect microscopic amounts of blood in the feces. however, they cannot indicate which part of the gastrointestinal tract is bleeding. acid-fast stains of fecal smears that reveal clumps of acid-fast rods usually indicate infection with mycobacterium paratuberculosis ( johne's disease). generally, individual acid-fast rods found on fecal examination are nonpathogenic. abdominocentesis is useful in discerning the causes of fluid distention in the abdomen. two methods can be used. the first technique involves tapping the lowest point of the abdomen slightly to the right of midline; it is useful in ruling out a ruptured bladder as the cause of general ascites (figure - ) . , the clinician should take care to avoid the prepuce in males. the second technique is useful if peritonitis is suspected. because localized peritonitis is more common than generalized peritonitis, four sites are tapped. the two cranial sites are slightly caudal to the xyphoid and medial to the milk veins on the left and right sides. the two caudal sites are slightly cranial to the mammary gland and to the left and right of midline. for either technique, manual restraint with sedation is recommended; the use of real-time ultrasonography may help locate fluid pockets. a -gauge needle or teat cannula can be used for fluid collection. the clinician should prepare the site using sterile technique and provide local anesthesia when employing a teat cannula. fluid should be collected in a small ethylenediamine tetra-acetic acid (edta) tube for analysis and a sterile tube for culture. abdominal fluid can be difficult to obtain because of the small amounts normally present in both sheep and goats. the clinician should minimize the ratio of edta to fluid because edta can falsely elevate protein levels. using edta tubes made for small animals or shaking excess edta out of large tubes resolves this problem. normal culture values are similar to those for cattle (clear, colorless to slightly yellow, to g/dl protein, less than , cells). cytologic examination . weigh g of feces and thoroughly mix with ml of water. this is the preferred method. however, if a gram scale is not available, feces can be added to the ml of water until the water level indicates ml. this approximates g of feces. . remove ml of well-mixed fecal-water suspension, add to ml of sheather's solution,* and mix well. is needed to characterize the cell population and assess for the presence of phagocytized bacteria. radiography of the abdomen can be performed in small ruminants using small animal techniques. in adults, the rumen normally fills the entire abdomen. radiography can detect gas distention of the small intestine, abdominal fluid, and foreign bodies. , contrast techniques are useful for diagnosing atresia of the rectum or colon. unlike in other small animals, contrast techniques are not practical for characterizing small intestinal problems in sheep and goats because the rumen dilutes and slows passage of the contrast media. ultrasonography ultrasonography can be used to provide better characterization of abdominal distention, internal and external abdominal masses, and gross lesions of the liver. ascites may be differentiated from fluid in the intestinal tract, and gas distention of the intestines can be differentiated from fluid distention. normal ultrasonographic examination of the liver in sheep has been described. the liver can be viewed on the right side from the seventh or eighth rib caudally to the thirteenth rib ( . ultrasonography can be used to perform biopsies of organs or masses and to locate pockets of fluid. laparoscopy is more commonly used as a reproductive tool, but it also can be used diagnostically as an alternative to exploratory laparotomy in small ruminants. , general anesthesia is recommended. the technique for laparoscopic exploration of the abdomen used for cattle can be modified for use in sheep and goats. the clinician inserts a cannula in the caudal abdomen and carefully inflates the abdomen with carbon dioxide (co ). with the animal restrained in dorsal recumbency and either sedated or anesthetized, the clinician places the cannula in the inguinal area as described for laparoscopic insemination in chapter . entrance on the right side allows visualization of most of the abdominal organs. the clinician should avoid the rumen when introducing the laparoscope into the abdomen. this procedure may be enhanced by lowering the head or rear of the animal, allowing better visualization of the entire abdomen. animals should be properly ventilated during this procedure because inflation of the abdomen and lowering of the head can put pressure on the diaphragm. exploratory laparotomy can be a valuable diagnostic tool in evaluating gastrointestinal diseases when other tests indicate abdominal disease. in some cases, therapeutic surgical techniques can be performed at the same time. the technique of exploratory laparotomy used in cattle can be adopted for sheep and goats as long as the clinician keeps in mind that these animals are more likely to lie down during surgery and standing surgery should only rarely be attempted. small ruminants should be heavily sedated or placed under general anesthesia during this procedure. they may show signs of postoperative pain, anorexia, and depression and should be treated accordingly with a nonsteroidal antiinflammatory drug (nsaid) (flunixin meglumine . to . mg/kg intravenously [iv] ). the decision to use perioperative and postoperative antimicrobial agents should be based on the conditions under which the surgery is performed and the diagnosis made at surgery. sheep the area should be clipped, but in goats alcohol can be applied to the overlying hair and skin. if the area is clipped, the clinician should apply a bland coupling material (e.g., methyl cellulose, vegetable oil) between the skin and the transducer. liver biopsy in sheep and goats is performed using the same technique and instruments as in cattle. however, sedation and ultrasound guidance are recommended. the biopsy can be performed in the ninth to tenth intercostal space slightly above an imaginary line from the tuber coxae to the point of the elbow (figure - ) . the site should be surgically prepared, and a local anesthetic ( % lidocaine hydrochloride) infused subcutaneously. a small scalpel blade is used to make a stab incision through the skin. a -gauge, . -cm liver biopsy instrument is inserted through the incision and the intercostal muscles and into the liver. the biopsy instrument should be directed toward the opposite elbow in most cases, but the use of real-time ultrasonography can help determine the direction and depth needed ( to cm). the clinician should avoid the vessels along the caudal border of the ribs. on reaching the liver, the clinician will note a slight increase in resistance. samples can be submitted for culture (in a sterile plastic or glass vial or tube), histopathology (in formalin at a Ϻ ratio of formalin to tissue); and/or mineral analysis (in a plastic tube). when performing a liver biopsy for mineral analysis, the clinician should rinse the biopsy site with distilled and deionized water after sterile preparation to minimize sample contamination. samples for mineral analysis should not be placed in formalin. the skin incision can be sutured, and aseptically prepared, the surgeon makes a stab incision in the skin and introduces a -gauge biopsy needle. bloat is less common in small ruminants than in cattle, with goats having the condition less commonly than sheep. bloat is the accumulation of either free gas or froth in the rumen, which causes rumen distention. the causes of bloat can be divided into three categories , : . frothy bloat-caused by diets that promote the formation of stable froth . free gas bloat-caused by diets that promote excessive free gas production . free gas bloat-caused by failure to eructate pathogenesis. frothy bloat is usually associated with the ingestion of legume forages or hay (particularly alfalfa) and with grazing on lush cereal grain pastures, but it also may occur with high-grain diets. in the case of frothy bloat from a finely ground diet (usually corn), mucoprotein released from rumen protozoa stabilizes the foam at a low ph. in legume-associated frothy bloat, plant chloroplasts released into the rumen trap gas bubbles. regardless of the form of frothy bloat, the small bubbles fill much of the rumen, preventing clearance of the rumen's cardia and resulting in a cessation of eructation. free gas bloat also occurs with grain diets, especially if the animals are not adapted to the diet. failure to eructate has a variety of causes. physical obstructions of the esophagus such as choke or swollen mediastinal lymph nodes can cause free gas bloat. any disease of the rumen wall can interfere with rumen contractions and eructation. hypocalcemia, endotoxemia, pain, peritonitis, and some pharmaceutical agents (especially xylazine) can all interfere with rumen function and eructation. , , , clinical signs. clinical signs of frothy bloat and free gas bloat from either food intake or physical obstruction of the esophagus are usually more severe and immediately life-threatening than bloat seen from rumen wall diseases and systemic influences. abdominal enlargement occurs, particularly in the dorsal left paralumbar fossa. this may be subtle in sheep or angora goats with full fleece. signs of colic and anxiety are common. the rumen may be either hypomotile or hypermotile. respiratory distress is evident, with some animals breathing through their mouths; death can ensue if the bloat is not treated. this condition is a medical emergency, and therefore diagnosis and treatment should occur almost simultaneously. if the animal is not in immediate danger of dying, an orogastric tube can be passed. most cases of free gas bloat are relieved with passage of the tube. a clinician should then take a thorough history and perform a complete physical examination to find the cause of the bloat. if the bloat is not relieved with an orogastric tube, the tube should be removed and examined for evidence of froth. frothy bloat can be treated with poloxalene ( mg/kg) or dioctyl sodium sulfosuccinate (dss) ( cc [ oz]) delivered by orogastric tube. the froth encountered in frothy bloat caused by the ingestion of finely ground grain has a ph of less than . . if frothy bloat occurs while animals are being fed concentrates, mineral oil ( ml) may work better. peanut oil ( to mg/kg), vegetable oil ( to ml), and hand soap ( ml) also have been recommended in emergency situations. if the animal is in severe respiratory distress, the clinician should insert a trocar or large needle into the rumen at the paralumbar fossa. if gas does not escape, or froth is seen coming out of the trocar, an emergency rumenotomy should be performed (see the rumenotomy section of this chapter). if several cases of bloat are encountered in a group of pastured animals, the entire group should be removed from the pasture and reintroduced slowly after gradual acclimation. if only one or two cases of bloat are encountered, the healthy animals can remain on the offending pasture, but grazing should be limited to ensure gradual acclimation. prevention. prevention of frothy bloat involves limiting access to offending pastures or feedstuffs; providing supplemental feed and providing poloxalene in mineral supplements; and adding ionophores to the ration or supplement. when grazing or consuming legumes as "greenchop," animals should be introduced to the feed or pasture slowly, preferably over to weeks. animals should be closely monitored after a frost and during the rapid growth phase of plants because legumes, particularly alfalfa, may be more likely to cause bloat at this time. certain varieties of legumes that are designed for intensive grazing systems (e.g., alfagraze) should be planted and managed in a manner that decreases the incidence of bloat (limited or creep grazing). feeding dry, stemmy hay for to hours before allowing access to the legume pasture also may help minimize bloat. grass-legume pastures in which legumes are limited to less than % of the forage are safer but can still pose a problem for animals that are selective grazers. grazing legumes with high leaf tannin concentrations (e.g., arrowleaf clover, kudzu) is usually safer because tannins help break down rumen foam. the inclusion of poloxalene ( to mg/kg daily) in the feed or mineral supplement is useful in preventing frothy bloat. if poloxalene supplements are used, keepers should feed them for to weeks before moving animals onto a problem pasture. free gas bloat from concentrate feeds can be controlled by slow introduction to these feeds to allow for rumen adaptation and by the inclusion of ionophores in the diet. monensin ( mg/head/day in ewes, mg/kg/day in goats) and lasalocid ( . to mg/kg/day in sheep and goats) both decrease the formation of free ruminal gas. by enhancing propionic acid formation, these drugs not only reduce the amount of methane produced in the rumen, they also improve the efficiency of nutrient assimilation from feedstuffs. bloat in lambs and kids can have the same causes as in adults but also can be caused by improper milk feeding. overfeeding, feeding of large infrequent meals, and feeding spoiled or cold milk have all been associated with bloat in lambs and kids. rapid overdistention of the abomasum and improper chemical or physical composition of milk replacers inhibit rumen motility, leading to bloat. even though the feeding of cold milk has been associated with bloat, the practice can be used effectively in orphan feeding programs. lambs and kids tend to limit their intake of cold milk after they have become accustomed to cold milk in a free-choice feeding system. milk is usually placed in the rumen when animals are tube-fed; this may result in milk spoilage. , simple indigestion is a mild form of upset of the reticulorumen caused by a change in feeding routine. it can be caused by an alteration in the type of feed or in the amount of feed offered. the most common causes of simple indigestion are the addition of grain to the diet, an increase in the amount of grain fed, and an increase in the energy density of the diet. examples of such dietary changes are replacing oats with corn or changing from whole to ground corn. if the changes are drastic, rumen acidosis can occur (see the following section). other common causes are changes in hay or pasture, consumption of moldy hay, and ingestion of weeds and toxic plants after overgrazing or droughts. clinical signs include mild anorexia that lasts for to days. mild diarrhea and bloat also may occur. rumen fluid ph can be unchanged, increased, or decreased depending on the inciting cause. most animals improve with no treatment. pathogenesis. rumen acidosis is caused by the rapid rumen fermentation of highly digestible carbohydrates that are ingested in excessive amounts. although corn is commonly implicated, other cereal grains (oats, wheat, barley) may be involved, particularly if they are finely ground. the smaller the particle size, the more quickly rumen bacteria are able to ferment the carbohydrates contained in the feed. the common name of this condition is "grain overload," but breads, candy, apples and other fruits, beets, and potatoes also can cause this condi-tion. rumen acidosis usually occurs in animals that have been fed predominantly forage-based rations and are suddenly given access to large amounts of highly fermentable concentrates or concentrated forms of energy. it also can occur in animals that have been receiving concentrates previously, if the amount is suddenly and drastically increased; if access is denied for a time, then suddenly returned (e.g., during weather changes and alterations in water availability); or if ration mixing errors occur (e.g., leaving out monensin and rumen buffers) as highly digestible carbohydrates are fermented, rumen ph drops. lactobacillus species, which are lactic acid producers, proliferate in the acidic rumen environment and further lower rumen ph. as the rumen ph drops, rumen protozoa and many of the lactate users begin to die. lactic acid production causes the osmotic pressure in the rumen to increase. fluid is drawn from the systemic circulation into the rumen, resulting in dehydration and possibly hypovolemic shock. lactate concentrations increase in the blood and may cause systemic lactic acidosis. the lactic acid in the rumen also is toxic to the rumen epithelium. damage to the epithelium can result in leakage of bacteria and toxins into the portal and systemic circulation. chronic sequelae to rumen acidosis include fungal rumenitis and occasionally liver abscesses. , liver abscesses are less commonly encountered in sheep and goats than in cattle. laminitis also can occur, but may be more of a problem in sheep than in goats. the severity of the disease depends on the composition of the feed, particle size, amount of feed consumed, and the period of adaptation to the diet. clinical signs. clinical signs vary with the amount and type of feed ingested and the time since ingestion. signs first appear to hours after ingestion of the offending feed; they vary from anorexia, depression, and weakness to a down animal suffering from severe circulatory shock. dehydration is usually severe and evidence of toxemia is present (e.g., injected mucous membranes, increased scleral injection). colic, bilateral ventral abdominal distention, rumen stasis, and a "splashy" feel to the rumen also may be present. diarrhea can develop, adding to dehydration. , , the diarrhea can range from a pastelike feces to very watery droppings with foam and occasionally pieces of grain easily recognized. dehydration, lactic acidosis, and toxemia result in neurologic signs, including ataxia, head pressing, opisthotonos, and seizures. the body temperature is initially elevated but may drop as the condition worsens or the animal becomes toxic. some animals develop polioencephalomalacia and appear blind. diagnosis. the rumen fluid ph may fall below . . the fluid itself is milky gray and particles of the inciting feed may be noticed. protozoa are usually reduced in number or absent, and large gram-positive rods (lactobacillus species) may be seen on gram's stain. clinicopathology is consistent with dehydration (increased pcv and total protein, prerenal azotemia) and metabolic acidosis. liver enzymes (gamma-glutamyl transpeptidase [ggt], aspartate aminotransferase [ast], lactate dehydrogenase [ldh]) may be elevated on serum biochemical analysis. , the leukogram can vary from normal to a degenerative left shift, depending on the severity of the case. urinalysis reveals an increased specific gravity. treatment. treatment is aimed at correcting cardiovascular shock, dehydration, acidosis, and toxemia and removing or neutralizing the offending feedstuffs. iv fluids containing % sodium bicarbonate should be administered. , oral fluids are contraindicated because they cannot be absorbed and may increase the rumen distention and discomfort of the animal. nsaids are indicated for toxemia (flunixin meglumine, . to . mg/kg iv). , oral administration of magnesium hydroxide and magnesium oxide ( g/kg) may neutralize the acidic ph and is sufficient in mild cases. however, if much of the feed is still in the rumen, these two alkalinizing agents will only work temporarily. oral antibiotics have been recommended to kill rumen microflora and stop fermentation. however, the authors of this chapter feel they are contraindicated because the gram-negative anaerobes that need to flourish to reestablish normal rumen microflora are susceptible to most antimicrobials effective against lactobacillus species. removing the substrate for the lactobacillus species is more effective. because orogastric tubes with large enough bores to reflux feedstuffs are too large for sheep and goats, rumenotomy is indicated in severe cases to remove the feed (see the section on rumenotomy in this chapter). after the rumen ph is cor-rected, transfaunation of the rumen microflora with about qt of rumen fluid from a small ruminant is beneficial (box - ). thiamine supplementation (vitamin b , mg/lb subcutaneously [sc] three times a day [tid] to four times a day [qid]) is indicated until rumen function returns. in certain instances, calcium may be indicated and can be included in the iv fluids (calcium gluconate). the clinician should avoid mixing calcium salts and sodium bicarbonate. bacterial leakage into the rumen wall, liver, and systemic circulation makes antimicrobial therapy necessary. the systemic antimicrobial agent of choice is penicillin (procaine penicillin g, , iu/kg im bid) because anaerobes are the most likely offending organisms. if treated aggressively, the prognosis for immediate survival is good. feed (grass hay only) and water should be limited until rumen contractions return to prevent overdistention of the rumen. the chronic sequelae discussed previously influence long-term survival. prevention. prevention involves introducing concentrate feeds slowly to allow rumen microflora adaptation. dietary change from a lower to a higher fermentable energy concentration should occur slowly and preferably over a -to -week period. in the case of animals being fed high-grain rations (e.g., club lambs, feedlot lambs, dairy goats), buffering agents can be added to the diet. rumen buffers may improve milk production, increase feed intake, and increase rate of gain. the crude fiber content should comprise a minimum of % of the diet's total digestible nutrients (tdn). for example, the tdn is %, the minimum acceptable crude fiber is %. crude fiber levels lower than this can be fed for short periods if the rumen is properly adapted, but problems may nevertheless occur. sodium bicarbonate is probably the most commonly used buffer; it can be offered free choice or included in the diet as % of dry matter intake. calcium carbonate or limestone (which both have low can be collected through a weighted orogastric tube. alternatively, fluid can be collected from any normal ruminant at slaughter. handling rumen contents collected from a fistulated cow or at slaughter can be strained through gauze or cheesecloth to separate the fluid from the fibrous contents. fluid collected through a weighted tube should be ready for storage. rumen fluid should ideally be administered immediately. however, it can be stored for to hours. the surface of the fluid should be covered with a layer of mineral oil to maintain an anaerobic environment and stored at refrigerator temperature. caution: do not store rumen fluid in a closed container because it may explode. rumen solubility) and magnesium oxide (which has poor palatability) also can be included in the feed. magnesium oxide should be limited to . % to . % of the dry matter intake. pathogenesis. reticulitis and rumenitis can result from chemical or mechanical damage to the mucosal lining of the reticulorumen. the most common cause of chemical damage is rumen acidosis. however, ingestion of caustic toxins also can damage the mucosa. mechanical damage can occur from ingested foreign bodies or the formation of rumen bezoars. in cattle, some viruses such as the ones that cause bovine virus diarrhea and infectious bovine rhinotracheitis can infect the rumen wall. similar viruses have yet to be identified in sheep and goats. after the mucosa has been damaged, secondary infection by bacteria or fungi can occur. previous treatment with oral antibiotics may predispose to fungal infections of the rumen wall, especially if the mucosa is already damaged. actinobacillosis, actinomycosis, and tuberculosis rarely affect the rumen wall. tumors of the rumen wall also have been reported. , not all of these causes of reticulitis and rumenitis have been reported in sheep and goats, but all are potential problems. clinical signs. the clinical signs of these diseases are vague. anorexia and forestomach hypomotility may be the only clinical signs. confirming a diagnosis also may prove difficult. samples of rumen fluid may only show changes associated with anorexia (alkaline ph, decreased numbers and motility of protozoa, prolonged mbr time; see table - for normal values). occasionally fungal organisms may be seen on diff quik stained slides of rumen fluid. in these cases a diagnosis of fungal rumenitis should be made. an exploratory laparotomy and rumenotomy may be required to diagnose foreign bodies or masses. rumen parakeratosis is characterized by dark, thickened, and clumped rumen papillae. it is seen mainly in feedlot lambs that consume finely ground or pelleted rations. the parakeratotic rumen papillae are fragile and predisposed to damage, which can increase the chances of rumenitis. treatment and prevention. treatment depends on the inciting cause. dietary changes should be made to decrease energy density and increase fiber intake. mild rumenitis may improve with time and supportive care (transfaunation, fluid support, high-quality feed). fungal rumenitis can be treated with thiabendazole ( mg/kg orally). severe changes may lead to scarring and permanent impairment of rumen function. traumatic reticuloperitonitis is not as common in small ruminants as in cattle, but it has been reported. goats are affected more commonly than sheep. this is probably because of the dietary habits of small ruminants; they tend to be selective grazers and do not "vacuum" the ground as cattle do. offending foreign bodies that cause traumatic reticuloperitonitis include pieces of wire and needles. , the clinical signs are identical to those in cattle and may include anorexia, depression, colic, signs of heart failure, and evidence of draining tracts from the chest cavity. treatment is usually difficult. rumen impaction can occur after dehydration, blockage of the omasal orifice by a foreign body, sand ingestion, or consumption of diets high in fiber and low in digestibility. clinical signs are nonspecific, but the firm rumen can usually be palpated in the left flank. the feces may be scant and dry. oral fluids containing magnesium sulfate ( g) may loosen impactions, but a rumenotomy is required in severe cases. to reduce rumen fill, sheep or goats should ideally have feed withheld for hours before rumenotomy. however, this is usually impossible because in most cases rumenotomy is an emergency procedure. the perioperative administration of antimicrobial agents is essential because even with meticulous technique some contamination of the incision site and possibly the peritoneal cavity is inevitable. because the rumen microflora is predominantly composed of anaerobic bacteria, penicillin ( , iu/kg) is the antimicrobial agent of choice and should be administered to hours before surgery. if the rumenotomy is being performed in an emergency situation, penicillin salts (potassium or sodium) that can be given iv provide therapeutic concentrations more rapidly than procaine penicillin. nsaids (flunixin meglumine, . to . mg/kg iv) also are recommended before surgery. if necessary, treatment of cardiovascular shock and dehydration with iv fluids also should begin before surgery and continue until the animal is rehydrated and in stable condition (see appendix ii). general anesthesia is recommended, but heavy sedation and local anesthetic infiltration of the incision site can be efficiently used (see chapter ) . the clinician should clip and surgically prepare a square area from cm in front of the last rib to the tuber coxae, and from the dorsal midline to the lower abdomen, encompassing the entire left paralumbar fossa. the surgeon makes a skin incision approximately cm longer than the width of the hand cm caudal and parallel to the last rib. the incision is continued through the muscle layers into the abdomen. because the abdominal wall is relatively thin, the surgeon should take care not to enter the rumen or bowel. the surgeon grasps the rumen wall and pulls it through the incision; suturing it to the skin with a simple continuous circular pattern around the entire incision. this forms a seal that minimizes rumen content contamination of the deep layers of the incision and peritoneal cavity. the rumen wall is then incised inside the circle of sutures. the incision in the rumen wall should be large enough for the surgeon to put his or her hand inside the rumen without traumatizing the rumen wall. after the rumen has been explored and emptied and the primary reason for doing the procedure has been completed, the surgeon closes the rumen wall in a continuous inverting pattern (cushing, lembert, or guard's rumen stitch) with absorbable suture ( catgut). the area should be rinsed with copious amounts of sterile isotonic fluids, and a new set of sterile instruments, sterile gloves, and surgical attire should be used for the remainder of the surgery. the surgeon then removes the suture securing the rumen to the skin and rinses the area again before performing routine closure of the abdominal muscles and subcutaneous layers with absorbable suture ( catgut) in simple continuous patterns, taking care to close dead space between layers. the skin is closed with a continuous pattern (ford interlocking) using a nonabsorbable suture material. the sheep or goat should be observed closely by the clinician for signs of complications, including peritonitis, incisional dehiscence, incisional hematoma, abscess, and hernia formation. penicillin therapy (procaine penicillin g, , iu/kg bid) should continue for at least days. the skin sutures can be removed to days after surgery. abomasitis and abomasal ulcers in adult sheep and goats are associated with rumen acidosis or chronic rumenitis but also can be caused by infections. [ ] [ ] [ ] [ ] finely ground feeds, pelleted rations, systemic stress, and feeding lush forages have all been implicated. anecdotal associations with mineral deficiency (copper) have gone unproved. clinical signs and diagnosis. this disease often goes unnoticed in mild cases, and the most common signs are anorexia and colic. no definitive antemortem diagnostic tests are available. fecal occult blood is often absent. occasionally dark stool, altered appetite (wood chewing), and bruxism are seen. therefore other causes of colic should be eliminated. diagnosis is based on clinical signs. effective therapy can be difficult. oral medications such as coating agents must first pass through the rumen, and therefore arrive at the abomasum diluted. iv (not oral) ranitidine ( mg/kg once a day [sid]) may be beneficial. herd problems of rumen acidosis may be addressed with buffers in the feed. a syndrome of abomasal hemorrhage, bloat, and ulceration is seen in lambs and kids to weeks of age. sarcina-like bacteria, clostridium falax, clostridium sordelli, and clostridium septicum have been isolated from many of these cases. [ ] [ ] [ ] [ ] c. septicum infections of the abomasum are commonly called braxy. the feeding of milk replacer free choice, iron deficiency, and bezoars have been implicated as predisposing factors. , clinical signs. the signs of this syndrome are severe, acute abdominal distention; colic; and death. [ ] [ ] [ ] [ ] diagnosis and treatment. the diagnosis of this condition is by postmortem examination. treatment in suspected antemortem cases is unsuccessful. prevention. adding formalin to milk replacers and vaccinating for clostridial diseases may decrease the occurrence of this disease. , lambs or kids on problem farms can be vaccinated for clostridium species during the first week of life with multivalent bacterins. similar to rumen impaction, abomasal impaction usually occurs when poor-quality roughage is fed, but it also can be seen with foreign body obstruction of the pylorus. , , goats appear to be more commonly affected than sheep, and boer goats are more commonly affected than angora goats. pregnant animals may be more prone to this condition. clinical signs and diagnosis. affected animals are usually anorexic. they have mild distention of the ventral abdomen, and in some cases the firm abomasum can be palpated through the abdominal wall on the right side. weight loss may be apparent. clinicopathologic evaluation may be normal, or mild hypochloremic metabolic alkalosis may be present, with elevated rumen chloride concentrations (more than meq/l). treatment. diet changes and mineral oil by mouth (po) are the most commonly employed treatments. abomasotomy can be attempted, but it has rarely been reported in small ruminants and does not usually improve the animals' long-term prognosis. when attempting abomasotomy, the clinician should perform the procedure with the animal in dorsal recumbency and under general anesthesia. the abomasum can best be visualized through an incision parallel and to the right of midline, caudal to the xyphoid process. the prognosis is poor. prevention. dietary manipulation to improve feed or forage quality is the best mode of prevention. abomasal emptying defect is a disease that presents similarly to abomasal impaction but is recognized only in suffolk sheep. the underlying cause is unknown. unlike abomasal impaction, this disease is associated with concentrate feeding and often occurs around lambing time. the clinical signs are chronic weight loss, abdominal distention, and anorexia. clinical pathology and rumen chloride levels are the same as described for abomasal impaction. on necropsy the abomasum is greatly distended, and the contents may be liquid or dry. treatment with laxatives, cathartics, motility modifiers, and abomasotomy has been mostly unsuccessful. [ ] [ ] [ ] azalea, laurel, and rhododendron toxicity members of the azalea, laurel, and rhododendron plant group produce andromedotoxins that alter sodium metabolism, resulting in prolonged nerve depolarization. these plants are cardiotoxic, but affected animals generally exhibit acute gastrointestinal upset. these evergreen shrubs produce thick, dark green leaves. they also have five-lobed, white to pink, saucer-shaped flowers that bloom around july. some of these plants are grown as ornamental shrubbery around homes, whereas others grow wild along streams, cliffs, and rocky slopes. they can be short or tall (as large as m) and can form thickets. all parts of these plants are toxic. clinical signs. animals browsing a new area, those fed clippings from trimmed azalea hedges, and underfed, hungry animals given access to these plants are likely candidates for intoxication. animals that ingest as few as two or three leaves may show signs of salivation, grinding teeth, nasal discharge, colic, epiphora, and acute digestive upset within hours of ingestion. as the intoxication progresses, animals become depressed and exhibit projectile vomiting, frequent defecation, and a slowed pulse. terminally intoxicated animals become paralyzed and comatose. some sheep and goats develop aspiration pneumonia secondary to intoxication. diagnosis. the diagnosis of this condition is usually based on clinical signs coupled with a history of ingestion of one of these plants and/or the discovery of these plants in the gastrointestinal tract. treatment. intoxicated animals may recover in to days without any therapy if the offending plants are removed from the diet. however, the administration of charcoal ( to g/kg po), atropine ( . to . mg/kg iv), other antiarrhythmic drugs, and iv fluids all may be indicated. to manage the aspiration pneumonia, the administration of antibiotics (penicillin , units/kg bid im) and oral magnesium hydroxide also may be beneficial. obviously, any existing dehydration should be corrected (see appendix ii). mountainous or hilly areas should be fenced. feeding shrubbery clippings is discouraged. diarrhea in lambs and kids is a complex, multifactorial disease involving the animal, the environment, nutrition, and infectious agents. decades of research have been devoted to the study of the pathophysiology of infectious diarrhea of calves; the pathology in lambs and kids is quite similar. despite improvements in management practices and prevention and treatment strategies, diarrhea is still the most common and costly disease affecting neonatal ruminants. [ ] [ ] [ ] [ ] some general preventive measures (e.g., improved sanitation) decrease disease no matter the cause. however, specific control measures such as vaccination require the definition of a specific cause of diarrhea. table - lists the agents most likely to cause diarrhea in lambs and kids, tissues or other samples required for diagnosis, and commonly employed test methods. the color and consistency of the feces and any gross lesions can appear similar no matter the cause. therefore laboratory identification of infectious agents and tissue histopathology are key to establishing a diagnosis. because autolysis and secondary bacterial invasion of the gut begins within minutes of death, necropsy samples taken immediately from euthanized lambs and kids yield the most reliable diagnostic material. mixed infections with two or more pathogens are common, and pathogens that are a problem on a farm change from year to year. , , in some cases an underlying nutritional deficiency or excess may occur concurrently with an infectious agent. therefore the clinician should be careful to take a variety of samples to ensure that all pathogens and predisposing factors involved are recognized; continued reevaluation of the causes of diarrhea is crucial. examination of several cases, with a focus on those in the acute phases, is important. although examination of antemortem fecal samples can be diagnostic, laboratory testing of tissue samples may yield better results. treatment and preventive measures specific to a particular disease are discussed with that disease in the following paragraphs. general supportive treatment and control measures are covered at the end of this section. four major pathogens cause diarrhea in lambs and kids during the first month of life: enterotoxigenic escherichia coli (etec), rotavirus, cryptosporidium species, and salmonella species. the relative prevalence of these infectious agents varies greatly among studies. this variance most likely results from differences in location, season, diagnostic techniques, and the occurrence of mixed infections. other, less common causes of diarrhea in neonates are giardia infections and nutritional diarrhea. pathogenesis. etec employs two virulence factors to cause disease. the first is the ability to attach and colonize the intestinal villi, which is accomplished via fimbria or pili. the most important fimbria in lambs are k and f . , the fimbrial antigens can be recognized from samples sent to most diagnostic laboratories and are im- portant in diagnosing this agent as a cause of diarrhea. after the organism attaches to the villi, it produces the second virulence factor, enterotoxin. enterotoxin interferes with the normal physiology of the gut, with resultant diarrhea. calves have an age-associated resistance, most likely related to the blocking of fimbrial attachment to the gut, so etec occurs mainly in calves less than a week old. , the mode of infection is fecal-oral. clinical signs. etec is seen in lambs and kids less than days of age but is most common at to days of age, so age-related resistance also may occur in these animals. , it usually presents as an outbreak in lambs and kids between and hours of age. because etec causes a "secretory" diarrhea, bicarbonate loss in the diarrhea leads to severe acidosis, with lambs and kids quickly becoming dehydrated and recumbent. however, many infected animals die before developing diarrhea. affected neonates are depressed, stop nursing, and may show excessive salivation. fluid sequestration in the abomasum causes a "splashing" sound on movement.this condition results in high mortality if animals are not treated promptly. diagnosis. fecal culture and serotyping for the k and f fimbrial antigens are the basis for diagnosis. because many nonpathogenic e. coli are normal gut inhabitants, simply culturing this organism is usually insignificant. occasionally the bacteria do not express the fimbrial antigens in culture, so etec cannot be ruled out if the culture is negative for k and f . histologic evidence of colonization of the small intestine can support a diagnosis. treatment. supportive care consisting of fluid therapy with either oral, iv, or sc administration of a polyionic solution is the mainstay of therapy. the use of oral antimicrobial agents is controversial. although antibiotics may kill the etec, they also may interfere with normal gut flora. if fluid support is provided, the diarrhea usually subsides without antibiotic treatment. still, oral neomycin ( to mg/kg bid) or trimethoprim sulfa ( mg/kg po) and systemic ampicillin ( to mg/kg im bid) or amoxicillin ( to mg/kg im tid) may be beneficial. nsaids are indicated to decrease inflammation of the gut and provide some analgesia. the use of flunixin meglumine ( to mg/kg im) has been shown to decrease fecal output in etec infections in calves and appears to be beneficial in lambs. prevention. it is recommended that clinicians vaccinate ewes and does with bovine etec vaccine before they give birth to increase passive immunity. , , monoclonal and polyclonal antibody products for calves may be beneficial during an outbreak if it can be given to lambs or kids within the first hours of life. the use of neomycin ( to mg/kg po bid) in lambs that appear normal may help stop the progression of an outbreak. shearing ewes prepartum to minimize fecal ingestion by neonates and ensuring that newborns ingest adequate colostrum both help decrease the incidence of this disease. making sure that ewes and does give birth at a . to . body condition score increases the chance of adequate colostrum manufacture by the dam. pathogenesis. lambs and kids are infected with group b rotaviruses, whereas most other animals and human beings are infected with group a rotaviruses. rotaviruses infect villus tip cells of the small intestine, which results in villus atrophy and malabsorptive diarrhea. clinical signs. rotavirus generally causes diarrhea in lambs and kids to days old, but older animals also can be affected. young animals can become very depressed and dehydrated. , , , diagnosis. detection of the organism by electron microscopy of fecal or colonic samples or by immunologic techniques on feces or tissue sections is the basis of diagnosis. , because these organisms are sloughed with the villus tip cells they infect, and viral antigens are complexed with the lambs' and kids' antibodies, tissue samples from acutely infected animals are best. rotavirus has been detected in animals without diarrhea, so other causes of diarrhea should be investigated as well. , treatment and prevention. rotavirus is treated with supportive care. prevention by vaccination of ewes and does with bovine rotavirus vaccines before they give birth is recommended to increase passive immunity. clinical signs. cryptosporidia can cause diarrhea in lambs and kids to days of age. , , affected animals are often active, alert, and nursing. the diarrhea is usually very liquid and yellow. diarrhea can vary from mild and self-limiting to severe, especially with mixed infections. , , , relapses are quite common, and this organism usually occurs as a component of mixed infections. diagnosis. acid-fast staining of air-dried fecal smears is a quick and easy method of diagnosis. examination under ϫ to ϫ magnification reveals round protozoa that have taken up the red color of the carbol fuchsin portions of the stain on a green background (figure - ). although they can be diagnosed by fecal flotation, their very small size ( - mm) makes this method difficult and subject to false negative results. , both immunologic and polymerase chain reaction (pcr) techniques have been developed to improve detection limits. , cryptosporidia also can be identified with histology. cryptosporidiosis is a zoonotic disease, and people can easily become infected from handling infected animals or feces. prevention. no consistently effective treatment for cryptosporidiosis in ruminants has been identified. anecdotal reports suggest that decoquinate and monensin sodium may be useful in control of cryptosporosis. decoquinate ( . mg/kg po) may be very useful in prevention of cryptospirosis in goats and possibly kids. during an outbreak affected animals should be isolated from the rest of the flock. no new animals should be added to a pen in which the disease has been diagnosed. keepers should depopulate pens in which the disease has been diagnosed and attempt to clean the environment. cryptosporidiosis can be particularly difficult to control because of the organisms' persistence in the environment and resistance to most chemical disinfectants. however, ammonia ( % to %) and formalin ( %) seem to be most effective. , feeders should be constructed to minimize fecal contamination. studies are currently underway to develop a vaccine for cryptosporidiosis in cattle. early results are favorable, and this may prove the best way to control the disease in the future. this is potentially a zoonotic disease, and therefore clinicians and keepers should exercise great caution when handling affected animals. pathogenesis. the bacterial genus salmonella has thousands of serotypes, and all can potentially cause diarrhea in animals. salmonella can cause diarrhea in lambs and kids of any age. , the microbes produce enterotoxins, are invasive, and cause severe inflammatory disease and necrosis of the lining of the small and large intestines. clinical signs. animals less than week old are more likely to die acutely without clinical signs, whereas animals older than week are more likely to have diarrhea. , , an acute onset of fever, depression, tenesmus, and shock is occasionally observed. salmonella-induced diarrhea is more likely to contain blood. this also is a zoonotic disease that warrants protective measures. a diagnosis of this condition is based on culture of the organism in feces or tissues and histologic examination of the small and large intestine. more sensitive pcr techniques for identifying salmonella species in feces are being developed. the diarrhea may occa-sionally contain fibrin, but many animals die before this is observed. clinicians may note leukopenia or leukocytosis in the cbc results. treatment. therapy for salmonella-induced diarrhea involves supportive care and possibly parenteral antimicrobial therapy. the use of antimicrobial agents is controversial and probably does not influence the gastrointestinal infection. however, because this is an invasive organism, parenteral use of antimicrobial agents may be beneficial in preventing septicemia. antimicrobial susceptibility patterns are difficult to predict for salmonella species, so antimicrobial therapy should be based on culture and sensitivity results. ceftiofur sodium ( . to . mg/kg im bid) or trimethoprim sulfadiazine ( mg/kg sc sid) can be administered until antimicrobial sensitivity results are known. prevention. latent carriers of salmonella can potentially shed organisms to other animals, particularly when they are stressed. newly introduced animals should be isolated for month, and fecal culture should be considered. bleach is an effective disinfectant to use during an outbreak. identification of carrier animals by fecal culture is recommended for herd problems. vaccine efficacy is questionable, and to date its effects have not been thoroughly evaluated in sheep and goats. giardia: giardia-induced diarrhea is more commonly seen in but not limited to -to -week-old lambs and kids. , the diarrhea is usually transient, but infected animals can continue to shed cysts for many weeks, even when they are clinically normal. , , therefore simply finding the agent in feces does not mean it is the cause of diarrhea, especially in older animals. however, these animals may be a source of infection for other animals and possibly humans. , iodine-stained wet mounts of feces or tissue is the classic method of diagnosing giardiasis, but more sensitive immunologic techniques are now available. , infected animals can be treated effectively with fenbendazole ( to mg/kg bid for days or sid for days). giardia has historically been treated with metronidazole ( mg/kg po sid for days). however, use of this drug class in food animals is currently illegal in the united states.this is potentially a zoonotic condition. infectious agents are not the only cause of diarrhea in neonates. nutritional problems can result in diarrhea, but these causes are overshadowed in the literature because the resulting diarrhea is usually mild and subsides without treatment. nutritional diarrhea is most common in orphaned animals as a result of keepers offering poorquality milk replacers, making mixing errors, or feeding large amounts infrequently (see chapter ) . diarrhea re- sulting from consumption of lush pasture or high-energy rations is a common occurrence. in most cases such diarrhea is self-limiting. the incidence of this form of gastric upset can be minimized by a slow introduction (over to weeks) to energy-dense diets. calves with infectious diarrhea that develop maldigestion or malabsorption can have secondary nutritional diarrhea from an inability to digest carbohydrates (lactose, xylose). , this has been reported in goats, and also is probably a cause of diarrhea in lambs. diarrhea resulting from primary lactose deficiency also has been reported in calves. calves on poor-quality milk replacers can develop an overgrowth of normal enteric e. coli, resulting in diarrhea. if lactose intolerance is suspected, decreasing the amount of lactose fed and using commercially available lactose enzymes may alleviate signs. the most common cause of diarrhea in older lambs and kids is nematode infestation. this condition is discussed later in this chapter in the section on causes of adult diarrhea. other major causes of diarrhea in older lambs and kids are c. perfringens and coccidiosis. c. perfringens types a, b, c, and d can all cause diarrhea in lambs and kids, but type d is the most common agent. , , pathogenesis. the disease occurs in peracute, acute, and chronic forms and is commonly called enterotoxemia or overeating disease. in the case of type c infection, a beta-toxin can cause acute hemorrhagic enteritis. type c infection is seen mostly in lambs or kids younger than weeks of age. an epsilon-toxin is responsible for pathology in type d infections. enterotoxemia is usually seen in rapidly growing feedlot lambs on high concentrate rations. it also is associated with other feeding changes, including changes in type of pasture. however, it occasionally occurs with no reported dietary changes, particularly in goats. , , this disease usually occurs in the fastest-growing and most well-conditioned animals. it can occur in vaccinated herds (again, more commonly in goats) so it should not be ruled out if a history of previous vaccination is present. clinical signs. the peracute form of clostridial infection is characterized by the rapid onset of severe depression; abdominal pain; profuse, bloody diarrhea; and neurologic signs. death occurs within hours of the onset of signs. sudden death may occur without signs of diarrhea. the onset of neurologic signs followed by sudden death is more common in sheep, whereas goats are more likely to show signs of diarrhea before death. similar but less severe signs are seen in the acute form of the disease. the chronic form occurs more commonly in goats. , diagnosis. antemortem diagnosis is based on clinical signs. at necropsy, c. perfringens can be cultured from intestinal tissue samples. however, the significance of a positive culture can be difficult to interpret because these organisms can be present in the gut normally and then proliferate after death. histologic examination of sections of the gut can be helpful. identification of the toxins (namely the epsilon-toxin) in intestinal contents is required for a definitive diagnosis. , because the toxin degrades within several hours of death, not finding the toxin does not preclude enterotoxemia as a diagnosis. treatment. treatment is rarely effective but consists mainly of aggressive supportive care. c. perfringens type d antitoxins ( to ml sc) can be administered to animals during an outbreak of enterotoxemia if clinical signs are noted before death. the antitoxin may be more effectively used as a preventive in the face of an outbreak. during an outbreak any animals that have not been vaccinated should be given the antitoxin and vaccinated with the toxoid simultaneously; those previously vaccinated should receive a booster vaccination. prevention. routine vaccination should start at to weeks of age and be followed by a booster to weeks later. however, on farms where the disease has become endemic, lambs or kids can be vaccinated and given antitoxin during the first week of life. yearly vaccination, preferably a few weeks before the ewes and dams give birth increases colostral immunity in neonates and improves prevention programs. goats may not respond as well to vaccination as sheep, so biannual or triannual vaccination is recommended, especially in problem herds. , vaccination with only c. perfringens types c and d and tetanus is superior to the use of more polyvalent clostridial vaccines. reducing the energy density of the diet and avoiding sudden dietary changes or alterations of the feeding routine are crucial to prevention. reducing internal parasites, particularly tapeworms, may further reduce the incidence of these disorders. pathogenesis. coccidiosis is a protozoan parasitic disease that is a common cause of diarrhea in lambs and kids. it also may cause subclinical production losses. clinical disease is often seen when some form of stress (e.g., dietary change, weather changes, parturition, weaning) is occurring on the farm or in the flock. eimeria species cause the disease in sheep and goats; each is infested with its own host-specific species. unlike cryptosporidium, which can be shed in feces in the infective stage, coccidia must sporulate outside the host to become infective. sporulation occurs under moderate temperatures and high moisture conditions. the nonsporulated and sporulated oocysts can survive a wide range of temperatures and may survive for years under certain conditions. clinical signs. lambs and kids are most susceptible to the problem at approximately to months of age, although younger animals may become infected. clinical disease is common after the stress of weaning, feed changes, or shipping. crowded conditions result in excessive manure and urine contamination, which is ideal for the buildup and sporulation of the oocysts. under these conditions, animals may be exposed to high numbers of infective organisms and develop diarrhea.the diarrhea in lambs and kids is usually not bloody, but it can contain blood or mucus and be very watery. anorexia, dehydration, weakness, rough hair coat, and death all may occur. weight loss is common, and constant straining can result in rectal prolapse. in severe cases the disease becomes protracted because of necrosis of the mucosal lining. even if these animals are treated appropriately, the diarrhea continues until the intestinal mucosa heals, which can take several days to weeks. permanent scarring can result in chronic poor development, even if the diarrhea subsides. , [ ] [ ] [ ] diagnosis. acute coccidiosis can be easily diagnosed from a direct smear or flotation of feces (figure - ) . in the chronic stages, most of the organisms have been shed and very low numbers are seen on fecal examination. because normal animals can shed small numbers of pathogenic species or large numbers of nonpathogenic species, interpretation of fecal examinations in the chronic stages of coccidiosis or in animals with diarrhea from other causes can be difficult. [ ] [ ] [ ] in these cases the clinician should rule out other diseases before making a diagnosis of coccidiosis. blood analysis may show both anemia and hypoproteinemia. treatment. treatment of affected animals with clinical signs includes supportive care and administration of coccidiostats. all animals in the group should be treated during an outbreak. the use of coccidiostats has little effect on the existing infection, but it does prevent the spread of the disease from continued exposure to infective organisms. many coccidiostats inhibit coccidia development and prevent disease if given prophylactically. they are of little value if they are given after the onset of clinical disease. sulfa drugs appear to be clinically beneficial, but they may simply decrease secondary or concurrent bacteria-induced diarrhea. because coccidia develop some resistance to coccidiostats, these drugs should be administered only before stressful events (e.g., shipping, weaning, parturition). the drugs listed in table - and trimethoprim sulfa ( mg/kg orally sid for days) are approved for use in the united states. , prevention. control involves improved sanitation and possibly the use of coccidiostats. preventing overcrowding decreases the buildup of manure and infective oocysts. exposure to sunlight and desiccation are two of the most effective means of killing the organisms. minimizing stress and optimizing nutritional intake also are important. coccidiostats available in the united states are shown in table - and appendix i. to avoid toxicity in growing animals, the clinician or keeper must carefully adjust dosages to the changing levels of feed intake as animals grow. all agents except amprolium should be fed for at least weeks. , this allows exposure and subsequent development of immunity to occur while preventing the detrimental effects of clinical disease. however, coccidia can become resistant to coccidiostats; fecal samples should be periodically evaluated after prolonged use of a particular product. anecdotal reports suggest amprolium resistance may occur on some farms. moreover, if amprolium is offered with a creep feed rich in thiamine, its ability to act as a thiamine antagonist may be compromised. year-round use of coccidiostats increases the potential for resistance. therefore they should be fed only during times of expected risk. the inclusion of lasalocid ( kg of % premix) or decoquinate ( kg of % premix) in kg of trace mineralized salt fed as the only source of salt for days prepartum can reduce the number of oocysts shed in ewe or dam feces. this practice can reduce the coccidia contamination of pasture and thereby remove a source of infection for kids and lambs. the benefits of administering lasalocid and monensin beyond coccidia control include increased feed efficiency, enhanced growth rate, and decreased incidence of free gas bloat. however, if coccidiostats are included in either mineral or feed supplements, inconsistent or depressed intake may result in subtherapeutic drug dosing. lambs are resistant to infection in the first few weeks of life. exposure to the protozoa during this time confers immunity and resistance to later infections. , adenovirus, caprine herpesvirus, coronavirus, campylobacter jejuni, yersinia species, and strongyloides papillosus can cause diarrhea in lambs and kids of various ages. , , enterohemorrhagic e. coli (ehec) and enteropathogenic e. coli (epec) also have been isolated in the feces of kids with diarrhea. , these e. coli types are k and f -negative. culture and serotyping of these organisms from feces and tissue samples with typical histopathologic lesions is diagnostic. although etec is not zoonotic, ehec and epec can potentially affect humans. although some causes of diarrhea have specific treatments, many animals need to be treated for dehydration and metabolic acidosis regardless of the inciting cause. animals with only mild diarrhea, especially mild nutritional diarrhea, may not require therapy unless they become dehydrated. if kids or lambs become less than % dehydrated and are only mildly depressed but still willing to nurse, they can be treated with oral electrolytes designed for calves. fluids can be administered by bottle or by tube if the animal will not nurse. the keeper or clinician should carefully adjust the amount of fluids for lambs and kids ( to ml, or to oz, as opposed to l in a calf ). because most electrolyte solutions designed for calves contain glucose, after they have been mixed they should be refrigerated and any leftovers discarded within hours. iv fluids may be needed to treat more severe dehydration. if the lamb or kid is too weak to stand, iv fluids are indicated. isotonic fluids containing electrolytes should be given to replenish losses. glucose can be added to fluids to make a % to . % solution. sodium bicarbonate also may be administered, especially if the dehydration is severe. a rule of thumb is to give one fourth of the calculated fluid need (see appendix ii) as isotonic bicarbonate ( . %). extra potassium ( to meq/l) can be added to fluids because most animals are severely dehydrated from diarrhea and depleted in potassium, even though their blood potassium levels may be elevated. if extra potassium is added, acidosis must be corrected concurrently. after correcting the dehydration, the keeper or clinician can offer oral electrolyte-enriched fluids to replace ongoing losses caused by continued diarrhea. removing milk or milk replacer from the diet is not recommended. young animals need nutrients, and even high-energy, glucose-containing electrolyte solutions are no substitute for milk. animals should continue to receive milk replacer in normal amounts or be allowed to nurse; they can be supplemented with oral electrolytes if necessary. animals being hand fed should be offered small amounts frequently to help minimize problems. electrolytes should never be mixed with milk, but should instead be given in separate feedings. if lactose deficiency is suspected, lactase drops or capsules (available in health food stores) can be added to milk or milk replacer. nsaids (flunixin meglumine, . to . mg/kg iv; ketoprofen, . to . mg/kg iv) are beneficial, especially if toxemia is involved, as in etec, enterotoxemia, and salmonellosis. it is the authors' opinion that antimicrobial agents should be reserved for proven outbreaks of salmonellosis and for animals with other causes of diarrhea that do not respond to fluid therapy and nsaids; these drugs should only be administered parenterally. the use of oral coating agents and antacids is popular, but it has not been shown to be beneficial and is not therapeutically logical in light of the pathogenesis of these diseases. probiotics may be beneficial in reestablishing the normal flora of the small intestine. the authors' rule of thumb is that nothing should be given orally except milk, oral electrolytes, and probiotics. ensuring adequate intake of high-quality colostrum and minimizing stress are important for prevention of all neonatal diseases. a normal lamb or kid will stand and nurse within minutes to hour of birth. the ingestion of colostrum within to hours is essential in preventing hypothermia and hypoglycemia and decreasing the incidence of various diseases. lambs or kids born as twins or triplets, weak or injured neonates, those born during severe weather, those born from a dam with dystocia, and those delivered by cesarean section are all candidates for colostrum supplementation. if supplemental colostrum is provided, it should be good-quality colostrum from females that have tested negative for johne's disease, ovine progressive pneumonia (opp), and caprine arthritis-encephalitis (cae). mixing colostrum from several cows decreases the incidence of the "cow colostrum-associated" hemolytic disease sometimes seen in lambs. if the lamb or kid is unable to nurse, it should be tube fed ml/kg of colostrum. the veterinarian or animal handler can sit comfortably holding the lamb or kid in sternal recumbency in the lap. a to french soft feeding tube is then lubricated, inserted into the side of the mouth, and passed slowly. if the tube is placed in the trachea, the lamb or kid will become uncomfortable and may shake and cough. the tube may be palpated on the left side of the throat. after the tube has been slowly passed to the thoracic inlet, colostrum can be administered by gravity flow (see chapter ) . prepartum shearing of the dam may decrease the ingestion of feces by lambs. good sanitation of lambing and kidding areas is paramount in management programs that stress prevention. the presence of organic matter interferes with the effectiveness of many disinfectants, so removal and proper disposal of feces, carcasses, and placentas are essential. when disposing of waste material containing either cryptosporidium or giardia, the keeper should be careful to avoid contaminating water sources. infected animals should be isolated to prevent spread of the infection throughout the flock. in general, infected animals should remain in the environment where the infection was first diagnosed, because it is already contaminated. removing pregnant ewes or dams to a clean area before lambing or kidding helps minimize the continued spread of disease. if possible, lambs and kids already born but not showing clinical signs should be removed to a third area. if "safe" pastures are maintained for internal nematode control, they are ideal for use in an emergency situation to control these diseases. although some animals may appear normal, they may be incubating and possibly shedding the infective agents of a disease. if such animals are moved with pregnant females, they can be a source of contamination in a clean area. if healthy lambs and kids cannot be moved to a third, relatively safe area, they should be left with the clinically infected animals because they have already been exposed. . schultheiss p: diarrheal disease in calves, large anim vet ( ) the differential diagnosis list for acute and chronic diarrhea in small ruminants is very long. the most common cause of diarrhea in adult sheep and goats is parasitism; another major cause is johne's disease. both of these diseases are discussed in the following sections. other causes of acute diarrhea include rumen acidosis, peritonitis, endotoxemia, and ingestion of toxins. the list of toxins that cause diarrhea also is very long, and often the diarrhea is not the primary clinical sign. some of the more common toxins that produce diarrhea are arsenic, toxic amounts of salt, levamisole, copper, oak, selenium, and pyrrolizidine alkaloids. salmonella species and chronic enterotoxemia can cause diarrhea in adult animals. coccidiosis can occur in adults under severe stress or in animals that possess limited immunity because of lack of exposure. hepatic and renal disease and copper deficiency are sometimes accompanied by chronic diarrhea, but weight loss is a more common sign in adults. etiology and pathogenesis. sheep and goats are infested with many of the same gastrointestinal nematode parasites as cattle, but these parasites tend to either be species-specific or have some amount of host specificity. sheep and goats are susceptible to the same nematodes and tend to share resistance to those that infect cattle and horses. the major gastrointestinal nematodes that parasitize pastured sheep and goats are haemonchus, ostertagia, trichostrongylus, cooperia, nematodirus, oesophagostomum, and bunostomum species. the acronym hotc comes from the first letter of each of the first four genera of parasites listed. the specific parasites that produce disease vary from flock to flock. climate usually determines which parasites are of clinical significance on a farm, and the weather determines when the parasites will be transmitted and infective. in much of the united states, haemonchus is the most significant parasite with respect to both clinical disease and anthelmintic resistance. most of these parasites affect the abomasum or small intestine of young, recently weaned animals and occasionally adult animals. sheep (and, to a lesser extent, goats) that are older than months may be less susceptible. overcrowding and overgrazing with concurrent pasture mismanagement and malnutrition usually increase susceptibility to these parasites. inadequate nutrient or protein intake may result in greater susceptibility. the life cycle appears to be similar in most of these parasite species. adults lay eggs that are passed in the feces; except for nematodirus species, the eggs hatch under favorable environmental conditions. the larvae go through several free-living developmental stages becoming infective. when the infective larvae are ingested by the host, the parasite completes its life cycle as an adult. trichuris eggs are the infective stage and can survive for extended periods in dry lots or barns. however, trichuris is associated with minimal pathology. during dry environmental conditions, fecal pellets tend to trap the nematode larvae, whereas in wet conditions, larvae are released onto the pasture. therefore drought conditions followed by rain can result in devastatingly high rates of pasture contamination as larvae that have remained in fecal pellets are released. very high environmental temperatures result in shorter survivability of some stages of infective larvae. most of the larvae have adapted the ability to over-winter, but can survive only for short periods outside the host during spring. nematodirus is an exception in that the developmental stages leading to infective larvae occur while the microbe is still encapsulated in the egg. however, compared with other species of parasite, nematodirus is of minor importance. nematodirus battus may pose a threat to young, newly weaned and therefore immunologically naive grazing lambs. the hookworm bunostomum also is different, as it may infect the host by either oral ingestion or percutaneous penetration. with the exception of the small intestinal parasite strongyloides, lambs or kids fed indoors or in dry pens tend to be free of parasites. clinical signs. all intestinal nematode infections produce similar signs, although infection with the more rarely encountered bunostomum may perhaps result in more profound anemia. if they infest the animal in sufficient numbers, all nematodes may cause poor growth, decreased feed conversion, decreased milk production, weight loss, diarrhea, anemia, ventral edema (bottle jaw), midline edema, and death. again, all these parasites can potentially result in disease, but haemonchus is the most devastating, particularly in more temperate regions. diagnosis. antemortem diagnosis of nematode infestation is made by examining the feces for nematode eggs. although a direct fecal smear can be examined, the mere presence of parasite eggs is not helpful in determining the parasite load of an animal or animals. quantifying of the epg of feces is the best way of estimating parasite loads. the quantitative mcmaster's technique for determining the epg of feces is shown in box - . common nematode eggs are shown in figure - . treatment and control program. after taking a thorough history of the previous parasite control program used on a farm and determining its effectiveness, the clinician can design and implement a new control program. , however, before deciding to implement a deworming program, the clinician should decide which parasites are in need of control and whether control of these parasites is cost effective in a particular flock. whenever possible, a dewormer that can reduce epg counts on the farm by % should be identified and used for at least a year. of all the parasite prevention programs, strategic deworming or a combination of strategic and tactical programs appear to produce the best results. , , strategic deworming is used when most of the parasites are inside the animals and not on the pasture. in northern climates, strategic deworming can best be carried out during the winter, when the nematode parasites are in a hypobiotic state. when environmental conditions are inhospitable for the survival of the infective larvae, some of the most pathogenic nematodes (e.g., haemonchus) may become hypobiotic; that is, they assume a state of arrested development. they may then mature to the adult stage when environmental conditions become conducive for the survival of their eggs or larvae. preventing or decreasing the numbers of maturing adults by killing the larvae before the periparturient rise in parasite egg production and pasture contamination is an excellent management tool. , , unfortunately, in warmer, more temperate to subtropical environments, this method is less effective because larvae can survive the environment for longer periods. the addition of a protein supplement overlapping the expected periparturient rise has been shown to decrease the number of parasite eggs shed around the time of parturition. however, the cost of the protein supplement may outweigh its benefits. a strategic program entails the use of an anthelmintic agent that is capable of killing encysted larvae. animals are then moved to parasite-free or safe pastures-areas in which the level of parasite contamination is too low to result in infection of grazing animals. examples of safe pastures include pastures where sheep or goats have not grazed for to months in the spring or fall, respectively (and depending on the climate); pastures used for hay production; new pastures (i.e., those used for corn, cotton, or other crops); and pastures grazed by horses or cattle. the use of safe pastures is paramount in any de-worming program. rotating pastures after less than months during the warm part of the year or less than months during cooler months is ineffective. however, if pastures are tilled and replanted, by the time new grazeable forage is available, infective parasite larvae will be dead or significantly decreased. an alternative to pasture rotation is to perform an initial strategic deworming before lambing or kidding and follow it with two to four more dewormings at week intervals throughout the lambing and kidding period. [ ] [ ] [ ] treating lambs or kids at weaning and moving them to a safe pasture is a form of strategic deworming. in lambs or kids to be sold at an early age, the administration of a single anthelmintic treatment followed by a move to a safe pasture may be all that is required. a "double treat and move" system is required for lambs kept for to weeks after weaning, particularly during the summer. , this form of strategic deworming requires two treatments to weeks apart as well as two safe pastures. in northern climates where animals are moved to a dry lot or barn for the winter, a strategic anthelmintic administration as animals are moved off pasture can help reduce the parasite burden through the winter. if this deworming is followed by minimal or no exposure to grazing areas and another dewormer is administered before the spring rise in fecal egg counts, the total parasite burden on spring pasture can be drastically reduced, effectively controlling parasites until summer or fall. tactical deworming programs are used to remove parasites from their hosts before they enter their reproductive phase and can contaminate the pasture. an example of tactical deworming is treating animals to days after a rain, particularly if the rain has followed a drought. parasite transmission is worse in most flocks during this time as pastures become heavily contaminated. mcmaster's counts of more than epg in the spring or more than epg in the fall warrant tactical deworming. , , opportunistic deworming and salvage deworming are usually less effective in long-term flock management. many times salvage deworming programs are used to save the lives of heavily parasitized animals. if animals are dewormed only after showing signs of parasitism (e.g., bottle jaw, anemia), animal and flock productivity have already been depressed. deworming during handling for other procedures (e.g., castration, vaccination, shearing) is an example of an opportunistic program. it is convenient but is not conducive to long-term flock health. flock work should be scheduled around parasite management programs, not vice versa. , suppressive deworming programs entail the use of anthelmintics at regular intervals, usually every to weeks. suppressive programs are labor-intensive, tend to be very expensive, fail to identify animals with superior immunity to parasites, and ultimately result in anthelmintic resistance despite initial effectiveness. , as a general rule, the more frequently deworming occurs, the more quickly resistance is attained to anthelmintics. after deworming, only resistant parasites remain to infect the animal and they are able to reproduce freely, resulting in proliferation of resistant strains. , using drugs that remain in tissues at inappropriately low concentrations and treating and retaining immunocompromised animals encourage the development of anthelmintic resistance. practices that ensure adequate dosages, proper treatment techniques, and appropriate types of anthelmintics should be emphasized. , the clinician should do everything in his or her power to minimize the incidence of anthelmintic resistance, both through their own actions and by counseling owners in proper use of deworming drugs. the product development market for anthelmintics is the cattle industry. the small sheep and goat markets simply use drugs made available for cattle. because most available anthelmintics are highly effective in controlling parasites, anthelmintic resistance in sheep and goats must be avoided. the anthelmintics that have been used previously on a flock, the route of administration (e.g., po, sc, im, pour-on), and the length of use should be determined. few dewormers are approved for use in sheep and goats, but many approved for use in cattle and horses may be effective. , if sheep graze with goats that harbor anthelmintic-resistant parasites, the sheep also may become infected. however, if sheep are allowed to graze while the goats browse and the two groups rarely mingle, less parasite movement will occur between these species. resistance to macrolides (e.g., ivermectin, doramectin, moxidectin) does occur. resistant worms are generally not very tolerant of cold temperatures and therefore resistance to this drug class in northern environments is not as large a problem as it is in more temperate or subtropical zones. although moxidectin is not approved for use in sheep and goats in the united states, it has been shown to be effective in cases where ivermectin resistance is encountered. still, this drug should be avoided until all other anthelmintics have failed. craig , has suggested that clinicians refrain from injecting or using pour-on macrolide preparations designed for cattle in small ruminants. this practice may enhance the development of resistant strains of some internal parasites because of inappropriately low drug absorption (with pour-on use) or long-term subtherapeutic levels (with injection). if resistance to tetrahydropyrimidines (e.g., morantel, pyrantel) occurs in a flock, levamisole also may be ineffective. morantel and levamisole resistance in parasites appears to be sex-linked. therefore if animals are not exposed to these drugs for several years, reversion to susceptibility can occur. , if resistance to one of the benzimidazole dewormers has been documented in a flock, some resistance to all members of that class is likely. benzimidazole-resistant haemonchus species appear to be more virulent, produce more eggs, cause greater environmental contamination, and survive in the environment as free-living larvae for longer periods. benzimidazole-resistant parasites apparently do not revert to susceptible forms, even over long periods. therefore the clinician or keeper should exercise caution to minimize resistance. benzimidazole efficacy can be improved by increasing dosages, dividing dosages into two treatments administered at -hour intervals, and instituting pretreatment fasting. if resistance to numerous classes of anthelmintics occurs on a farm, combining two of the resistant classes of dewormers (fenbendazole and levamisole) has proven effective. when using combined dewormers, the clinician should administer the full therapeutic dosage of each. anthelmintics are metabolized at different rates by sheep and goats. goats may require larger dosages of some dewormers than sheep. craig has suggested that if no dose rate is known for a particular anthelmintic for sheep or goats, the animals should be treated at twice the suggested cow dosage. pour-on anthelmintics designed for cattle tend to be of limited value when used topically on either goats or sheep. table - . to maximize a parasite control program, anthelmintics that appear effective should be used for only year before a new class of deworming drug is used. more frequent rotation (after less than year) of anthelmintic agents hastens resistance and should be avoided whenever possible. whenever a flock is dewormed, animals should be treated based on the heaviest animal in the group and not on the group's average weight. underdosing can hasten the formation of parasitic resistance and therefore should be avoided. holding the sheep or goats in a dry lot overnight or feeding only dry hay for to hours before and hours after deworming appears to improve the efficacy of some orally administered anthelmintic agents (benzimidazole). limiting feed intake before deworming slows the rate of passage of ingesta through the bowel, enhancing drug effectiveness. , , feed should never be withheld from sick or debilitated animals or late-term females. , , most dewormers may effectively control adult or larval parasites but are ineffective against eggs. therefore animals should be kept on a dry lot for as long as days after deworming, then moved to a safe pasture. use of this procedure minimizes parasite egg contamination of the new pasture because most of the egg-contaminated feces is voided within hours of deworming. if more than one dosage appears on the drug label, the larvacidal dose should be used (fenbendazole at mg/kg rather than mg/kg). anthelmintic effectiveness can be determined by comparing a mcmaster's fecal epg on the day of deworming with one taken to days later. if less than a % drop in epg is found, anthelmintic resistance exists and the animals should be switched to another class of dewormer. although it is a controversial method, the authors have used this technique to identify anthelmintic effectiveness for many years and on many farms and ranches. , the authors randomly collect feces from % to % (or a minimum of animals) of the sheep or goats on the farm. a composite sample is prepared by combining equal amounts of stool from all animals. craig has suggested that combining stool samples from many animals alters the accuracy of the tests because great individual variation in fecal egg counts occurs among animals. composite egg counts more accurately reflect parasite burdens in groups of young animals, and individual fecal examinations are more accurate in adults. , still, the authors prefer to use composite samples unless obvious differences in stool character or body condition score exist among the sampled animals. anthelmintic resistance can be minimized by using drugs that reduce fecal egg counts by %. pre-and postdeworming changes in epg should be evaluated yearly or whenever resistance is suspected. in vitro methods of assessing flock parasite resistance also are available at some diagnostic laboratories. in most in vitro tests, larvae are hatched from collected feces and the sensitivity of different anthelmintics is determined by larval exposure. these tests are very accurate but tend to be quite expensive. the most effective method to prevent anthelmintic resistance is to not use deworming drugs at all. one of the most overlooked management procedures is the identification and selection of parasite-resistant sheep and goats. some breeds or familial lines within breeds have excellent parasite resistance (e.g., gulf coast native and barbados sheep, some strains of spanish, pygmy, and tennessee myotonic-fainting goats). one study comparing boercrossed goats with non-boer crosses found that the boer crosses had significantly more parasite infestations. only a small number of flock members contribute the greatest amount of environmental parasite contamination because susceptible animals shed the most eggs in their feces. animals with the lowest epg in a flock may be those that possess the most parasite resistance. salvage deworming programs should generally be avoided, but they may be used as aggressive selection criteria. that is, animals that do well with little or no deworming, particularly those grazing heavily contaminated pastures, should be identified and retained in the breeding flock. those that become infected should be dewormed to salvage them or save their lives and then sold when possible. proper record keeping and identification of all animals is paramount in selecting for parasite resistance. , this aggressive approach can yield excellent results if it is carefully implemented, but devastating losses can occur if it is poorly managed. when introducing new animals to a flock, keepers should have biosecurity programs in place to limit the introduction of new or potentially anthelmintic-resistant parasites. new flock additions should be kept in a dry lot for weeks and dewormed at least twice with two different classes of dewormers during this period. the effectiveness of the anthelmintic agent used should be deter-mined by fecal examination before the animal is allowed contact with the rest of the flock. other nontraditional chemical methods of parasite control are used by some owners. some appear to be worthless (e.g., diatomaceous earth), but others (e.g., nematophagous fungi, herbal dewormers) may prove effective in some situations. pathogenesis. the most common gastrointestinal tapeworm of sheep and goats seen in north america belongs to the genus moniezia. cestodes (tapeworms) are usually of more concern to owners than clinicians, who generally consider them only incidental low-grade pathogens, particularly in adult animals. still, several to -foot-long tapeworms can compete with the host for nutrients, hinder normal gut motility, and excrete some toxic wastes into the host's gastrointestinal tract. mature tapeworm eggs are passed in the feces individually or protected in proglottides, which are usually visible to the owner. the eggs embryonate and infect a mite, a small pasture-living arthropod that serves as the intermediate host. a sheep or goat ingests the mite while grazing, allowing the tapeworm to complete its life cycle. clinical signs. tapeworms may rarely cause disease in lambs and kids less than months of age. anecdotal reports suggest a cause-effect relationship between heavy tapeworm infestation and an increased incidence of c. perfringens enteritis, digestive disturbances (e.g., diarrhea, constipation), poor condition, and anemia. ulceration at the site of attachment may be seen on necropsy. rarely species of trypanosoma, the fringed tapeworm, may cause liver condemnation. a presumptive diagnosis can be made by finding proglottides in the stool, eggs on direct smears, or eggs on fecal flotations (see figure - ).treatment with albendazole ( mg/kg), fenbendazole ( to mg/kg), or praziquantel ( to mg/kg) may be effective either with a single treatment or with daily therapy (e.g., fenbendazole daily for to days). because of the free-living nature of the arthropod intermediate host, animals are readily reinfected after treatment, which may give rise to the false assumption that the therapy was ineffective. again, tapeworm infestation may result in disease, but often it is easier to blame the tapeworm segment seen in the stool as a cause of disease than to implicate the unseen thousands of hotc complex parasites in the abomasum and small intestine of the animal. , johne's disease johne's disease (also called paratuberculosis) is a chronic wasting and diarrheal disease caused by the bacteria my-cobacterium avium subspecies paratuberculosis. transmission of the organism is primarily by the fecal-oral route. young animals are more susceptible to infection than adults. it can be transmitted through milk and placenta. pathogenesis. bacterial shedding in feces and milk and transplacental transmission is more common in animals showing clinical signs. [ ] [ ] [ ] therefore the offspring of infected animals and especially the offspring of animals showing clinical signs are most likely to acquire the infection. after an animal is exposed, it will either clear the organism or develop a chronic, persistent infection. the infection is most commonly isolated to the ileal regions of the small intestine, where it causes granulomatous thickening of the intestine and subsequent malabsorptive diarrhea. infected animals may be asymptomatic for years. clinical signs. morbidity rates are low (approximately %), but for every animal with clinical signs, several exist in the subclinical state, and may be a source of both horizontal and vertical transmission. both sheep and goats appear to remain asymptomatic until to years of age. the most consistent clinical sign in sheep and goats is chronic weight loss. chronic diarrhea occurs in approximately % of cases. signs may appear with or be exacerbated by stress, especially after parturition. , hypoproteinemia and chronic mild anemia are the only consistent clinicopathologic findings. because of their low protein levels, infected animals can develop submandibular edema. diagnosis is by culture of the organism from feces. unfortunately, this testing takes between and weeks, but it can detect % to % of clinically infected goats. sheep strains of johne's disease and some goat variant strains seem to be more difficult to culture in media used to identify cattle strains of the disease.therefore fecal culture in sheep and goats appears to be of limited benefit. , a relatively inexpensive and easily performed method of identifying approximately % of all clinically infected animals is acid-fast staining of fecal smears. , a pcr test of feces also is available, but its sensitivity is lower than that of fecal culture. good diagnostic results can be obtained with serologic testing for antibodies (e.g., agar gel immunodiffusion [agid], enzyme-linked immunospecific assay [elisa], complement fixation) in animals showing clinical signs. the specificity of all the serologic tests is greater than % in sheep and goats with signs of clinical disease, although the sensitivity is not as high. [ ] [ ] [ ] therefore a positive serologic test in an animal showing clinical signs indicates that the animal has johne's disease. however, the disease cannot be ruled out with a negative test. sheep and goats appear to respond differently in regard to the formation of antibodies. sheep tend to develop antibodies in the later stages of the disease, whereas antibodies may be detected much earlier in the goat. the agid test appears to be the best serologic test currently available. , the elisa and complement fixation tests can cross-react with corynebacterium pseudotuberculosis, making them of limited value in flocks with caseous lymphadenitis infections. , necropsy diagnosis is based on the finding of thickened, corrugated intestines, especially in the area of the ileum. acid-fast staining of impression smears (taken from the ileum and ileocecal lymph nodes) can help yield a quick diagnosis. the staining of numerous clumps of acid-fast rods is highly suggestive of johne's disease. prevention. johne's disease has no effective treatment, so prevention and control are imperative. however, preventing the introduction of johne's disease into a herd can be difficult. because animals with subclinical infection may not shed the organism or may shed only small quantities of it, fecal culture is helpful only if a positive culture is obtained. the sensitivity of serologic tests of animals with subclinical disease is low and variable among flocks. , negative test results in subclinically infected animals are common. however, the specificity of serologic tests remains high, and therefore a positive test is a valid reason to not purchase an animal. because johne's disease also occurs in cattle, supplemental colostrum supplies should come only from dairy herds with no history of johne's disease. after johne's disease is diagnosed in a herd, several control measures can be taken. sanitation is important because the organism is highly resistant in the environment (able to survive more than year under most conditions). reduced stocking rates, frequent cleaning of pens, and use of automatic waterers decrease fecal transmission. keepers should cull the offspring of infected animals. culling animals based on the results of agid tests or fecal culture of the flock is recommended. animals should be tested at least once a year. more frequent testing as resources allow speeds the identification of infected animals. a vaccine for cattle is only available in some locales and clinicians or keepers may require official permission to use it. vaccine use does not eliminate infection, but it can decrease herd prevalence, delay the onset of clinical signs, and decrease cross-transmission by infective bacterial shedding in the feces. any cause of intestinal obstruction that occurs in other ruminants may occur in sheep and goats. most of these diseases produce abdominal discomfort and occasionally abdominal distention. diagnosis can be difficult because rectal palpation cannot be performed on small ruminants. abdominal radiographs and ultrasonography may help differentiate these diseases, but exploratory surgery may be required to obtain a definitive diagnosis and select appropriate treatment. intussusception is more common in young animals, but it can occur in adults. it occurs when one segment of the intestine telescopes into an adjacent segment. any portion of the intestine can intussuscept, but the ileum and ileocecal junction are the most common areas involved. when intussusception occurs, the lumen of the intestine narrows to the point of obstruction. the initiating cause is not always known. , it is associated with an intestinal mass in adults and enteritis in young animals. oesophagostomum infestations have been implicated as a cause in sheep. clinical signs. the initial complaint is colic (manifested as kicking at the abdomen, repeated rising and lying down, and vocalization) followed by low-grade pain. true colic signs are variable in lambs and kids. in some cases, after the initial colic episode subsides, animals show no evidence of pain until the abdomen becomes distended. the time between the initial intussusception and abdominal distention depends on where the blockage occurs. intussusception of the ileal area may take several days to cause bilaterally symmetric abdominal distention. fecal output is scant, and what little there is may be dark or tarry, or may contain mucus. dehydration becomes evident, hypochloremic metabolic alkalosis may develop and rumen chloride levels may increase with obstructions of the duodenum. diagnosis. abdominocentesis may yield fluid compatible with a transudate (increased protein concentration and leukocyte numbers). radiography and ultrasonography reveal fluid-distended intestinal loops. occasionally the intussusception itself can be visualized with ultrasonography or palpable through the abdominal wall. if the disease is not treated, intestinal rupture and peritonitis can occur. treatment. surgical correction is required. if the intussusception is corrected early, the prognosis is good in the absence of peritonitis. fluid support is needed to correct dehydration and metabolic abnormalities. fluids should be administered iv until rumen function returns. ringer's solution with added calcium (approximately ml calcium borogluconate per liter) and potassium ( to meq/l) is a good choice for fluid therapy. ingested foreign bodies or bezoars can obstruct portions of the intestines. , the signs are similar to those of obstruction caused by intussusception and depend on the part of the intestine that is blocked. in some cases the obstructing body can be seen with radiography or ultrasonography. surgical removal is required for treatment. cecal volvulus and torsion of the root of the mesentery occur sporadically in sheep and goats. , extreme abdominal pain, rapid abdominal distention, and circulatory collapse are typical signs. immediate surgical correction and circulatory support are needed. atresia of the colon, rectum, and anus can all occur as congenital problems. the clinical sign of progressive abdominal distention usually is noted in the first week of life. atresia of the anus can be diagnosed on physical examination, but atresia of the colon and rectum may require contrast radiography for a definitive diagnosis. surgical establishment of anal patency can be performed for atresia ani. a permanent colostomy may be required for atresia of the colon and rectum. atresia of the anus and rectum are considered heritable in cattle. in the authors' experience, atresia ani is more common in sheep than in goats. if surgical correction of atresia ani is attempted, the animal should be neutered or kept out of the breeding program because of the potential genetic basis for this condition. occasionally a slight bulge in the skin may occur where the anus should be located, especially in male lambs. ultrasonography can be used to locate a fecesfilled rectum. for surgical correction, the clinician should locate the area where the anus should be, prepare it with sterile technique, and infiltrate it with a local anesthetic. the surgeon then makes a circumferential incision to remove the overlying skin covering the rectum. an alternative is to make an x-shaped incision into the rectum. treated animals should be given mineral oil, dss, or stool softeners as needed. ileus of the small intestine is a pseudo-obstruction that occurs when there is an absence of intestinal motility. the animal's failure to pass ingesta leads to signs similar to intussusception. the cause of ileus is usually unclear, but it often occurs secondary to systemic diseases. the same elements that cause rumen stasis may potentially result in intestinal stasis and ileus. symptomatic treatment with nsaids for pain and inflammation and fluids for dehydration is usually curative. however, if signs persist, surgical exploration is indicated to rule out true obstructive diseases. pathogenesis. infection of the peritoneal lining of the abdominal cavity may lead to septic peritonitis. common causes include uterine tears; rupture of the rumen or abomasum secondary to rumenitis, abomasitis, or abomasal ulcers; trocarization of the rumen for bloat; and rupture of the intestine secondary to obstruction. clinical signs. signs depend on the severity of the condition. abdominal discomfort and distention, dehydration, injected mucous membranes, depression, and death can all occur in cases of peritonitis. the presence of a fever is variable, both heart rate and respiration rate are usually elevated, and respiratory effort may be guarded. animals may be febrile early, but have a normal to low body temperature as the condition progresses. abdominal ultrasound can be useful in locating pockets of fluid for abdominocentesis, which usually yields fluid with increased protein concentration and leukocyte numbers. on occasion, intracellular bacteria are observed on cytologic examination. the presence of extracellular bacteria is not diagnostic because accidental enterocentesis can occur. culture of abdominal fluid and subsequent antimicrobial sensitivity tests are indicated for the implementation of proper treatment. the causative organisms vary depending on the source of the bacteria. rumen bacteria are typically gram-negative anaerobes, and e. coli and other enteric bacteria are common if the intestine is the source of infection. exploratory surgery may be required to diagnose a gastrointestinal rupture. the cbc can be normal but often shows an inflammatory leukogram and, in severe cases, a degenerative left shift. treatment. treatment includes the prescription of appropriate antimicrobial agents, the administration of nsaids for pain and endotoxemia, and fluid support for dehydration. the prognosis is guarded, especially if an intestinal rupture has occurred. pathogenesis, clinical signs, and diagnosis. rectal prolapse is more common in sheep than in goats. this evagination of the rectal mucosa and rectal structures (and possibly the descending colon) is usually associated with excessive straining. straining is seen in lambs with diarrhea caused by coccidiosis, salmonella, or dietary imbalances, in ewes or ewe lambs with vaginal prolapse, in males with urolithiasis, and in animals grazing lush forage (particularly legumes such as alfalfa and clover). it also can occur secondary to chronic coughing, short tail docking, and the use of growth implants. [ ] [ ] [ ] rabies also can cause chronic straining and rectal prolapse. [ ] [ ] [ ] [ ] [ ] regardless of the cause, after the rectal mucosa becomes everted and exposed, irritation of the mucosa causes further straining, which exacerbates the problem. venous drainage of the prolapse may be compromised, but the arterial supply usually remains intact and contributes to the swelling. rectal prolapses are graded as type i to iv, based on the portion of rectum and distal colon that is everted. a description of these grades in shown in table - . treatment. correction may be cost prohibitive for feedlot lambs, and immediate slaughter is recommended. in more valuable animals, very mild, early cases can be treated with frequent application of hemorrhoidal ointment designed for humans and manual replacement of the prolapsed mucosa into the anus. the authors try to avoid applying purse-string sutures in the anus because they tend to serve as a nidus and result in further straining. however, if less aggressive therapies do not relieve the problem in hours, a purse-string suture may become necessary, particularly in type i and ii prolapse. in all cases and modes of treatment, restricting feed for to hours and administering mineral oil is recommended. dusty feedstuffs (concentrates, pellets, hay) should be avoided because they may contribute to coughing, which exacerbates this condition. adding molasses to feeds and lightly wetting hay may help reduce problems with dust. purse-string suture is easily performed. the prolapsed tissue and perineal area are washed with mild soap and lubricated with petroleum jelly or hemorrhoidal ointment before the prolapse is replaced. , after replacing the prolapsed mucosa, the clinician inserts a tubular object (syringe case, wooden dowel, gloved finger) into the rectum. he or she then places a purse-string suture of nonabsorbable suture material ( - nylon) in the skin around the anus, tightens it around the tubular object, and ties it off. the suture should be placed around the anus using a cutting needle, and entering and exiting at the o'clock position. tying the knot above the anus ensures that less fecal soiling of the suture will occur. the clinician should tie the suture in a bow knot to allow easy identification over the next few days and then remove the tubular object. the suture should be tight enough to prevent prolapse but loose enough to allow feces to pass. the clinician should regularly reevaluate the animal and if possible loosen the purse-string suture at -hour intervals until no tension exists. after a full day of no tension, the suture can be removed. if animals continue to strain, an epidural anesthetic can be administered. petroleum jelly and hemorrhoid gels should be placed on the anus daily. , the injection of counterirritants around the rectum ( ml or less of lugol's iodine) either alone or in conjunction with anal purse-string suturing is a quick and inexpensive treatment. , , the clinician inserts an -gauge needle ( cm) deeply into the skin around the anus at , , and o'clock. an injection at the o'clock position should be avoided because swelling around the urethra can result in obstruction. for more severe cases, submucosal resection or rectal amputation of tissue may be necessary. , rectal amputation can be performed with a prolapse ring or suture technique. prolapse ring usage is a salvage technique. the clinician inserts the prolapse ring into the rectum and places an elastrator band or suture around the area to be amputated to induce vascular compromise and necrosis of tissue. if a ligature is used, it should be tightened to allow purchase on the tube or ring. a fibrosis is induced just proximal to the band or suture, and mucosa subsequently grow across the areas. strictures, peritonitis, and abscesses are possible complications, but this technique may be useful as a field procedure. submucosal resection can be performed under epidural analgesia after the prolapse and the perineal area have been surgically prepared. the clinician places two spinal needles ( to cm) at -degree angles to each other to mm distal to the anal sphincter and through the entire prolapse. a circular incision is made to mm distal to the spinal needles through the mucosa and around the outside of the anus. another circular incision is made just distal to the caudal extent of the prolapse into the point where the mucosa reflects on itself on the innerside of the prolapse. the clinician connects these two incisions with a longitudinal incision parallel to the prolapse and dissects the mucosa between the circumferential incisions. the mucosal edges are then sutured with a simple interrupted pattern using a suitable absorbable suture material. after completely suturing the mucosal surfaces, the clinician removes the two spinal needles and places a purse-string suture in the anal sphincter. placement of the suture and follow-up care are the same as described for the purse-string suture technique. submucosal resection decreases the incidence of both peritonitis and stricture formation compared with other surgical techniques, but it is expensive. in all of these techniques, a caudal epidural anesthetic ( % lidocaine, . ml per kg) is recommended to decrease straining and ease pain from the procedures. , a xylazine epidural ( . to . mg/kg as sufficient [qs] to ml with % lidocaine) may give longer relief (approximately to hours) from straining than lidocaine. an alcohol epidural also may prevent straining for extended periods. either isopropyl alcohol or ethanol can be used to demyelinate the motor and sensory nerves. this type type i small, circular amount of submucosal swelling good prognosis if there is no damage to mucosa; protrudes through anus; probing reveals a pocket purse-string suture, iodine injection, submucosal or fornix just inside anus resection type ii slightly more circular submucosal and mucosal good prognosis if treated quickly and no mucosal swelling, possibly containing retroperitoneal rectal damage; purse-string suture, iodine injection, tissue from anus; probing reveals a pocket just submucosal resection, rectal amputation inside anus type iii complete prolapse containing part of the if there is vascular injury to the descending colon, retroperitoneal structures of the rectum and the prognosis is guarded to poor; submucosal descending colon; probing reveals a fornix just resection or rectal amputation are the methods of inside anus; the affected portion of the descending choice colon does not prolapse through the anus type iv the descending colon appears as a tube, and has if there is vascular injury to the descending colon, intussuscepted through the rectum and anus; prognosis is poor; abdominal exploration may unlike the previous types, in this case a probe be required to determine the extent of damage to or finger can be inserted into the prolapse through the descending colon the anal sphincter for a distance of to cm of anesthesia can be permanent and therefore should be used only for animals intended for slaughter. because of the potential for some loss of sciatic nerve function, the clinician should perform a test injection of a local anesthetic ( % lidocaine) before using alcohol. if the epidural appears effective and no ataxia or muscle weakness of the rear limbs occurs, the clinician can inject a mixture of equal parts of lidocaine and alcohol into the sites where the test epidural was performed. possible problems with alcohol epidural anesthesia include injection site necrosis, sciatic nerve dysfunction, and the inability to void feces. regardless of the type of epidural used, the clinician clips, washes, and dries the area before placing a small needle ( -to -gauge [ . cm]) in the most cranial yet moveable intracaudal vertebral space-usually c to c or c to c . the needle is placed on the dorsal midline, with the needle degrees to the skin and the hub moved slightly caudal, and then slowly advanced. liver abscesses usually occur as a result of chronic rumenitis in cattle, but they are rare in sheep and goats. they can occur in feedlot lambs and kids and other animals fed rations high in grain. in lambs and kids, septicemia or extension of an umbilical vein infection can cause liver abscesses. in most cases, however, liver abscesses are an incidental finding. weight loss, anorexia, depression, and decreased production (growth, milk) may occur. in adults, corynebacterium pseudotuberculosis is the most common cause. actinomyces pyogenes and fusobacterium necrophorum also are cultured from abscesses. [ ] [ ] [ ] liver enzymes may or may not be elevated. diagnostic ultrasonography of the liver may help detect abscesses, especially if they are numerous and widespread. however, no specific treatment or control measure is available. many of the preventive protocols used for feeder cattle apply to the control of abscesses in sheep and goats. these include slowly introducing concentrates into the diet, offering long-stemmed hay free choice, and including rumen buffers (alkalinizing agents) and antimicrobial agents in the feed. pathogenesis. fatty liver occurs in conjunction with pregnancy toxemia in ewes and does during the last month of gestation. it is most common in thin or obese ewes or does with a single large fetus, twins, or triplets. during late gestation, particularly in obese females, the abdominal space is filled with accumulated fat and an ever-expanding uterus. because of the lack of rumen space, these females have difficulty consuming enough feedstuffs to satisfy energy requirements. in most management systems, late gestation occurs during the winter months, when less pasture is available and poorer-quality feedstuffs are offered. energy requirements for ewes and does carrying twins or triplets is greatly increased during the final months of gestation because % to % of fetal growth occurs during this time. ewes with twins require % more energy, and those with triplets need % to % more dietary energy. pregnancy toxemia also occurs in association with anorexia caused by other diseases (foot rot, opp, cae) or sudden stresses (feed or weather changes, predator attacks, hauling). whatever the initiating cause, a period of anorexia and lack of sufficient energy intake result in a negative energy balance. these animals begin to mobilize body stores of fat and transport them to the liver. in the liver, fat is catabolized to glycerol and free fatty acids (ffas). ffas can be used in the citric acid cycle (krebs cycle) as an energy source, but not in the direct formation of glucose. anorexic animals have less ruminal substrate available for production of the glucose precursor propionic acid. however, oxaloacetate, which is an integral part of the citric acid cycle, is removed from the cycle and converted into glucose. depletion of oxaloacetate inhibits the normal citric acid cycle's function, inhibiting the use of ffas. as the pool of ffas increases, they are converted to ketone bodies or repackaged into lipoproteins. because ruminants are not efficient at transporting lipoproteins out of the liver and back to the adipose stores, the lipoproteins overwhelm the liver's ability to handle this massive buildup, resulting in a fatty liver. because less substrate is available for glucose formation, more oxaloacetate is "cannibalized" from the citric acid cycle, further inhibiting the body's ability to use ffas. this in turn causes the continued accumulation of ketone bodies. hypoglycemia, hy-perketonemia, and potentially uremia and death can occur. clinical signs. animals suffering from fatty liver or pregnancy toxemia become anorexic and depressed, display altered behavior, and become recumbent. some are constipated, grind their teeth, have a ketone smell to their breath, and suffer from dystocia. neurologic signs include blindness, circling, incoordination, star-gazing, tremors, and convulsions. , death can occur if the condition is left untreated. in the case of in-utero fetal death, maternal septicemia-endotoxemia and death are common sequelae. diagnosis. diagnosis is based on clinical signs, the presence of multiple fetuses, and typical clinicopathologic findings. cbc results may be normal or show an eosinophilia, neutropenia, and lymphocytosis. these animals may or may not be hypoglycemic, but ketoacidosis, hypocalcemia, and hypokalemia are common. , liver enzymes are usually within normal limits but occasionally may be increased. azotemia, both from dehydration and secondary renal disease, is a common finding, and a fatal uremia may occur. blood concentrations of ß-hydroxybutyric acid greater than mmol/l are consistent with pregnancy toxemia. urinalysis will be positive for both ketones and protein. urine is collected from sheep by holding the nares and from does by frightening them and then allowing them a perceived escape when they stop, squat, and void. although not commonly performed, liver biopsy can help determine the extent of fatty infiltration. this syndrome must be differentiated from hypocalcemia, hypomagnesemia, polioencephalomalacia, encephalitis, lead toxicity, and cerebral abscesses. treatment. very early cases (before the animal exhibits recumbency) may be treated with oral or iv glucose. a balanced electrolyte solution with extra calcium ( ml of a % calcium borogluconate per liter), potassium ( to meq/l), and % dextrose is needed. in some cases, sodium bicarbonate is valuable in treating acidosis (see appendix ii). energy intake must be increased, and propylene glycol can be administered ( to ml every hours) as a glucose precursor. rumen transfaunation and supplementation with vitamin b complex (including vitamin b , biotin, niacin, and thiamine) also are recommended. after females become recumbent, treatment must be very aggressive. removal of the fetuses is crucial in these cases. chemically inducing parturition (by administering . to mg of prostaglandin f a or . mg/ kg of cloprostenol in does and to mg of dexamethasone in ewes) and giving the ewe or doe medical support (fluids, b vitamins, glucose) while waiting is a useful protocol in some cases. unfortunately, during the time before parturition, endotoxemia from dead fetuses further compromises the female. for this reason, the authors recommend immediate cesarean section on depressed moribund animals (see chapters and ) . the owner should be forewarned of the poor prognosis for animals already in a moribund state. fluid support during and after surgery is crucial. regardless of the therapeutic plan, the animal should be offered a palatable, energy-rich, highly digestible feedstuff. the keeper and clinician should take care to minimize the risk of a confounding disease during convalescence (e.g., lactic acidosis, polioencephalomalacia). prevention. fatty liver and pregnancy toxemia can be prevented through proper nutrition. maintaining animals in proper body condition throughout the year and making sure energy and protein levels are adequate in late gestation (see chapter ) are two key preventive measures. , the owner/manager should be taught to assess body condition in individual animals and should maintain emergency stores of feed in case of severe weather or natural disasters. the requirement for energy may be one and a half to two times maintenance for single fetuses and two to three times maintenance for multiple fetuses. prevention of concurrent disease that may further increase energy demands or cause anorexia (e.g., intestinal parasitism, foot rot) is crucial. the keeper should take care to increase the grain portion of the diet slowly because anorexia from rumen upset can lead to this disease. ewes should be offered . to kg of a cereal grain (corn, oats, barley, or a combination) every day during the final months of gestation; does can be offered ⁄ to kg of grain. keepers should maintain ewes and does at a body condition score of . to (see chapter ) throughout gestation and evaluate the animals' energy every to weeks. ultrasonography can help identify females with multiple fetuses. these animals should be separated into groups and fed accordingly. ultrasonographic determination of fetal numbers is best accomplished between days and after breeding with a . mhz transducer; a mhz transducer produces better results between days to . either type of transducer may be of value and these windows of time may be expanded by the ability of the operator (see chapter ) . determination of fetal numbers may be enhanced by shearing the hair or fiber in front of the udder, applying a coupling substance to the skin, and viewing as much of the abdomen as possible, building a mental image of its structures and the number of fetuses while systematically moving from one side of the posterior abdomen to the other. keepers and clinicians should ensure that ewes are healthy and free of chronic diseases (e.g., opp, cae, foot rot, chronic parasitism) and that a good-quality trace mineral salt mixture is available free choice. the addition of lasalocid ( . to mg/kg/day) or monensin ( mg/kg/day) to the feed or mineral mixture enhances the formation of the glucose precursor propionic acid and improves the efficiency of feed use. however, monensin should be used with caution because toxicity may occur; the agent should comprise no more than ppm of the complete diet. the inclusion of niacin ( g/head/day) in a feed supplement or mineral mixture helps prevent pregnancy toxemia. supplementation with lasalocid, monensin, or niacin should begin to weeks before the females give birth. shearing in the last trimester also is recommended in ewes. many sheep producers routinely clip the wool around the vulva. if complete body shearing is performed, the incidence of fatty liver or pregnancy toxemia may be decreased. sheared sheep require less energy to walk and graze. sheared ewes also tend to shiver on cold days, exercising the enzyme systems that promote the more efficient use of ffas as energy substrate. these ewes tend to seek shelter during cold weather, which may decrease lamb losses resulting from hypothermia. obviously, if ewes are to be shorn, keepers should make adequate shelter available. keepers should avoid hauling or moving females during late gestation. proper predator control measures should be maintained. good hoof care programs should be in place on farms or ranches where grazing is the predominant form of nutrient intake. sheep and goats should have their teeth checked to ensure good dentition before the breeding season. animals with poor teeth should be culled. measuring serum b-hydroxybutyric acid concentrations is useful in assessing energy status in ewes. values of . to . mmol/l suggest a negative energy balance. keepers should take steps to correct the problem by feeding better-quality, more digestible feedstuffs. white liver disease is a form of fatty liver disease reported only in angora and angora-cross goats and sheep. it is associated with cobalt deficiency. pathogenesis. cobalt is needed by rumen microflora to produce cyanocobalamin, or vitamin b , which is a coenzyme for methylmalonyl-coa mutase. in turn, this enzyme is needed to convert propionate to glucose through the krebs cycle. cobalt deficiency leads to the accumulation of methylmalonyl-coa, or methylmalonic acid, which is converted to branched chain fatty acids that accumulate in the liver. , high-grain diets that are fermented to propionate coupled with deficient or marginal cobalt intake may predispose to this condition. , white liver disease has not been reported in the united states, but ill thrift from cobalt deficiency has been observed. it is therefore possible that the disease goes unrecognized. clinical signs. signs are most commonly seen in young animals, and include ill thrift, anorexia, and diar-rhea; sheep may exhibit photosensitization. clinicopathologic findings include a macrocytic, normochromic anemia and hypoproteinemia. , diagnosis. abnormal serum or liver concentrations of vitamin b or liver cobalt are the basis of diagnosis. liver cobalt concentrations on a dry matter basis of . Ϯ . ppm were reported in goats with white liver disease, compared with . Ϯ . ppm in controls. treatment and prevention. sheep can be treated with oral cobalt ( mg/head/day) or vitamin b injections. the condition can be prevented by including cobalt in the ration by feeding a good-quality trace mineral salt. both fasciola hepatica and fascioloides magna can infest sheep and goats. the disease occurs along the gulf coast and in the pacific northwest and great lakes areas. clinical signs. f. hepatica infestation usually causes acute disease in sheep and goats but can present as a chronic condition. chronic disease is the result of the mature flukes in the bile ducts and is manifested in depressed growth and milk production. acute disease occurs when large numbers of immature flukes migrate at once, particularly in animals with limited immunity to flukes. signs include anorexia, depression, weakness, dyspnea, anemia, ascites, colic-like signs, dry feces, and sudden death. the clinical signs are identical to those of nematode infestations (i.e., chronic weight loss, ill thrift, diarrhea, anemia, hypoproteinemia). similar but more severe signs occur with f. magna infection, which is usually fatal. , , diagnosis. antemortem diagnosis of fluke infestation can be difficult. finding eggs in feces is diagnostic for f. hepatica. eggs are only produced by adults and not in great numbers, so a negative fecal test cannot preclude acute or chronic fascioliasis. fluke eggs do not float in routine fecal flotation methods used for nematode diagnosis; a sedimentation technique should be used for suspected fluke infestations. to perform a sedimentation test, the clinician mixes to g of feces with ml of tap water and strains the mixture through a tea strainer into a beaker. the sediment can be examined minutes later under a dissecting microscope. eggs are light yellow to golden and have an operculum at one end (see figure - ). f. magna does not mature, so eggs are not produced and fecal examination is of no value. most fluke infestations are discovered by finding the flukes at necropsy or slaughter. an elisa test may be available in the future. , , cbcs of affected animals may indicate eosinophilia and anemia. increased liver enzymes and hypoalbuminemia also are occasional findings. because antemortem diagnosis is difficult, the clinician should institute fluke treatment after ruling out other differential diagnoses if the possibility of fascioliasis exists. if fascioliasis is diagnosed at necropsy, the remaining animals in the herd should be treated. because flukicides available in the united states are highly effective only against mature flukes, the timing of treatment is important. in the southern portions of north america the snails are ingested in the spring and the flukes migrate in the summer and mature in the fall. in cooler, northern climates, snails may remain active during summer, so flukes can mature in the fall and into the winter. clinicians should begin treatment in the southern united states in the late summer or early fall. a single treatment in late winter or early spring is commonly used in the northern climates of north america. albendazole ( - mg/kg orally) and clorsulon ( mg/kg orally, mg/kg sc) are very effective against adult f. hepatica. , , clorsulon has no efficacy against nematode parasites but is highly efficacious against both adult and late-stage immature flukes. albendazole ( mg/kg orally) is somewhat useful in controlling f. magna at weeks after infestation, and clorsulon is effective only at very high dosages. [ ] [ ] [ ] unfortunately, neither agent can kill % of f. magna, and only a few remaining flukes can be fatal. control of fluke infestations is difficult, although timely treatment of animals can decrease infec-tions in successive years. decreasing exposure is the key to control. eliminating the snail is impractical, but fencing off low-lying areas may prevent ingestion. depending on local fluke life cycles, keepers should avoid grazing animals on areas with high fluke populations during peak infection times. areas where water stands or flows over grazing pastures, streams, and irrigation ditches (particularly those with clay soil) are high-risk zones. cysticercus tenuicollis is the larval stage of the dog tapeworm taenia hydatigena, of which sheep and goats are intermediate hosts. the larval stage migrates through the liver, then attaches to the liver or other abdominal organs and causes black, winding tracts and cysts in the liver. acute disease occurs only with large numbers of cysticerci and is characterized by depression and weakness resulting from liver damage. the chronic cystic stage is usually asymptomatic. no treatment is available and control is problematic because it requires treating infestation in dogs and preventing contact with dogs. , , pathogenesis. copper (cu) toxicosis is more common in sheep than in goats. goats appear closer to cattle than sheep in their ability to store and handle cu and resist toxicosis. toxicity results from chronic accumulation in the liver from the ingestion of excess cu in relation to molybdenum (mo) or sulfate in the diet. in sheep, a cuto-mo ratio greater than Ϻ leads to the accumulation of excess cu. the most common sources of excess cu in sheep and goats are trace mineral mixtures and feeds formulated for cattle or horses. clinical signs are often absent during the chronic accumulation phase. acute disease is seen when cu is suddenly released from the liver in large amounts. stress usually precipitates this acute phase. acute release and subsequent high blood cu concentrations cause an acute hemolytic crisis, resulting in anemia, hemoglobinuria, and acute renal failure. existing hepatic disease (such as that caused by liver flukes) may predispose animals to this condition. some breeds seem to be prone to cu absorption and storage problems (merino sheep), whereas others tend to be more resistant and prone to deficiency (pygmy goats) (see chapters and ). clinical signs. anorexia, depression, diarrhea, and weakness are all signs of cu toxicity. many affected animals are found dead with hemolysis and icterus. signs of abdominal pain and diarrhea are sometimes present. port wine-colored urine is evidence of hemoglobinuria. hemoglobinemia produces icterus of the mucosal membranes and fever. diagnosis. on clinicopathologic examination, anemia, hemoglobinemia, hyperbilirubinemia, increased liver enzymes, and azotemia are present. urinalysis reveals hemoglobinuria and isosthenuria. the combination of azotemia and isosthenuria indicates acute renal failure. definitive diagnosis of acute disease requires measurement of cu concentrations in serum. normal blood cu concentrations are approximately to mg/dl in sheep and goats. , , these concentrations increase to -fold with an acute hemolytic crisis. on necropsy, kidney cu concentrations are the most diagnostic because liver concentrations may be normal from release into the bloodstream. generally kidney concentrations greater than ppm and liver concentrations greater than ppm on a dry matter basis are diagnostic. , if tissue copper is reported in wet weight, the conversion to dry tissue weight can be estimated by multiplying the tissue concentration by a factor of . treatment. treatment of acutely affected animals is often futile. it consists of supportive therapy for the acute renal failure and anemia and attempts to lower liver cu stores. fluid therapy for the acute renal failure (see appendix ii) is of therapeutic value, and a blood transfusion may be needed if the pcv drops precipitously. ammonium tetrathiomolybdate ( . mg/kg iv or . mg/kg sc on alternate days for three treatments) is the most economical agent for treatment for acute cases. in valuable animals, d-penicillamine ( to mg/kg bid or mg/kg sid po for days) increases urinary cu excretion. trientine is used in human beings, but has shown variable results in sheep. treatment of the remainder of the flock should include the administration of ammonium molybdate ( to mg/head/day po) and sodium thiosulfate ( to mg/head/day po) for weeks. stress should be minimized, so keepers and clinicians should delay routine maintenance procedures such as deworming and hoof trimming until after treatment. the offending source of cu should be eliminated. prevention. avoiding high dietary cu (more than ppm), a high cu-to-mo ratio (greater than Ϻ ) in the feed, cu-containing foot baths, and other sources of cu is crucial. including supplemental mo in the diet to lower the cu-to-mo ratio to Ϻ to Ϻ is beneficial. this requires to ppm of mo in many instances. often too much emphasis is placed on the trace mineral component of the diet. the clinician should be aware that even if no cu is added to the trace mineral mixture and the element does not appear on the product label, the mineral mixture may still contain cu. many components of mineral mixes are contaminated with cu (zinc sulfate may contain ppm of cu, dicalcium phosphate may contain more than ppm of cu). therefore the clinician needs to perform a dietary analysis to find and correct the problem. pathogenesis. the liver is vulnerable to toxic insult because one of its major functions is detoxification. the most common plants that are gastrointestinal and liver toxins are shown in table - . clinical signs depend on the cause. acute, severe toxicity is more common with chemical toxicosis, whereas plant toxins usually cause chronic disease. a thorough history is important and in many cases inspection of the animals' environment is required. clinical signs. the clinical signs of toxic hepatitis can be vague. animals may only show anorexia and depression. icterus is more common with hemolytic diseases and is not always seen with liver disease. photosensitivity is a common clinical feature in ruminants and hepatoencephalopathy also can occur. clinicopathologic data are more helpful in diagnosing acute toxicity. serum ast and ldh levels can increase with hepatocellular necrosis but are not liverspecific, so muscle injury and disease must be ruled out. these enzymes also increase if serum is not separated from a blood clot in a timely fashion. increased levels of alkaline phosphatase (ap) and ggt indicate biliary stasis. ap also is not liver-specific, but increased serum levels of ggt are very specific for liver disease. ggt also increases in some hepatocellular diseases, so testing for its normal concentrations is important. unfortunately, all of these enzymes can be normal with liver disease, especially if it is chronic. hyperbilirubinemia, hypoglycemia, low blood urea nitrogen (bun), and hypoalbuminemia are not always evident as classically taught. if hepatoencephalopathy is suspected, blood ammonia concentrations may be elevated. blood ammonia analysis may be impractical in the field because the blood should be kept on ice, and the test should be performed within minutes of collection. to enhance the accuracy of blood ammonia analysis, the clinician should collect blood from a normal control animal for comparison. ammonia concentrations three times those of the control animal are diagnostic. liver biopsy remains the most valuable tool in diagnosing liver disease. although clotting dysfunction may occur in liver disease, it is an uncommon complication in ruminants and should not discourage the clinician from performing a liver biopsy. treatment. if the intoxication is caught in the acute stage, activated charcoal ( g per adult animal) can be given. supportive care, especially fluid support with dextrose solutions, is the mainstay of therapy. low-protein diets may suppress ammonia production temporarily, but they can be detrimental over time depending on the production status of the animal. if photosensitization occurs, animals should be housed indoors if possible, and broad-spectrum (systemic or topical) antibiotics may be necessary to control secondary bacterial dermatitis. corticosteroids (dexamethasone . to mg/kg iv or im) may be indicated in early cases of photosensitization to decrease inflammation. neurologic signs can be controlled with phenobarbital (initial dose: to mg/kg iv diluted in saline and administered over minutes; subsequent doses: to mg/kg iv diluted in saline, as needed up to tid). diazepam (valium) is contraindicated in hepatoencephalopathy because it may worsen signs. congenital hyperbilirubinemia, or black liver disease, occurs in mutant corriedale sheep (dublin-johnson syn-drome). this is a genetically recessive condition. it is characterized by an abnormality in the excretion of conjugated bilirubin and phylloerythrin and is often seen in animals consuming green forage. clinical signs include anorexia, photodermatitis, and icterus. liver biopsy of affected animals reveals dark to black granules in otherwise normal hepatocytes. the syndrome first manifests itself in lambs around months of age. a similar condition occurs in southdown lambs around months of age (gilbert's syndrome). this too is a recessive condition that causes decreased hepatic uptake of phylloerythrin and bilirubin, with concurrent renal failure. signs include icterus, photodermatitis, and ulceration around the ears and mouth. a liver biopsy reveals normal hepatic tissue. in both of these conditions, animals should be kept out of sunlight and fed minimal various tumors of the liver, including fibrosarcoma, lymphosarcoma, and cholangiocellular carcinoma, have been reported. , the use of ultrasonography and ultrasound-guided liver biopsy may aid in diagnosis. the umbilicus is an opening in the ventral abdominal wall that allows passage of the umbilical vessels and allantoic stalks. this opening should close within a few day of birth. the failure of this opening to close properly is termed umbilical hernia. the hernial sac has an inner peritoneal layer and an outer layer of skin. these hernias are probably of genetic origin but may occur as sequelae to umbilical remnant infection. the opening in the abdominal wall is perceived as a ring on palpation. if the clinician can insert more than one finger into the hernial ring or if the hernia persists for more than to weeks, surgical intervention is indicated. penning. clamps or rubber bands may be of value for closing small hernias (those less than cm in diameter). the clinician should either lightly sedate the animal or infiltrate the skin around the hernia with a local anesthetic ( % lidocaine). the animal should be placed on its back and held by a technician-helper. any viscera prolapsing into the hernial sac should be replaced into the abdomen. the clinician then inserts two metal pins (baby diaper pins can be used) through the skin and on opposite sides of the hernial ring, just on the edge of the linea alba. the pins should be placed deep enough to sit next to the abdominal wall. slight tension is placed on the skin in the center of the umbilical sac, pulling it away from the abdomen. when the clinician is confident that all viscera have been cleared from the hernial sac, he or she places an elastrator band between the pins and the abdominal wall. this results in ischemic necrosis of the skin. the skin will slough and the abdominal defect will heal in to days. lambs should be given tetanus prophylaxis. this procedure and other clamping techniques are useful in females and some males. however, urine scalding of the skin may occur in some males. clinicians should closely monitor animals that have undergone clamping. surgical resection. in cases in which the hernial ring is larger than cm, surgical intervention should be carried out. animals can be sedated and then infiltrated with a local anesthetic or placed under general anesthesia. the area around the hernia is clipped and surgically prepared. the clinician opens the hernial sac and introduces a finger into the abdomen to ensure that no viscera have adhered to the inner lining of the ring and that no enlarged or infected umbilical remnants are present. he or she then carefully excises the ring and closes the defect in the abdominal wall. this closure can be made by simply opposing the abdominal wall with a horizontal mattress pattern stitch (absorbable suture). an alternate closure of the abdominal wall is to suture the peritoneal lining in a separate pattern and close the abdominal wall defect so one side of the defect is pulled to overlap the other side. the upper free edge is sutured to the opposite wall with a near-far-far-near pattern. the authors choose not to employ surgical techniques that slow this procedure. the subcutaneous tissue can be closed with simple interrupted pattern using absorbable suture and the skin should be closed with whatever pattern the clinician prefers. animals should be given tetanus prophylaxis and antibiotics. they should be closely monitored for signs of sepsis and surgical failure. exercise should be limited for to days after surgery. infections of the umbilical arteries (omphaloarteritis) and veins (omphalophlebitis) and urachal disease can occur because of failure or partial failure of passive transfer of colostral antibodies and subsequent sepsis. contamination of the umbilicus, retracting of these structures after stretching and breaking, and chemical damage (from strong tincture of iodine) to the amniotic remnants are other possible causes. , , dipping the umbilicus with iodine or iodine-chloriodine substances is a common practice. aggressive use of these chemicals may precipitate serious inflammation of the cord. excessive torsion of the umbilical cord, distention of the proximal urachus, and some genetic factors may all be associated with patent urachus, which also may occur as a sequela to omphaloarteritis or omphalophlebitis. clinical signs and diagnosis. the clinical signs include umbilical swelling, pain, and occasionally drainage or discharge of the umbilical stump. palpation and transabdominal ultrasonographic evaluation reveal an enlarged cord-like structure ascending from the umbilicus cranially (umbilical vein) or caudally (urachus or um-bilical artery). ultrasonographic evaluation may indicate an abscess or thickened tissue. patent urachus is associated with dermatitis, urine scalding of the ventral abdomen, and urine dribbling. if the urachus becomes infected it may leak urine intraperitoneally or subcutaneously. both of these developments may be identified with abdominal palpation, ballottement, ultrasonographic evaluation, and, when indicated, paracentesis. the cbc may indicate neutrophilia. blood culture is indicated if sepsis occurs simultaneously. occasionally infection of the internal structures may occur with no outward umbilical swelling. deep abdominal palpation and/or the use of real-time ultrasound are necessary to attain a diagnosis. animals with umbilical infections also may have signs of septicemia, anorexia, depression, joint distention, and fever. treatment. if a patent urachus occurs without inflammation of the associated tissues, it can be cauterized daily with iodine or silver nitrate. however, if it remains patent for more than days, it should be surgically closed. the animal should be placed under general anesthesia (see chapter ) . the area around the umbilicus should be clipped and surgically prepared, and the animal should be placed on a broad-spectrum antimicrobial agent to hours before surgery. the clinician opens the abdomen lateral to the umbilicus and digitally explores the adjacent area for adhesion formation. the urachus should be identified and followed to the urinary bladder. after this, the clinician should amputate the urachal attachment to the bladder and close the bladder with a double-layered inverting pattern (cushing). the abdominal wall, subcutaneous tissue, and skin are closed as described for umbilical hernia repair. on occasion some cases of omphalophlebitis-omphaloarteritis can be treated medically. prolonged antibiotic therapy with a broad-spectrum antimicrobial agent (ceftiofur . mg/kg sid or oxytetracycline mg/kg sc every hours) may be attempted. however, if medical therapy is ineffective, the infected umbilical remnants should be marsupialized or excised. the authors prefer more aggressive, surgical removal of the umbilical rem-nants. as with urachal surgery, the abdomen should be opened lateral to the umbilicus. depending on the severity of infection and the amount of tissue involved, the clinician may need to perform extensive dissection of necrotic tissue and possibly intestinal resection. if the infection of the umbilical vein extends to and involves the liver, marsupialization of the umbilical vein is an effective method of therapy. , the clinician can pull the vein to the most cranial portion of the abdominal incision and suture it to the muscle layers and skin before closing the abdomen as described for umbilical hernia repair. however, a preferable method is to close the abdominal wall, pull the transected umbilical vein through, and suture it to a separate stab incision. this may help minimize the incidence of abdominal wall herniation. only monofilament, absorbable, non-gut suture material should be used. the venous stump should be flushed daily with antiseptic solution ( % chlorhexidine, . % povidone iodine), and the animal should be maintained on antibiotics for more than days. the venous stump usually closes within a month. prevention. umbilical infections can be prevented or drastically reduced by ensuring adequate intake of goodquality colostrum. lambs and kids also should be only minimally stressed (particularly during the first to days of life) to enhance colostral absorption. in some management scenarios, proper dipping of the navel with non-caustic materials also helps reduce the incidence of this disease. indigestion in ruminants bloat or ruminal tympany diseases of the goat on the effect of xylazine on forestomach motility in sheep anorexia during febrile conditions in dwarf goats: the effect of diazepam, flurbiprofen and naloxone bloat in kids experimentally induced lactic acidosis in nubian goats: clinical, biochemical, and pathological investigations diagnosis of enteric disease in small ruminants ruminal lactic acidosis in sheep and goats biochemical alterations in serum and cerebrospinal fluid in experimental acidosis in goats commonly encountered diseases of goats generalized aspergillosis in dairy sheep rumen papillomas in sheep effect of monensin on development of ruminal parakeratosis in fattening lambs, zentralblatt fur veterinarmedizin lactic acidosis foreign body syndrome in goats-a report of five cases traumatic gastritis in sheep and goats goat medicine references . sherman dm: causes of kid morbidity and mortality: an overview, proceedings of the fourth international conference on goats enteric infections in young goats and their control enteritis and diarrhea goat medicine infectious gastrointestinal diseases of young goats occurrence of cryptosporidia, rotaviruses, coronavirus-like particles and k ϩ escherichia coli in goat kids and lambs jensen and swift's diseases of sheep escherichia coli in domestic animals and humans development of resistance with host age to adhesion of k ϩ escherichia coli to isolated intestinal epithelial cells references . smith bp: alterations in alimentary and hepatic function control programs for gastrointestinal nematodes in sheep and goats epidemiology of internal parasites: effects of climate and host reproductive cycles on parasite survival anthelmintic resistance: the selection and successful breeding of superior parasites control and prevention of specific diseases of sheep and goats herd rp: control of periparturient rise in worm egg counts of lambing ewes production medicine and health programs for goats the effects of dietary protein on establishment and maturation of nematode populations in adult sheep nematode infections-cattle, sheep, goats, swine parasites affecting goats in the southeast, proceedings of goat production and marketing opportunities in the south grazing management strategies for the control of parasitic diseases in intensive sheep production systems goat medicine helminth parasites of the gastrointestinal tract. nematode infections in cattle, sheep, goats, and swine parasite control programs in sheep and goats epidemiology and control of trematodes in small ruminants use of anthelmintic combinations against multiple resistant haemonchus contortus in angora goats preliminary investigation of anthelmintic efficacy against gi nematodes of goats and susceptibility of goat kids to gastrointestinal nematode infection johne's disease in sheep and goats paratuberculosis in small ruminants, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference paratuberculosis in small ruminants, deer, and south american camelids comparison of the absorbed elisa and agar gel immunodiffusion test with clinicopathologic findings in ovine clinical paratuberculosis paratuberculosis in a large goat herd serodiagnosis of paratuberculosis in sheep by use of agar gel immunodiffusion corynebacterium pseudotuberculosis infection in sheep and the complement fixation test for paratuberculosis obstructive intestinal diseases intussusception in goats goat medicine duodenal obstruction by a phytobezoar in a goat general surgical techniques for small ruminants: part ii, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference rectal prolapse in ruminants and horses jensen and swift's diseases of sheep rectal prolapse in food animals. part : cause and conservative management rectal prolapse in food animals. part ii: surgical options, comp cont ed pract vet : , . references . fetcher a: liver diseases of sheep and goats goat medicine a retrospective study of hepatic abscesses in goats: pathological and microbiological findings hepatic lipidosis jensen and swift's diseases of sheep hepatic lipidosis associated with cobalt deficiency in omani goats epidemiology of internal parasites: effects of climate and host reproductive cycle on parasite survival epidemiology and control of trematodes in small ruminants efficacy and safety of albendazole against experimentally induced fasciola hepatica infections in goats efficacy of an injectable ivermectin/clorsulon combination against fasciola hepatica in sheep albendazole treatment of experimentally induced fascioloides magna infection in goats efficacy of clorsulon against fascioloides magna infection in sheep evaluation of clorsulon against fascioloides magna in cattle and sheep clinical biochemistry of domestic animals metals and other inorganic compounds interpreting a bovine serum chemistry profile: part i large animal internal medicine large animal internal medicine neonatal conditions, with emphasis on equine neonate umbilical hernia, umbilical abscess, and auricle fistula general surgical techniques for small ruminants: part ii, proceedings of the small ruminants for the mixed animal practitioner, western veterinary conference key: cord- - ztyj z authors: backovic, marija; rey, felix a title: virus entry: old viruses, new receptors date: - - journal: curr opin virol doi: . /j.coviro. . . sha: doc_id: cord_uid: ztyj z the long-sought entry receptors for rubella, sindbis and respiratory syncytial viruses (rv, sv and rsv), together with the missing measles virus (mv) receptor for infection of epithelial cells, were identified in . these have been major developments in the field of virus entry. in addition, was rich in new information about the interactions of mv, rsv and phleboviruses with dc-sign during infection of dendritic cells, a crucial step allowing the virus to breach the epithelial barrier and gain access to the lymph nodes. this faciliates dissemination to susceptible tissues where it can develop a vigorous and sustained replication, to eventually target specific organs from which it can propagate into the environment and efficiently infect new hosts, closing the merry-go-round of the virus cycle. the long-sought entry receptors for rubella, sindbis and respiratory syncytial viruses (rv, sv and rsv), together with the missing measles virus (mv) receptor for infection of epithelial cells, were identified in . these have been major developments in the field of virus entry. in addition, was rich in new information about the interactions of mv, rsv and phleboviruses with dc-sign during infection of dendritic cells, a crucial step allowing the virus to breach the epithelial barrier and gain access to the lymph nodes. this faciliates dissemination to susceptible tissues where it can develop a vigorous and sustained replication, to eventually target specific organs from which it can propagate into the environment and efficiently infect new hosts, closing the merry-go-round of the virus cycle. a key step in the virus cycle is the process of entry into a new cell, during which the viral genome is translocated across the membrane of the target cell. this process requires specific interactions with a cell surface receptor, or more often, several surface receptors. this can lead to mere 'attachment', resulting in accumulation of virus particles at the cell surface, and the cellular molecules involved are termed attachment factors. a different set of interactions at the cell surface results in an active entry process, either by inducing a conformational change in the virus particle that is necessary for entry, or by inducing uptake of the virion into an endosomal compartment. in this second case, the local environment of the endosome -its acidity, or the presence of cellular proteases, termed convertases -activates the virion to interact with the membrane in such a way that the viral genome is delivered across into the cytoplasm. the cell surface molecules involved in these active processes are called entry receptors. each virus has a different set of cellular surface molecules with which it interacts for entry, and the identification of these receptors is a major goal in contemporary virology. understanding the nature of the receptors may have important implications for understanding tissue tropism and host range, and sometimes the pathogenicity of a virus. beyond the fundamental question of how a virus translocates its genome into the cytoplasm once it is in front of a target cell, an additional important question is how it reaches that particular cell, or the particular tissues in which it can sustain the infection to levels that are sufficient to ensure its propagation to other organisms. such cells are not necessarily at the anatomical site of entry into a susceptible host. the cells with which a virus interacts early during infection of a new organism therefore may be different to those where it sustains the infection, which in turn may be different to the tissues used in the final stages, from which it can efficiently propagate to other hosts. in addition, the receptors involved in each case may also be different. many viruses were found to target dendritic cells for dissemination within a new organism, because these cells are often among the first to be encountered. some viruses, like measles virus, target the tracheobronchial airways at a late stage, such that aerosols can efficiently propagate them through the air. other viruses, like the arthropod-borne viruses (arboviruses) induce high viremia to ensure infection of a biting insect or tick -the vectors that transmit and perpetuate the virus in nature. once in the vector, the virus journey is such that at the late stages it targets the salivary glands to be efficiently transmitted upon a new bite to another vertebrate. in the past year, there have been considerable new findings about the nature of some of the receptors involved, and about the way viruses initially target dendritic cells in order to be transported across the epithelial barrier and for downregulating the immune response of the host. dendritic cells (dcs) are the sentinels of the immune system, activating the innate response and making the link between antigens and lymphocytes to trigger the adaptive immune response [ ] . immature dcs are present at peripheral tissues, deliberately exposed by the organism to invading pathogens. dcs also detect aberrant self-molecules circulating in the extracellular environment -for instance glycoproteins secreted from tumor cells with an aberrant glycosylation pattern. antigen capture by dcs induces their maturation concomitant with migration to the lymph nodes, where the processed antigens are presented to t cells. many viruses have evolved to specifically rely on dcs for their own benefit, either by reprogramming dcs in order to interfere with an efficient immune response by the host organism, and/or by taking advantage of the induced migration of dcs across the epithelium to reach the lymph nodes, from which the newly synthesized virus can gain access to other susceptible tissues. there are several types of dcs in the body, organized into several phenotypic and functional subsets [ ] . all of them express pattern-recognition receptors at their surface, and among these, toll-like receptors and specific lectins for detecting high-mannose glycans. in mammals, the glycoproteins found in the extracellular environment normally have glycan chains that are extensively modified by specific glycosyl-transferases that reside in the golgi apparatus (see [ ] for a recent review). during glycoprotein synthesis, an initial mannose-rich core glycan is attached to the protein cotranslationally, upon translocation of the nascent polypeptide chain across the er membrane after interaction with the signal recognition particle. the mannose residues of this core glycan are then cleaved sequentially in the golgi apparatus, and are replaced by nacetylglucosamine, glucose and other sugar residues to form complex and/or hybrid glycans. the mature glycoproteins exiting the golgi thus essentially have complex n-glycans, with only an internal core of mannose residues left from the initial core glycan [ , ] . high mannose oligosaccharides are therefore rarely found circulating in the extracellular environment in mammals under normal conditions [ ] . however, they can become abundant in the case of certain tumors [ ] , when the malignant transformation affects the glycosylation pattern. similarly, viral proteins are often heavily glycosylated, and the glycosyl-transferase enzymes responsible for processing the high mannose sugars become saturated in the virus-infected cell. this results in high mannose glycans present at the surface of viral proteins, in spite of being synthesized in mammalian cells. several related, ca + -dependent type c lectins specific for high-mannose glycans [ ] are displayed at the surface of different types of dcs. the most studied is dc-sign (cd ) [ ] , which is expressed on myeloid-lineage dcs [ ] [ ] [ ] [ ] [ ] , in activated b cells [ , ] and in some macrophages [ , ] . the related l-sign (cd ) [ ] is displayed on liver sinusoidal endothelial cells, in the lung and lymph nodes [ ] [ ] [ ] . a similar lectin is langerin (cd ) [ , ] , present at the surface of langerhan cells, which constitute a distinct population of immature dcs derived from the bone marrow, located in the epidermis and in stratified mucosal epithelia [ ] [ ] [ ] . all three lectins have been reported to be involved in interaction with viruses. these are just a few examples among many other lectins at the surface of the various types of dcs that have been described (for a review, see [ ] ). dc-sign is a homo-tetrameric type ii membrane protein and c-type lectin. crosslinking of dc-sign at the cell surface by multiple high-mannose sites on the virion triggers uptake of the bound particle, resulting in antigen capture (see [ ] for a recent review). dc-sign is used by many viruses for attachment to dcs, including mv [ ] , hiv- (see the recent review in [ ] ), hcv [ ] [ ] [ ] , influenza a viruses [ , ] , the herpes simplex virus type [ ] , the human cytomegalovirus [ ] , ebola virus [ ] , the sars coronavirus [ , ] , the human t-cell leukemia virus type [ , ] , and arboviruses belonging to the flavivirus genus -dengue virus [ , ] and west nile virus [ ] ) -and to the alphavirus genus, like sv [ ] and aura virus [ ] . in this review, we focus on recent insights for understanding the mechanism by which dc-sign signaling induces the uptake of the virus particle by the cell, beyond the initial attachment step. we thus discuss the role of dc-sign in promoting infection of dcs by mv and rsv and the impact on the immune response of the host. furthermore, we discuss the implications of the newly identified entry receptors for mv, rsv, rv and sv, which were major highlights of the year . bunyaviruses are negative-sense single-stranded rna viruses with a segmented genome. many of them cause severe illnesses in humans, such as encephalitis and hemorrhagic fever (reviewed in [ ] ). they are classified in five genera (hantavirus, nairovirus, orthobunyavirus, phlebovirus and tospovirus), all being arboviruses except for hantaviruses, which are transmitted by rodents. helenius and collaborators [ ] studied two members of the phlebovirus genus as model systems to explore interactions with dc-sign: the non-pathogenic uukuniemi virus (uukv) transmitted by infected ticks, in parallel to the human pathogenic rift valley fever virus (rvfv), which is transmitted by infected mosquitoes. they show that both viruses infect dcs in a dc-sign-dependent manner. raji cells, normally poorly infectable by phleboviruses, become susceptible to infection upon transfection with dc-sign. dc-sign binds to both viruses directly via their high-mannose n-glycans. on the opposite side of the membrane, the cytosolic tail of dc-sign contains several sequence motifs involved in signaling, including the classical dileucine (ll) motif, which serves as signal for its endocytotic internalization. they show that uukv entry into dc-sign expressing cells occurs via endocytosis, and dc-sign is internalized together with the virion. when the ll motif in dc-sign is mutated, the virus still binds to the cells, but is not internalized and there is no infection. this is in contrast to an earlier study that found that the endocytosis motifs of dc-sign had no effect on dengue virus uptake [ ] . using total internal reflection fluorescence (tirf) microscopy, the authors were able to directly observe the clustering of dc-sign molecules -tagged with a monomeric version of the enhanced green fluorescence protein (megfp) -at the cell surface upon uukv binding. this suggests that binding of virions can create a receptor-rich microdomain to induce its endocytosis. indeed, dc-sign cross-linking with antibodies at the cell surface had been shown to induce efficient endocytosis of the lectin in dcs [ ] , so it is proposed that the uukv-mediated clustering of dc-sign leads to activation of signaling pathways that trigger endocytic uptake of dc-sign and bound virus. these findings appear to establish dc-sign not just as an attachment factor, but also as a true receptor required for entry of phleboviruses and their uptake into an endosomal compartment. this is the first report describing a role for dc-sign in virus entry that goes beyond its involvement in virus attachment, as documented before for other viruses. the above studies highlight an early interaction constituting the first stage of the infection of a new organism. nothing is known about other potential receptors that are necessary to sustain phlebovirus infection in the host and allow its dissemination in nature. for instance, for the arbovirus sv, which was also shown to interact with dcs via dc-sign for reaching the lymph nodes [ ] , the studies discussed below have now identified an entry receptor -nramp -that is more likely to be used in these further stages of the infection, and perhaps at all stages, since it is very well conserved between humans and mosquitoes. similarly, phleboviruses infect a wide spectrum of tissues, most of which do not express dc-sign, and so there must be other entry receptors for other tissues both in the mammalian and arthropod hosts. nevertheless, dc-sign is clearly identified as a critical player in the initial transmission after bites by insects or ticks infected by phleboviruses. mv is the type species of the morbilivirus genus in the paramyxovirinae subfamily, which together with the pneumovirinae subfamily makes up the paramyxoviridae family of negative-sense, single-stranded rna viruses. mv disease is characterized by fever, running nose, coughing, and a typical macular rash covering most of the body. importantly, mv infection leads to a generalized immunosuppression that can cause serious complications. for a long time, laboratory-adapted strains of mv were known to use cd for entry [ ] , whereas wild type strains use the signaling lymphocyte activation molecule (slam, also termed cd ) [ ] , although it was suspected that additional receptors were involved. slam/cd functions as a lymphotropic receptor for both clinical and laboratory adapted isolates of mv [ ] . cd is a complement regulatory protein, ubiquitously expressed with the exception of erythrocytes, while slam/cd is expressed on activated b and t cells, monocytes and dcs. cd + dcs, alveolar macrophages and lymphocytes are the first cells to be infected by mv, although cd appears not to be abundant at the surface of dcs. epithelial cells become important for later stages of infection during which the virus is spread via aerosol droplets. this year, in addition to the identification of nectin as a new entry receptor for wild type mv for epithelial cells (reviewed below), new insight into the role of dc-sign to allow cd -mediated entry of mv into dcs [ ] has been brought forth. although it was known that mv interacts with dc-sign for entry into dcs, the mechanism was not clear, since ectopic expression of dc-sign in cho cells was not sufficient to allow cell entry [ ] . schneider-schaulies and colleagues [ ] now show that the interaction of mv with dc-sign at the cell surface signals via the mitogenactivated protein (map) kinases erk and , resulting in the activation of cellular sphingomyelinase (smase) activities. the corresponding cellular enzymes process sphingomyelin lipids present in the membrane to yield phosphocholine and ceramides, and the latter accumulate to form clusters in the plasma membrane. both neutral and acid smase (nsm and asm), become activated as a consequence of dc-sign signaling induced by interactions with mv particles. nsm is anchored at the cytoplasmic leaflet of the plasma membrane, whereas asm is soluble, and is stored in intracellular compartments (reviewed in [ ] ). the authors find that this internal compartment, which is apparently induced to fuse with the plasma membrane, also contains cd /slam anchored in the membrane. this fusion event results in exposure of cd to the extracellular environment, together with the activated asm that degrades sphingolipids. the presence of activated nsm and asm at either side of the plasma membrane leads to the formation of ceramide-rich platforms that concentrate the receptor and allow virus entry. human airway epithelial cells are cd negative, yet wild-type mv can efficiently infect them. this feature propelled the search for an mv receptor in epithelial cells. in , the richardson [ ] and cattaneo [ ] laboratories independently identified adherens junction molecule nectin -also called 'poliovirus receptor-like ', or pvrl -as mv receptor for epithelial cells. both groups used comparative microarray analysis of susceptible versus non-susceptible tumor cell lines to identify the receptor. nectin is expressed during phases of rapid cellular growth, and it is highly abundant in placenta during embryogenesis, as well as in cancer cells. this is in line with the observation that mv efficiently infects adenocarcinoma cells derived from breast, lung and colon cancers, and with reports that lytic mv infection causes regression of certain tumors [ ] . in addition, human smooth-airway epithelial cells, normally expressing low levels of nectin , could be infected by mv independently of cd and slam only when grown in serum-containing medium, which increases nectin expression. antibodies specific for human nectin and transient knockdown of nectin using sirna abolished mv infection of these cells. nectin is a member of the nectin family of ca +independent immunoglobulin-like cell adhesion molecules. they are composed of an extracellular ectodomain, containing three sequential ig-like domains, a single tm domain and a c-terminal cytoplasmic tail (reviewed in [ ] ). the ectodomain is organized such that the n-terminal domain, which is furthest from the membrane, is a variable-type ig domain, whereas the other two are of the constant c -type ig domains, resulting in a 'v-c-c' organization, where v and c are the variable and constant domains, respectively. antibodies directed against the v domain block infection, whereas antibodies directed against the other two domains do not, or block to a much lower extent [ ] . using a fusion protein with a c-terminal fc, the authors tested inhibition with v-fc and vcc-fc, and found that both efficiently inhibited infection. affinity measurements by surface-plasmon resonance indicated that the affinity of the two forms is very similar, measuring a dissociation constant of nm, which is an affinity about -fold higher than that of a similar soluble construct of cd /slam. nectins are localized, together with cadherins, at the adherens junction between cells, forming homotypic or heterotypic interactions in trans to establish and maintain cell-cell connections. members of the nectin family function as receptors for poliovirus (pvr or cd ) and herpes simplex viruses (nectins and ) [ ] [ ] [ ] . the new reports now clearly establish the involvement of nectin in entry of mv into airway epithelial cells. rsv is a common cause of respiratory infections, in particular bronchiolitis, which can be fatal in infants and in the elderly. in contrast to mv and rv, there is no effective vaccine for rsv, and the use of passive immunoprophylaxis with anti-rsv-specific antibodies is expensive and limited to high-risk patients (reviewed in [ ] ). rsv is the type species of the pneumovirus genus in the pneumovirinae subfamily of the paramyxoviridae. like mv, the rsv virion contains two major envelope glycoproteins, called f and g. in addition, it contains a small hydrophobic (sh) protein, which is thought to play the role of ion channel and not to be involved in entry. the f proteins are homologous in all members of the paramyxoviridae family, and are responsible for the membrane fusion reaction. contrary to mv, for which receptor binding by h triggers the fusogenic conformational change in f [ , ] , rsv f does not require g to induce membrane fusion [ ] . g appears to mainly play a role in attachment, and both g and f were shown to interact with gags [ , ] . in addition, f binds to icam- [ ] and g to annexin ii [ ] although these interactions are not essential for entry. while the primary tissues targeted in the lungs are the airway epithelial cells, many reports describe rsv infection of dcs [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in the past year johnson and colleagues showed that the g glycoprotein interacts both with dc-sign and l-sign, although antibodies directed against the lectins do not block infection of dcs [ ] . they show that the interactions of g with the lectin signals via erk and phosphorylation to produce an immunomodulatory effect, and that inhibition of these interactions leads to significantly higher levels of ifn-a, mip- a, and mip- b secretion during the maturation process induced by rsv infection of immature dcs [ ] . in the past year, the first clear identification of a molecule with all the characteristics of an entry receptor for rsv was reported [ ] . indeed, hegele and colleagues performed a virus overlay protein binding assay [ ] that led to the identification of cell surface nucleolin as an rsv entry receptor. nucleolin and rsv f were coprecipitated, demonstrating a direct interaction. rsv infection was decreased in neutralization experiments using anti-nucleolin antibodies, in competition experiments in which the virus was preincubated with nucleolin before addition to the cell cultures, and upon rna interference to silence the expression of cellular nucleolin. nonpermissive sf insect cells became susceptible to rsv infection upon expression of human nucleolin, and rna-mediated knockdown of lung nucleolin in mice was associated with a significant reduction in rsv infection, demonstrating nucleolin involvement in the rsv cycle in vivo. nucleolin also plays a role in entry of another paramyxovirus, the human parainfluenza virus (hpiv- ) [ ] . nucleolin has a dual localization, and is found both at the cell surface and in the nucleus. it is an rna and proteinbinding multi-functional molecule (reviewed in [ ] ) involved in diverse processes such as ribosome biogenesis, chromatin decondensation and transport of nucleolar proteins between nucleus and cytoplasm [ ] . intracytoplasmic vesicles shuttle nucleolin between the cell surface and the nucleolar pool [ ] [ ] [ ] . the cellsurface bound nucleolin has not been well characterized so far. it has been shown to associate with intracellular actin filaments, but because the nucleolin sequence does not contain any potential membrane-anchoring domains, it was proposed to connect to actin via intermediary transmembrane proteins [ ] . nucleolin is expressed on many tissues and its use as rsv entry receptor does not explain the restricted tropism of rsv, which may be rather specified by other yet unknown co-receptors or intracellular cofactors. rv, together with the alphaviruses, belongs to the togaviridae family of positive-stranded rna viruses, and encodes two envelope glycoproteins, e and e . rv e is responsible both for receptor binding and for inducing membrane fusion. rv is the etiological agent of rubella disease, and also of the congenital rubella syndrome (crs), which is associated with intra-uterine infection of the fetus during early pregnancy. rubella disease is also called 'german measles', because its symptoms are similar to the ones caused by mv. similar to mv, rv propagates efficiently by aerosols, but the specific receptors that are targeted by rv are not known. it is possible that dcs play a role in dissemination of rv, although this has not been investigated. rv enters cells by receptor-mediated endocytosis [ , ] . as with its alphavirus cousins, exposure of the virion to low ph in the endosome leads to a fusogenic conformational change in the membrane fusion protein e [ ] . rv infects a wide range of human derived cell lines, indicating that the receptor is likely to be a ubiquitous molecule, or that the virus can interact with different receptors depending on the cell type. in contrast to protease treatment, which had no effect, phospholipase or glycosidase treatment of susceptible cells was reported to decrease rv infection, suggesting that the receptor may not be a protein [ ] . however, tien and colleagues have recently identified 'myelin oligodendrocyte glycoprotein' (mog) as a putative rubella receptor by pull-down experiments of host cell proteins that bind to recombinant rv e [ ] . mog is a highly conserved type i transmembrane protein and a minor component of myelin ( . %). it is present at the surface of mature oligodendrocytes in the central nervous system (cns), and is suspected to function in maintenance of myelin sheaths (reviewed in [ ] ). tien and colleagues demonstrated that the soluble mog ectodomain binds rv particles and blocks binding of the virus to cells [ ] . expression of mog in t cells, which are normally refractory to rv infections, were shown to render them permissive for rv entry and replication. finally, they showed that antibodies directed against mog block rv infection. most research interest on mog has been driven by its role as an autoantigen in demyelinating diseases such as multiple sclerosis (ms). the mog immunodominant epitope that maps to residues - [ , ] is responsible for binding to both t cells and demyelinating antibodies. the crystal structure of the mog ectodomain revealed a classical ig-variable domain fold [ ] , which is found in other adhesion molecules. mog is found as a dimer when purified from cns tissue [ ] , and the crystal packing of the ectodomain indicates contacts that are compatible with mog dimerization in trans to glue the myelin sheaths together [ ] . molecular mimicry between rv protein e and mog had been reported as a cause of the autoimmune response resulting in demyelination, characteristic of ms [ ] . residues - of mog, which map to the signal sequence, had been found to exhibit close similarity to rv e residues - [ ] . however, this particular e sequence is only found in one partial sequence of the structural region of the m rv strain [ ] (accession code aaa . ). numerous sequences corresponding to rv are available in the databank and none of them contains the mentioned e peptide. furthermore, closer inspection reveals that the mimicry e peptide sequence is not present in the complete sequence of the structural polyprotein gene of the same rv m strain, deposited later [ ] (accession code p ). these observations strongly suggest that the reported mimicry was due to a sequencing artifact in the initial submission of the m polyprotein sequence. demyelination appears to be at least one of the underlying pathologies of crs, and rv replication in the brain has been demonstrated in crs [ ] . it is likely that rv infection via mog expressed by oligodendrocytes directly contributes to this process, rather than an autoimmune effect triggered by molecular mimicry as postulated earlier. however, rv has a wide cell tropism, and after initially infecting the nasopharyngeal lymphoid tissues, the virus is transmitted to other hosts through airborne droplets and aerosols. expression levels of mog reported in lymphoid tissue are very low [ ] , but as suggested by cong et al. may be sufficient for infection to occur. more likely, additional receptors in tissues where mog is not expressed exist (which would explain the wide tropism of the virus), and remain to be identified. sv is, together with semliki forest virus, one of the beststudied alphaviruses, a group of arboviruses that includes serious human pathogens such as chikungunya virus, ross river virus or venezuelan equine encephalitis virus (reviewed in [ , ] ). they form a genus within the togaviridae family of positive-strand rna viruses, which also includes the rubivirus genus with rv as only member. alphavirus particles have two envelope glycoproteins, e and e , with e /e heterodimers forming a surface icosahedral glycoprotein shell that encases the viral membrane. alphaviruses enter cells by endocytosis. e is responsible for receptor binding, inducing receptormediated endocytosis, while e drives membrane fusion in response to the low ph of the endosome (reviewed in [ ] ). fusion of alphavirus virions with artificial liposomes can be induced in vitro by exposing purified virions to low ph [ ] , indicating that the fusion trigger is low ph and not an interaction with the receptor. alphaviruses are transmitted by mosquitoes and infect a wide range of insect and mammalian cells. as discussed above, sv interacts with dc-sign for infection of dcs [ ] , which are used for translocation of the epithelial barrier and subsequent dissemination within the organism. the high-affinity laminin receptor [ ] and heparan sulfate [ ] have been reported to enhance, but not to be essential for alphavirus infection into certain cell types. in the past year, cherry and collaborators identified the 'natural resistance-associated macrophage protein' (nramp) as an sv entry receptor [ ] . they performed a genome-wide rnai screen in drosophila melanogaster cells, which are not a natural host for sv. they found that dnramp, the drosophila ortholog of nramp, is required for virus infection. flies mutant for dnramp were protected from the virus. dnramp was shown to physically bind to virus particles, and this interaction was necessary for entry. nramps constitute a family of highly conserved proteins, found in bacteria as well as in insects and mammals. they function as proton gradient driven transporters of divalent metal ions such as fe + . they are integral membrane proteins, spanning the membrane times (reviewed in [ ] ). there are two nramp genes in mammals, nramp and nramp . only nramp is localized at the plasma membrane of cells of peripheral tissues [ ] . high fe + concentrations downregulate nramp expression (reviewed in [ ] ), and as anticipated, iron treatment attenuated sv infection both in mosquito and mammalian cells. this indicated that nramp is also a receptor in the relevant vector host, the mosquito. direct binding of sv to the receptor was demonstrated by coprecipitation of nramp with sv particles. this interaction was lost at high iron concentrations due to decreased presence of nramp in the membrane. although the authors discuss that the conservation of e among different alphaviruses is important ( %), this conservation across alphaviruses concerns the residues that either form the hydrophobic core or are otherwise not exposed on the virion [ ] . the surface exposed residues available for interactions with receptor show, on the contrary, a high degree of variation, in line with the observation that nramp is specific for sv and does not interact with other alphaviruses. the identification of nectin as a receptor for mv closes an important chapter in understanding the biology of this virus. additional recent data have shown that as it enters a new host, it initially infects alveolar macrophages and dcs present in the airways, using cd [ , , ] . however, the fact that cd is not readily available at the surface of these cells was a puzzle, and understanding the role of dc-sign in making cd available for infection is now an important step forward. infection of dcs thus has a dual effect: downtuning the immune response of the host, while at the same time providing a means for transport across the epithelial barrier. dc infection allows the virus to reach the lymphatic organs, where it replicates vigorously and subsequently propagates into lymphoid and myeloid cells of the trachea, located right under the epithelial cell layer. infection of the latter cells through the basolateral side would then result in virus release into the lumen of the tracheobronchial airways, allowing propagation in the environment via aerosols. the mv studies lead the way: rsv has the same primary tropism as mv, targeting dcs to interfere with the immune defense of the host and to breach the epithelial barrier. however, the difference in the case of rsv is that it does not seem to actively involve dc-sign for entry, but rather the interactions with the g glycoprotein induce the necessary signaling to dampen the immune response so that the infection can proceed. the tissue distribution of nucleolin being very broad, it is not understood what restricts the rsv tissue tropism. a scenario similar to that of mv remains possible, although this needs to be investigated further. similarly, rv is very efficiently propagated in aerosols, so that it must reach the airways at late stages of the infection. however, little is known about the overall journey of this virus within the host organism before propagation to other hosts, and receptors other than mog are very likely important. in addition, no experiments have been reported to understand whether rv also targets dcs. the identified mog receptor does, however, shed light on some of the complications of crs, during which the virus disseminates into the cns and causes demyelination. in the case of sv, it was known that many different cell types were susceptible to the infection, from insect to humans, and the fact that the nramp family is so conserved is compatible with this feature. however, many other arboviruses have a very broad tropism as well, and the fact that the use of nramp is restricted to sv among all the alphaviruses was surprising. it is possible that there are other conserved surface molecules that are as conserved as nramp and could be exploited by other arboviruses for entry; this remains therefore an important area of study. the connection between sv infection and iron metabolism is interesting, and brings out another parallel: the relation between the pathogenicity of newworld arenaviruses targeting the transferrin receptor as entry receptor, and iron metabolism in the host [ ] . another important feature for sv and other arboviruses is the use of dc-sign to efficiently infect dcs. the new finding of the past year was that, in the case of the phleboviruses, dc-sign appears to act as a bona-fide entry receptor, and not just as an attachment factors as reported for all the other viruses that target dcs. however, the data discussed above about the interactions between mv and dc-sign suggest a possible reinterpretation: it cannot be ruled out that the interaction of phleboviruses with dc-sign does not trigger the same type of signaling, bringing out an as-yet unidentified entry receptor from an internal compartment into ceramide-enriched clusters at the plasma membrane. this would be compatible with the dc-sign clustering observed in that study, leading to uptake of the virion. taken together, the various virus/host systems analyzed in parallel in this review suggest interesting approaches to spark additional research in each of them, with the mv system as paradigm. in particular, these data highlight the role of dcs in spreading and aggravating the infectionacting as a 'trojan horse' as described in the case of hiv. indeed, dcs appear to be the achilles heel of the host organism, being deliberately exposed to anything that is foreign. the fact that vertebrates have evolved to take this risk witnesses about its importance: the organism is ready to pay the price, because in the absence of dcs the toll would be much higher. the viruses that are being studied today are essentially those that cause disease, which in turn are those that appear to have managed to cope with dcs for their own benefit. fortunately, the great majority of viruses to which an organism is exposed go unnoticed because of the efficient detection by the strategic dc network distributed throughout the body. papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest dendritic cells and the control of immunity nomenclature of monocytes and dendritic cells in blood nabi ir: glycosylation, galectins and cellular signaling intracellular functions of n-linked glycans role of n-glycosylation in trafficking of apical membrane proteins in epithelia the development of retrosynthetic glycan libraries to profile and classify the human serum n-linked glycome high-mannose glycans are elevated during breast cancer progression drickamer k: the c-type lectin superfamily in the immune system multiple modes of binding enhance the affinity of dc-sign for high mannose n-linked glycans found on viral glycoproteins sequence and expression of a membrane-associated c-type lectin that exhibits cd -independent binding of human immunodeficiency virus envelope glycoprotein gp dc-sign, a dendritic cellspecific hiv- -binding protein that enhances trans-infection of t cells identification of dc-sign, a novel dendritic cell-specific icam- receptor that supports primary immune responses hiv gp receptors on human dendritic cells diversity of receptors binding hiv on dendritic cell subsets hiv- envelope triggers polyclonal ig class switch recombination through a cd -independent mechanism involving baff and c-type lectin receptors dc-sign on b lymphocytes is required for transmission of hiv- to t lymphocytes dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin/cd is abundant on macrophages in the normal human lymph node and is not required for dendritic cell stimulation of the mixed leukocyte reaction constitutive and induced expression of dc-sign on dendritic cell and macrophage subpopulations in situ and in vitro segmented helical structure of the neck region of the glycanbinding receptor dc-signr a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans trimeric structure of langerin structural basis for langerin recognition of diverse pathogen and mammalian glycans through a single binding site the skin: initiation and target site of immune responses. recent results cancer res signalling through c-type lectin receptors: shaping immune responses c-type lectin dcsign: an adhesion, signalling and antigen-uptake molecule that guides dendritic cells in immunity measles virus targets dc-sign to enhance dendritic cell infection role of dc-sign and l-sign receptors in hiv- vertical transmission l-sign (cd l) is a liver-specific capture receptor for hepatitis c virus dc-sign and l-sign are high affinity binding receptors for hepatitis c virus glycoprotein e hepatitis c virus glycoproteins interact with dc-sign and dc-signr reading pc: n-linked glycosylation facilitates sialic acidindependent attachment and entry of influenza a viruses into cells expressing dc-sign or l-sign dc-sign mediates avian h n influenza virus infection in cis and in trans dendritic cells mediate herpes simplex virus infection and transmission through the c-type lectin dc-sign human cytomegalovirus binding to dcsign is required for dendritic cell infection and target cell trans-infection ctype lectins dc-sign and l-sign mediate cellular entry by ebola virus in cis and in trans dcsign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus ph-dependent entry of severe acute respiratory syndrome coronavirus is mediated by the spike glycoprotein and enhanced by dendritic cell transfer through dc-sign dc-sign facilitates fusion of dendritic cells with human t-cell leukemia virus type -infected cells dcsign mediates cell-free infection and transmission of human tcell lymphotropic virus type by dendritic cells dendritic-cellspecific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquitocell-derived dengue viruses dc-sign (cd ) mediates dengue virus infection of human dendritic cells west nile virus discriminates between dc-sign and dc-signr for cellular attachment and infection dcsign and l-sign can act as attachment receptors for alphaviruses and distinguish between mosquito cell-and mammalian cell-derived viruses virus-receptor mediated transduction of dendritic cells by lentiviruses enveloped with glycoproteins derived from semliki forest virus fields virology dc-sign as a receptor for phleboviruses this paper reports the first characterization of dc_sign as involved beyond cell-surface attachment. the authors use tirf microscopy to follow dc_sig recruitment at the surface of dcs, and to follow the subsequent virion uptake by the cell dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals distribution and lateral mobility of dc-sign on immature dendritic cellsimplications for pathogen uptake the human cd molecule is a receptor for measles virus (edmonston strain) slam (cdw ) is a cellular receptor for measles virus dc-sign mediated sphingomyelinase-activation and ceramide generation is essential for enhancement of viral uptake in dendritic cells the authors report the mechanism of entry of mv into dcs, through dc-sign signaling that leads to accumulation of ceramides at the viral membrane and redistribution of the cd receptor into the cell surface for virus uptake biological roles of acid and neutral sphingomyelinases and their regulation by nitric oxide tumor cell marker pvrl (nectin ) is an epithelial cell receptor for measles virus adherens junction protein nectin- is the epithelial receptor for measles virus report the identification of the missing receptor for measles virus, allowing for infection of epithelial cells reprogrammed viruses as cancer therapeutics: targeted, armed and shielded the immunoglob ulin-like cell adhesion molecule nectin and its associated protein afadin virus entry: old viruses, new receptors backovic and rey www.sciencedirect.com current opinion in virology cellular receptor for poliovirus: molecular cloning, nucleotide sequence, and expression of a new member of the immunoglobulin superfamily the ectodomain of a novel member of the immunoglobulin subfamily related to the poliovirus receptor has the attributes of a bona fide receptor for herpes simplex virus types and in human cells entry of alphaherpesviruses mediated by poliovirus receptor-related protein and poliovirus receptor viral and host factors in human respiratory syncytial virus pathogenesis measles virus glycoprotein complex assembly, receptor attachment, and cell entry paramyxovirus membrane fusion: lessons from the f and hn atomic structures molecular biology of human respiratory syncytial virus functional analysis of recombinant respiratory syncytial virus deletion mutants lacking the small hydrophobic and/or attachment glycoprotein gene identification of linear heparin-binding peptides derived from human respiratory syncytial virus fusion glycoprotein that inhibit infectivity blocking intercellular adhesion molecule- on human epithelial cells decreases respiratory syncytial virus infection isolation and characterisation of potential respiratory syncytial virus receptor(s) on epithelial cells alpha and lambda interferon together mediate suppression of cd t cells induced by respiratory syncytial virus respiratory syncytial virus infection of monocyte-derived dendritic cells decreases their capacity to activate cd t cells differential response of dendritic cells to human metapneumovirus and respiratory syncytial virus persistent of respiratory syncytial virus in human dendritic cells and influence of nitric oxide primary human mdc , mdc , and pdc dendritic cells are differentially infected and activated by respiratory syncytial virus differentiation and immune function of human dendritic cells following infection by respiratory syncytial virus effect of aging on cytokine production in response to respiratory syncytial virus infection inhibition of toll-like receptor -and -mediated alpha/beta interferon production in human plasmacytoid dendritic cells by respiratory syncytial virus and measles virus respiratory syncytial virus g glycoprotein interacts with dc-sign and l-sign to activate erk and erk this paper shows that dc-sign is not involved in rsv entry into dcs, but that the rsv envelope glycoprotein g specifically interacts with dc-sign to signal the downregulation of the innate immunity reaction this is the first report about an entry receptor for the human respiratory syncytial virus identification of alpha-dystroglycan as a receptor for lymphocytic choriomeningitis virus and lassa fever virus role of nucleolin in human parainfluenza virus type infection of human lung epithelial cells nucleolin: a multifaceted protein major nucleolar proteins shuttle between nucleus and cytoplasm cell-surface nucleolin is a signal transducing p-selectin binding protein for human colon carcinoma cells the cell surface expressed nucleolin is a glycoprotein that triggers calcium entry into mammalian cells the cell-surface-expressed nucleolin is associated with the actin cytoskeleton entry and uncoating of enveloped viruses pathway of rubella virus infectious entry into vero cells conformational change of rubella virus spike proteins induced by -mercaptoethanol role of membrane phospholipids and glycolipids in the vero cell surface receptor for rubella virus identification of the myelin oligodendrocyte glycoprotein as a cellular receptor for rubella virus this is the first report concering an entry receptor for rubella virus the structure and function of myelin oligodendrocyte glycoprotein the n-terminal domain of the myelin oligodendrocyte glycoprotein (mog) induces acute demyelinating experimental autoimmune encephalomyelitis in the lewis rat predominance of the autoimmune response to myelin oligodendrocyte glycoprotein (mog) in multiple sclerosis: reactivity to the extracellular domain of mog is directed against three main regions the crystal structure of myelin oligodendrocyte glycoprotein, a key autoantigen in multiple sclerosis kerlero de rosbo n: preparation of highly purified human myelin oligodendrocyte glycoprotein in quantities sufficient for encephalitogenicity and immunogenicity studies antibodies directed against rubella virus induce demyelination in aggregating rat brain cell cultures rubella virus cdna. sequence and expression of e envelope protein nucleotide sequence and in vitro expression of rubella virus s subgenomic messenger rna encoding the structural proteins e , e and c neurological aspects of rubella virus infection myelin/oligodendrocyte glycoprotein-deficient (mogdeficient) mice reveal lack of immune tolerance to mog in wildtype mice togaviridae: the viruses and their replication. in fields virology understanding the alphaviruses: recent research on important emerging pathogens and progress towards their control alphavirus entry and membrane fusion low-ph-dependent fusion of sindbis virus with receptor-free cholesterol-and sphingolipidcontaining liposomes high-affinity laminin receptor is a receptor for sindbis virus in mammalian cells adaptation of sindbis virus to bhk cells selects for use of heparan sulfate as an attachment receptor natural resistance-associated macrophage protein is a cellular receptor for sindbis virus in both insect and mammalian hosts the long sought entry receptor for sindbis virus is reported here. this is the first receptor identified for an alphavirus the nramp family of metal-ion transporters iron regulatory proteins and the molecular control of mammalian iron metabolism glycoprotein organization of chikungunya virus particles revealed by x-ray crystallography measles virus infection of alveolar macrophages and dendritic cells precedes spread to lymphatic organs in transgenic mice expressing human signaling lymphocytic activation molecule (slam, cd ) early target cells of measles virus after aerosol infection of non-human primates key: cord- -g oes authors: nemzek, jean a.; lester, patrick a.; wolfe, a. marissa; dysko, robert c.; myers, daniel d. title: biology and diseases of dogs date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: g oes historically, the dog played an important role as a laboratory animal in biomedical research. although numbers are declining, the use of dogs continues to be common in pharmacokinetics and cardiovascular studies. the normal biology of the dog as both a laboratory and a companion animal has been well studied and reference values are presented here as a clinical and experimental resource. this provides the necessary background to discuss the spontaneous diseases, including infectious and neoplastic conditions, prevalent in purpose bred as well as random source dogs used in biomedical research. in addition, diseases and conditions that arise secondary to the housing and experimental manipulation of dogs is discussed with emphasis on treatment and prevention. laboratory animal medicine mellitus. a comprehensive but concise review of the use of the dog as a research subject is available in gay ( ) . the breed of dog most commonly bred for use in biomedical research is the beagle. some commercial facilities also breed foxhounds or other larger dog breeds for use in surgical research studies. some specific breeds with congenital or spontaneous disorders have also been maintained by research institutions (see examples below). random-source dogs used in research are most frequently mongrels or larger dog breeds (e.g., german shepherd, doberman pinscher, labrador and golden retrievers) that are used for surgical research and/or training. according to a computerized literature search for "beagle" for the years - , a significant portion of the biomedical scientific publications identified were in the fields of pharmacology or toxicology. especially common were studies focusing on pharmacokinetics, alternative drug delivery systems, and cardiovascular pharmacology. other common areas of research using beagles were dental and periodontal disease and surgery, orthopedic surgery, skeletal physiology, and imaging studies. other research areas that utilized beagles included canine infectious disease, prostatic urology, and ophthalmology. most large-sized dogs (either purpose-bred or randomsource) are used in biomedical research because of their suitability for surgical procedures. anesthetic protocols and systems for dogs are well established and the organs of larger dog breeds are often an appropriate size for trials of potential pediatric surgical procedures. surgical canine models have been used extensively in cardiovascular, orthopedic, and transplantation research. there are also some unique spontaneous conditions for which dogs have proven to be valuable animal models. a colony of gray collies had been maintained at the university of washington (seattle) for the study of cyclic hematopoiesis. this condition is manifested by periodic fluctuations of the cellular components of the blood, most notably the neutrophil population. these dogs can be used to study the basic regulatory mechanisms involved with hematopoiesis, as well as possible treatments for both the human and the canine conditions (brabb et al., ) . golden retrievers affected with muscular dystrophy have been used as models of duchenne muscular dystrophy in human children. duchenne muscular dystrophy is caused by an absence of the muscle protein dystrophin, inherited in an x-linked recessive manner. the dystrophy in golden retrievers is caused by the absence of the same protein and is inherited in the same way. the clinical signs (such as debilitating limb contracture) are also similar between the canine and human conditions (kornegay et al., ) . other genetic disorders studied in dog colonies include hereditary canine spinal muscle atrophy (cork, ) and narcoplepsy in doberman pinschers (ripley et al., ) . bedlington terriers have been used to study copper storage diseases (such as wilson's disease) and the development of spontaneous diabetes mellitus and hypothyroidism has been studied in several breeds of dogs for comparisons with the human conditions. although historically the dog has been a common laboratory animal, their use in research has waned over the past years. according to the u.s. department of agriculture (usda), animal and plant health inspection service ( , ) , the number of dogs used in research has declined from , in to , in (prior to the previous edition of this text) and , in . this decrease was caused by a variety of factors, including (but not limited to) decreased availability, local restrictive regulations, conversion to other animal models (such as livestock or rodents), increased cost, and shift in scientific interest from pathophysiology to molecular biology and genetics. dogs used for research are generally segregated into two classes: purpose-bred and random-source. purposebred dogs are those produced specifically for use in biomedical research; they are intended for use in long-term research projects and/or pharmacologic studies in which illness or medication would require removal from the study. usually these dogs are either beagles or mongrel foxhounds, although other breeds may be available. purpose-bred dogs typically receive veterinary care throughout their stay at the breeding facility. they are usually vaccinated against rabies virus, canine distemper virus, parvovirus, adenovirus type , parainfluenza virus, leptospira serovars canicola, icterohaemorrhagiae, grippotyphosa, and pomona, and bordetella bronchiseptica (jasmin, personal communication). purpose-bred dogs are also usually treated prophylactically for intestinal helminths and ectoparasites, and possibly given a heartworm preventative. random-source dogs are not bred specifically for use in research. they may be dogs bred for another purpose (e.g., hunting and racing) or stray dogs collected at pounds or shelters. the health status of these dogs can be the same quality as purpose-bred dogs, or it can be an unknown entity. random-source dogs that have been treated and vaccinated in preparation for use in research are termed conditioned dogs. these dogs are then suitable for long-term studies or terminal preparations that require unperturbed physiologic parameters. conditioned dogs are often tested for heartworm antigen because of the implications that infestations can have on cardiovascular status and surgical risk. nonconditioned random-source dogs are useful only in a limited number of research studies, such as nonsurvival surgical training preparations and tissue/organ harvest. options for procurement of dogs for biomedical research typically include purchase from a usdadesignated class a or class b licensed dealer or directly from a municipal pound. the requirements for usda licensure are detailed in code of federal regulations (cfr), title , chapter ( - - edition), subchapter a, animal welfare, . definitions, and . requirements and application (office of the federal register, ) . briefly, class a licensees are breeders who raise all animals on their premises from a closed colony. class b licensees purchase the dogs from other individuals (including unadopted animals from municipal pounds) and resell them to research facilities. there are additional regulations that apply to class b dealers (such as holding periods and record-keeping documentation) because of the public concern that stolen pets could enter biomedical research facilities in this manner. in december , the national institutes of health (nih) issued notice not-od- - entitled notice regarding nih plan to transition from use of usda class b dogs to other legal sources (national institutes of health, ) . this nih policy begins in the fiscal year and prohibits the procurement of dogs from class b dealers using nih grant funds. from that point forward, dogs on nihfunded studies will have to be obtained from class a vendors, privately owned colonies (such as institutional breeding colonies), or client-owned animals (e.g., animals participating in veterinary clinical trials). the best resource for identification of possible vendors are online 'buyer's guide' sites or 'buyer's guide' issues of trade periodicals. online sites include the buyer's guide of the american association of laboratory animal science (http://laboratoryanimalbuyersguide.com), and the trade journals lab animal (http://guide.labanimal. com) and animal lab news (http://www.alnmag.com/ content/buyers-guide). a 'buyer's guide' typically lists sources for both purpose-bred and random-source dogs, and denotes such features as pathogen-free status, health status, and availability of specific breeds and timed pregnant females. some suppliers also have separate advertisements within issues of the journals. federal regulations promulgated by the animal and plant health inspection service, usda, in response to the animal welfare act ( cfr . , . , and . [g] ) are described in cfr chapter ( - - edition), subchapter a, animal welfare (office of the federal register, ) . regulations pertaining specifically to the care of dogs used in research are found in subpart a, specifications for the humane handling, care, treatment, and transportation of dogs and cats of part (standards) of subchapter a. particular attention should be paid to section . c (primary enclosures-additional requirements for dogs) because the space required for housing dogs is calculated using body length rather than weight (a parameter used for other species and also for dogs in the national research council (nrc) guidelines). section . (exercise for dogs) describes the requirements that dealers, exhibitors, and facilities must follow in order to provide dogs with sufficient exercise. the institute for laboratory animal research (ilar) has written the guide for the care and use of laboratory animals (national research council, ) . the 'guide' is the primary document used by institutional animal research units to develop their programs and by animal care evaluation groups, such as the association for assessment and accreditation of laboratory animal care international (aaalac international), to facilitate site visits and inspections. the primary difference between the th and th editions of the 'guide' (national research council, regarding the care of dogs is the notation that "enclosures that allow greater freedom of movement and unrestricted height (i.e., pens, runs, or kennels) are preferable." the ilar committee on dogs authored dogs: laboratory animal management (national research council, ) . this publication describes "features of housing, management, and care that are related to the expanded use of dogs as models of human diseases" and includes "an interpretive summary of the animal welfare regulations and the requirements of the public health service policy on humane care and use of laboratory animals." the reader is encouraged to use these publications to obtain further information on care and husbandry of dogs in the biomedical research setting. the information presented in the tables represents a range of normal values that can vary depending on the analytical method, as well as the age, breed, and sex of the animal. cohen, covance laboratories, inc., cumberland, va ( ) . physiological data for a mixed population of dogs of both sexes. fig. . demonstrates the normal weights and corresponding ages for both male and female beagle and hound dogs. tables . and . feature hematology data from beagles of both sexes from two commercial facilities. tables . and . list serum chemical data for beagles of both sexes from two commercial facilities. representative blood gas, coagulation data, and normal urinalysis parameters can be found in tables . - . , respectively. finally, the reviews in arterial and venous blood gas anaylses (rieser, ) and the manual of canine and feline cardiology (tilley et al., ) are excellent resources. good nutrition and a balanced diet are essential to the health, performance, and well-being of the animal. the nrc of the united states national academy of sciences is the leading provider of nutrient recommendations for dogs and provides average requirements needed to maintain growth and prevent deficiencies (subcommittee on dog and cat nutrition, ) . the nrc publications form the basis for the association of american feed control officials (aafco) nutrient profiles, which are updated periodically (baldwin et al., ) . the aafco is an advisory body comprising state representatives from across the united states. it provides a mechanism for developing and implementing uniform and equitable laws, regulations, standards, and enforcement policies, and establishes nutrient profiles for cat and dog foods (dzanis, ; thatcher et al., ) . additional resources should be consulted for details on the nutritional requirements for dogs of all ages (dzanis, ; subcommittee on dog and cat nutrition, ; baldwin et al., ; thatcher et al., ; hand et al., ) . recommendations for feeding the appropriate amount of diet are determined by the dog's metabolic requirements. the maintenance energy requirement (mer) is the amount of energy used by a moderately active adult animal in a thermoneutral environment. the mer for most breeds may be calculated using the following equation: mer (metabolizable kcal/day) = bw × . × kj de, where bw = body weight (kg), kj = kilojoules, de = digestable energy (kienzle and rainbird, ) . in-depth overviews of diets used in biomedical research are available in diet-specific literature. open-formula diets have defined concentrations of all ingredients and the information is publicly available. this allows researchers to control for this important environmental variable and enables retrospective analysis of possible diet composition effects on research results (barnard et al., ) . open-formula diets occasionally may require changes in formulation to maintain nutrient composition or meet changing nutrient requirements. these changes in quantitative ingredient formulation are made public when open-formula diets are modified. in contrast, closed-formula diets are commercially available, balanced diets that meet and label the minimum requirements for protein and fat and the maximum values for ash and fiber; however, the exact composition of ingredients may vary from batch to batch. ingredient composition varies as the manufacturer applies a leastcost strategy, referring to formulating diets to maximize profit by using the least-expensive ingredients. although the ingredients are listed, the quantitative ingredient formulation is not publicly available and can vary without public disclosure, due to proprietary nature of commercial diets produced and marketed under vendor trade names. closed-formula diets have also been referred to as as 'fixed formula' or 'constant nutrition' (labdiets, pmi nutrition international, st. louis, missouri) by manufacturers (barnard et al., ) . in fixed-formula diets, the quantitative ingredient formulation does not change; however, this information is proprietary and therefore laboratory animal medicine not disclosed publically (barnard et al., ) . semipurified and purified diets provide the strictest control of ingredients and are formulated from purified components: amino acids, lipids, carbohydrates, vitamins, and minerals. although purified and semipurified diets do differ in the types of ingredients used, the terms are generally used to mean the same thing. purified-ingredient diets are generally 'open' formulas, meaning that they are published and available to the scientific community. the animal care provider should be aware of the manufacture date of the diet, which should be clearly visible on the bag. as a general rule, diets are safe for consumption up to months following the manufacture date when stored at room temperature. refrigeration may prolong the shelf-life, but the best strategy is to feed only fresh diets and use each lot based on the date of manufacture. specifications for feeding and watering of dogs are provided in the regulations of the animal welfare act. management of a breeding colony requires broad knowledge of the dog's anatomy, reproductive physiology, and behavioral needs during breeding, gestation, and parturition. although a comprehensive discussion of the biology of canine reproduction is beyond the scope of this chapter, essential features of the broad topics noted above are presented. from dr. asheley wathen, covance laboratories, inc., madison, wi, and dr. kimberley cohen, covance laboratories, inc., cumberland, va ( ) . overall health, body condition, nutrition, and age greatly influence reproductive efficiency (gavrilovic et al., ; johnson, ) . therefore, only normal, healthy animals in excellent body condition should be used in breeding programs. beagles between and . years of age have the best conception rates and litter size with the lowest neonatal mortality. after years of age, conception rates and litter size decline and neonatal mortality increases (johnson, ) . the vagina is a long, musculomembranous canal that extends from the uterus to the vulva. during physical examination, the gloved finger or examination instrument should be introduced through the dorsal commissure of the vulva, avoiding the deep ventral clitoral fossa. examination should proceed at an angle of approximately ° until the instrument or fingertip has passed over the ischial arch, after which it can be directed further craniad toward the cervix. the uterus consists of the cervix, uterine body, and uterine horns. the cervix is an abdominal organ, located approximately halfway between the ovaries and the vulva. when the bitch is in proestrus and estrus, the cervix can be distinguished during abdominal palpation as an enlarged, turgid, walnut-shaped structure. female dogs are monoestrous, typically nonseasonal, spontaneous ovulators that have a spontaneous luteal phase approximately days longer than the ± days of pregnancy followed by obligate anestrus. puberty (beginning of the first estrus) occurs between and months in most breeds. the time of onset positively correlates with the body size (concannon, ) . the canine cycle is divided into four phases: proestrus, estrus, diestrus, and anestrus. the duration of laboratory animal medicine proestrus is - days with an average of days and reflects the follicular phase rise in estrogen. during this stage, the vulva is enlarged and turgid, and a serosanguinous vaginal discharge is present (concannon, ) . estrus may be from to days in duration but generally lasts days. the endocrine feature of estrus is the first abrupt increase in progesterone (> ng/ml), which occurs concomitantly with the luteinizing hormone (lh) surge % of the time, followed by ovulation within - h. the vulva is softer and smaller than in proestrus. the vaginal discharge persists and may remain serosanquinous or become straw colored. diestrus begins approximately days after the onset of standing heat. the end of this stage is days later, which would be coincident with whelping if the bitch had become pregnant. defined behaviorally as starting when estrous behavior ceases (concannon, ) , diestrus represents the peak of serum progesterone. anestrous may last from to days and is the stage of reproductive quiescence. it is characterized by an absence of ovarian activity and serum progesterone levels of less than ng/ml. the onset of puberty in the male ranges from to months of age and is affected by breed, season, nutrition, and disease status. this process is initiated by the secretion of lh from the anterior pituitary, which stimulates the production of testosterone by the interstitial or leydig's cells. at this time, the testicular growth is rapid, the seminiferous tubules begin to differentiate, and sertoli cells form the blood-testis barrier. secretion of follicle-stimulating hormone (fsh) by the anterior pituitary stimulates the production of other key hormones by the sertoli cells, including, inhibin, androgen binding protein, and estrogen. fsh stimulates spermatogenesis in the presence of testosterone, whereas inhibin and estrogen provide negative feedback to the pituitary gland to decrease fsh production. spermatogenesis in the dog is completed in days, with subsequent maturation of sperm occurring in the epididymis for approximately days. thus, the entire process from the initiation of spermatogonial mitosis to the delivery of mature sperm to the ejaculate is days. a breeding soundness exam should be conducted to assess the probability of a male dog's successful production of offspring. factors affecting male fertility include libido, ability to copulate, testicular size, and quality semen production. supression of sexual behavior and problems with libido may occur in dogs due to early weaning, isolation, or inherited abnormalities. animals with poor hind limb conformation or trauma to the back may be unable to properly mount the female. there is a positive correlation between scrotal circumference and the number of sperm produced. finally, the quality of sperm is assessed by motility, morphology, volume, and concentration. an ejaculate ( ml) that contains approximately million progressively motile sperm without significant morphological abnormalities is a good indicator of normal male fertility. complete anatomy of the bitch and dog can be found in miller's anatomy of the dog (evans and de lahunta, ) . cells of the vaginal epithelium mature to keratinized squamous epithelium under the influence of estrogen. because of the rise in estrogen throughout proestrus, with peak levels occurring just prior to the onset of standing heat, the vaginal smear can be used as an indicator of the bitch's readiness for breeding. the smear will not confirm the presence of ovulation nor is it of prognostic value in normal bitches during anestrus. the percentage of vaginal epithelial cell cornification is an index of estrogen secretion by the ovarian follicles. cornification occurs approximately days prior to the estrogen peak and days prior to standing heat. as cornification of vaginal epithelial cells proceeds, the cells become larger, with more angular borders. the nuclear/ cytoplasmic ratio decreases until the nuclei reach a point where they no longer take up stain (coincident with the onset of estrus). the cells appear 'anuclear' and are classified as 'cornified' or 'anuclear squames.' the vaginal cytology smear of the bitch changes from predominantly cornified to noncornified days after ovulation. the day of this change is the first day of diestrus. other epithelial cell types noted on vaginal cytology include superficial cells (large, angular cells with small nuclei); intermediate cells (round or oval cells with abundant cytoplasm and large, vesicular nuclei); and parabasal cells (small round or elongated cells with large, well-stained nuclei, and a high nuclear/cytoplasmic ratio). based on vaginal cytology, the estrous cycle is classified as follows: although vaginal cytology is a useful tool, observation of behavioral estrus is the best criterion to use in breeding management. during proestrus, the male is attracted to the bitch and will investigate her hindquarters, but she laboratory animal medicine will not accept breeding. estrus is characterized by proactive receptivity to mounting by males and increased male-seeking behavior (concannon, ) . during this stage, the bitch will exhibit 'flagging,' or elevation of her tail with muscular elevation of the vulva to facilitate penetration by the male. in order to maximize the conception rate and litter size, it is recommended to breed the bitch on days , , and of the standing heat. due to the long life span of canine sperm, fertilization occurs in the oviduct up to days after coitus. the ovulated oocyte is a primary oocyte that must undergo two meiotic divisions before fertilization can occur. this overall maturation process takes approximately days. after maturation, the oocyte remains viable for - days. optimal conception rates tend to occur when the bitch is bred from days before to days after ovulation; best litter size is achieved when the bitch is bred days after ovulation. implantation is evident by areas of local endometrial edema - days after breeding. there is no correlation between the number of corpora lutea and the number of fetuses in the corresponding uterine horn, suggesting transuterine migration of embryos. the dog has endotheliochorial placentation. the endothelium of uterine vessels lies adjacent to the fetal chorion, mesenchymal, and endothelial tissues, so that maternal and fetal blood are separated by four layers. the canine placenta is also classified as zonary, indicating the placental villi are arranged in a belt, and deciduate, reflecting that maternal decidual cells are shed with fetal placentas at parturition. the length of gestation is - days. luteal progesterone is responsible for maintaining pregnancy and canine corpora lutea retain their structural development throughout gestation. serum progesterone rises from less than ng/ml in late proestrus to a peak of - ng/ml during gestation, and then declines to - ng/ml just prior to parturition. progesterone is essential for endometrial gland growth, secretion of uterine milk, attachment of the placentas, and inhibition of uterine motility (johnson, ; verstegen-onclin and verstegen, ) . pregnancy detection can be performed by several methods. abdominal palpation of the uterus may be most informative at approximately days after breeding. the embryos and chorioallantoic vesicles form a series of ovoid swellings and are approximately inches in length at - days. by day , the uterus begins to enlarge diffusely and the vesicles become difficult to identify by palpation. radiology can be used to confirm pregnancy and facilitate determination of gestational age, beginning days after the lh surge (lopate, ) . bitches in which a difficult whelping is anticipated should be radiographed in late pregnancy to determine the litter size and to evaluate the size of the fetal skulls in relation to the bony maternal birth canal. ultrasonography can be used to confirm pregnancy beginning on days - , at which point the gestational sacs will be approximately cm in diameter, and until parturition (shille and gontarek, ; lopate, ) . ultrasonography can assess fetal viability by visualizing fetal heartbeats and fetal movement beginning on gestational days - and , respectively (lopate, ) . it can also predict gestational age using the inner diameter of the chorionic cavity in early pregnancy and the biparietal diameter in late pregnancy luvoni and beccaglia, ) . however, ultrasonography for determination of gestational age is most accurate at day of pregnancy when using correction factors for small (< kg) and large (> kg) body weight dogs (kutzler et al., ) . thermal support should be provided prior to parturition. dogs housed on grated flooring should be provided with mats and those on solid floors would benefit from blankets placed in a corner of the primary enclosure. shavings are discouraged because they may adhere to the umbilical cord and predispose to ascending infections. heat lamps may be placed h prior to parturition and remain until all neonates demonstrate vigorous suckling behavior. however, the use of heat lamps necessitates strict supervision in order to prevent thermal burns. if possible, whelping bitches should be housed in a quiet corridor in order to decrease periparturient stress, especially in primiparous or young mothers. monitoring of parturition is important, but human intervention should be minimal in order to prevent stressinduced cannibalism. an abrupt drop in body temperature to less than °f indicates impending parturition within - h. the process of parturition has been divided into three stages. stage of labor lasts - h and is characterized by uterine contractions and cervical dilation. during this stage, the bitch may appear restless, nervous, and anorexic. other common clinical signs include panting and increased pulse rate (johnson, ) . fetal expulsion occurs during stage , which lasts approximately - h. as the fetus engages the cervix, there is release of oxytocin, referred to as the ferguson reflex, which strengthens the uterine contractions and may elicit abdominal contractions as well. the bitch is able to inhibit this stage of labor if disturbed. the chorioallantois ruptures either during passage of each neonate through the birth canal or by the bitch's teeth at birth. interestingly, posterior presentation is common in dogs but does not predispose to dystocia. the time interval between deliveries of each pup is irregular, but the average is less than h between pups. veterinary assistance laboratory animal medicine is necessary if the bitch remains in stage for more than h without delivering the first pup, or for more than h before delivering subsequent pups. during stage of labor, the placentas are expelled either immediately or within min of delivery of each pup. if two pups are delivered from alternate uterine horns, then the birth of both puppies may precede expulsion of the respective placentas. the bitch will lick the newborn vigorously to remove the membranes from its head and to promote respiration. she will also sever the umbilical cord. the bitch may ingest the placentas, although they confer no known nutritional benefit and may induce a transient diarrhea. the peripartum use of oxytocin is required only in the event of uterine inertia, stillbirths, or agalactia. oxytocin should not be used in the event of systemic illness or abnormalities precluding vaginal delivery. indications for its use include lack of delivery h after onset of stage labor, greater than h of unproductive stage labor, inadequate contractions, or abnormal vaginal discharge. in these cases, radiographs are recommended to assess fetal size in relation to the birth canal and any possible obstructions, followed by . - . iu of oxytocin intramuscularly or subcutaneously. the oxytocin can be repeated - min after the first dose for a total of two doses (plunkett, ) . in some cases, treatment with . - . ml/kg of % calcium gluconate, delivered slowly iv while monitoring closely for bradycardia, and % dextrose iv may be indicated. uterine involution occurs during anestrus within - weeks of parturition. during this time, a greenish to red-brown vaginal discharge, or lochia, is considered normal. the presence of an odiferous, purulent discharge, accompanied by systemic signs of illness, indicates metritis or pyometra. desquamation of the endometrium begins by the th postpartum week, with complete repair by months. newborn puppies are easily sexed by examination of the anogenital distance. in female puppies, the vulva is evident a short distance from the anus, whereas the prepuce of male puppies is nearly adjacent to the umbilicus. eyes are open at approximately days, and ears are patent at approximately - days. solid food can be introduced between . and weeks of age, and puppies can be weaned at - weeks. artificial insemination (ai) is indicated when the male is physically incapable of mounting or penetrating the bitch, when there are vaginal abnormalities such as a vaginal-vestibular stricture, narrow vagina, vaginal septum, and vaginal hyperplasia, or if there is a behavioral incompatibility between the male and female dogs (kutzler, ) . semen is collected using a plastic centrifuge tube and rubber latex artificial vagina. the male is introduced to the scent of an estrous bitch and manually stimulated. the first two fractions are collected followed by a sufficient amount of the third fraction (predominantly of prostatic fluid) to bring the total semen volume to - ml. the semen can be introduced into the cranial vagina or directly into the uterus either through trans-cervical catheterization with a norwegian ai catheter or utilizing fiberoptic endoscopy. use of the norwegian ai catheter for intrauterine insemination of frozen-thawed, fresh, and chilled-extended semen results in significantly higher whelping rates than intravaginal insemination (linde-forsberg et al., ; thomassen and farstad, ) . for trans-cervical insemination, the bitch is either standing on all four legs or standing with hindquarters raised. the ai catheter and guiding tube are inserted into the vestibulum as far as the pseudocervix. firm abdominal palpation is then used to locate and fix the cervix in the other hand, at which point the catheter is further inserted along the dorsal vaginal fold until the cervical opening is located and semen is deposited into the uterus lumen (thomassen and farstad, ) . surgical and laparoscopic ai has been used successfully for intrauterine and intratubal insemination; however, these techniques are invasive and require anesthesia. therefore, the nonsurgical techniques mentioned above are recommended, as these approaches are less invasive and can be completed without anesthesia in nonsedated or sedated dogs depending on the experience of the personnel and personality of the dogs. ai with freshly collected sperm can be done on days , , and of standing heat or on days of maximal vaginal cornification. the viability of frozen-thawed sperm is significantly reduced compared to fresh or chilled sperm that may live up to or days in the reproductive tract of the bitch; frozen-thawed sperm live only a few hours. therefore, the ova must be mature and insemination with frozen-thawed semen must be done - days after ovulation in the bitch as determined by serum progesterone concentrations (thomassen et al., ) . false pregnancy (pseudocyesis), a stage of mammary gland development and lactation associated with nesting or mothering behavior, is common in the bitch. the condition occurs after the decline in serum progesterone toward the end of diestrus. there is no age or breed predisposition. pseudopregnancy does not predispose the bitch to reproductive disease or infertility. a comprehensive review of canine pseudocyesis exploring its cause, clinical features, and treatments is covered by c. gobello (gobello et al., ) . reproductive performance in the bitch is optimal prior to years of age. cycling does not completely cease; however, after - years of age, bitches demonstrate significant decreases in conception rate and the number of live pups whelped. by - years of age, pathologic conditions of the uterus, such as cysts, hyperplasia, atrophy, and neoplasia, are extremely common. dogs prefer living in a social environment. dogs have well developed olfactory glands, vision, and auditory and tactile senses that allow them to gain environmental cues and information from other dogs and humans (field and jackson, ; joint working group on refinement, ) . much of their instinctive behavior is dependent on learning to interact with other members of their species. beagles have been a popular animal model because of their docile nature. they are easily handled and, for the most part, respond favorably to repetitive manipulations such as body weight measurements, physical examination, electrocardiograms (ecgs), oral gavage, and venipuncture. although sexually mature by - months of age, dogs are not socially mature until - months of age. the socialization process should begin early during development, when puppies are receptive to conspecific and human contact. for example, from to weeks of age, puppies are most capable of learning about how to interact with other dogs. between weeks and , puppies are most capable of learning how to interact with people. by - weeks of age, dogs voluntarily wander and explore new environments. thus, early handling and mild stress (such as vaccination) appear to be extremely beneficial components of a dog's social exposure. canid social systems use signals and displays that minimize the probability of outright aggression. these behavior patterns are most likely elicited during distressful situations, such as strange environments, being handled by strange people, or encountering new animals. an excellent, illustrated discussion of normal canine behavior patterns can be found in the canine behavior section of the manual of clinical behavioral medicine for dogs and cats (overall, ) . by virtue of the dog's status as a companion animal, there are many veterinary publications and reference texts on the diagnosis, medical management, pathology, and epidemiology of its disorders. the authors of this chapter have chosen to emphasize those diseases that are more frequently encountered in the research setting, especially infectious diseases associated with the use of random-source dogs and conditions seen frequently in the beagle. for more thorough and detailed discussion of these diseases, as well as those not discussed in this chapter, the reader should consult standard veterinary textbooks. etiology canine infectious respiratory disease (cird) is a highly contagious illness and several organisms have been incriminated including bordetella bronchiseptica; streptococcus equi subsp. zooepidemicus; canine parainfluenza virus (cpiv); canine influenza virus (civ); canine respiratory coronavirus; canine adenovirus type (cav- ); canine herpesvirus; canine reovirus types , , and ; and mycoplasma and ureaplasma. naturally occuring infection can result in coinfection by two or more organisms (garnett et al., ; ford, ) . clinical signs cird can be subdivided into mild or severe forms. the mild form is more common and is characterized by an acute onset of a loud, dry, hacking cough. increased formation of mucus sometimes results in a productive cough, followed by gagging or retching motions. cough may be elicited by tracheal palpation and may be more frequent with excitement or exercise. otherwise, dogs are typically asymptomatic. mild tracheobronchitis usually lasts - days, even when untreated. the severe form results from poor general health, immunosuppression, or lack of vaccination. secondary bronchopneumonia can occur and can be the determinant of severity (sherding, ) . animals are clinically ill and may be febrile, anorexic, and depressed. productive cough and mucopurulent naso-ocular discharge are more common than in the mild form. epizootiology and transmission the natural reservoir for b. bronchiseptica is the respiratory tract (bemis, ) , and it is very easily spread by aerosol and direct contact. transmission is heightened by confined housing of multiple animals. bordetella bronchiseptica is highly infectious with an incubation period of - days. pathogenesis the most common clinical isolates are cpiv and b. bronchiseptica (mochizuki et al., ) . however, b. bronchiseptica is often recovered from clinically healthy animals (chalker et al., ) . during clinical infection, b. bronchiseptica attaches to the cilia of the upper airway epithelium, causing suppurative tracheobronchitis and bronchiolitis. infections with cpiv laboratory animal medicine or cav- alone are usually subclinical but can cause necrotizing tracheobronchiolitis (dungworth, ) . diagnosis and differential diagnosis diagnosis is often based on clinical signs and known history; however, cough elicited by tracheal palpation may be inconsistent and should not be used for definitive diagnosis. presumptive diagnosis can be made by isolation of b. bronchiseptica or mycoplasma by nasal swabs. viral isolation or paired serology is often impractical and expensive. if cough persists for more than days, other disease conditions should be considered. differential diagnoses include civ, canine distemper virus, pneumonia, heartworm disease, tracheal collapse, mycotic infections, and diseases resulting in tracheal compression (johnson, ) . prevention prevention is best achieved by avoiding exposure to infected animals. dogs should be vaccinated prior to or upon admission to the animal facility. intranasal vaccines protect against infection and disease and can be given to dogs as young as weeks of age (greene and levy, ) . combination vaccines for b. bronchiseptica, cav- , and cpiv are preferred. vaccinations should be boostered every months when multiple animals are housed in a confined area. control staff must practice proper hygiene to prevent transmission by fomites. sanitation, proper ventilation, and proper humidity are critical for control. symptomatic animals should be isolated and kennels should be disinfected with agents such as bleach, chlorhexidine, or quaternary ammonium chloride. treatment bordetella bronchiseptica is sensitive to potentiated sulfas, chloramphenicol, quinolones, tetracyclines, gentamicin, and kanamycin. use of antibiotics is indicated when severe or persistent clinical signs occur and should be continued for days. for severe or unresponsive infection, treatment should be based on bacterial culture/sensitivity patterns. nebulized gentamicin or kanamycin may be helpful in severe cases. antitussives should be avoided if the cough is productive; however, their use is indicated if coughing is causing discomfort or interfering with sleep. bronchodilators such as aminophylline, theophylline, or terbutaline can be helpful in reducing reflex bronchoconstriction. research complications due to the altered respiratory tract physiology, infected animals should not be used for pulmonary studies. etiology β-hemolytic lancefield's group c streptococcus (s. equi ssp. zooepidemicus) is a gram-positive, non-spore-forming coccus that causes pneumonia and sepsis in dogs. clinical signs clinical signs vary based on the organ system affected. pneumonic disease is typically associated with sudden onset of clinical signs including coughing, weakness, fever, dyspnea, and hematemesis. the rapid progression of disease is similar to that seen in humans with toxic shock syndrome (tss) caused by streptococcus pyogenes. peracute death has been reported in research and shelter dogs (bergdall et al., ; pesavento et al., ) . epizootiology and transmission streptococcus equi ssp. zooepidemicus is not considered a commensal of healthy dogs as most of the β-hemolytic commensal organisms belong to group g, specifically streptococcus canis. asymptomatic carriers are suspected to be the route by which infection enters populations. streptococcus equi ssp. zooepidemicus is considered an opportunistic pathogen and stressful factors such as transport can predispose to disease (priestnall et al., ) . pathologic findings in peracute cases, hemorrhage from the mouth and nose and within the pleural cavity can be the most striking lesion. ecchymotic and petechial hemorrhages can be noted on several organs ( fig. . ). 'bull's-eye' lesions may be observed on the pleural surface of affected lung lobes. histologic lesions can include fibrino-suppurative, necrotizing, and hemorrhagic pneumonia. gram-positive cocci can be found in intracellular clusters throughout the lung ( fig. . ), tonsils, and spleen of affected animals (bergdall et al., ; priestnall and erles, ) . pathogenesis predisposing factors such as transport stress and viral coinfection have been shown to contribute to the virulence of s. zooepidemicus (priestnall and erles, ) . due to the similarities with the clinical signs seen in human cases of tss, superantigens are thought to contribute to the virulence of s. zooepidemicus in cases of acute hemorrhagic pneumonia. these superantigens work by bypassing the conventional mechanisms of antigen presentation and binding to major histocompatibilitity complex class ii receptors. as a result, there is a hyperactive proinflammatory response and an 'avalanche' of cytokines including interleukin β (il- β), interleukin (il- ), and tumor necrosis factor alpha (tnf-α). three novel superantigen-encoding genes have been identified from a case of acute fatal hemorrhagic pneumonia, szef, szen, and szep. however, it is currently unclear what effect these superantigens have in vivo priestnall et al., ) . while superantigens have been detected in some isolates, there is not enough data to determine if superantigens play a role in the pathogenesis (byun et al., ; kim et al., ) . diagnosis and differential diagnosis definitive diagnosis is based on bacterial culture of nasal swabs or transtracheal lavage. polymerase chain reaction (pcr) can be done on post-mortem lung tissue. bacterial pneumonias or bacteremias can be caused by other pathogenic streptococcus spp., staphylococcus spp., escherichia coli, pasteurella multocida, pseudomonas spp., klebsiella pneumoniae, and b. bronchiseptica. nonbacterial causes of respiratory disease include rodenticide intoxication, coagulopathies, heartworm disease, pulmonary thromboembolism, ruptured aneurysm, and left-sided congestive heart failure. prevention and control there is no vaccine for prevention of s. zooepidemicus. the organism has been isolated from the environment during active outbreaks (pesavento et al., ) , so dogs diagnosed with s. zooepidemicus should be quarantined and any potential fomites (e.g. food bowls, enrichment) should be properly disinfected. treatment antibiotic therapy should be based on culture and sensitivity. resistance to doxycycline and tetracycline has been demonstrated (garnett et al., ; pesavento et al., ) . research complications dogs with severe hemorrhagic pneumonia or systemic disease are not appropriate for research study. the association between epizootics of this disease and transportation supports operational policies that require adequate acclimation periods for animals upon arrival. etiology serovars canicola, bratislava, and grippotyphosa result in renal or hepatic disease, whereas serovars icterohaemorrhagiae and pomona predominantly result in hepatic disease . clinical signs canine leptospirosis can present as subclinical, acute, or chronic disease. clinical signs in acute infection can be nonspecific and include lethargy, depression, abdominal discomfort, stiffness, anorexia, vomiting, muscle tenderness, and pyrexia. clinical signs can be related to renal failure including polyuria and polydipsia, with or without azotemia, oliguria, or anuria. leptospirosis can also lead to hepatic failure with signs such as icterus or bleeding abnormalities. peracute leptospirosis is characterized by shock, vascular collapse, and rapid death. uveitis, abortions, stillbirths, and pulmonary hemorrhage have also been associated with leptospirosis (klopfleisch et al., ; van de maele et al., ) . bivalent vaccines against the most common canine serovars, icterohaemorrhagiae and canicola, have resulted in the increased prevalence of other serovars including grippotyphosa, pomona, bratislava, and autumnalis. increased movement of wild animal reservoirs (rats, raccoons, skunks, opossums) into urban/suburban areas have also contributed to the greater prevalence of previously uncommon serovars (sykes et al., ) . transmission occurs primarily through environmental contact, although direct transmisson between hosts may also occur. leptospires passing from urine into water is the most common route of contamination (goldstein, ) . leptospirosis is a zoonotic disease. pathologic findings the kidneys consistently have gross and microscopic lesions. in the acute phase, the kidneys are swollen with subcapsular and cortical ecchymotic hemorrhages. petechial or ecchymotic hemorrhages and swelling of the lungs may also be noted. hepatic lesions during the acute phase consist of diffuse hemorrhage and necrotic foci (searcy, ) . in chronic stages of leptospirosis, the kidneys become small and fibrotic. endothelial cell degeneration and focal to diffuse lymphocytic-plasmacytic interstitial nephritis are the characteristic histopathological findings. pathogenesis the severity and course of leptospirosis depend on the causative serovar as well as the age and immune status of the dog. infection occurs after the leptospires penetrate a mucous membrane or abraded skin. the organisms then invade the vascular space and multiply rapidly, reaching the renal tubular epithelium several days postinfection. acute or progressive renal failure leading to oliguria or anuria may occur. nephritis may or may not be accompanied by hepatitis, uveitis, pulmonary hemorrhage, and meningitis. disseminated intravascular coagulation is often a secondary complication. diagnosis and differential diagnosis paired serology for the microscopic agglutination test is the most reliable means of definitive diagnosis, and successive serum sampling should be done - days after the first sample. pcr can be used to identify active infection early in the disease when serologic testing is negative or in previously vaccinated animals (sykes et al., ) . differential diagnoses include other causes of acute renal failure and hepatitis. prevention and control according to the american animal hospital association's vaccination guidelines, vaccination for leptospirosis is recommended based on geographic location and exposure risk (welborn et al., ) . both quadrivalent and bivalent inactivated bacterins are available. quadrivalent bacterins protect against canicola, icterohaemorrhagiae, grippotyphosa, and pomona serovars, whereas bivalent bacterins cover only canicola and icterohaemorrhagiae. immunization does not prevent the development of the carrier state or protect against other serovars. control requires preventing contact with wildlife reservoirs as well as identification of carrier animals. treatment doxycycline is the drug of choice as it can eliminate renal colonization. if vomiting or allergic reactions prohibit treatment with doxycycline, ampicillin or other penicillins should be utilized. aggressive fluid therapy and supportive care may also be needed. research complications due to the zoonotic potential, dogs with clinical leptospirosis should not be used in research studies. etiology campylobacter spp. are thin, curved or spiral, microaerophilic, thermophilic motile gram-negative rods. many species of campylobacter have been isolated from normal and diarrheic animals; however, the most common pathogenic species include campylobacter jejuni ssp. jejuni and c. coli (marks et al., ) . clinical signs most adult animals infected with c. jejuni are asymptomatic carriers; clinical signs are most commonly noted in dogs that are less than months of age (greene, ; burnens et al., ) . in cases of clinical illness, mild and intermittent mucoid or watery diarrhea, with or without frank blood, is most commonly noted. signs typically last - days but can persist for several months. tenesmus, inappetance, vomiting, and a mild fever may accompany the diarrhea (marks et al., ) . bacteremia and cholecystitis secondary to c. jejuni have also been documented in dogs (fox, ) . epizootiology and transmission the role of campylobacter spp. as a primary pathogen has been questioned; it may require a coenteropathy to produce disease (sherding and johnson, ) . stress or immunosuppression may make animals more susceptible. transmission is via the fecal-oral route, mostly through contaminated food or water. campylobacter jejuni can be zoonotic with immunocompromised individuals at greatest risk. pathologic findings lesions depend on the mechanism of the enteropathy (van kruiningen, ) . enterotoxin production results in dilated, fluidfilled bowel loops, with little or no histopathologic alteration. cytotoxin-mediated disease results in a friable, hemorrhagic mucosal surface. histologically, the mucosa is ulcerated with lymphoplasmacytic infiltration. translocation can result in edema and congestion of the lamina propria with focal accumulation of granulocytes. epithelial hyperplasia and decreased goblet cell numbers are also noted. campylobacter jejuni may be visualized between enterocytes with warthin-starry silver-stained sections. pathogenesis clinical disease may be produced by several different mechanisms as campylobacter spp. have a variety of virulence factors including enterotoxins, cytotoxins, and adherence or invasion properties. campylobacter jejuni can cause an erosive enterocolitis by invasion of epithelium and production of the cytolethal distending toxin (cdt) (fox, ; van kruiningen, ) . in addition, c. jejuni can produce illness via translocation to regional lymph nodes causing a mesenteric lymphadenitis. diagnosis and differential diagnosis fresh feces (per rectum) can be used for presumptive diagnosis by demonstration of highly motile, curved or spiral organisms with dark-field or phase-contrast microscopy. gram-stained c. jejuni appear as gull-winged rods. definitive diagnosis requires isolation of the organism (sherding and johnson, ) . culture requires selective isolation media, and growth is favored by reduced oxygen tension and a temperature of °c. a pcr multiplex assay for differentiation of c. jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus ssp. fetus has been developed (wang et al., ) . any disorder that can cause diarrhea in dogs should be considered as a differential diagnosis. prevention and control proper environmental sanitation, waste disposal, and food storage can prevent campylobacteriosis. in enzootic situations, group housing should be avoided. outbreaks are controlled by isolation and treatment of affected individuals. treatment antibacterial treatment should be considered in severely ill dogs. erythromycin, neomycin, enrofloxacin, clindamycin, and doxycycline are all effective. resistance to quinolones and ciprofloxacin has been documented (acke et al., ) . treatment should be a minimum of - days with bacterial cultures repeating and weeks after treatment. research complications dogs with clinical campylobacteriosis have temporary derangements to digestive and absorptive functions. etiology helicobacters are gram-negative, microaerophilic, spiral bacteria that infect the gastrointestinal tract. helicobacter spp. can be separated into gastric and enterohepatic groups. the gastric helicobacters commonly identified in dogs are referred to as non- (haesebrouck et al., ; joosten et al., ) . the most common enterohepatic species found in dogs include h. bilis, h. canis, and h. cinaedi (castiglioni et al., ; dewhirst et al., ; fox, (haesebrouck et al., ; fox, ) . clinical signs most infections are subclinical in the dog. gastric infections may present with vomiting, diarrhea, and fever, accompanied by anorexia, pica, or polyphagia. enterohepatic helicobacters have been linked with inflammatory bowel disease in experimental animal models. heavy infections in dogs have been associated with inflammatory lesions of the large intestine (castiglioni et al., ; nguyen et al., ) . epizootiology and transmission the epizootiology and transmission of helicobacter spp. in the dog remain unknown. both oral-oral and fecal-oral routes for transmission have been suggested in humans, but transmission via canine saliva is a less reliable source of infection (craven et al., ) . enterohepatic infections of pet dogs are as high as % (castiglioni et al., ) . prevalence of gastric helicobacter infections in colony or shelter dogs can be as high as - % (fox, ; hermanns et al., ) . pathologic findings gastritis is usually mild and characterized by reduced mucus content of the surface epithelium with vacuolation, swelling, karyolysis, and karyorrhexis of parietal cells. multifocal infiltrates of plasma cells and neutrophils occur around blood vessels and between gastric pits (hermanns et al., ) . intestinal lesions include mild to moderate lymphoplasmacytic infiltration as well as crypt dilation and crypt hyperplasia (castiglioni et al., ) . pathogenesis gastric helicobacters are urease positive, which assists with survival in the acidic environment of the stomach (kusters et al., ; uberti et al., ) . enterohepatic helicobacters are urease negative and typically reside in the lower intestine. the mechanism by which enterohepatic helicobacters colonize the liver is thought to be through portal circulation after uptake by enterocytes or through retrograde movement from the intestine into the bile duct (fox, ) . diagnosis and differential diagnosis organisms may be demonstrated with histopathology on endoscopic or surgical biopsy tissue samples. warthin-starry silver stain may increase the sensitivity for histopathologic diagnosis. culture may be difficult depending on the helicobacter spp. for species that produce urease, a positive urease test on a gastric biopsy specimen may give a presumptive diagnosis. the urea breath test has been successfully used to diagnose helicobacter spp. in laboratory beagles with a sensitivity and specificity of % (kubota et al., ) . western blot has been used to detect serum antibodies to enterohepatic species and pcr can be used to detect helicobacter spp. in fecal samples (oyama et al., ; wadström et al., ) . any causes of acute or chronic vomiting and diarrhea in the dog are differential diagnoses. prevention and control until more is known about the epizootiology and transmission of helicobacter spp., specific recommendations cannot be made for prevention and control. treatment for gastric species, combination therapy of amoxicillin ( mg/kg q h), metronidazole ( mg/kg q h), and sucralfate ( . - . mg/kg q h) has proven to be most effective (hall and simpson, ) . replacing sucralfate with famotidine, omeprazole, or bismuth subsalicylate may also be effective (marks, ; jenkins and bassett, ; denovo and magne, ) . recurrence rates within days of treatment can be as high as % (anacleto et al., ) . treatment of enterohepatic helicobacters may depend on species susceptibility. aminoglycosides have been successful in treating h. cinaedi, but resistance to fluoroquinolones has been documented (tomida et al., ) . combination therapy of amoxicillin, clarithromycin, metronidazole, and omeprazole in medicated chow has been successful in eliminating various enterohepatic helicobacters from mice (del carmen martino-cardona et al., ) . long-term antibiotic treatment at a minimum of days is suggested for enterohepatic and gastric helicobacters. research complication dogs used in gastrointestinal physiology or oral pharmacology studies should be free from helicobacteriosis. etiology parvoviral enteritis in dogs is caused by canine parvovirus strain (cpv- ) of the family parvoviridae, genus protoparvovirus, species carnivore protoparvovirus . currently, there are three antigenic variants, a, b, and c. parvoviruses are nonenveloped, single-stranded dna viruses. clinical signs while parvoviral infection can affect the gastrointestinal tract, bone marrow, myocardium, and nervous tissues, the most common manifestation of disease is acute enteritis. clinical signs usually appear days after fecal-oral inoculation and include anorexia, fever, depression, vomiting, and profuse intractable diarrhea which may become hemorrhagic. excessive fluid and protein losses through the gastrointestinal tract result in rapid and severe dehydration. dogs can develop severe leukopenia with a total leukocyte count of cells/μl or less. repeated hemograms may provide prognostic value, as rebounds in leukocyte counts are indicative of impending recovery. terminally ill dogs may develop hypothermia, icterus, or disseminated intravascular coagulation due to endotoxemia. epizootiology and transmission parvovirus can infect dogs of any age, but puppies between and weeks of age are particularly susceptible. puppies less than weeks of age are protected by passive maternal antibody. strain cpv- c has been associated with severe disease in adult vaccinated dogs (calderon et al., ) . pathogenesis canine parvovirus has an affinity for rapidly dividing cells of the intestine and causes acute enteritis with intestinal crypt necrosis and villus atrophy. the virus also has tropism for the bone marrow and lymphoid tissues; thus, leukopenia and lymphoid depletion accompany the intestinal destruction. diagnosis and differential diagnosis parvovirus can be detected with a commercially available fecal enzyme-linked immunosorbent assay (elisa). due to intermittent and brief shedding of the virus, fecal elisas can have false-negative results. pcr can be used to confirm an elisa result and to differentiate the viral strain. at necropsy, diagnosis is based on gross and histopathologic evidence of necrosis and dilatation of intestinal crypt cells with secondary villous collapse. prevention and control parvoviral-positive animals should be quarantined for at least days as the infectious virus is shed for several days after onset of clinical signs. although pcr has been used to detect viral dna in feces for up to weeks (decaro et al., ) , it is currently unknown if the material being shed at this time is still infectious. disinfection of exposed areas with dilute bleach ( : ) or a commercial disinfectant is essential for elimination of the virus. six-week-old puppies should be vaccinated every - weeks with a modified live vaccine until at least weeks of age. treatment treatment is largely supportive and aimed at restoring fluid and electrolyte balance. antimicrobial therapy is recommended due to intestinal compromise and risk of sepsis. early nutritional support continued throughout the disease has been shown to decrease recovery times (mohr et al., ) . research complications infection with parvovirus precludes the use of a particular dog in an experimental protocol. due to the significant discomfort of the animal, as well as the intensive therapy required, humane euthanasia is usually chosen in a research setting. etiology rabies virus is a lyssavirus belonging to the family rhabdoviridae. clinical signs clinical progression of neurologic disease occurs in three stages. the first, prodromal, stage is characterized by a change in species-typical behavior. the loss of the instinctive fear of humans by a wild animal is a classic sign of impending rabies. in the second, furious, stage, animals are easily excited or hyperreactive to external stimuli and will readily bite at inanimate objects. the third, paralytic, stage is characterized by incoordination and ascending ataxia of the hindlimbs due to viral-induced damage of motor neurons. death due to respiratory failure usually occurs after onset of the third stage. wild animals such as raccoons, skunks, and bats are common reservoirs of infection for domestic animals, which in turn are the principal source of infection for humans. transmission occurs primarily by contact with infected saliva, usually via bite wounds. pathogenesis the incubation period for rabies is - weeks to the onset of clinical signs but can range from week to year. bites to the head and neck result in shorter incubation periods due to the close proximity to the brain. following infection, the virus migrates centripetally via peripheral nerve fibers to neurons within the brain, resulting in neurologic dysfunction. on reaching the brain, the virus migrates centrifugally to the salivary glands, thus enabling shedding and subsequent transmission. diagnosis and differential diagnosis definitive diagnosis is based on immunofluorescence of the virus in negri bodies of hippocampal cells. submission of the whole, unfixed brain, including the cerebellum and proximal brain stem, should be done within h of collection. the tissue should be kept refrigerated as freezing can cause delays in testing. differential diagnoses laboratory animal medicine include pseudorabies, canine distemper, bacterial meningitis, and toxicants that affect neurologic function. prevention and treatment puppies should be vaccinated by weeks of age, again at year, and then annually or triennially, depending on state and local laws. research complications immuno-prophylaxis is recommended for animal care and research personnel who may have work-related risks of exposure. due to risk of human exposure, animals with suspected infection should be humanely euthanized and brain tissue should be submitted for confirmation. giardiasis giardia lamblia, also known as g. duodenalis and g. intestinalis, is a binucleate flagellate protozoan that usually causes subclinical infestation of the small intestine. clinical disease is usually seen in young dogs and the characteristic sign is voluminous, light-colored, foul-smelling, soft to watery diarrhea, which is the result of malabsorption and hypersecretion. giardia has a direct life cycle with infection resulting after consuming cyst-contaminated food or water. the change in ph between the stomach and duodenum activates excystation and trophozoites then attach to the enterocytes. for diagnosis, direct fecal smears are considered best for observing trophozoites and zinc sulfate centrifugation is preferred for detection of cysts. a commercial elisa kit is licensed for use in dogs, but the positive predictive value is poor and zinc sulfate centrifugation techniques should be used in conjunction with elisa (rishniw et al., ) . pcr assays are also available for diagnosing giardiasis. differential diagnoses for giardiasis include bacterial and protozoal enteritis, coccidiosis, and whipworm infestation. metronidazole at - mg/kg po q h for - days is effective at treating giardiasis as well as other enteric protozoans, which may be potential differential diagnoses or coinfections. albendazole, fenbendazole, pyrantel, and praziquantel are also effective. coccidiosis intestinal coccidia associated with enteropathy in dogs include isospora canis, i. ohioensis, i. neorivolta, i. burrowsi, and hammondia heydorni (dubey and greene, ) . coccidian oocysts can be found in feces of clinically healthy dogs, as well as animals with diarrhea. clinically affected animals are young or immunosuppressed and develop diarrhea, which can vary from soft to watery and may contain blood or mucus. vomiting, dehydration, lethargy, and weight loss can also be seen. coccidia oocysts are typically spread by fecal-oral transmission, but dogs can ingest monozoic cysts in intermediate host tissues. the coccidian life cycle is both sexual and asexual, and results in the release of unsporulated eggs, which sporulate under appropriate environmental conditions. other causes for diarrhea should be excluded before a coccidial etiology is implicated. treatment may not be necessary, as infections are typically self-limiting and clinically insignificant. treatment may help to limit the number of oocysts shed in a kennel-housing situation and may be necessary in cases of protracted clinical illness. possible choices for treatment include daily administration of sulfadimethoxine ( - mg/kg po q h for - days) or trimethoprim sulfa ( mg/kg po q h for days). ascarids roundworms of dogs are most often toxocara canis; however, toxascaris leonina can also affect dogs. clinical illness is usually only seen in young animals with large worm burdens. diarrhea, vomiting, dehydration, and abdominal discomfort with vocalization can be seen. puppies may have a classical 'potbellied' appearance. heavy infestations can cause intussusception and/or intestinal obstruction. puppies that experience lung migrations of larval worms can develop fatal pneumonia. toxascaris canis can infect dogs by transplacental migration, transmammary migration, or ingestion of infective eggs. the infective stage of t. canis is the third-stage larva (l ). in transplacental infections, puppies may be born with l larvae in their lungs (sherding, ) . for diagnosis, large ( - μm in diameter) and relatively round ascarid eggs can be seen by standard fecal flotation methods. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention (hall and simpson, ) . most anthelmintics are effective for treatment. puppies should be treated early and often (every other week until weeks of age) because of the possibility of prenatal or neonatal infection. pregnant bitches can be treated with extended fenbendazole therapy ( mg/kg po once a day from day of gestation through day of lactation). hookworms the most common and most pathogenic hookworm of dogs is ancylostoma caninum. ancylostoma braziliense can also be found in dogs, but only a. caninum infestation typically results in clinical illness. puppies with hookworm infections can present as anemic with bloody diarrhea or melena. other clinical signs include lethargy, anorexia, dehydration, vomiting, and poor weight gain. these signs are a direct result of the worms' consumption of blood and body fluids. infective larvae (l ) are ingested from the environment and develop directly in the intestinal tract. infestation can also be transmammary, from ingestion of a paratenic host, and, less often, by transplacental migration. on histological sections, embedded worms with mouthparts may be identified. diagnosis is made by identification of eggs or larvae by either fecal flotation or direct smear. a differential diagnosis of parvovirus should be considered for puppies with bloody diarrhea, and autoimmune hemolytic anemia should be considered in young dogs with anemia. pyrantel pamoate is the anthelmintic of choice because it is safest in young ill animals. monthly administration of milbemycin or ivermectin plus pyrantel pamoate is recommended for prevention and control (hall and simpson, ) . due to transplacental or milkborne infection, puppies should be treated q weeks from to weeks of age. whipworms trichuris vulpis, the canine whipworm, can cause acute or chronic large intestinal diarrhea. the adult worm resides in the cecum or ascending colon. most infections are subclinical, but in symptomatic cases, the typical clinical sign is diarrhea with blood and/or mucus. abdominal pain, anorexia, and weight loss may also be seen. dogs may have eosinophilia, anemia, and/or hypoproteinemia on clinical hematology. trichuris vulpis has a direct life cycle with eggs passed in the feces. the penetration of the adult worm into the enteric mucosa, and the associated inflammation, can lead to diarrhea. factors that influence development of clinical symptoms are the number and location of adult whipworms; the severity of inflammation, anemia, or hypoproteinemia in the host; and the overall condition of the host. whipworm infestation is diagnosed by the presence of barrel-shaped, thickwalled eggs with bipolar plugs on fecal flotation. adult worms intermittently release eggs; therefore, negative results do not exclude infection. differential diagnoses for whipworm infestation include giardiasis, coccidiosis, and bacterial enteritis. fenbendazole, oxibendazole, and milbemycin have all been recommended for treatment of whipworms. treatment for whipworm infestation should be at monthly intervals for months (jergens and willard, ) . several species of cestodes parasitize the small intestine of dogs. the most common is dipylidium caninum. other species include taenia pisiformis and, more rarely, echinococcus granulosus, multiceps spp., mesocestoides spp., and spirometra spp. most cestode infestations are subclinical, but severe infestations with dipylidium can cause diarrhea, weight loss, and poor growth. the cestode requires an intermediate host, which for d. caninum are fleas and lice. ingestion of these arthropods results in transmission of the tapeworm. definitive diagnosis is usually made by the identification of egg capsules or proglottids (tapeworm segments) on the surface of the feces or around the anus. the most significant means to limit cestode infestation is to control flea and/or louse exposure. praziquantel at - . mg/kg orally or subcutaneously is the standard treatment for cestodiasis, especially taenia or echinococcus species. fenbendazole, mebendazole, or oxfendazole may also be effective against d. caninum (hall and simpson, ) . demodicosis canine demodicosis is caused by demodex canis, a commensal mite that lives in the hair follicles and is passed from dams to nursing pups. localized demodicosis is typically asymptomatic, but disease can present with variable and nonspecific clinical signs, such as alopecia, erythema, pruritus, crusts, and hyperpigmentation. it can occur anywhere on the body but is often seen on the feet and face, and around the ears (demanuelle, a). generalized demodicosis can develop in juvenile or adult populations and is indicative of an underlying immunosuppressive disorder. demodex has a characteristic 'cigar shape' and can be identified from deep skin scrapings mounted on mineral oil (campbell, ; noli, ) . differential diagnoses include dermatophytosis, allergic contact dermatitis, and seborrheic dermatitis. the primary differential diagnosis for generalized demodicosis is primary bacterial pyoderma, which is also a common secondary complication of generalized demodicosis. ivermectin at - μg/kg and oral milbemycin at - mg/kg/day are effective treatments. treatment duration can be extensive and must be accompanied by repeated skin scrapings. sarcoptic mange canine sarcoptic mange is caused by sarcoptes scabiei var. canis, which is zoonotic. the most common clinical sign is an intense pruritus, usually beginning at sparsely furred areas of the ear pinnae, elbows, ventral thorax, and abdomen. lesions are characterized by alopecia and yellowish dry crusts with a macular papular eruption. these lesions may be exacerbated by excoriation due to the pruritic nature of the condition. adult mites, mite eggs, or mite feces may be observed on superficial skin scrapings, but diagnosis may be difficult because multiple skin scrapings may yield negative results. even if scrapings are negative, a therapeutic trial should be initiated if the clinical signs and history suggest a sarcoptes etiology. demonstration of anti-mite ige either in the serum or via an intradermal antigen test can be used as a diagnostic aid (campbell, ) . histologic examination is nondiagnostic; however, suggestive lesions include small foci of edema, exocytosis, degeneration, and necrosis . an important differential diagnosis is flea allergy dermatitis (fad). unless antiparasitic therapy would interfere with research objectives, all dogs with sarcoptic mange should be treated. in addition, their kennel mates should also be treated due to the contagious nature of the disease and its zoonotic potential. the usual means of treatment is either ivermectin at - μg/kg q days or milbemycin at mg/kg q days for three oral doses . ticks ticks are obligate arachnid parasites that require vertebrate blood as their sole food source. genera that more commonly infest dogs in the united states include species of rhipicephalus, dermacentor, amblyomma, and ixodes. the primary significance of tick infestation is vector-borne infectious diseases, including rocky mountain spotted fever (rickettsia rickettsii), lyme disease (borrelia burgdorferi sensu stricto), thrombocytic anaplasmosis (anaplasma platys), and canine monocytic ehrlichiosis (ehrlichia canis). ticks alone cause minimal signs unless the dog develops a hypersensitivity reaction leading to a more granulomatous response at the bite location (merchant and taboada, ) . some species (primarily dermacentor andersoni and d. variabilis) produce a salivary neurotoxin that causes an ascending flaccid paralysis (malik and farrow, ) . uncomplicated tick bites and tick-bite paralysis are diagnosed by identification of the tick and clinical signs of paralysis. dogs with tick-bite paralysis usually show improvement within h of tick removal, with complete recovery within h (malik and farrow, ) . formamidines (amitraz), pyrethroids, and phenylpyrazoles (fipronil) are available as spot-ons, collars, sprays, and foggers to treat tick infestations in both the animal and the environment (halos et al., ; beugnet and franc, ) . differential diagnoses for tick-bite paralysis include botulism, snakebite, polyradiculoneuritis, and idiopathic polyneuropathy (malik and farrow, ) . fleas the most common flea to infest dogs is ctenocephalides felis felis, the cat flea (sousa, ) . flea infestations usually cause foci of alopecia and pruritus. dogs that are hypersensitive to antigenic proteins in flea saliva develop severe fad, which features papules, crusting, and excoriations over the lumbosacral region, flanks, thighs and abdomen. these animals may require oral corticosteroids to relieve clinical signs (muller et al., ) . secondary bacterial and fungal infections can also develop. fleas can also transmit other parasitic diseases, such as dipylidium tapeworms. flea infestations and fad are definitively diagnosed by observing the fleas on the host's skin; however, the presence of flea excrement can support a presumptive diagnosis (demanuelle, b) . treatment of flea infestations should use an integrated pest management (ipm) approach that targets adult fleas, immature stages, and environmental contamination in order to limit the risk of chemoresistance. combining ovicidal treatments, such as lufenuron and selamectin, with adulticidal treatments, such as fipronil, spinosad, selamectin, and imidacloprid, is recommended (halos et al., ; beugnet and franc, ; dryden et al., ) . certain chemicals (i.e., imidacloprid and selamectin) have both adulticidal and larvacidal abilities, but the principles of ipm preclude the use of one product solely for both adulticidal and larvacidal properties (schwassman and logas, ). differential diagnoses include mite and louse infestations, bacterial folliculitis, and allergic or atopic conditions that present with skin lesions in dogs. canine dermatophytoses are commonly caused by microsporum spp., trichophyton spp., and epidermophyton spp. (moriello and deboer, ) . uncomplicated infections are characterized by circular areas of alopecia and crusting with or without follicular papules, usually around the face, neck, and forelimbs. dermatophytes infect the hair shaft and follicle, as well as the surrounding skin. infected hairs become brittle and broken shafts remain infective in the environment for months. dermatophytoses are zoonotic and easily transmitted to other animals through the environment or by direct contact. definitive diagnosis is made using dermatophyte test medium for culture. hair and crust material from infected sites can be plucked and placed on culture; however, the 'toothbrush' method is more effective for sampling multiple sites. the brush is used to comb hairs and scales from several infected sites and then pressed into the culture media. media plates should be visually inspected daily for days. positive cultures will become red at the same time as growth of a fluffy white colony. microscopic examination of hairs and scales to visualize fungal elements can be done using skin scrapings in % koh or mineral oil; however, this method is not very sensitive. topical and systemic therapy should be initiated together after all suspected areas are clipped to reduce spreading of contaminated fragile hairs. wholebody topical therapies with antifungal shampoos, rinses, and creams are recommended rather than spot treatment. systemic therapy can be achieved with griseofulvin, ketoconazole, itraconazole, or fluconazole. due to the highly infective nature of this disease, animals should be isolated and the environment thoroughly disinfected. chlorhexidine and virkon ® s are ineffective at clearing environmental spores, but lime sulfur ( : ), enilconazole ( . %), and bleach ( : ) are effective across many strains of microsporum canis (moriello and deboer, ) . although the incidence of hypothyroidism in the canine population is not high (kemppainen and clark, ) , deficiency in thyroid hormone can significantly laboratory animal medicine affect basal metabolism and immune function. because these factors are important in many biomedical research studies, it is imperative that laboratory animal veterinarians be able to recognize, diagnose, and treat this problem. etiology primary hypothyroidism affects the thyroid gland directly, whereas secondary hypothyroidism has indirect effects through dysfunction of the pituitary gland (seguin and brownlee ) . both of these causes result in a gradual loss of functional thyroid tissue (avgeris et al., ; kemppainen and clark, ) . the majority of cases of canine hypothyroidism are due to lymphocytic thyroiditis, an autoimmune disorder, or idiopathic atrophy of the thyroid gland. lymphocytic thyroiditis is the major cause of hypothyroidism in laboratory beagles and appears to be familial in that breed (tucker, ; beierwaltes and nishiyama, ; manning ) . rarely, congenital defects or nonfunctional tumors may cause hypothyroidism (peterson and ferguson, ; kemppainen and clark, ) . clinical signs because it affects metabolism in general, hypothyroidism can produce a large number of clinical signs referable to many organ systems. an individual dog with hypothyroidism may have one or any combination of clinical signs. hypothyroidism reduces the dog's metabolic rate, which then produces such signs as obesity, lethargy, cold intolerance, and constipation. additionally, hypothyroidism can produce several dermatologic abnormalities, including nonpruritic, bilaterally symmetrical alopecia, hyperpigmentation, seborrhea, and pyoderma (avgeris et al., ; peterson and ferguson, ; panciera, ) . several clinicopathologic abnormalities have also been reported in a large percentage of hypothyroid dogs. these aberrations include increased serum cholesterol and triglycerides due to a decrease in lipolysis and decreased numbers of low-density lipopolysaccharide receptors (peterson and ferguson, ; panciera, ) . normocytic, normochromic, nonregenerative anemia may be seen in approximately one-half of the cases (avgeris et al., ) . increased serum alkaline phosphatase and creatine kinase have also been reported in a significant number of hypothyroid dogs (peterson and ferguson, ; panciera ) . neurologic signs of hypothyroidism, which include lameness, foot dragging, and paresis, may be caused by several mechanisms such as segmental nerve demyelination or nerve entrapment secondary to myxedema (peterson and ferguson, ) . mental impairment and dullness have also been reported in hypothyroid dogs, secondary to atherosclerosis and cerebral myxedema (peterson and ferguson, ) . hypothyroidism has been implicated in other neurological abnormalities such as horner's syndrome, facial nerve paralysis, megaesophagus, and laryngeal paralysis; however, these conditions do not always resolve with treatment (bichsel et al., ; panciera, ) , and a true causal relationship with hypothyroidism has not been completely defined (panciera, ) . myopathies associated with hypothyroidism are caused by metabolic dysfunction and atrophy of type ii muscle fibers and can present with signs similar to neurological disease (peterson and ferguson, ) . hypothyroidism can also cause abnormalities of the cardiovascular system including bradycardia, hypocontractility, increased vascular volume, and atherosclerosis (seguin and brownlee ) . abnormalities that may be detected by ecg include a decrease in p-and r-wave amplitude (peterson and ferguson, ) and inverted t waves (panciera, ) . these ecg abnormalities are caused by lowered activity of atpases and calcium channel function. an association between hypothyroidism and von willebrand disease has been suggested. however, the relationship is probably one of shared breed predilection and not a true correlation. contradictory studies have shown either deficient (avgeris et al., ) or normal (panciera and johnson , ; avgeris et al., ) von willebrand factor antigen and bleeding times in hypothyroid dogs. most importantly, hypothyroidism does not appear to cause overt, clinical von willebrand disease. however, it may exacerbate existing subclinical von willebrand disease (seguin and brownlee, ) . epizootiology the prevalence of hypothyroidism in the general canine population is reportedly less than % (panciera, ) . the disorder occurs most often in middle-aged, larger breed dogs (avgeris et al., ) , and reports suggest a higher incidence of hypothyroidism in spayed, female dogs (panciera, ; peterson and ferguson, ) . doberman pinschers and golden retrievers appear to have a higher incidence of hypothyroidism compared with other breeds (panciera, ; peterson and ferguson, ; scarlett, ) . there have been several reports about hypothyroidism in laboratory colonies of beagles (manning, ; tucker, ; beierwaltes and nishiyama, ) . diagnosis and differential diagnosis because of the large number of clinical manifestations in dogs, the recognition of hypothyroidism is not always straightforward. likewise, the diagnosis of hypothyroidism can be difficult because of the lack of definitive diagnostic tests available for the dog. a complete understanding of the diagnosis of hypothyroidism requires a familiarity with thyroid hormone metabolism and function that is beyond the scope of this writing. for additional information, the reader is referred to one of several manuscripts available (peterson and ferguson, ; ferguson, ) . currently, the ability to diagnose hypothyroidism relies heavily on the measurement of serum total t (thyroxine) and free t (peterson and ferguson, ; ferguson, ) . t serves primarily as a precursor for t and is heavily protein bound. free t represents the laboratory animal medicine unbound fraction that is available to the tissues (peterson and ferguson, ) . the measurement of total t carries a sensitivity of around % and can be used as a good screening tool. with the measurement of both serum total t and free t , hypothyroidism can usually be ruled out if the values are within the normal range or higher. if both hormone concentrations are low, it is highly likely that the patient has hypothyroidism, and a therapeutic trial may be in order (peterson and ferguson, ) . however, nonthyroidal illnesses and some drugs (e.g., glucocorticoids, anticonvulsants, phenylbutazone, salicylates) can falsely lower these values (peterson and ferguson, ; ferguson, ) . therefore, low values do not always indicate that hypothyroidism is present and animals should not be treated solely on the basis of serum hormone levels if clinical signs are not present. if the clinical signs are equivocal or only total t or free t is decreased, further diagnostic testing is warranted (peterson and ferguson, ) . although t is the most biologically active form of thyroid hormone, the measurement of serum t levels is an unreliable indicator of hypothyroidism (peterson and ferguson, ; ferguson, ) . serum t can be falsely lowered by many nonthyroidal illnesses and many drugs (see above). in addition, t may be preferentially released and conversion of t to t may be enhanced by the failing thyroid (peterson and ferguson, ; ferguson, ) , particularly early in the disease. in one study, t was within normal limits in % of the hypothyroid dogs (panciera, ) . autoantibodies can be responsible for false elevations in the concentrations of t and t found in these respective assays. it has been recommended that free t , measured by equilibrium dialysis, be assayed in dogs that are suspected of hypothyroidism and have autoantibodies with normal or high t and t . autoantibodies have been found in less than % of the samples submitted to one laboratory (kemppainen and behrend, ) . other means of diagnosing hypothyroidism have been described. in humans, endogenous thyroid-stimulating hormone (tsh) levels provide reliable information on thyroid status, and an assay is available for dogs. however, endogenous tsh levels can be normal in some dogs with hypothyroidism and high tsh levels have been noted in normal dogs and sick animals that are actually euthyroid. it is therefore recommended that tsh levels be considered along with other information (clinical signs, t ) prior to diagnosis and treatment (kemppainen and behrend, ) . tsh stimulation testing using exogenous bovine tsh provides a good and reliable method for establishing a diagnosis. unfortunately, the availability and expense of tsh limit the use of this diagnostic tool (peterson and ferguson, ; ferguson, ) . another drawback of tsh testing is that the test must be postponed for weeks if thyroid supplementation has been given (peterson and ferguson, ) . when tsh is available for testing, there are several recommendations for dosage, routes of administration, and sampling times. one recommendation is . u of tsh per pound of body weight (up to a maximum of u) to be administered iv. for this protocol, blood samples are taken prior to administration of tsh and h after. a normal response to the administration of tsh should create an increase of t levels at least μg/dl above the baseline levels or an absolute level that exceeds μg/dl (peterson and ferguson, ; wheeler et al., ) . treatment the treatment of choice for hypothyroidism in the dog is l-thyroxine (sodium levothyroxine). a recommended dosing regimen is . - . mg/kg once a day (avgeris et al. ). if drugs that decrease thyroxine levels are being administered concurrently, it may be necessary to divide the thyroxine dose for twice daily administration. after the supplementation has begun, the thyroid hormone level should be rechecked in - weeks, and blood samples should be drawn - h after the morning pill. a clinical response is usually seen in - weeks and would include weight loss, hair regrowth, and resolution of other signs (panciera, ) . ecg abnormalities also return to normal (peterson and ferguson, ) . for dogs with neurologic signs, the prognosis is guarded, because the signs do not always resolve with supplementation (panciera, ) . weight gain and eventual obesity are frequent findings in dogs in the research environment. because obesity can adversely affect several body systems as well as general metabolism, the laboratory animal veterinarian must address obesity and its potential effects on animal welfare and research results. etiology obesity is defined as a body weight - % over the ideal. in general, obesity occurs when the intake of calories exceeds the expenditure of energy, the result of overeating or eating an unbalanced diet. overeating is a common cause of obesity in pet dogs and may be triggered by boredom, nervousness, or conditioning (macewen, ) . in addition, pet animals are often subjected to unbalanced diets supplemented with high-fat treats. in the laboratory animal setting, overeating is less likely than in a household because access to food is more restricted, and diets are usually a commercially prepared balanced ration. however, obesity can still be a problem if specific guidelines for energy requirements are not followed. in addition, the necessary caging of dogs in the research environment and limitation to exercise reduces energy expenditure. it is also important to realize that other factors may predispose dogs to obesity, even when guidelines for caloric intake and energy laboratory animal medicine expenditure are followed (butterwick and hawthorne, ) . as in humans, genetics plays an important role in the development of obesity in dogs, and certain breeds are more predisposed toward obesity. in a study of dogs visiting veterinary clinics in the united kingdom, labrador retrievers were most likely to be obese. other breeds affected included cairn terriers, dachshunds, basset hounds, golden retrievers, and cocker spaniels. the beagle was also listed as a breed predisposed to obesity in the household environment (edney and smith, ) . several metabolic or hormonal changes are also associated with obesity. it has been well established that neutering promotes weight gain. in one study, spayed female dogs were twice as likely to be obese compared with intact females (macewen, ) . the authors proposed that the absence of estrogen promotes an increase in food consumption. a similar trend toward obesity was found in castrated male dogs (edney and smith, ). in addition, hypothyroidism and hyperadrenocorticism may present with obesity as one of the clinical signs (macewen, ) . differential diagnosis the diagnosis of obesity is somewhat subjective and relies on an estimate of ideal body weight. the ideal body condition for dogs is considered to be achieved when the ribs are barely visible but easily palpated beneath the skin surface. when the ribs are not easily palpated and/or the dog's normal function is impaired by its weight, the animal is considered obese. there are few objective, quantifiable methods for establishing this diagnosis. ultrasound has been evaluated for measurement of subcutaneous fat in dogs, and measurements taken from the lumbar area can be used to reliably predict the total body fat (wilkinson and mcewan, ) . after a diagnosis of obesity has been made, additional diagnostic tests should be performed to determine if there is an underlying cause for the problem. a complete physical exam should be performed to look for signs of concurrent disease and to establish if obesity has adversely affected the individual. serum thyroid hormones should be evaluated (see above), and serum chemistry may reveal an increased alkaline phosphatase associated with hyperadrenocorticism. treatment restricting food intake readily treats obesity, and this is easily done in the research setting. it has been suggested that a good weight loss program involves restriction of intake to % of the calculated energy requirement to maintain ideal body weight. it has been shown that restriction of calories down to % produces no adverse health effects. however, t levels will decrease in direct proportion with caloric intake. ideally, weight loss will occur at a rate of - % of body weight per week (laflamme et al., ) . with more severe calorie restriction and more rapid weight loss, the individual is more likely to have rebound weight gain after restrictions are relaxed. there has been a great deal of attention in humans as to the correct diet to encourage weight loss. likewise, the type of diet fed to dogs has been examined. as mentioned above, the restriction of calories is most important, and feeding less of an existing diet can do this. alternatively, several diet dog foods are available, and there is some evidence that these diets are superior to simple volume restriction (macewen, ) . there has been much concern about the addition of fiber to the diet as a method for reducing caloric intake while maintaining the volume fed. studies in dogs have examined the addition of both soluble and insoluble fibers to calorie-restricted diets. these studies have shown that the addition of fiber does not have an effect on satiety in dogs and therefore does not have a beneficial effect in weight loss protocols (butterwick et al., ; butterwick and markwell, ) . research complications it is important to control weight gain in research animals because of the association of obesity with metabolism. although an association between obesity and reproductive, dermatologic, and neoplastic problems has been reported (macewen, ) , this relationship is not consistently apparent (edney and smith, ) . joint problems including osteoarthritis and hip dysplasia have also been related to obesity (macewen, ; kealy et al., ) . in addition, diabetes mellitus has been linked to obesity and obesity-induced hyperinsulinism in several experimental models (macewen, ) . a recent study demonstrated metabolic disease, typified by hyperinsulinemia and hypoadiponectinemia, in approximately % of obese dogs (tvarijonaviciute et al., ) . research that requires anesthesia may be complicated by a greater risk of cardiovascular diseases (edney and smith, ) including hypertension and compromise to the respiratory tract. etiology in the laboratory setting, the majority of traumatic wounds will be small in size and quickly observed. occasionally, dogs may sustain minor trauma during transport or have a small, previously undetected, chronic wound upon arrival at the facility. when dogs are group housed, they may sustain bite wounds during early socialization periods. under these conditions, proper initial treatment will lead to uncomplicated wound healing. complications such as infection and delayed healing arise when wounds are not noticed immediately or when the basic principles of wound management are not followed. clinical signs the signs and appearance of a traumatic wound will vary with the cause and the duration of time since wounding. abrasions, sustained by shear forces, are partial thickness skin wounds characterized laboratory animal medicine by minimal bleeding or tissue disruption. puncture wounds have a small surface opening but penetrate into deep tissues with the potential for contamination. lacerations are wounds caused by sharp separation of skin that may extend to deeper tissues. acute wounds are characterized by bleeding tissue, sharp edges and no obvious devitalization. they have variable degrees of contamination. chronic wounds generally do not exhibit active bleeding and will have curled or rounded edges. these wounds often have necrotic tissue and are considered contaminated. treatment to aid decision making about wound therapy, several classification systems have been developed for traumatic injuries. at one time, decisions about wound therapy were largely based upon the length of time since wounding, or the concept of a 'golden period.' it is now recognized that several factors must be considered prior to initiating wound care, including (but not limited to) the type and size of the wound, the degree of wound contamination, and the competence of the host's defense systems (swaim, ; waldron and trevor, ) . one of the most widely used classification systems is based upon wound contamination and categorizes wounds as clean, clean-contaminated, contaminated, or dirty (see table . ). the vast majority of the wounds seen in the laboratory setting will fall into the clean and clean-contaminated categories. these wounds may be treated with the basic wound care described below and primary closure of the wound. contaminated and dirty wounds require more aggressive therapy. postsurgical infections or complications of initial therapy would be considered dirty wounds. when in doubt as to the classification of a wound, the worst category should be presumed in order to provide optimal therapy and reduce the chance for complications. the initial treatment of a wound is the same regardless of its classification. when first recognized, the wound should be covered with a sterile dressing until definitive treatment can be rendered. bleeding should be controlled with direct pressure; tourniquets are discouraged because of the complications that may arise with inappropriate placement (swaim, ) . it is best to avoid using topical disinfectants in the wound until further wound treatment (culture, debridement, lavage) has been performed (swaim, ) . anesthesia or analgesia may be necessary and the choice of agent will depend on the size and location of the wound as well as the preference of the clinician. if the wound is contaminated or dirty, bacterial cultures, both aerobic and anaerobic, should be performed. then a water-soluble lubricant gel may be applied directly to the wound to prevent it from further contamination during the hair removal process. a wide margin of hair should be clipped and a surgical scrub performed around the edges of the wound. povidone-iodine alternating with alcohol or chlorhexidine gluconate scrub alternating with water is most often recommended for surgical preparation of the skin surface (osuna et al., a, b) . simple abrasions that involve only a partial thickness of the skin do not generally require further treatment. full-thickness wounds require further attention, including irrigation with large quantities of a solution delivered under pressure. several irrigation solutions have been recommended (lozier et al., ; waldron and trevor, ; sanchez et al., ) , but type may not be as important as the volume and pressure of delivery. it has been suggested that psi is required to obtain adequate tissue irrigation, and this may be achieved by using a -ml syringe with an -or -gauge needle (waldron and trevor, ) . for wounds that are contaminated or dirty, debridement is an important part of initial therapy. debridement usually proceeds from superficial to deeper layers. skin that is obviously necrotic should be removed. although it is often recommended to remove skin back to the point at which it bleeds, this may not be feasible with large wounds on the limbs. in addition, other factors such as edema or hypovolemia may reduce bleeding in otherwise viable skin (waldron and trevor, ) . if one is unsure about tissue viability in areas that are devoid of waldron and trevor ( ) . extra skin, the tissue may be left (swaim, ; waldron and trevor, ) , and nonviable areas will demarcate within - days (waldron and trevor, ) . necrotic fat should be resected liberally, because it does not have a large blood supply and will provide an environment for infection. often, resection of subcutaneous fat is necessary to remove debris and hair that could not be removed during wound irrigation. damaged muscle should also be liberally resected (swaim, ) . the wound should be irrigated several times during debridement and again after completion. after initial wound treatment, the options concerning wound closure must be weighed. the principles of basic surgery are discussed in several good texts, and readers are encouraged to pursue additional information. primary wound closure is defined as closure at the time of initial wound therapy and is the treatment of choice for clean and clean-contaminated wounds. closure is performed in two or more layers, carefully apposing tissues and obliterating dead space. if dead space will remain in the wound, a drain should be placed. subcutaneous closure should be performed with absorbable suture such as polydioxanone, polyglactin , or polyglycolic acid. it is best to use interrupted sutures and avoid leaving excess suture material in the wound. it may be necessary to choose tension-relieving suture patterns, such as horizontal mattress. skin closure is generally performed with nylon ( - or - ). in situations where gross contamination cannot be completely removed, closure of the wound should be delayed or avoided. after debridement and irrigation, the wound should be bandaged. the wound may be covered by a nonadherent dressing such as vaseline-impregnated gauze (swaim, ) . the contact layer is covered by cotton padding, and the entire bandage is covered by a supportive and protective layer. the bandages should be changed once or twice daily, depending upon the amount of discharge coming from the wound. wound closure within - days of wounding (prior to the formation of granulation tissue) is considered delayed primary closure. when the wound is closed after days, this is considered secondary closure (waldron and trevor, ) . second-intention healing involves allowing the wound to heal without surgical intervention. this type of healing is often used on limbs when there is an insufficient amount of skin to allow complete closure (swaim, ) . it is important to note that second-intention healing will take longer than with surgical repair, and, in the case of large wounds, it will be more expensive because of the cost of bandaging materials. several factors must be weighed when considering the use of antibiotics in traumatic wound care, including the classification and site of the wound, host defenses, and concurrent research use of the animal. when wounds are clean or clean-contaminated, antibiotics are seldom necessary unless the individual is at high risk for infection. when wounds have been severely contaminated or are dirty, antibiotics are indicated and the type of antibiotic will ultimately depend on culture and sensitivity results. until such results are available, the choice of antibiotic is based on the most likely organism to be encountered. topical application of bacitracin, neomycin sulfate, and polymixin b combinations may be used in wounds with minor contamination. in skin wounds with more extensive contamination, staphylococcus spp. are generally of concern, whereas pasteurella multocida should be considered in bite wounds. when systemic antibiotics are necessary, cephalosporins, amoxicillinclavulanate, and trimethoprim sulfas are often recommended for initial antibiotic therapy (waldron and trevor, ) . prevention in facilities with good husbandry practices and a diligent staff, potentially injurious equipment or surfaces are identified quickly. appropriate attention to surgical technique and to initial wound care will generally reduce the occurrence of postprocedure wound infection. etiology pressure sores (decubital ulcers) can be a problem in long-term studies and housing situations that require chronic skin contact with hard surfaces. decubital ulcers often develop over a bony prominence such as the elbow, tuber ischii, tarsus, or carpus. the compression of soft tissues between hard surfaces results in vascular occlusion, ischemia, and ultimately tissue death (swaim and angarano, ) . several factors that increase pressure at the site and/or affect the integrity of the skin will predispose an individual to develop pressure sores, including poor hygiene, self-trauma, low-protein diet, preexisting tissue damage, muscle wasting, inadequate bedding, and ill-fitting coaptation devices (swaim and angarano, ) . clinical signs initially, the skin will appear red and irritated. over time, constant trauma can result in full-thickness skin defects and can progress to necrosis of underlying tissues. the severity of the sores may be graded from i to iv according to the depth of the wound and the tissues involved, from superficial skin irritation to involvement of underlying bone (waldron and trevor, ) . epizootiology the problem usually occurs in large dog breeds, but any type of dog can be affected. prevention and control minimizing or eliminating predisposing factors is important to both the prevention and treatment of this condition. if a dog will experience long periods of recumbency, adequate bedding or padding must be provided. recumbent animals should be moved frequently, ideally every h, to prevent continuous compression on a specific laboratory animal medicine area (waldron and trevor, ) . skin hygiene is of the utmost importance when trying to prevent or treat pressure sores. the skin should be kept clean and dry at all times. if urine scalding is a problem, the affected area should be clipped, bathed, and dried thoroughly at least once or twice daily. finally, an appropriate diet to maintain body weight will minimize compressive forces experienced over areas susceptible to ulceration (swaim and angarano, ) . treatment the treatment of pressure sores must involve care of the wound and attention to the factors causing the wound. the extent of initial wound management will largely depend on the depth of the wound. for simple abrasions and small wounds involving the skin only, simple wound cleansing and openwound management provide adequate treatment. when wounds involve deeper tissues, including fat, fascia, or bone, more aggressive diagnostics and therapy must be performed. the affected area should be radiographed to assess bone involvement and the wound should be cultured. all of the damaged tissue should be debrided and basic wound management guidelines should be followed (see above). when a healthy granulation bed has formed over the entire wound, a delayed closure over a drain may be performed (swaim and angarano, ) . with extensive lesions, reconstruction with skin flaps may be necessary (waldron and trevor ) . bandaging should be performed on all full-thickness wounds; however, it is important to remember that illfitting or inadequately padded bandages or casts may worsen the problem. the area over the wound itself should not be heavily padded. the wounded area should be lightly covered and then a doughnut, created from rolled gauze or towel, should be fitted around the wound, in order to displace pressure over a larger area and onto healthier tissue. the doughnut is then incorporated into a padded bandage. if a cast has been applied to the area for treatment or research purposes, a hole can be cut over the wound to reduce pressure in that area and allow treatment of the wound (swaim and angarano, ) . bandages should be removed at least once or twice a day to allow wound care. etiology an acral lick granuloma is a skin lesion caused by self-trauma. in a few cases, the self-trauma is due to initial irritation caused by an identifiable neurologic or orthopedic condition (tarvin and prata, ) . allergy may also be a source of irritation that leads to self-trauma. however, the majority of cases begin because of repetitive licking by dogs that are confined and lack external stimuli (swaim and angarano, ) . it has been theorized that the self-trauma promotes the release of endogenous endorphins, which act as a reward for the abnormal behavior (dodman et al., ) . the laboratory environment could promote the abnormal behavior and lead to acral lick granuloma. epizootiology the lesions associated with acral lick granuloma are seen most often in large dog breeds, particularly dobermans. however, any type of dog can be affected (walton, ) . clinical signs early lesions appear as irritated, hairless areas usually found on the distal extremities (swaim and angarano, ) . the predilection for the limbs may be due to accessibility or possibly a lower threshold for pruritus in these areas. as the lesions progress, the skin becomes ulcerated and the wound develops a hyperpigmented edge. the wounds may partially heal and then be aggravated again when licking resumes. diagnosis and differential diagnosis acral lick granulomas must be differentiated from several other conditions, including bacterial or fungal infection, foreign bodies, and pressure sores. in addition, mast cell tumors and other forms of neoplasia can mimic the appearance of acral lick granuloma. many of the aforementioned problems can be ruled out by the history of the animal. however, a complete history may be unavailable in the laboratory setting. fungal cultures and allergy testing may aid in diagnosis. biopsy of the affected area would rule out neoplasia. an uncomplicated acral lick granuloma would feature hyperplasia, ulceration, and fibrosis without evidence of infection or neoplasia (walton, ) . prevention and control behavior modification and relief of boredom are important aspects of preventing (and treating) acral lick granuloma. environmental enrichment including exercise, co-housing and various toys is already a basic requirement and may be increased to combat self injurious behaviors. treatment several treatments have been reported for acral lick granuloma and the selection of a treatment should be based on the underlying cause. one of the most important aspects of treatment is to break the cycle of self-trauma. mechanical restraint with an elizabethan collar is one of the easiest methods to accomplish this goal. several direct treatments have been examined, including intralesional and topical steroids, perilesional cobra venom, acupuncture, radiation, and surgery (swaim and angarano, ; walton, ) . opioid antagonists have been applied as treatments for acral lick granulomas and self-injurious behaviors with the theory that this will block the effects of endogenous opioids. naltrexone and nalmefene have been used successfully to reduce excessive licking behaviors and resolve associated lesions. however, lesions did recur after the drugs were discontinued (dodman et al., ; white, ) . the topical administration of a mixture of flunixin meglumine, steroid, and dimethyl sulfoxide has also been shown to be effective (walton, ) . in addition, psychoactive drugs have been suggested to relief of laboratory animal medicine boredom or anxiety. these have included phenobarbital, megestrol acetate, and progestins. however, side effects have been reported (swaim and angarano, ) . other behavior-modifying medications such as clomipramine may be effective in the treatment of compulsive anxiety disorder. the potential for effects that could interfere with experimental results must be determined prior to initiation of treatment. it is important to note that none of the above-mentioned treatments have been successful in all cases. the overall prognosis for acral lick granuloma should be considered guarded since the lesions often recur when treatment is discontinued. etiology hygromas are fluid-filled sacs that develop as a result of repeated trauma or pressure over a bony prominence. the area over the olecranon is most frequently affected, but hygromas have been reported in association with the tuber calcis, greater trochanter, and stifle (newton et al., ) . epizootiology elbow hygromas are most frequently reported in large and giant breeds of dogs, less than years of age (johnston, ; white, ; cannap et al., ) . elbow hygromas are seen infrequently in the laboratory animal setting because the commonly affected breeds are seldom used in research. however, the housing environment of research dogs, especially cage bottoms and cement runs, may predispose them to hygromas. for this reason, laboratory animal veterinary and husbandry staff should be familiar with this condition. clinical signs the clinical presentation will depend upon the chronicity of the problem. a dog with an elbow hygroma usually presents with a painless, fluctuant swelling over the point of the elbow without signs of lameness. the condition may be unilateral or bilateral. over a long period of time, elbow hygromas may become inflamed and ulcerated. if the hygroma becomes secondarily infected, the animal may exhibit pain and fever (johnston, ; white, ) . pathology the fluid-filled cavity in the hygroma is lined by granulation and fibrous tissue. hygromas lack an epithelial lining and therefore are not true cysts. the fluid within the cavity is a yellow or red serous transudate. this fluid is less viscous than joint fluid and elbow hygromas do not communicate with the joint (johnston, ) . treatment the treatment of elbow hygromas should be conservative whenever possible. conservative management of the elbow hygroma is aimed at relieving the source of pressure at the point of the elbow. in early and mild cases, simply providing padding to cover hard surfaces will result in resolution of the hygroma. a soft padded bandage or doughnut bandage around the affected site may also be of benefit. neoprene/polyester sleeves that cover the elbows and fit over the shoulders are also available as an option for either prevention or treatment of hygromas (cannap et al., ) . more aggressive therapies, including needle drainage and injection of corticosteroids into the hygroma, have been described but are not recommended due to the risk of infection (johnston, ) . surgical options should be reserved for complicated or refractory cases. even simple excision can be associated with complications such as wound dehiscence and ulceration (johnston, ) due to the location of the bony prominence at the surgical site. this issue may be avoided by using a skin advancement flap (white, ) that allows intact, healthy skin to cover the boney prominence. a muscle advancement flap has also been described (green et al., ) . regardless of the method used to treat an elbow hygroma, recurrence of the problem is likely unless the predisposing factors are identified and relieved. etiology in the research environment, corneal ulcers are most often associated with direct trauma, contact with irritating chemicals, or exposure to the drying effects of air during long periods of anesthesia. chronic or recurrent corneal ulcers may also be associated with infection or hereditary causes in some breeds of dogs; however, these would be rare in the laboratory setting. clinical signs the signs of corneal ulceration are blepharospasm, epiphora, and photophobia. the eye may appear irritated and inflamed. in minor cases, the cornea may appear normal however, in cases of deeper ulceration, the cornea may appear roughened or have an obvious defect. in addition, the periocular tissues may be swollen and inflamed because of self-inflicted trauma from rubbing at the eye. diagnosis a tentative diagnosis of corneal ulcer or abrasion may be based on the clinical signs. a definitive diagnosis of corneal ulcers is made by the green appearance of the cornea when stained with fluorescein dye. when a corneal ulcer has been diagnosed, the eye should be inspected for underlying causes such as foreign bodies, abnormal eyelids, or abberant cilia. treatment the treatment of corneal ulcers will depend on the depth and size of the affected area, as well as the underlying cause. superficial abrasions are generally treated with topical application of antibiotics. a triple antibiotic ointment that does not contain steroids given three times a day for - days usually provides adequate treatment. simple corneal ulcers are restained with fluorescein after days and should show complete healing at that time. if the ulcer is not healed, this may indicate that the ulcer has an undermined edge impeding proper healing. topical anesthetic should be applied to the eye, and a cotton-tipped applicator can be rolled over the surface of the ulcer toward its edge. this will remove the unattached edge of the cornea and healing should progress normally after debridement. deep ulcers may require further debridement and primary repair. in such cases, a third eyelid or conjunctival flap may be applied to the eye until experienced help can be obtained. in all cases, an elizabethan collar or other restraint may be necessary to prevent additional trauma to the eye. ulcers caused by entropion, ectropion, or dystichiasis will not resolve until the condition is repaired, and descriptions for this can be found elsewhere. prevention the proper application of lubricant eye ointment at the time of anesthesia will prevent drying due to exposure and may also protect the eye from scrub solutions applied near the eye. early treatment of superficial ulcers should prevent self-trauma and progression of the wound. etiology research protocols often require the placement of chronic implants. implants such as cardiac or other biomedical devices may be the primary focus of the research study. implants may also be used as chronic monitoring devices, for delivery of compounds, or to collect serial samples. infection may occur at the time of implant. alternatively, the implant may serve as a nidus after hematogenous spread from other sources. one of the most common sources of infection is from colonization of the device from an external component, which is a frequent complication with indwelling catheters. the actual incidence of complications associated with indwelling vascular catheters in dogs is unknown. one study (hysell and abrams, ) examined the lesions found at necropsy in animals with chronic indwelling catheters, which included traumatic cardiac lesions, visceral infarcts, and fatal hemorrhages. these lesions were primarily associated with catheter-induced trauma or secondary to embolization of fibrin. in a veterinary clinical setting, infections in peripheral catheters were more likely when the catheters were used for blood collection immediately after placement and when a 't' connector rather than a 'y' connector was used. . intestinal access ports have been used to study the pharmacokinetics of drugs at various levels in the intestinal tract. these catheters are usually vascular access ports with several modifications to allow secure placement in bowel (meunier et al., ) . the most frequently reported complication associated with these catheters is infection around the port site (meunier et al., ; kwei et al., ) . clinical signs dogs with implant infections may not exhibit signs initially . localized swelling around the implant may occur. in the case of indwelling catheters, signs may include redness and swelling of the skin around the external port or discharge from the skin wound. vascular access ports may develop fluctuant subcutaneous abscesses. in more severe cases, systemic signs may be noted (bach et al., ; hysell and abrams, ) . the systemic signs of infection are covered elsewhere in this chapter. treatment the treatment of catheter infections almost invariably requires removal of the catheter, as demonstrated in both dogs and monkeys (ringler and peter, ; darif and rush, ). superficial wound irritation or infection may be treated locally with antibiotic ointment, sterile dressing changes, and efforts to minimize catheter movement; however, more extensive problems require aggressive therapy. localized abscesses or sinus tracts may be managed by establishing drainage and copious flushing. aerobic and anaerobic cultures of blood and locally infected sites should be performed prior to initial treatment (ringler and peter, ) . systemic antibiotic therapy should be initiated for a -day period. the choice of drug will ultimately be based on previous experience and culture results. if retention of a catheter is important, the catheter lumen may be safely disinfected with chlorine dioxide solution (dennis et al., ) . the solution is removed after min and replaced with heparinized saline. all of the extension lines and fluids used with an infected catheter should be discarded. the blood cultures should be repeated days after the antibiotic therapy has ceased. if bacteria are still cultured, the catheter must be removed. prevention it is highly desirable to prevent complications that may result in loss of an implanted device. catheters and other implants should be made of nonthrombogenic material and be as simple as possible. a catheter with extra ports or multiple lumens requires additional management and supplies more routes for infection. the initial placement of an indwelling catheter must be done under aseptic conditions by individuals who are familiar with the procedure. intravenous catheters that are used for delivery of drugs or blood sampling should be positioned in the vena cava and not in the right atrium, thereby minimizing trauma to the tricuspid valve. ideally, catheters are secured to reduce movement and irritation of the skin, which may predispose to infection around external ports. the use of vascular access ports that lie entirely under the skin eliminates many problems with infection. it has also been found that long extension tubing connected to the port may actually reduce the potential for infection of the catheter (ringler and peter, ) . for intestinal access ports, catheter security may be improved with a synthetic cuff added to the end of the catheter allowing better attachment to the intestine (meunier et al., ) . after any catheter placement, animals should be observed daily for signs of either local or systemic infection. the catheter entry site should be disinfected, coated with antibiotic ointment, and rebandaged every other day. once a month, the catheter line may be disinfected with chlorine dioxide. in addition, a solution of the antibiotic ceftazidime used on alternate days with the heparin locking solution has been shown to effectively reduce infections in indwelling vascular catheters (bach et al., ) . throughout the life of the catheter, injections into and withdrawals from the catheter should be done in a sterile manner, and the number of breaks in the line should be kept to a minimum. periodically, the placement of an indwelling catheter may be verified by radiography. when placed and managed correctly, catheters and ports of any kind may remain in place for months without complications. etiology sepsis is defined as the systemic response to infection caused by bacteria (gram negative and/or gram positive), fungi, or viruses. in laboratory animals, sepsis is most often seen as a complication of surgical procedures or associated with chronic implants. sepsis may also be seen as a complication of infectious diseases such as parvovirus. clinical signs the signs of sepsis can vary, depending on the source of the infection and the stage of the disease. early in the course, dogs may present with signs of a hyperdynamic sepsis, including increased heart rate, increased respiratory rate, red mucous membranes, and a normal-to-increased capillary refill time. systemic blood pressure and cardiac output will be increased or within the normal range. the animals will often be febrile. later in the course of the syndrome, the animals may show classic signs of septic shock including decreased temperature, pale mucous membranes, and a prolonged capillary refill time. cardiac output and blood pressure are decreased as shock progresses. peripheral edema and mental confusion have also been reported (hauptman and chaudry, ) . pathogenesis the pathophysiology of sepsis is complex and is mediated by immune responses involving mediators such as cytokines, eicosinoids, complement, superoxide radicals, and nitric oxide. the body responds to overwhelming infection with an attempt to optimize metabolic processes and maximize oxygen delivery to tissues. however, if inflammation is left unchecked, the system may be unable to compensate, and the result is cardiovascular collapse. diagnosis in general, a presumptive diagnosis of sepsis is made based on the occurrence of several in a group of signs, including altered body temperature, increased respiratory and/or heart rate, increased or decreased white blood cell (wbc) count, increased number of immature neutrophils, decreased platelet count, decreased blood pressure, hypoxemia, and altered cardiac output. however, extreme inflammation without infection (e.g., pancreatitis, trauma) may create similar signs. one study examined the diagnosis of sepsis in canine patients at a veterinary hospital based on easily obtainable physical and laboratory findings. that study found that septic individuals had higher temperatures, wbc counts, and percentage of band neutrophils than nonseptic individuals, whereas platelet counts were lower in the septic dogs. there were no differences in respiratory rate or glucose levels between the groups. using these criteria, the results had a high sensitivity and a tendency to overdiagnose sepsis (hauptman et al., ) . ultimately, the presence of a septic focus simplifies diagnosis greatly; however, the focus may not be obvious. if the signs of sepsis are evident but the focus is not, several areas should be evaluated for infection, including the urinary, reproductive, respiratory, alimentary, and cardiovascular systems, as well as the abdominal cavity (kirby, ) . treatment the treatment of sepsis has three aims. the first aim is to support the cardiovascular system. all septic animals should be treated with fluids to replace deficits and to maximize cardiac output. crystalloids are most frequently used to maintain vascular volume, primarily because of their low cost. colloids offer the advantage of maintaining volume without fluid overload and may have other positive effects on the cardiovascular system. acid-base and electrolyte imbalances should also be addressed. after the animal has stabilized, the treatment of sepsis should be aimed at removing the septic focus. obvious sources of infection should be drained or surgically removed. if an implant is infected, it should be removed. antibiotic therapy should also be instituted. the choice of antibiotic will ultimately depend upon the results of culture; however, the initial choice of antibiotics is based on previous experience, source of infection, and gram stains. the organisms associated with sepsis are often gram-negative bacteria of gastrointestinal origin or are previously encountered nosocomial infections. ideally, the antibiotic chosen for initial therapy should be a broad-spectrum, bactericidal drug that can be administered intravenously. second-or thirdgeneration cephalosporins provide good coverage, as does combination therapy with enrofloxacin plus metronidazole or penicillin. finally, the treatment of sepsis is aimed at blocking the mediators of the systemic response. this category of sepsis treatment is the focus of much research. several studies have examined the effects of steroids, nonsteroidal anti-inflammatory drugs, and antibodies directed against endotoxin, cytokines, or other mediators of the inflammatory response; however, none of these treatments have proven greatly effective in clinical trials. consequently, there is no 'magic bullet' for the treatment of sepsis at this time. successful therapy remains dependent on aggressive supportive care coupled with identification and elimination of the inciting infection. etiology in research animals, aspiration may occur accidentally during the oral administration of various substances or by the misplacement of gastric tubes. aspiration of gastric contents may also occur as a complication of anesthesia. in pet animals, aspiration is often seen as a result of metabolic and anatomical abnormalities; however, such occurrence would be rare in the research setting. pathogenesis aspirated compounds can produce direct injury to lung tissue, but more importantly, the aspiration provokes an inflammatory response, probably mediated by cytokines. the result is a rapid influx of neutrophils into the lung parenchyma and alveolar spaces. the inflammation leads to increased vascular permeability with leakage of fluid into the alveolar spaces and can eventually lead to alveolar collapse. if the condition is severe, it may result in adult respiratory distress syndrome and respiratory failure. it should be noted that infection is not present in the early stages of this condition but may complicate the problem after - h. clinical signs the severity and clinical manifestation of aspiration lung injury are dependent upon the ph, osmolality, and volume of the aspirate. the signs of aspiration lung injury may include cough, increased respiratory rate, pronounced respiratory effort, and fever. when respiration is severely affected, the oxygen saturation of blood will be decreased. the diagnosis of this problem is based on witness of aspiration, history consistent with aspiration, and/or the physical findings. classically, radiographs of the thorax demonstrate a bronchoalveolar pattern in the cranioventral lung fields. however, these lesions may not appear for several hours after the incident of aspiration. in addition, the location of the lesions may be variable, depending on the orientation of the animal at the time of aspiration. treatment the treatment of aspiration lung injury is largely supportive and depends upon the severity of the inflammation and the clinical signs. if the aspiration is witnessed, the mouth and, idealy, the upper airway should be cleared of residual material. when small amounts of a relatively innocuous substance (e.g., barium) have been aspirated, treatment may not be necessary. when severe inflammation is present, systemic as well as localized therapy may be necessary. oxygen therapy may be instituted; however, the concentration and time frame are controversial, because lung injury may be exacerbated by long-term administration of oxygen at high concentrations (nader-djahal et al., ) . fluid therapy may also be necessary in severe cases; however, cardiovascular support should be performed judiciously as fluid overload could lead to an increase in pulmonary edema. the use of colloids is also controversial because of the increase in vascular permeability that occurs in the lungs. several studies have addressed the use of anti-inflammatory agents to reduce lung injury associated with aspiration; however, none are used clinically in human or veterinary medicine at this time. corticosteroids are contraindicated (raghavendran et al., ) . in humans, antibiotics are reserved for cases with confirmed infection, in order to prevent the development of antibiotic-resistant pneumonia. it has been suggested that dogs should be immediately treated with antibiotics when the aspirated material is not acidic or has potentially been contaminated by oral bacteria associated with severe dental disease. amoxicillin-clavulanate has been recommended as a first line of defense, reserving enrofloxacin for resistant cases (hawkins, ) . the presence of pneumonia should be verified by tracheal wash and cultures. prevention aspiration of drugs and other compounds may be avoided through careful administration of oral medications by experienced individuals. likewise, gavage or orogastric administration of liquids should be performed by experienced individuals, and the procedure should be aborted if coughing or other respiratory signs occur. the aspiration of stomach contents can largely be avoided by appropriate fasting prior to anesthesia for at least h for food and h for water. if appropriate fasting times are not observed, anesthesia should be postponed whenever possible, particularly if intended procedures require manipulation of the viscera or head-down positioning of the dog. if anesthesia cannot be avoided, it should be rapidly induced and the dog should be intubated. during recovery from anesthesia, the endotracheal tube should be removed with the cuff partially inflated and with the dog in a head-up position (haskins, ) . based on the source of energy, burn wounds may be categorized into four groups: thermal, chemical, radiation, and electrical. in laboratory animals, accidental burns are usually the result of thermal injury (heating pads, water bottles), chemicals (strong alkalis, acids, disinfectants, drugs), or experimental irradiation protocols. etiology inappropriate use of external heating devices is the most common cause of burns in laboratory animal medicine. the insult to the skin results in desiccation of the tissue and coagulation of proteins. in addition, the severely injured area is surrounded by a zone of vascular stasis, which promotes additional tissue damage. even small burns can result in significant inflammation that could affect the outcome of some research investigations and cause considerable discomfort to the animal. the proper and immediate treatment of burn wounds can reduce the effects of the injury on both the individual and the research. clinical signs the clinical signs vary with the depth, location, and surface area of burn injury. classification systems for thermal burns are generally based on the depth of the injury, varying from superficial involvement of only epidermis to complete destruction of skin and subcutaneous tissues (bohling, ) . superficial burns appear erythematous and inflamed. in some cases, matting of the overlaying hair with exudate may be the first sign of a previously undetected skin lesion. progressive hair and skin loss may be evident over the first few days after injury (johnston, ) . although blistering is a characteristic of partial thickness burns in humans, this is rarely seen in dogs (bohling, ) . uncomplicated, superficial burn wounds heal by reepithelialization within - days. deeper burn wounds are characterized by a central area of nonviable tissue surrounded by edematous, inflamed tissues. a thick eschar, composed of the coagulated proteins and desiccated tissue fluid, develops over deep burn wounds. these wounds heal by granulation under the eschar, which will eventually slough. the amount of pain associated with burns depends upon several factors including the depth and area of the wound, procedural manipulations, and movement at the affected site (bohling, ) . pain associated with superficial burn wounds usually subsides in - days. theoretically, deep burns destroy nerve endings and result in less pain than superficial burns. however, inflammatory pain may still be present due to the tissue reaction around the necrotic site. in addition, sharp procedural pain and breakthrough pain have been described in humans during the healing phases of burn injuries and should be considered as potential complications in dogs as well (bohling, ) . severe and widespread accidental burn injury can result in clinical signs associated with multiple organs including the pulmonary, gastrointestinal, hematopoietic, and immune systems. in addition, extensive burn injury can predispose to infection and even sepsis. this type of injury with the associated complications would be extremely rare in the laboratory setting. treatment appropriate and timely treatment of a burn wound will reduce the extent of tissue damage and associated pain. thermal injuries should be immediately exposed to cool water ( °c) to reduce edema and pain. exposure to very cold water and ice does not improve outcomes (bohling, ) . topical wound dressings are recommended in the early stages of treatment for both partial-and full-thickness burns that are of small size. systemic antibiotics are unable to penetrate eschar and are not adequately distributed through the abnormal blood supply of burned tissues. therefore, a thin film of a water-soluble, broad-spectrum antibiotic ointment should be applied to the wound surface. silver sulfadiazine has a broad spectrum, penetrates eschar, and is often the preparation of choice for burn wound therapy. povidone-iodine ointment will also penetrate thin eschar and provides a broad spectrum. mafenide has a good spectrum that covers gram-negative organisms well and is often used to treat infected wounds, although it has been associated with pain upon application (demling and lalonde, ) . once a topical antibiotic has been applied, a nonadherent dressing should be placed on the wound. burn wounds covered in such a manner tend to epithelialize more rapidly and are less painful than uncovered wounds (demling and lalonde, ; bohling, ) . after the initial treatment, burn wounds should be gently cleansed two to three times a day, followed by reapplication of the topical antibiotic and rebandaging (demling and lalonde, ) . systemic antibiotics are indicated in cases where local or systemic infection is present and their ultimate selection should be based on culture results. burn wounds can be extremely painful, and analgesia should be instituted immediately and adjusted accordingly throughout the treatment period. surgical intervention may be necessary in some cases. with small or moderately sized wounds, the eschar over the burn wound may actually impede wound contraction and reepithelialization. in such cases, once the eschar has become fully defined, a complete resection may improve wound healing. with large and severe burn wounds, repeated debridement by surgery or other means might be necessary. in the laboratory setting, a decision to pursue extensive surgical intervention would be dependent upon full consideration of the effects on animal welfare and research results. prevention thermal burns can be prevented in the research setting. electric heating pads and heat lamps should be avoided if possible. only heated water blankets or circulating warm air devices should be used to provide warmth to the animals. in rare instances, heated water blankets have also caused burns; therefore, these devices should be carefully monitored. as a precaution, a thin towel may be placed between the animal and the water blanket. basic fire prevention precautions should be taken particularly around oxygen sources and flammable agents etiology chemical injury may be due to skin contact with concentrated solutions such as disinfectants or inadvertent exposure to laboratory chemicals. in addition, perivascular injection of certain drugs (pentobarbital, thiamylal, thiopental, thiacetarsemide, vincristine, vinblastine, and doxorubicin) have been associated with extensive tissue damage (swaim and angarano, ; waldron and trevor, ) . the mechanism of action will vary depending upon the ph, osmolality, and chemical composition of the agent and may include oxidation, reduction, disruption of lipid membranes, or other reactions (bohling, ; swaim, ; waldron and trevor, ) . clinical signs surface contact with chemicals may result in mild irritation and redness of superficial layers of the skin. however, many agents may cause progressive injury until the chemical reaction has been neutralized. this may result in tissue necrosis and secondary infection. the immediate signs of perivascular injection are withdrawal of the limb or other signs of discomfort and swelling at the injection site. the area may appear red, swollen, and painful as inflammation progresses. there may eventually be necrosis of the skin around the injection site. in cases of doxorubicin extravasation, signs may develop up to a week after the injection, and the affected area may progressively enlarge over a -to -month period. this is because the drug is released over time from the dying cells (swaim and angarano, ) . treatment in cases of skin contact with chemical agents, the affected area should be thoroughly and repeatedly lavaged with warm water to dilute or remove the substance. the material safety data sheet for the substance should be consulted for any possible neutralization protocol. additional treatment will depend upon the severity of the tissue damage and will follow the same guidelines as for the thermal injury described earlier. for the treatment of perivascular injections, dilution of the drug with subcutaneous injections of saline is recommended. in addition, steroids may be infiltrated locally to reduce inflammation. topical application of dimethyl sulfoxide (dmso) may also be helpful in reducing the immediate inflammation and avoiding the development of chronic lesions. the addition of lidocaine to subcutaneous injections of saline has been used in cases of thiacetarsemide injection (hoskins, ) . the local infiltration of hyaluronidase accompanied by warm compresses has been suggested for perivascular vinblastine (waldron and trevor, ) and for doxirubicin. the use of dmso or another free radical scavenger, dexrazoxane, infused at the site has also been suggested for doxorubicin toxicities. despite these treatments, necrosis of skin may be observed and would require serial debridement of tissues with secondary wound closure or skin grafting. in cases of doxorubicin extravasation, early excision of affected tissues is advocated to prevent the progressive sloughing caused by sustained release of the drug from dying tissues (swaim and angarano, ) . in all cases, the condition can be painful and analgesia should be addressed. prevention prior to the use of any substance, the investigator should be aware of its chemical composition and the potential for problems. the material safety data sheets should be available for all compounds and storage recommendations followed closely. for intravenous administration of toxic compounds, insertion of an indwelling catheter is extremely important. prior to the injection, the catheter should be checked repeatedly for patency by withdrawal of blood and injection of saline. any swelling at the catheter site or discomfort by the subject indicates that the catheter should not be used. access to a central vessel such as the cranial or caudal vena cava is preferred over the use of peripheral vessels. when peripheral catheters are used, the injection should be followed by a vigorous amount of flushing with saline or other physiological solution and removal of the catheter. additional injections are best given through newly placed catheters in previously unused vessels. the repeated use of an indwelling peripheral catheter should be approached cautiously and done only out of necessity. etiology radiation burns are generally a complication of therapeutic administration and are a result of free oxygen radical formation (waldron, ) . the severity of radiation burns and their treatment will depend upon the dose, frequency, total surface area, and location of the radiation. damage to epithelial layers of the skin can lead to desquamation. direct injury to fibroblasts results in decreased collagen production and poor wound healing. in addition, there may be fibrosis of blood vessels (pavletic, ) and subsequent hypoxia causing necrosis of deeper tissues. clinical signs the tissues most often affected are the skin and mucus membranes. with superficial injury, affected skin may exhibit hair loss and erythema, and produce a clear exudate. the intensity of the inflammation may increase for - weeks after the completion of radiation treatment. deeper and more serious injury manifests with subcutaneous fibrosis and can lead to disfigurement . the skin and underlying deep structures including the bone may become necrotic over several weeks (pavletic, ) . these deeper injuries are prone to infection due to their lack of blood supply. systemic signs such as vomiting are rare in dogs unless there has been direct radiation treatment to organs . treatment with superficial skin burns, the wound should be kept clean and should be covered if possible. in cases of oral mucous membrane damage, there may be special feeding requirements. when wounds are ulcerated, avascular tissues should be excised. treatments with silver sulfadiazine, mafenide acetate, or other topical agents are recommended to control infection. in addition, infection is avoided by closure of the wound as soon as possible. the goal of surgery is to cover the wound with healthy tissue to promote vascularization of the area. in some cases, this may require muscle and/ or skin grafts. prevention radiation burns can be limited by selection of appropriate, fractionated therapy and application of shielding to reduce exposure. prompt treatment of the injuries can reduce the occurrence of infection. since radiation is associated with poor wound healing, complications may arise when additional procedures are required. it is recommended to wait at least week (pavletic, ) or even longer (laing, ) prior to administering radiation to a surgical site. after radiation, routine surgeries should be avoided for - months (pavletic, ). the prevalence of cancer in the general canine population has increased over the years (dorn, ) . this can be attributed to the longer life spans resulting from improvements in nutrition, disease control, and therapeutic medicine. because of these changes, cancer has become a major cause of death in dogs (bronson, ) . in a lifetime cancer mortality study of intact beagles of both sexes, albert et al. ( ) found death rates similar to the death rate of the at-large dog population (bronson, ) . approximately % of the male beagles died of cancer. the majority of the tumors were lymphomas ( %) and sarcomas ( %), including hemangiosarcomas of the skin and fibrosarcomas. of the female beagles dying of cancer ( % of the population studied), three-quarters had mammary cancer ( %), lymphomas ( %), or sarcomas ( %). of the sarcomas in females, one-third were mast cell tumors. in addition to these tumors that cause mortality, the beagle is also at risk for thyroid neoplasia (hayes and fraumeni, ; benjamin et al., ) . because of the popularity of the beagle as a laboratory animal, discussion of specific neoplasms will focus on the tumors for which this breed is at risk, as well as tumors that are common in the general canine population. a complete review of clinical oncology in the dog is beyond the scope of this chapter but can be found elsewhere (withrow et al., ) . fine-needle aspirates are generally the first diagnostic option for palpable masses, because they can easily be performed in awake, cooperative patients. this technique allows for rapid differentiation of benign and neoplastic processes. in cases where cytologic results from fine-needle aspirates are not definitive, more invasive techniques must be used. needle-punch or core biopsies can also be performed in awake patients with local anesthesia. an instrument such as a tru-cut ® needle (travenol laboratories, inc., deerfield, illinois) is used to obtain a -mm × - . -cm biopsy of a solid mass. a definitive diagnosis may be limited by the size of the sample acquired using this technique. incisional and excisional biopsies are utilized when less invasive techniques fail to yield diagnostic results. excisional biopsies aid in histopathological examination and are the treatment of choice when surgery is necessary, because the entire mass is removed. surgical margins should extend at least cm around the tumor and cm if mast cell tumors are suspected (morrison et al., ) . incisional biopsies are performed when large softtissue tumors are encountered and/or when complete excision would be surgically difficult or life-threatening. when performing an incisional biopsy, always select tissue from the margin of the lesion and include normal tissue in the submission. etiology lymphomas are a diverse group of neoplasms that originate from lymphoreticular cells. canine lymphoma represents - % of canine tumors and a majority ( %) of canine hematopoetic disease (ettinger, ; vail and young, ) . whereas retroviral etiologies have been demonstrated in a number of species (e.g., cat, mouse, chicken), conclusive evidence of a viral etiology has not been established in the dog. in humans, data implicate the herbicide , -dichlorophenoxyacetic acid as a cause of non-hodgkin's lymphoma, but studies in dogs with similar conclusions have come under scrutiny (macewen and young, ) . in addition, tobacco smoke, environmental chemicals, and waste emissions are considered possible risk factors (marconato et al., ; gavazza et al., ) clinical signs multicentric high-grade lymphoma (mhgl) accounts for the majority of reported cases of canine lymphoma. depending upon grade, immunophenotype, and location involved, dogs with mhgl usually present with painless, enlarged lymph nodes and nonspecific signs such as anorexia, weight loss, polyuria, polydypsia, fever, and lethargy. when the liver and spleen are involved, generalized organomegaly may be felt on abdominal palpation. less commonly, dogs develop alimentary, mediastinal, cutaneous, and extranodal lymphomas. alimentary lymphoma is associated with vomiting and diarrhea, in addition to clinical signs associated with mhgl. dogs with mediastinal lymphoma often present with respiratory signs (dyspnea and exercise intolerance) secondary to pleural effusion or cranial vena caval syndrome. hypercalcemia is most frequently associated with this form of lymphoma and may result in polyuria, polydypsia, and weakness. cutaneous lymphoma is an uncommon epitheliotrophic form of lymphoma. it is often referred to as mycosis fungoides and is typically of a cd + t-cell immunophenotype. it varies in presentation from solitary to generalized and may mimic any of a number of other inflammatory skin disorders including oral mucosal lesions. the lesions may occur as erythema, plaques, erosions, scales, nodules, crusts, hypopigmentation, and alopecia (fontaine et al., ) . approximately half of the cases are pruritic. a number of extranodal forms of lymphoma have been reported, including tumors affecting the eyes, central nervous system, kidneys, or nasal cavity. clinical presentation varies, depending on the site of involvement (e.g., nervous system: seizures, paresis, paralysis). epizootiology the incidence of lymphoma is highest in dogs - years old, accounting for % of cases. although the neoplasm generally affects dogs older than year, cases in puppies as young as months have been reported (dorn et al., ) . no gender predilection has been reported. diagnosis and pathologic findings a fine-needle aspirate is initially performed on accessible lymph nodes. thoracic radiographs and abdominal ultrasound ± fine needle aspiration of the liver or spleen can be used if mediastinal or abdominal involvement is suspected. additional staging can be determined through complete blood counts, serum biochemistry, flow cytometry for immunotyping, bone marrow aspiration, or surgical lymphadenectomy and histology. enlarged neoplastic lymph nodes vary in diameter from to cm and are moderately firm. some may have areas of central necrosis and are soft to partially liquefied. the demarcation between cortex and medulla is generally lost, and on cut section, the surface is homogenous. the spleen may have multiple small nodular masses or diffuse involvement with generalized enlargement. the enlarged liver may have disseminated pale foci or multiple large, pale nodules. in the gastrointestinal tract, both nodular and diffuse growths are observed. these masses may invade through the stomach and intestinal walls. flow cytometry and lymphoblastic markers (cd ) can aid in diagnosis and subtyping of tumors. in addition, positron emission tomography is being explored for detection of extranodal and metastatic lymphoma (leblanc et al., ; marconato, ; elstrom et al., ) . classification of lymphoma types is based upon cytological, morphological, and immunological characteristics using the kiel classification criteria (vail and young, ) . histologically, the most common lymphomas are classified as intermediate to high grade and of large-cell (histiocytic) origin. the neoplastic lymphocytes typically obliterate the normal architecture of the lymph nodes and may involve the capsule and perinodal areas. lymphoma subtypes can be further characterized based upon genetic, molecular, and immunological criteria (ponce et al., ) . pathogenesis all lymphomas regardless of location should be considered malignant. a system for staging lymphoma has been established by the world health organization. the average survival time for dogs without treatment is - weeks. survival of animals undergoing chemotherapy is dependent on the treatment regimen as well as the form and stage of lymphoma (macewen and young, ) . median survival time with aggressive therapy is generally less than months. hypercalcemia is a paraneoplastic syndrome frequently associated with lymphoma. the pathogenesis of this phenomenon is not fully understood but may be a result of a parathormone-like substance produced by the neoplastic lymphocytes. differential diagnosis differential diagnoses for multicentric lymphoma include systemic mycosis; salmon-poisoning and other rickettsial infections; lymph node hyperplasia from viral, bacterial, and/or immunologic causes; and dermatopathic lymphadenopathy. alimentary lymphoma must be distinguished from other gastrointestinal tumors, foreign bodies, and lymphocytic-plasmacytic enteritis. in order to make a definitive diagnosis, whole lymph node biopsies and full-thickness intestinal sections for histopathologic examination may be needed. treatment therapy for lymphoma primarily consists of one or a combination of several chemotherapeutic agents. in addition, radiation therapy and bone marrow transplantation have been utilized. the treatment regimen is based on the staging of the disease, the presence of paraneoplastic syndromes, and the overall condition of the patient. although treatment may induce clinical remission and prolong short-term survival, most treatment is palliative and aimed at improving quality of life. a thorough discussion of therapeutic options for the treatment of lymphomas in the dog can be found elsewhere (chun, ; marconato, ) . future directions include development of molecular and cellular targeted therapies to enhance traditional chemotherapy treatment, prolong remission, and treat immunologic subtypes of lymphoma (e.g., t-cell lymphoma). research complications given the grave prognosis for lymphoma with or without treatment, euthanasia should be considered for research animals with significant clinical illness. etiology mast cells are derived from cd + bone marrow progenitor cells. neoplastic proliferations of mast cells are the most commonly observed skin tumor of the dog and may account for up to % of canine skin laboratory animal medicine tumors (bostock, ; welle et al., ) . mast cells are normally found in the connective tissue beneath serous surfaces and mucous membranes, and within the skin, lung, liver, and gastrointestinal tract. current research has linked mast cell tumor development to multifactorial causes including breed predisposition and a genetic component, chronic inflammation, and mutations in the surface growth factor, c-kit (ma et al., ; reguera et al., ; webster et al., ) . clinical signs well-differentiated mast cell tumors are typically solitary, well-circumscribed, slow-growing, -to -cm nodules in the dermis and subcutaneous tissue. alopecia may be observed, but ulceration is not usual. poorly differentiated tumors grow rapidly, may ulcerate, and may cause irritation, inflammation, and edema to surrounding tissues. mast cell tumors can be found on any portion of the dog's skin but frequently affect the trunk and hind limb extremeties along with perineal and preputial areas. the tumors usually appear to be discrete masses, but they frequently extend deep into surrounding tissues. abdominal organs are rarely involved but may be associated with anorexia, vomiting, melena, abdominal pain, and gastrointestinal ulceration. mast cell tumors have also been reported in extracutaneous areas such as the salivary glands, larynx, nasopharynx (london and thamm, ) and conjunctiva (fife et al., ) . mast cell tumors within the perineal, preputial, or inguinal areas are associated with a greater predilection for recurrence or metastasis (misdorp, ) . epizootiology these tumors tend to affect middleaged dogs but have been observed in dogs ranging from months to years (pulley and stannard, ) . pathologic findings because of the substantial variation in histologic appearance of mast cell tumors, a classification and grading system described by patnaik et al. ( ) has become widely accepted. in this system, grade i has the best prognosis and are well differentiated, with round to ovoid, uniform cells with distinct cell borders. the nuclei are round and regular, the cytoplasm is packed with large granules that stain deeply, and mitotic figures are rare to absent. grade ii (intermediately differentiated) mast cell tumors have indistinct cytoplasmic boundaries with higher nuclear-cytoplasmic ratios, fewer granules, and occasional mitotic figures. grade iii (anaplastic or undifferentiated) mast cell tumors have the worst prognosis. the cells contain large, irregular nuclei with multiple prominent nucleoli and few cytoplasmic granules. mitotic figures are much more frequent. cells are pleomorphic with indistinct borders. in addition to associated skin lesions (e.g., ulceration, collagenolysis, necrosis, and infection), mast cell tumors have been associated with gastric ulcers in the fundus, pylorus, and/or proximal duodenum, most likely secondary to tumor production of histamine. histamine stimulates the h receptors of the gastric parietal cells, causing increased acid secretion. gastric ulcers have been observed in large numbers (> %) of dogs with mast cell tumors (howard et al., ) . pathogenesis although all mast cell tumors should be considered potentially malignant, the outcome in individual cases can be correlated with the histologic grading of the tumor. grade iii tumors are most likely to disseminate internally. this spread is usually to regional lymph nodes, spleen, and liver, and less frequently to the kidneys, lungs, and heart. diagnosis and differential diagnosis using fineneedle aspiration, mast cell tumors can be distinguished cytologically from other round cell tumors (such as histiocytomas and cutaneous lymphomas) by using toluidine blue to metachromatically stain the cytoplasmic granules red or purple. mast cell granules can also be stained with wright's, giemsa, and romanowsky stains. in addition, mast cells may contain tryptase, chymase, or both (fernandez et al., ) . histological evaluation is generally required for grading. examination of regional lymph nodes may be warranted if metastatic or systemic disease is suspected. in addition, radiographs and ultrasound with guided aspirates of the liver, spleen, or sublumbar lymph nodes can be used to determine metastatic disease. treatment depending upon the grade, initial treatment for mast cell tumors is generally wide surgical excision ( -cm margins), which may be followed by radiation, chemotherapy, or glucocorticoid therapy. aspiration or surgical removal of regional lymph nodes is recommended if lymphatic tumor drainage is suspected. if the tumor is not completely resectable or is grade ii or iii (moderately to undifferentiated), then debulking surgery and adjunct therapy may be used. treatment algorithms are outlined elsewhere (withrow et al., ) research complications because of the potential for systemic release of substances such as histamine, vasoactive substances, heparin, eosiniphilic chemotactic factor, and proteolytic enzymes, along with the possibility of delayed wound healing and tumor recurrence, dogs with mast cell tumors are not good candidates for research studies. grade i mast cell tumors may be excised, allowing dogs to continue on study; however, monitoring for local recurrence should be performed monthly. grade ii tumors are variable; animals that undergo treatment should be monitored for recurrence monthly, and evaluation of the buffy coat should be performed every - months for detection of systemic mastocytosis. because of the poor prognosis for grade iii tumors, treatment is unwarranted in the research setting. etiology also known as infectious or venereal granuloma, sticker tumor, transmissible sarcoma, and contagious venereal tumor, the canine transmissible venereal tumor (ctvt) is transmitted horizontally to the genitals by coitus (nielsen and kennedy, ) . ctvt is a 'parasitic-like' tumor that appears to have originated from dogs or wolves thousands of years ago and despite immense mutation, ctvt adapted, survived, and spread across multiple continents making it the oldest known continuously passaged somatic cell line (rebbeck et al., ; murchison et al., ; murgia et al., ) . it has been described as a round cell tumor of histiocytic origin. although this tumor has been reported in most parts of the world, it is most prevalent in tropical or temperate climates (macewen, ) . clinical signs the tumors are usually cauliflowerlike masses on the external genitalia, but they can also be pedunculated, nodular, papillary, or multilobulated. these friable masses vary in size up to cm, and hemorrhage is frequently observed. in male dogs, the lesions are found on the caudal part of the penis from the crura to the bulbus glandis or on the glans penis. less frequently, the tumor is found on the prepuce. females typically have lesions in the posterior vagina at the junction of the vestibule and vagina. when located around the urethral orifice, the mass may protrude from the vulva. these tumors have also been reported in the oral cavity, skin, and eyes. epizootiology and transmission ctvts are most commonly observed in young, sexually active dogs. transmission takes place during coitus when injury to the genitalia allows for exfoliation and transplantation of the tumor. genital to oral to genital transmission has also been documented (nielsen and kennedy, ) . extragenital lesions may be the result of oral contact with previously traumatized areas. pathologic findings histologically, cells are arranged in compact masses or sheets. the cells are round, ovoid, or polyhedral, and have large, round nuclei with coarse chromatin. the cytoplasm is eosinophilic with small vacuoles arranged in a 'string of pearls' pattern. pathogenesis tumor growth occurs within - months after mating or implantation, and then growth generally slows. metastasis is rare (< - % of cases) but may involve the superficial inguinal and external iliac lymph nodes as well as distant sites. spontaneous regression may occur within - months of tumor development. diagnosis and differential diagnosis transmissible venereal tumors have been confused with lymphomas, histiocytomas, mast cell tumors, and amelanotic melanomas. however, cytological examination of impression smears, swabs, and fine-needle aspirates generally provide a definitive diagnosis. although not usually required, histopathology of a biopsy from the mass can aid in diagnosis. prevention thorough physical examinations prior to bringing new animals into a breeding program should prevent introduction of this tumor into a colony. control removing affected individuals from a breeding program should stop further spread through the colony. treatment surgery and radiation can be used for treatment, but chemotherapy is the most effective. vincristine ( . - . mg/m ) iv once weekly for four to six treatments will induce remission and cure in greater than % of the cases (macewen, ) . research complications experimental implantation of ctvts has been shown to elicit formation of tumor-specific igg (cohen, ) . this response may occur in natural infections and could possibly interfere with immunologic studies. etiology dogs are susceptible to a wide variety of mammary gland neoplasms, most of which are influenced by circulating reproductive steroidal hormones. clinical signs single nodules are found in approximately % of the cases of canine mammary tumors. the nodules can be found in the glandular tissue or associated with the nipple. masses in the two most caudal glands (fourth and fifth) account for a majority of the tumors. benign tumors tend to be small, well circumscribed, and firm, whereas malignant tumors are larger, invasive, and coalescent with adjacent tissues. inflammatory mammary carcinomas may mimic mastitis or severe dermatitis and must be ruled out to prevent misdiagnosis. epizootiology mammary tumors are uncommon in dogs under years of age with the incidence rising sharply after that. the median age at diagnosis is - years. a longitudinal study of a large beagle colony showed that significant risk for development of mammary tumors begins at approximately years of age (taylor et al., ) . mammary tumors occur almost exclusively in female dogs, with most reports in male dogs being associated with endocrine abnormalities, such as estrogen-secreting sertoli cell tumors. pathologic findings the t (tumor size), n (lymph node involvement), and m (metastasis) system is commonly used to stage mammary tumors. based on histologic classification of mammary gland tumors, approximately half of the reported tumors are benign (fibroadenomas, simple adenomas, and benign mesenchymal tumors), and half are malignant (solid carcinomas, tubular adenocarcinomas, papillary adenocarcinomas, anaplastic carcinomas, sarcomas, and carcinosarcomas) (bostock, ) . histopathologic grades are scored based upon tubule formation, nuclear pleomorphism, and mitosis (elston and ellis, ) . extensive discussions of classification, staging, and histopathologic correlations can be found elsewhere (moulton, ; sorenmo et al., ) . pathogenesis mammary tumors of the dog develop under the influence of hormones. receptors for both estrogen and progesterone can be found in - % of tumors. malignant mammary tumors typically spread through the lymphatic vessels. metastasis from the first, second, and third mammary glands is to the ipsilateral axillary or anterior sternal lymph nodes. the fourth and fifth mammary glands drain to the superficial inguinal lymph nodes where metastasis can be found. many mammary carcinomas will eventually metastasize to the lungs and extraskeleton. diagnosis and differential diagnosis both benign and malignant mammary tumors must be distinguished from mammary hyperplasia, mastitis, and severe dermatitis. cytological evaluation from fine-needle aspirates correlates well with histological examination of benign and malignant tumors (simon et al., ) . radiographs and possibly ultrasound should be performed to rule out metastatic disease prior to surgery. prevention the lifetime risk of developing mammary tumors can effectively be reduced to . % by spaying bitches prior to the first estrus (schneider et al., ) . this is commonly done in the general pet population at months of age. the protective effects of early spay rapidly decrease after several estrus cycles. dogs spayed prior to the first estrus had a risk of . %, whereas dogs spayed after the first and second estrus had risks of % and %, respectively. treatment surgery is the treatment of choice for mammary tumors, because chemotherapy and radiation therapy have not been reported to be effective. the extent of the surgery is dependent on the area involved. single mammary tumors should be surgically removed with -cm lateral margins or margins wide enough for complete resection. deep margins may include removing sections of abdominal fascia or musculature en bloc with mammary tumor. multiple mammary tumors should be removed via regional or unilateral chain mastectomies. bilateral, staged mastectomies are reserved for more aggressive tumors. there is insufficient evidence at this time to recommend routine complete unilateral or bilateral chain mastectomies. at the time of surgery, axillary lymph nodes are removed only if enlarged or positive on cytology for metastasis. sorenmo et al. ( ) provide a thorough review of canine mammary gland neoplasia. research complications treatment of early-stage or low-grade mammary tumors may be rewarding, allowing dogs to continue on study. if removed early enough, malignant masses could yield the same results. all dogs should be monitored regularly for recurrence and new mammary tumors. beagles are subject to many of the inherited and/ or congenital disorders that affect dogs in general. in a reference table on the congenital defects of dogs (hoskins, ) , disorders for which beagles are specifically mentioned include brachyury (short tail), spina bifida, pulmonic stenosis, cleft palate-cleft lip complex, deafness, cataracts, glaucoma, microphthalmos, optic nerve hypoplasia, retinal dysplasia, tapetal hypoplasia, factor vii deficiency, pyruvate kinase deficiency, pancreatic hypoplasia, epilepsy, gm gangliosidosis, globoid cell leukodystrophy, xx sex reversal, and cutaneous asthenia (ehlers-danlos syndrome). other defects observed include cryptorchidism, monorchidism, limb deformity, inguinal hernia, diaphragmatic hernia, hydrocephaly, and fetal anasarca. each of these other congenital defects occurred at less than . % incidence. etiology benign prostatic hyperplasia (bph) is an age-related condition in intact male dogs. the hyperplasia of prostatic glandular tissue is a response to the presence of both testosterone and estrogen. clinical signs bph is often subclinical. straining to defecate (tenesmus) may be seen because the enlarged gland impinges on the rectum. urethral discharge (yellow to red) and hematuria can also be presenting clinical signs. epizootiology and transmission bph typically affects older dogs (> years), although glandular hyperplasia begins as early as years of age. approximately % of inact male dogs will develop bph by years of age (smith, ) . pathologic findings in the early stages of bph, there is hyperplasia of the prostatic glandular tissue. this is in contrast to human bph, which is primarily stromal in origin. eventually, the hyperplasia tends to be cystic, with the cysts containing a clear to yellow fluid. the prostate becomes more vascular with a honeycomb appearance (resulting in hematuria or hemorrhagic urethral discharge), and bph may be accompanied by mild chronic inflammation. pathogenesis bph occurs in older intact male dogs because increased production of estrogens (estrone and estradiol), combined with decreased secretion of androgens, sensitizes prostatic androgen receptors to dihydrotestosterone. the presence of estrogens may also increase the number of androgen receptors, and hyperplastic prostate glands also have an increased ability to metabolize testosterone to α-dihydrotestosterone (kustritz and klausner, ) mediating bph. diagnosis and differential diagnosis bph is diagnosed in cases of nonpainful symmetrical swelling of the prostate gland in intact male dogs, with normal hematologic profiles and urinalysis that may be characterized by hemorrhage. a prostatic biopsy can be performed to confirm diagnosis. differential diagnoses include squamous metaplasia of the prostate, para-prostatic cysts, bacterial prostatitis, prostatic abscessation, and prostatic neoplasia (primarily adenocarcinoma). these differential diagnoses also increase in frequency with age and, except for squamous metaplasia, can also occur in castrated dogs. as such, these conditions do not necessarily abate or resolve with castration. prevention castration is the primary means for prevention of benign prostatic hyperplasia. treatment the first and foremost treatment for bph is castration. in pure cases of bph, castration results in involution of the prostate gland detectable by rectal palpation within - days. for most dogs in research studies, this is a viable option to rapidly improve the animal's condition. the alternative to castration is hormonal therapy, primarily with estrogens. this may be applicable in cases where semen collection is necessary from a valuable breeding male (e.g., genetic diseases). if the research study concerns steroidal hormone functions, then neither the condition nor the treatment is compatible. finasteride, a synthetic α-reductase inhibitor, has been used in dogs to limit the metabolism of testosterone to α-dihydrotestosterone. treatment at daily doses of . - . mg/kg orally for weeks was shown to reduce prostatic diameter and volume without affecting testicular spermatogenesis (sirinarumitr et al., ) . upon discontinuation of finasteride, the prostate generally returns to its pretreatment size within several months (smith, ) . gonadotropin-releasing hormone analogs such as desorelin inhibit production of testosterone and estrogen via negative feedback on the hypothalamus-pituitary axis. this is available in a sustained release subcutaneous implant, which has demonstrated efficacy in reducing prostatic size in dogs (junaidi et al., ) . however, medical therapy has not shown to be as advantageous as castration. research complications bph can cause complications to steroidal hormone studies, in that the condition may be indicative of abnormal steroidal hormone metabolism, and neither castration nor estrogen therapy is compatible with study continuation. the development of tenesmus as a clinical sign may also affect studies of colorectal or anal function. research model older dogs with benign protastic hypertrophy are used in research to evaluate the use of ultrasonic histotripsy as a precise nonsurgical urethralsparing alternative to prostate surgery (lake et al., ; hall et al., ; schade et al., ) . etiology juvenile polyarteritis syndrome (jps), also known as steroid-responsive meningitis-arteritis, is a painful disorder seen in young beagles (occasionally reported in other breeds) caused by a systemic necrotizing vasculitis. the cause of the vasculitis has not been established but appears to have an autoimmune-mediated component and may have a hereditary predisposition. clinical signs clinical signs of jps include fever, anorexia, lethargy, and reluctance to move the head and neck. the dogs tend to have a hunched posture and/ or an extended head and neck. most dogs seem to be in pain when touched, especially in the neck region. neurological examination may reveal proprioceptive deficits, paresis, or paralysis. the syndrome typically has a course of remissions and relapses characterized by - days of illness and - weeks of remission (scott-moncrieff et al., ) . there may be a component of this condition that is subclinical, given that vasculitis has been diagnosed postmortem in beagles that had no presenting signs. epizootiology and transmission jps typically affects young beagles ( - months), with no sex predilection. jps has been reported in other breeds including sibling welsh springer spaniels (caswell and nykamp, ) . pathologic findings on gross necropsy, foci of hemorrhage can be seen in the coronary grooves of the heart, cranial mediastinum, and cervical spinal cord meninges (snyder et al., ) . local lymph nodes may be enlarged and hemorrhagic. histologically, necrotizing vasculitis and perivasculitis of small to mediumsized arteries are seen. these lesions are most noticeable where gross lesions are observed, but they may be seen in other visceral locations. arterial fibrinoid necrosis leading to thyroid gland hemorrhage and inflammation was also reported (peace et al., ) . the perivasculitis often results in nodules of inflammatory cells that eccentrically surround the arteries. the cellular composition of these nodules is predominantly neutrophils, but it can also consist of lymphocytes, plasma cells, or macrophages (snyder et al., ) . fibrinous thrombosis of the affected arteries is also seen. a subclinical vasculitis has also been diagnosed in beagles post mortem; it is not known whether this subclinical condition is a different disorder or part of a jps continuum. this subclinical vasculitis often affects the coronary arteries (with or without other sites). pathogenesis the initiating factors for jps are unknown. it was once presumed to be a reaction to test compounds by laboratory beagles, but this may have been coincident to the fact that the beagle is the breed most often affected with jps. immune mediation of jps is strongly suspected, because the clinical signs have a cyclic nature and respond to treatment with corticosteroids, and the affected dogs have elevated α -globulin fractions and abnormal immunologic responses. there may be hereditary predisposition, given that pedigree analysis has indicated that the offspring of certain sires are more likely to be affected, and breeding of two affected dogs resulted in one in seven affected pups (scott-moncrieff et al., ) . diagnosis and differential diagnosis differential diagnoses include encephalitis, meningitis, injury or degeneration of the cervical vertebrae or disks, and arthritis. in the research facility, the disorder may be readily confused with complications secondary to the experimental procedure, or with postsurgical pain. beagles with jps that were in an orthopedic research study were evaluated for postsurgical complications and skeletal abnormalities prior to the postmortem diagnosis of systemic vasculitis (authors' personal experience). prevention and control no prevention and control measures are known at this time. treatment clinical signs can be abated by administration of corticosteroids. prednisone administered orally at . mg/kg, q h, was associated with rapid relief of clinical symptoms. maintenance of treatment at an alternate-day regimen of . - . mg/kg was shown to relieve symptoms for several months. however, withdrawal of corticosteroid therapy led to the return of clinical illness within weeks. research complications because of the potentially severe clinical signs and the need for immunosuppressive treatment, jps is often incompatible with use of the animal as a research subject. it is unknown whether subclinical necrotizing vasculitis causes sufficient aberrations to measurably alter immunologic responses. etiology interdigital cysts are chronic inflammatory lesions (not true cysts) that develop in the webbing between the toes. the cause for most interdigital cysts is usually not identified unless a foreign body is present. bacteria may be isolated from the site, but the lesions may also be sterile (hence the synonym 'sterile pyogranuloma complex'). clinical signs dogs with interdigital cysts are usually lame on the affected foot, with licking and chewing at the interdigital space. exudation may be noticed at the site of the lesion. the lesion appears as a cutaneous ulcer, usually beneath matted hair, with possible development of sinus tracts and purulent exudate. epizootiology and transmission interdigital cysts are common in a variety of canine breeds, including german shepherds. beagles have been affected in the research setting. interdigital cysts usually occur in the third and fourth interdigital spaces (bellah, ) . a retrospective study of beagles housed in a research industry setting, linked development of interdigital cysts to body codition score, age, location of cyst, and type of caging, and may result from chronic interdigital dermatitis (kovacs et al., ) . pathologic findings histopathologically, interdigital cysts are sites of chronic inflammation, typically described as pyogranulomatous. pathogenesis initial development of the cysts is unknown, except for those cases in which a foreign body can be identified. diagnosis and differential diagnosis bacterial culture swabs and radiographs should be taken of the cysts to rule out bacterial infection and radiopaque foreign bodies or bony lesions, respectively. a biopsy should be taken if neoplasia is suspected. treatment if a foreign body is associated with the lesion, then removal is the first order of treatment. if biopsy of the site provides a diagnosis of sterile pyogranuloma complex, then systemic corticosteroid therapy (e.g., prednisolone at mg/kg q h) can be initiated and then tapered once the lesion heals. interdigital cysts that are refractory to medical therapy require surgical removal. excision includes the removal of the lesion and the interdigital web, and a two-layer closure of the adjacent skin and soft tissues is recommended (bellah, ) . the foot should be put in a padded bandage and a tape hobble placed around the toes to reduce tension when the foot is weight-bearing. the prognosis for idiopathic interdigital cysts is guarded, because the cysts tend to recur (bellah, ) . research complications research complications from the cysts are minimal, unless the dogs need to be weight-bearing for biomechanic or orthopedic studies. treatment with systemic steroids could be contraindicated with some experimental designs. etiology 'cherry eye' is a commonly used slang term for hyperplasia and/or prolapse of the gland of the nictitating membrane (third eyelid). this is not considered a congenital anomaly, but there is breed disposition for this condition, including beagles. a specific etiology is not known. clinical signs the glandular tissue of the nictitating membrane protrudes beyond the membrane's edge and appears as a reddish mass in the ventromedial aspect of the orbit. excessive tearing to mucoid discharge can result, and severe cases can be associated with corneal erosion. pathologic findings typically, the glandular tissue is hyperplastic, possibly with inflammation. rarely is the tissue neoplastic. pathogenesis prolapse of the gland may be a result of a congenital weakness of the connective tissue band between the gland and the cartilage of the third eyelid (helper, ) . prevention hyperplasia of the third eyelid cannot be prevented, but dogs that develop this condition unilaterally should have the other eye evaluated for potential glandular prolapse. preventative surgical measures might be warranted. treatment corticosteroid treatment (topical or systemic) can be used to try to reduce the glandular swelling. however, surgical reduction or excision of the affected gland is typically required to resolve the condition. in the reduction procedure, the prolapsed gland is sutured to fibrous tissue deep to the fornix of the conjunctiva (helper, ). if reduction is not possible (as with deformed nictitating cartilage) or is unsuccessful, removal of the gland can be performed. such excision is fairly straightforward and can be done without removal of the nictitating membrane itself. the gland of the third eyelid is important in tear production; although the rest of the lacrimal glands should be sufficient for adequate tear production, keratoconjunctivitis sicca is a possible consequence after removal of the gland of the nictitating membrane. research complications in most cases, research complications would be minimal, especially if treated adequately. either the presence of the hyperplastic gland or its removal might compromise ophthalmologic studies. antimicrobial resistance profiles and mechanisms of resistance in campylobacter jejuni isolates from pets life span and cancer mortality in the beagle dog and human studies of distribution and recurrence of helicobacter spp. gastric mucosa of dogs after triple therapy plasma von willebrand factor concentration and thyroid function in dogs catheter-related infections in long-term catheterized dogs. observations on pathogenesis, diagnostic methods, and antibiotic lock technique aaha nutritional assessment guidelines for dogs and cats open-and closed-formula laboratory animal diets and their importance to research comparison of the accuracy of two ultrasonographic measurements in predicting the parturition date in the bitch dog thyroiditis: occurrence and similarity to hashimoto's struma surgical management of specific skin disorders bordetella and mycoplasma respiratory infections in dogs and cats associations between lymphocytic thyroiditis, hypothyroidism, and thyroid neoplasia in beagles diagnostic exercise: peracute death in a research dog insecticide and acaricide molecules and/ or combinations to prevent pet infestation by ectoparasites neurologic manifestations associated with hypothyroidism in four dogs veterinary surgery: small animal neoplasms of the skin and subcutaneous tissues in dogs and cats neoplasia of the skin and mammary glands of dogs and cats pancreatic adenocarcinoma in two grey collie dogs with cyclic hematopoiesis variation in age at death of dogs of different sexes and breeds comparison of campylobacter carriage rates in diarrheic and healthy pet animals advances in dietary management of obesity in dogs and cats effect of amount and type of dietary fiber on food intake in energy-restricted dogs effect of level and source of dietary fiber on food intake in the dog an outbreak of fatal hemorrhagic pneumonia caused by streptococcus equi subsp. zooepidemicus in shelter dogs molecular characterization of canine parvovirus strains in argentina: detection of the pathogenic variant cpv c in vaccinated dogs external parasites: identification and control orthopedic coaptation devices and small-animal prosthetics enterohepatic helicobacter spp. in colonic biopsies of dogs: molecular, histopathological and immunohistochemical investigations intradural vasculitis and hemorrhage in full sibling welsh springer spaniels respiratory disease in kennelled dogs: serological responses to bordetella bronchiseptica lipopolysaccharide laboratory animal medicine do not correlate with bacterial isolation or clinical respiratory symptoms lymphoma: which chemotherapy protocol and why? detection of humoral antibody to the transmissible venereal tumor of the dog laboratory animal management: dogs reproductive cycles of the domestic bitch hereditary canine spinal muscular atrophy: an animal model of motor neuron disease. can evaluation of the helicobacteraceae in the oral cavity of dogs management of septicemia in rhesus monkeys with chronic indwelling catheters clinical and virological findings in pups naturally infected by canine parvovirus type glu- mutant eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d client information series: canine demodicosis client information series: fleas and flea allergy dermatitis management of the burn wound burn trauma chlorine dioxide sterilization of implanted right atrial catheters in rabbits current concepts in the management of helicobacter associated gastritis discordant s and s rrna gene phylogenies for the genus helicobacter: implications for phylogenetic inference and systematics use of narcotic antagonists to modify stereotypic selflicking, self-chewing, and scratching behavior in dogs epidemiology of canine and feline tumors epizootiologic characteristics of canine and feline leukemia and lymphoma evaluation of the ovicidal cctivity of lufenuron and spinosad on fleas' eggs from treated dogs enteric coccidiosis the respiratory system the association of american feed control officials dog and cat food nutrient profiles: substantiation of nutritional adequacy of complete and balanced pet foods in the united states study of obesity in dogs visiting veterinary practices in the united kingdom pathological prognostic factors in breast cancer. i. the value of histological grade in breast cancer: experience from a large study with long-term follow-up utility of fdg-pet scanning in lymphoma by who classification principles of treatment for canine lymphoma miller's anatomy of the dog update on diagnosis of canine hypothyroidism immunohistochemical and histochemical stains for differentiating canine cutaneous round cell tumors the laboratory canine canine conjunctival mast cell tumors: a retrospective study canine cutaneous epitheliotropic t-cell lymphoma: a review canine infectious respiratory disease helicobacter-associated gastric disease in ferrets, dogs, and cats enteric bacterial infections hemorrhagic streptococcal pneumonia in newly procured research dogs association between canine malignant lymphoma, living in industrial areas, and use of chemicals by dog owners reproductive patterns in the domestic dog-a retrospective study of the drever breed the dog as a research subject a review of canine pseudocyesis leptospirosis surgical treatment of an elbow hygroma utilizing microvascular free muscle transfer in a newfoundland bacterial diseases immunoprophylaxis leptospirosis gastric helicobacters in domestic animals and nonhuman primates and their significance for human health non-helicobacter pylori helicobacter species in the human gastric mucosa: a proposal to introduce the terms h. heilmannii sensu lato and sensu stricto diseases of the small intestine histotripsy of the prostate: dose effects in a chronic canine model flea control failure? myths and realities small animal clinical nutrition operating room emergencies shock evaluation of the sensitivity and specificity of diagnostic criteria for sepsis in dogs pulmonary parenchymal disease canine thyroid neoplasms: epidemiologic features magrane's canine ophthalmology helicobacter-like organisms: histopathological examination of gastric biopsies from dogs and cats current veterinary therapy x: small animal practice congenital defects of the dog gastric and duodenal ulcers in dogs with mastocytoma complications in the use of indwelling vascular catheters in laboratory animals helicobacter infection diseases of the large intestine pregnancy management in the bitch tracheal collapse. diagnosis and medical and surgical treatment hygroma of the elbow in dogs thermal injuries joint working group on refinement factors contributing to the contamination of peripheral intravenous catheters in dogs and cats diversity in bacterium-host interactions within the species helicobacter heilmannii sensu stricto morphological study of the effects of the gnrh superagonist deslorelin on the canine testis and prostate gland five-year longitudinal study on limited food consumption and development of osteoarthritis in coxofemoral joints of dogs cvt update: interpretation of endocrine diagnostic test results for adrenal and thyroid disease etiopathogenesis of canine hypothyroidism maintenance energy requirement of dogs: what is the correct value for the calculation of metabolic body weight in dogs? outbreak and control of haemorrhagic pneumonia due to streptococcus equi subspecies zooepidemicus in dogs kirk's current veterinary therapy xii: small animal practice an emerging pulmonary haemorrhagic syndrome in dogs: similar to the human leptospiral pulmonary haemorrhagic syndrome? tarsal joint contracture in dogs with golden retriever muscular dystrophy an epidemiological study of interdigital cysts in a research beagle colony value of the ( )c-urea breath test for detection of gastric helicobacter spp. infection in dogs undergoing endoscopic examination pathogenesis of helicobacter pylori infection prostatic diseases semen collection in the dog accuracy of canine parturition date prediction using fetal measurements obtained by ultrasonography chronic catheterization of the intestines and portal vein for absorption experimentation in beagle dogs evaluation of weight loss protocols for dogs problems in wound healing associated with chemotherapy and radiation therapy histotripsy: minimally invasive technology for prostatic tissue ablation in an in vivo canine model fdg-pet imaging in canine lymphoma and cutaneous mast cell tumor comparison of fertility data from vaginal vs intrauterine insemination of frozenthawed dog semen: a retrospective study withrow & macewen's small animal clinical oncology estimation of gestational age and assessment of canine fetal maturation using radiology and ultrasonography: a review effects of four preparations of . % chlorhexidine diacetate on wound healing in dogs the prediction of parturition date in canine pregnancy clustering of activating mutations in c-kit's juxtamembrane coding region in canine mast cell neoplasms transmissible venereal tumors kirk's current veterinary therapy : small animal practice canine lymphoma and lymphoid leukemias the staging and treatment of multicentric highgrade lymphoma in dogs: a review of recent developments and future prospects association between waste management and cancer in companion animals tick paralysis in north america and australia thyroid gland and arterial lesions of beagles with familial hypothyroidism and hyperlipoproteinemia bacterial gastroenteritis in dogs and cats: more common than you think enteropathogenic bacteria in dogs and cats: diagnosis, epidemiology, treatment, and control dermatologic aspects of tick bites and tick-transmitted diseases a chronic access port model for direct delivery of drugs into the intestine of conscious dogs mast cells and canine mast cell tumours: a review etiologic study of upper respiratory infections of household dogs effect of early enteral nutrition on intestinal permeability, intestinal protein loss, and outcome in dogs with severe parvoviral enteritis determination of strain variability of microsporum canis to disinfectants cutaneous fungal infections diagnosis of neoplasia tumors of the mammary gland transmissible dog cancer genome reveals the origin and history of an ancient cell lineage clonal origin and evolution of a transmissible cancer notice regarding nih plan to transition from use of usda class b dogs to other legal sources (not-od- - ) guide for the care and use of laboratory animals surgical closure of elbow hygroma in the dog colitis and colon cancer in waspdeficient mice require helicobacter species tumors of the genital system practical laboratory methods for the diagnosis of dermatologic diseases animals and animal products, subchapter a, parts , , and comparison of three skin preparation techniques in the dog comparison of three skin preparation techniques in the dog. part : clinical trial in dogs manual of clinical behavioral medicine for dogs and cats. mosby-year book identification of and screening for human helicobacter cinaedi infections and carriers via nested pcr identification of three novel superantigen-encoding genes in streptococcus equi subsp. zooepidemicus, szef, szen, and szep hypothyroidism in dogs: cases ( - ) plasma von willebrand factor antigen concentration in dogs with hypothyroidism plasma von willebrand factor antigen concentration and buccal bleeding time in dogs with experimental hypothyroidism canine cutaneous mast cell tumor: morphologic grading and survival time in dogs atlas of small animal wound management and reconstructive surgery what's your diagnosis? fever and leukocytosis in a young beagle. canine juvenile polyarteritis syndrome (beagle pain syndrome) a clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by streptococcus equi subsp. zooepidemicus thyroid diseases urogenital emergencies emergency procedures for the small animal veterinarian prognostic significance of morphological subtypes in canine malignant lymphomas during chemotherapy streptococcus zooepidemicus: an emerging canine pathogen characterization of pneumonia due to streptococcus equi subsp. zooepidemicus in dogs tumors of the skin and soft tissue aspiration-induced lung injury origins and evolution of a transmissible cancer canine mast cell tumors express stem cell factor receptor arterial and venous blood gas analyses dogs and cats as laboratory animals hypocretin levels in sporadic and familial cases of canine narcolepsy comparison of giardia diagnostic tests in diagnosis of naturally acquired canine chronic subclinical giardiasis effects of chlorhexidine diacetate and povidone-iodine on wound healing in dogs genetic evidence for an east asian origin of domestic dogs urethral-sparing histotripsy of the prostate in a canine model factors influencing canine mammary cancer development and postsurgical survival how to treat common parasites safely muller and kirk's small animal dermatology systemic necrotizing vasculitis in nine young beagles thomson's special veterinary pathology, second ed. mosby-year book thyroid and parathyroid glands diseases of the small bowel canine infectious tracheobronchitis (kennel cough complex) diseases of the intestines the use of ultrasonography for pregnancy diagnosis in the bitch cytologic examination of fine-needle aspirates from mammary gland tumors in the dog: diagnostic accuracy with comparison to histopathology and association with postoperative outcome effects of finasteride on size of the prostate gland and semen quality in dogs with benign prostatic hypertrophy canine prostatic disease: a review of anatomy, pathology, diagnosis, and treatment pathologic features of naturally occurring juvenile polyarteritis in beagle dogs development, anatomy, histology, lymphatic drainage, clinical features, and cell differentiation markers of canine mammary gland neoplasms withrow & macewen's small animal clinical oncology nutrient requirements of dogs and cats (nutrient requirements of domestic animals) trauma to the skin and subcutaneous tissues of dogs and cats chronic problem wounds of dog limbs acvim small animal consensus statement on leptospirosis: diagnosis, epidemiology, treatment, and prevention lumbosacral stenosis in dogs mammary neoplasia in a closed beagle colony complete mitochondrial genomes of ancient canids suggest a european origin of deomestic dogs artificial insemination in canids: a useful tool in breeding and conservation artificial insemination with frozen semen in dogs: a retrospective study of years using a non-surgical approach manual of canine and feline cardiology comparative evaluation of agar dilution and broth microdilution methods for antibiotic susceptibility testing of helicobacter cinaedi thyroiditis in a group of laboratory dogs: a study of beagles obesity-related metabolic dysfunction in dogs: a comparison with human metabolic syndrome pro-inflammatory properties and neutrophil activation by helicobacter pylori urease of agriculture, animal and plant health inspection service annual report animal usage by fiscal year. available from: <www leptospirosis in dogs: a review with emphasis on clinical aspects thomson's special veterinary pathology, second ed. mosby-year book endocrinology of pregnancy in the dog: a review immunoblot analysis as an alternative method to diagnose enterohepatic helicobacter infections management of superficial skin wounds psychodermatosis colony multiplex pcr assay for identification and differentiation of campylobacter jejuni, c. coli, c. lari, c. upsaliensis, and c. fetus subsp. fetus the role of c-kit in tumorigenesis: evaluation in canine cutaneous mast cell tumors aaha canine vaccination guidelines canine mast cell tumours: a review of the pathogenesis, clinical features, pathology and treatment serum concentrations of thyroxine and , , ′-triiodothyronine before and after intravenous or intramuscular thyrotropin administration in dogs surgical treatment of specific skin disorders naltrexone for treatment of acral lick dermatitits in dogs use of ultrasound in the measurement of subcutaneous fat and prediction of total body fat in dogs mammal species of the world: a taxonomic and geographic reference withrow & macewen's small animal clinical oncology the authors thank dr. bambi jasmin of marshall farms for contributions on preventive health practices for class a dogs. key: cord- -qos vu r authors: dejnirattisai, wanwisa; webb, andrew i.; chan, vera; jumnainsong, amonrat; davidson, andrew; mongkolsapaya, juthathip; screaton, gavin title: lectin switching during dengue virus infection date: - - journal: j infect dis doi: . /infdis/jir sha: doc_id: cord_uid: qos vu r dengue virus receptors are relatively poorly characterized, but there has been recent interest in c-type lectin molecules, dendritic cell–specific intercellular adhesion molecule (icam- )–grabbing nonintegrin (dc-sign) and its close homologue liver/lymph node–specific icam- –grabbing integrin (l-sign), which can both bind dengue and promote infection. in this report we have studied the interaction of dengue viruses produced in insect cells, tumor cell lines, and primary human dendritic cells (dcs) with dc-sign and l-sign. virus produced in primary dcs is unable to interact with dc-sign but remains infectious for l-sign–expressing cells. skin-resident dcs may thus be a site of initial infection by insect-produced virus, but dcs will likely not participate in large-scale virus replication during dengue infection. these results reveal that differential glycosylation of dengue virus envelope protein is highly dependent on cell state and suggest that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. the global prevalence of the dengue virus (denv) has grown dramatically in recent decades, and it is now endemic in . countries, with some . billion people at risk of infection. dengue is an arthropod-borne flavivirus that can be subdivided into the major serotypes (den- -den- ). most dengue infections either are asymptomatic or lead to a self-limiting febrile illness, dengue fever. in some cases the illness is more severe, leading to dengue hemorrhagic fever (dhf) with severe plasma leakage and bleeding that can be life threatening. dhf is more common in individuals undergoing secondary heterologous dengue infection than those suffering primary infections. there has been considerable work to understand the mechanism of severe disease, but the increased frequency during secondary infection and the occurrence of severe symptoms at a time when virus loads are falling sharply imply that it likely results from immunopathology driven by the acquired immune responses, rather than from direct viral cytopathology [ ] [ ] [ ] [ ] . levels of a number of cytokines, such as interferon c and tumor necrosis factor a [ , ] , have been shown to correlate with disease severity, and a storm of inflammatory cytokine secretion has been proposed to lead to the vascular leak characteristic of dhf. recent reports have identified monocytes as a major cell target of viral replication, and heparin sulfates, dc-sign, mannose receptor, and other glycoproteins have been proposed as cellular receptors for denv [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . dc-sign polymorphism has been shown to be associated with the disease severity [ ] . clec a has also recently been described as a proinflammatory receptor for denv that contributes to lethal disease in a mouse model [ ] . as dengue virus circulates between hosts, humans and insects, it has to be adapted to replicate and infect both species. the majority of studies of dengue infection and tropism use viruses produced in insect cell lines such as c / or mammalian tumor cell lines such as vero cells. infection of humans occurs in a stepwise fashion: initial infection of cells with insect virus, followed by sequential infection of cells by mammalian-produced virus. we were interested to determine whether there were any differences in the tropism of viruses produced in primary nontransformed human cells. the dengue- strain, , was grown in c / cells, vero cells, and monocyte-derived dendritic cells (dcs). cell-free supernatants were used either neat or after concentration by ultracentrifugation at , rpm for h at °c, and the virus pellet was resuspended in . % fetal bovine serum (fbs)/ leibovitz l- . to concentrate large volumes of low-titer denv supernatant, denv were precipitated with % polyethylene glycol (sigma) before ultracentrifugation. u were maintained in % fbs/roswell park memorial institute medium (rpmi). nih/ t , and dc-sign and l-sign nih/ t cell lines were obtained through the aids research and reference reagent program (division of aids, national institute of allergy and infectious diseases, from drs thomas d. martin and vineet n. kewal ramani) and maintained in % fbs/ dulbecco's modified eagle's medium. all media were supplemented with mmol/l l-glutamine, u/ml penicillin, and u/ml streptomycin. the viral titers were determined by a focus-forming assay. briefly, virus was serially diluted and incubated with vero cells for hours at °c. the monolayers were then overlaid with . % carboxymethylcellulose and incubated at °c for days. virus foci were stained with anti-e antibody followed by peroxidase-conjugated anti-mouse immunoglobulin (ig) and visualized by the addition of , #-diaminobenzidine tetrahydrochloride substrate. dcs were cultured as described elsewhere [ ] . human cd monocytes were cultured in rpmi with ng/ml rhugm-csf (first link) and ng/ml rhuil- (ebioscience). the appropriate phenotype of immature dc (ie, lacking cd but expressing major histocompatibility complex class i and class ii and dc-sign) was confirmed. cells were infected with denv at a multiplicity of infection (moi) of for h. cells were washed twice in facs wash (fw; phosphate-buffered saline [pbs] containing . % bovine serum albumin, % fbs, % human serum, . % nan ). cells were fixed with % paraformaldehyde/pbs for minutes and permeabilized with . % saponin in fw for minutes. cells were then incubated with anti-ns mab or with anti-e mab followed by anti-mouse igg/pe (dakocytomation) in . % saponin/fw. cells were resuspended in fw and analyzed by facscan (becton dickinson). data were analyzed by using flowjo software (tree star). t and t were incubated with u/ml heparin for minutes at room temperature before being infected with denv. cells were incubated with denv produced from different cell types at equal amounts of e protein (measured by enzymelinked immunosorbent assay) for h at room temperature. after washing with ice-cold fw, cells were fixed with % paraformaldehyde/pbs, and surface-bound virus was detected with an anti-e mab and anti-mouse igg/pe followed by facs analysis. the pcdna -ll-sign plasmids expressing (n ) and (n ) tandem neck region repeats or a mixture of n and n plasmids (ratio : ) were transfected into t cells using the fugene (roche). l-sign expression was verified with l-sign-specific antibody (clone , r&d systems). denv supernatant was precleared with protein a-agarose for one h at °c. bead-free supernatant was incubated with lg of g at °c for h followed by protein a-agarose for h. the beads were washed with . % tween/pbs times and eluted with nonreducing loading buffer. the sample was run on nonreducing % sodium dodecyl sulfate (sds) polyacryramide gels and electroblotted onto nitrocellulose membrane (amersham). glycan types on denv proteins were determined using the dig glycan differentiation kit (roche). endoglycosidase h (endo h) or n-glycosidase f (pngase f; new england biolabs) was performed as described elsewhere [ ] . digested proteins were separated by % sds polyacryramide gels and analyzed by western blot. e protein was detected by anti-e mab followed by peroxidase-conjugated anti-mouse igg ab. following the bite from an infected mosquito, the host first encounters virus produced in the mosquito, and following this initial inoculation subsequent rounds of infection are driven by virus produced by host cells. to study these distinct stages of pathogenesis, we compared viruses from c / (insect cells) and virus produced from primary human monocyte-derived dendritic cells. viral supernatants were titered using a focus-forming assay on vero cells, and equal amounts of titered virus were used to infect vero, t, or dcs at an moi of . the percentage of infected cells was monitored by cytofluorometry staining of the intracellular nonstructural dengue antigen ns , which is produced only following productive infection. insect-derived virus was equally competent at infecting the cell types with high efficiency ( figure a-c) . surprisingly, dc-produced virus was not able to reinfect dcs but was nevertheless fully competent at infecting t and vero cells. the lack of infectivity of dc-produced virus on dcs was also shown using a different mab, g , which reacts with an epitope on dengue envelope protein ( figure c , lower panel). a time course of infection was performed where dcs or vero cells were infected with virus produced in c / , vero, or dcs, and infection was monitored by facs or using a focus-forming assay to measure infectious virus harvested from the supernatants of infected cells ( figure d -e). at , , and h the dc-produced virus showed a much reduced ability to infect dcs when compared with virus produced in c / or vero cells. the experiments we have described above used the dengue serotype strain . to see whether these results could be generalized, we checked infection of both dcs and vero cells with dengue serotype strain new guinea c, serotype strain hawaii, serotype strain h , and serotype strain h . infection efficiency was measured by facs at and hours following infection with virus produced in either c / cells or dcs (figure a-b) . in all cases dc-produced virus was much less infectious for dcs than insect-produced virus, whereas infection of vero cells was roughly equal. it is known that mature dcs are relatively resistant to dengue infection, so to rule out the possibility of any dc-produced cofactors that might induce maturation or any other form of resistance to infection we performed a mixing experiment whereby dc-produced and c / -produced viruses were used to infect dcs. in these experiments using mixed viruses the dcs were again susceptible to infection, presumably by the c / produced virus fraction ( figure c ). the lack of reinfection of dcs by dc-produced virus may reflect a difference in the surface binding interaction of dc-versus insect-produced viruses to dcs. to test this vero cells and dcs were incubated with denv produced from the different sources, and binding to the cells was then assessed by staining with the anti-e mab g . virus produced in c / cells could bind to dcs, while binding of dc virus back onto dcs was almost completely absent ( figure d ). conversely, both dc-and insect-produced viruses were able to bind to vero cells. to formally prove that the loss of infection of dcs was a result of the loss of affinity of dc-produced virus for dc-sign, we went on to test infection on t cells expressing dc-sign and included in these assays the related c-type lectin l-sign ( figure a ), which has also been reported to be a receptor for dengue virus. insect-derived virus could efficiently infect both dc-sign-and l-sign-expressing cells when compared with control t cells ( figure b ). similar to the lack of observable infection of dcs, the dc-derived virus showed a much lower level of infection on dc-sign-expressing t cells, with , % infection. surprisingly, however, this virus progeny was still able to infect cells expressing l-sign, with up to % infection observed. previous reports with dengue virus have suggested that the virus shows equal tropism for both dc-sign and l-sign. these reports were from virus produced in tumor cell lines and not from primary cells. we tested the infectivity of virus produced in vero cells, and in agreement with these previous reports dengue produced in this tumor cell line was able to efficiently infect t cells via either dc-sign or l-sign ( figure c ). finally, we tested the binding of insect-and dc-produced virus to the t transfectants ( figure d ). in agreement with the dc binding experiments dc-produced virus was unable to bind to dc-sign-expressing dcs, whereas both insect-and dc-derived viruses could bind to l-signexpressing cells. to gain insight into the glycosylation profiles of denv produced in c / , vero, and dc, purified virus was tested against a panel of lectins with differing carbohydrate-binding specificities by western blot ( figure a ). among the plant lectins tested, gna is the only one that selectively interacts with high-and pauci-mannose-type n-glycans. sna and maa bind to sialic acid terminally linked to galactose by a , linkage and a , linkage, respectively, which may occur on complex-type n-glycans. dsa recognizes repeating n-acetyllactosamine (galactosyl b , n-acetylglucosamine) sequences; these may also occur on complex-type n-glycans. pna, unlike dsa, binds preferentially to b , -linked terminal galactose, such as galactosyl b , n-acetylgalactosamine, a sequence commonly occurring on o-glycosylated proteins and gangliosides. virus produced in c / cells bound exclusively to gna, implying that it contained predominantly high-or pauci-mannose n-glycans consistent with the glycosylation patterns seen in insect cells. vero-produced virus was bound by of the lectins tested-gna, sna, and dsa-suggesting that the virus contained a variety of high-mannose and complex-or hybrid-type n-glycans with or without a , -linked sialic acids, possibly on polynacetyllactosamine outer chains. the dc-produced virus was bound only by sna and dsa, indicating that there was a lack of high-mannose or hybrid n-glycans and that only complex-type n-glycans were present. all of the lectin binding signals occurred at the same position on the gels as e protein in the region of kda; we did not see any signal at kda where prm would be expected to migrate. however, this could be due to a low level of prm on these viruses. finally, we assessed the n-linked glycan on the envelope protein by digestion with either endo h, which will remove high-mannose simple glycans containing or more terminal mannose residues, or pngase f, which removes all simple or complex n-linked glycan ( figure b ). dc-produced virus showed a mobility shift of around or kda corresponding to the addition of or complex carbohydrates at positions and . this was completely resistant to digestion with endo h, indicating the presence of complex low-mannose carbohydrate. for both insect-and vero-produced virus, envelope protein migrating at or kda higher than the pngase f-digested material indicated, as with dc-produced virus, that envelope had either or added sugars. following endo h digestion a complex pattern of bands was revealed, indicating that some of the added sugars were endo h sensitive and therefore contained . terminal mannose residues, whereas others were endo h resistant and contained more heavily processed structures, which is in agreement with a recent report [ ] . we conclude from these experiments that in c / , vero cells, and dcs both n-linked glycosylation sites can be used but that a single site is used in % of cases. envelope from dc-produced virus contains complex, highly processed endo h-resistant carbohydrate. however, in both c / and vero, a proportion of the sugar is high mannose and therefore will allow interaction with dc-sign, which is consistent with the results from lectin blotting shown above. l-sign contains a tandem repeat in its neck region, nterminal to the carbohydrate recognition domain, which can be of variable length between and repeats. l-sign exists as tetramers, and previous studies have suggested that heterogeneity in the tandem repeat region of the l-sign neck region may contribute to severe acute respiratory syndrome (sars) susceptibility by altering the viral env-receptor affinity [ ] . to determine whether heterogeneous l-sign neck lengths affect denv infection levels, we examined the effects of a single length repeat and compared it with cells expressing different-length l-sign alleles simultaneously. we examined this by transient transfection, and t cells were used for these assays as they can be transiently transfected to a high level. t were transfected with l-sign containing or repeats singly or together in equal amounts. l-sign expression was confirmed by surface staining and flow cytometry. equal expression of alternate-length l-sign constructs was also confirmed by western blotting. transfectants were then infected with dengue virus, and infection was monitored by intracellular staining together with an antibody specific to l-sign to reveal transfected cells. all transfectants were equally infected with no reduction in cells expressing both l-sign alleles, suggesting that heterotetrameric l-sign was as effective as homotetrameric l-sign at promoting infection ( figure ). finally, although dc-produced virus cannot reinfect dcs efficiently, we were interested to determine whether dc-produced virus was still able to infect cells by antibody-dependent enhancement (ade), which would allow it to replicate by infection of fc receptor-expressing cells. c / -and dc-derived viruses were incubated with increasing levels of pooled convalescent dengue immune serum and subsequently used to infect u , a monocyte cell line that expresses the fc receptor and which shows relatively low infectivity without the presence of enhancing antibodies. viruses produced in both dcs and insect cells were susceptible to enhancement, over the same range of antibody concentrations, showing that dc-produced virus could exploit ade to replicate in individuals undergoing a secondary dengue infection ( figure a ). in a final series of experiments we investigated whether dc-produced virus could be induced to infect primary human dcs by ade ( figure b ). dc-produced virus could be enhanced to infection by . -fold, whereas the already high level of infection of dcs by insectproduced virus was not enhanced further by dengue serum. during natural dengue infection, mosquito saliva containing denv particles is injected into the skin during a blood meal. skin-resident immature dcs have been proposed to be the primary site of infection for insect-derived denv [ ] . the primary receptor on dcs for dengue virus is believed to be dc-sign. dc-sign binds n-linked high-mannose oligosaccharides, including glycans with terminal fucose residues that include the blood group lewis x and lewis a eptitopes [ ] . l-sign, like dc-sign, binds to intercellular adhesion molecule (icam- ) and is thought to establish cellular interactions with icam- -expressing t cells. l-sign is able to capture a variety of viruses ( [ ] [ ] [ ] [ ] [ ] . dc-sign and l-sign preferentially bind pyranose sugars, in particular mannose. a study comparing l-sign-and dc-sign-specific ligands using glycan arrays revealed a restricted repertoire of glycan ligands for l-sign, namely, the high-mannose-type n-glycans. in contrast, dc-sign bound to fucosylated glycans in addition to highmannose-type n-glycans [ ] . the dengue e protein has potential n-linked glycosylation sites at positions and . cryo-electron microscopy reconstructions of dengue virus complexed to soluble dc-sign show interaction with the glycan at position [ ] . there have been several reports on the n-linked glycosylation of dengue envelope. some reports suggest the use of both sites, whereas others suggest that only one is used [ , [ ] [ ] [ ] . we show that in c / , vero cells, and dcs either one or both sites are used for the dengue serotype . although both glycosylation sites can play important roles in infectivity and viral replication, functional studies have confirmed the importance of asn- for infection of dc-sign-expressing cells [ ] . wnv e protein contains a single n-linked glycosylation site at position that is absent from some virus strains [ ] . both dengue and wnv contain a single n-linked site in prm, and although prm is cleaved by furin during viral maturation, a substantial fraction of uncleaved prm is found on some dengue viruses, particularly that produced in insect cells, and such partially cleaved viruses can still be infectious. for wnv the n-linked site on prm can also mediate interaction with l-sign, and in common with glycan at position this also showed differential specificity for l-sign when expressed in mammalian cell lines, perhaps mediated by the presence of n-acetylglucosamine-terminated structures [ ] . the difference in the binding specificities of dengue viruses produced in vero versus primary dendritic cells was somewhat surprising and likely related to the expression of high-mannose moieties in vero. a number of tumor cell lines express high-mannose sugars, and we have previously described the generation of a monoclonal antibody that recognizes a variety of tumor cell types and which binds to high-mannose moieties [ ] . the differential affinity of dc-sign for ligands expressed by tumor cell lines versus primary cells has been observed before and led some to speculate that dc-sign may participate in tumor surveillance [ ] . there appears to be a further added level of complexity as the glycosylation profiles may vary in a given cell line depending on the exact position of the n-linked site within the polypeptide chain [ ] . in humans, l-sign expression is restricted to endothelial cells beneath the subcapsular sinus in lymph nodes [ ] , sinusoidal endothelial cells in the liver [ , ] , alveolar and endothelial cells in the lung [ ] , and capillaries in the villous lamina propria of terminal ileum and peyer patches [ ] . dengue antigens have been demonstrated in the sinusoidal tissue of the liver and vascular endothelium of the lung and spleen and may provide an explanation for the unique pathology observed in these organs [ , ] . the accumulation of antigen in l-signexpressing tissue may also result in an increase in localized transinfection in a similar way that l-sign is thought to be responsible for the capture of hcv from the blood and transmission to hepatocytes or in the case of hiv, to cd t cells [ , ] . we conclude that skin-resident dcs are a likely target for initial infection by dengue virus injected by the infecting mosquito; however, subsequent dissemination of the virus to monocytes and other cell types will no longer use dc-sign as a primary receptor and may rely in part on a shift to l-sign as the primary lectin receptor. the differential glycosylation of the denv e protein during replication in primary mammalian cells suggests that studies of virus tropism using virus prepared in insect cells or tumor cell lines should be interpreted with caution. cross-reacting antibodies enhance dengue virus infection in humans immunodominant t-cell responses to dengue virus ns are associated with dhf immunopathological mechanisms in dengue and dengue hemorrhagic fever original antigenic sin and apoptosis in the pathogenesis of dengue hemorrhagic fever multiplex cytokine profile from dengue patients: mip- beta and ifn-gamma as predictive factors for severity high levels of stnfr p and tnf alpha in dengue-infected patients monocytes, but not t or b cells, are the principal target cells for dengue virus (dv) infection among human peripheral blood mononuclear cells phenotyping of peripheral blood mononuclear cells during acute dengue illness demonstrates infection and increased activation of monocytes in severe cases compared to classic dengue fever dengue virus infectivity depends on envelope protein binding to target cell heparan sulfate dc-sign (cd ) mediates dengue virus infection of human dendritic cells the mannose receptor mediates dengue virus infection of macrophages dengue virus binding to human hepatoma hepg and simian vero cell surfaces differs dengue virus entry into liver (hepg ) cells is independent of hsp and hsp heat shock protein and heat shock protein are components of dengue virus receptor complex in human cells identification of grp (bip) as a liver cell expressed receptor element for dengue virus serotype bacterial lipopolysaccharide inhibits dengue virus infection of primary human monocytes/macrophages by blockade of virus entry via a cd -dependent mechanism dendritic cell-specific intercellular adhesion molecule -grabbing non-integrin (dc-sign)-mediated enhancement of dengue virus infection is independent of dc-sign internalization signals dendritic-cell-specific icam -grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses a variant in the cd promoter is associated with severity of dengue disease clec a is critical for denguevirus-induced lethal disease a complex interplay among virus, dendritic cells, t cells, and cytokines in dengue virus infections histidine in the dengue virus type m protein has an important role in virus assembly n-linked glycans on dengue viruses grown in mammalian and insect cells homozygous l-sign (clec m) plays a protective role in sars coronavirus infection human skin langerhans cells are targets of dengue virus infection the dendritic cell-specific c-type lectin dc-sign is a receptor for schistosoma mansoni egg antigens and recognizes the glycan antigen lewis x a dendritic cell-specific intercellular adhesion molecule -grabbing nonintegrin (dc-sign)-related protein is highly expressed on human liver sinusoidal endothelial cells and promotes hiv- infection l-sign (cd l) and dc-sign (cd ) mediate transinfection of liver cells by hepatitis c virus the location of asparagine-linked glycans on west nile virions controls their interactions with cd (dendritic cell-specific icam- grabbing nonintegrin) human cytomegalovirus binding to dc-sign is required for dendritic cell infection and target cell trans-infection dc-sign and dc-signr interact with the glycoprotein of marburg virus and the s protein of severe acute respiratory syndrome coronavirus structural basis for distinct ligandbinding and targeting properties of the receptors dc-sign and dc-signr cryo-em reconstruction of dengue virus in complex with the carbohydrate recognition domain of dc-sign the envelope glycoproteins of dengue and dengue viruses grown in mosquito cells differ in their utilization of potential glycosylation sites both e protein glycans adversely affect dengue virus infectivity but are beneficial for virion release essential role of dengue virus envelope protein n glycosylation at asparagine- during viral propagation envelope protein glycosylation status influences mouse neuroinvasion phenotype of genetic lineage west nile virus strains enhanced immune recognition of cryptic glycan markers in human tumors the b homolog butyrophilin btn a is a novel ligand for dc-sign dynamic populations of dendritic cell-specific icam- grabbing nonintegrin-positive immature dendritic cells and liver/lymph node-specific icam- grabbing nonintegrin-positive endothelial cells in the outer zones of the paracortex of human lymph nodes dc-signr, a dc-sign homologue expressed in endothelial cells, binds to human and simian immunodeficiency viruses and activates infection in trans cd l (l-sign) is a receptor for severe acute respiratory syndrome coronavirus expression of dc-sign by dendritic cells of intestinal and genital mucosae in humans and rhesus macaques lethal antibody enhancement of dengue disease in mice is prevented by fc modification localization of dengue virus in naturally infected human tissues, by immunohistochemistry and in situ hybridization we thank t. feizi and yan liu for advice and critically reading the manuscript. key: cord- -ujflw b authors: newcomer, benjamin w.; cebra, chris; chamorro, manuel f.; reppert, emily; cebra, margaret; edmondson, misty a. title: diseases of the hematologic, immunologic, and lymphatic systems (multisystem diseases) [image: see text] date: - - journal: sheep, goat, and cervid medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ujflw b nan in this chapter, multisystemic diseases are discussed in small ruminants (sheep, goats, and cervids). these include diseases of the hematologic, immunologic, and lymphatic systems. in general, species will be discussed together, but when pertinent data are available, each species will be considered separately. the terms "cervid" and "deer" have been used interchangeably in parts of this chapter by the authors. an adequate volume of blood for hematologic and biochemical analysis is best obtained from the jugular vein. a docile animal may be restrained in a standing position or tipped up (sheep only) with the head turned away from the jugular vein to be used. wilder ones, such as some cervids, may require restraint devices or chemical sedation. ideally, the animal should be restrained by someone other than the blood collector, although the same person may be able to both restrain a sheep and collect blood if the animal is tipped up or a halter is used (see chapter ) . the animal should be at rest, with minimal excitement. the collector parts or clips the wool or hair to visualize the jugular vein and then uses the hand not holding the needle to apply digital pressure proximally just above the thoracic inlet to block blood movement through the vein. the vessel may take a second or more to distend after pressure is applied. the collector may then use the needlebearing hand to "strum" the vessel and cause the blood to oscillate. if in doubt about whether the distended vessel is the jugular vein, the collector can release the hand placing pressure on the vessel and observe whether the distended vessel disappears; if it does, the distended vessel was probably the jugular vein. the collector should avoid vessels that pulsate because these are likely to be the carotid arteries. the area should be cleaned with alcohol or other disinfectant, water, or a clean, dry gauze sponge. an -or -gauge, -to . -inch needle is usually adequate to collect blood from an adult, whereas a -gauge needle may be used in a neonate. the skin of adults or males may be thicker and more difficult to penetrate with the needle. a syringe or evacuated tube attached to a vacutainer (becton dickinson inc., rutherford, nj) can be used to collect blood. the needle should be plunged through the skin into the vein at an approximate -degree angle. the blood should not come out of the vessel in pulsatile waves; this is suggestive of an arterial stick. after aseptically obtaining an adequate volume of blood, the collector removes the needle and releases the pressure on the vessel near the thoracic inlet. pressure should be applied to the site of puncture for a minute or more to prevent extravascular leakage of blood and hematoma formation. the blood should be carefully transferred to a vial containing the appropriate anticoagulant to prevent red blood cell (rbc) rupture. goat erythrocytes are small and particularly prone to hemolysis. to minimize this problem, goat blood should be collected with a needle and syringe, not a vacutainer. white blood cell (wbc) differential distribution, individual blood cell staining characteristics, and morphology may be assessed by microscopic examination of a stained blood film. the differential distribution provides more information than total wbc count because inflammatory conditions in artiodactyls often result in a shift in neutrophil populations toward more degenerate, toxic, or immature forms without changing the overall wbc count. the preferred anticoagulant for a complete blood count (cbc) is ethylenediaminetetraacetate (edta), and tubes should be filled to ensure the proper blood-to-anticoagulant ratio. blood samples should be processed as soon as possible after collection. if a delay is anticipated, the blood sample should be refrigerated ( ° c) and an air-dried blood smear should be made because prolonged contact of blood with edta causes changes in wbc morphology and the separation of some rbc parasites. blood can be refrigerated for hours and still yield an accurate cbc. a reference range for hematologic data for sheep and goats is provided in table . (see appendix , tables and ) . goats tend to have a low mean corpuscular volume (mcv) because of their small erythrocytes. sheep and goats younger than months old tend to have lower hematocrit, rbc count, hemoglobin, and plasma protein concentrations, as well as a higher total wbc count. neonates often have a high hematocrit at birth that decreases with colostral ingestion. lactating animals may have decreased hematocrits, rbc counts, and hemoglobin concentrations. animals grazing at high altitude (mountain goats and bighorn sheep) tend to have increased rbc counts, hematocrits, and hemoglobin concentrations. interpreting hematologic changes in cervids is more complex. restraint method affects a variety of parameters in non-acclimated individuals. physical restraint yields red cell counts and hematocrit and hemoglobin concentrations that are to % higher than animals immobilized chemically. , neutrophil, lymphocyte, monocyte, and total white cell counts are also to % higher in physically restrained cervids (see appendix , tables and ) . adult deer also have seasonal variations in their hemogram. red cell numbers and related values are highest during midsummer and late winter. white cells, especially neutrophils, are also highest in midsummer, and platelet counts are highest in spring and fall. these changes may relate to diet or to seasonal activities, such as antler growth and rutting conflicts, which increase the chance of trauma. red cell stickling has also been reported in a variety of deer species. this appears to relate to a mutation in hemoglobin's b-globin component, similar to the disorder in people, but no pathologic role has been described. bone marrow aspirates and core biopsy samples taken from sites of active erythropoiesis can be useful to evaluate erythrocyte production and determine the cause of anemia and other hemogram abnormalities. the sites of biopsy include the sternebrae, femur, and ileum. the procedure should be done under chemical sedation or anesthesia (see chapter ) . the area over the biopsy site is clipped and surgically prepared; the sampler should wear sterile gloves to maintain asepsis. aspirates can be obtained by inserting a sterile needle attached to a -or -cc syringe containing one or two drops of edta through the bone and into the bone marrow. drawing back on the syringe plunger several times may aid in the procurement of an acceptable sample; such a sample may consist of as little as . ml of bone marrow. if the sample is going to be processed immediately, no anticoagulant is required. core biopsies are obtained using a jamshidi or westerman-jensen biopsy needle. the skin is incised with a scalpel and the biopsy needle is inserted into the bone and turned several times to obtain a core sample. more than one site may be used. the sampler then closes the skin with sutures or staples. biopsy samples are preserved by placing them in % neutral buffered formalin solution. impression smears can be made from these samples by gently rolling them on a clean glass slide before placing them in the formalin solution. information obtained from bone marrow samples includes subjective data regarding cell density, megakaryocyte numbers, abnormal cells, maturation patterns of rbcs and wbcs, and the ratio of erythroid to myeloid cells. prussian blue stain can be used on bone marrow to demonstrate iron stores. bone marrow aspirates and biopsies are painful and invasive procedures. therefore, animals should be placed on antibiotics and antiinflammatory drugs prophylactically. blood cultures can be useful in diagnosing bacteremia in an intermittently or persistently febrile animal or one with numerous sites of organ infection. ideally, the clinician should obtain the sample before instituting antimicrobial therapy. however, if this is impossible, antimicrobial therapy should be discontinued to hours before sampling. samples should be taken before and during febrile episodes. the jugular vein is most commonly used to attain a blood culture. as described previously, the skin over the jugular vein should be clipped and surgically prepared. the person collecting the blood sample should wear sterile gloves and use a sterile needle and syringe. blood samples should be placed immediately in a blood culture flask. the chances of attaining a positive culture from bacteremic animals increase with the size of the sample up to about ml, but adding more than the recommended amount to any single culture vial may overwhelm the capacity of the specialized antibiotic-absorbing resins within the flasks. the clinician should change the needle on the sample syringe after collecting the blood and before putting the sample in the culture medium. samples should be refrigerated until they can be sent to a diagnostic laboratory, where aerobic and sometimes anaerobic cultures are made. as an alternative to hematologic testing, comparing conjunctival color to swatches on a standardized famacha chart has been used as a rapid and inexpensive assessment of anemia in whole flocks, primarily to assess the impact of haemonchus contortus and other blood-sucking parasites. , results from a number of trials have yielded fair to good sensitivity to packed cell volume and h. contortus load in both sheep and goats. similar to body condition scoring systems, it is essential to calibrate assessors to ensure consistency when using this system. also, some breeds hematocrit (%) - - hemoglobin (g/dl) - . [ ] [ ] [ ] [ ] [ ] red blood cell count ( /ml) - . [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mean corpuscular volume (fl) - - mean corpuscular hemoglobin concentration (g/dl) - [ ] [ ] [ ] [ ] [ ] [ ] [ ] platelet count ( /ml) . - . . - . total white blood cell count (/ml) - , - , segmented neutrophils (/ml) - - band neutrophils (/ml) lymphocytes (/ml) - - monocytes (/ml) - - eosinophils (/ml) - - basophils (/ml) - - total plasma protein (g/dl) . - . . - . fibrinogen (mg/dl) - read differently on the cards, and use of an electronic color analyzer, while more expensive and less field-friendly, may detect anemia earlier (see chapter , figure . a, b, and chapter ) . easy use of this technique in deer is limited by their intractability and has not been reported. the most common and significant abnormality of the hemogram is anemia. anemia occurs most commonly after blood loss, hemolysis, or chronic disease. blood loss is usually covert and commonly caused by gastrointestinal or external parasites. overt blood loss is usually caused by major trauma such as that caused by dog bites, severe lacerations, male rivalry fighting, or complications of castration or dehorning. cbc values appear normal immediately after acute blood loss. however, after a few hours of fluid redistribution, anemia and hypoproteinemia are evident. evidence of red cell regeneration (macrocytosis, reticulocytosis, and nucleated red cells) should appear within a day or two of the blood loss. hemolysis occurs most commonly after ingestion of toxic plants, rbc parasitism, intravenous (iv) injection of hypotonic or hypertonic agents, contact with bacterial toxins, water intoxication, or immune-mediated destruction of opsonized erythrocytes. ingested toxins include sulfur compounds from onions and brassica plants (kale and canola), [ ] [ ] [ ] [ ] nitrates, nitrites, and copper. [ ] [ ] [ ] [ ] except for that caused by copper, hemolysis usually occurs within a day or two after ingestion. copper toxicosis can occur after acute overingestion but more commonly is seen in animals that are chronically overfed copper and suffer some stressful event. goats are more tolerant of excess copper than sheep are, and certain breeds of sheep, particularly the suffolk, are highly sensitive to copper toxicosis (see chapters and ) . hemolytic bacterial toxins include those from clostridium perfringens type a, clostridium haemolyticum, and leptospira interrogans. , intraerythrocytic parasites include anaplasma species, mycoplasma (eperythrozoon) species, and babesia species. [ ] [ ] [ ] [ ] [ ] immune-mediated rbc destruction is very uncommon except with parasitemia, the administration of certain drugs (penicillin), or bovine colostrum to small ruminant neonates. rapid reduction of plasma osmolality can lead to osmotic lysis of erythrocytes. this can occur locally as a sequela to rapid iv injection of hypotonic substances or after ingestion of a large quantity of water following a period of water deprivation and dehydration (water intoxication). selenium and copper deficiency have also been associated with heinz body anemia. parasite infestation, opsonization, and ingestion of toxic plants typically cause extravascular hemolysis. in these cases, damaged erythrocytes are removed by cells of the reticuloendothelial system, resulting in anemia, pallor, weakness, depression, icterus, and dark urine. bacterial toxins, changes in plasma osmolality, and copper toxicosis cause intravascular hemolysis, resulting in the additional signs of hemoglobinemia and hemoglobinuria. other signs such as fever, neurologic symptoms, and sudden death may be seen with specific diseases. signs of regeneration should be seen on the hemogram to days after the onset of hemolysis. anemia that is not related to the loss or destruction of erythrocytes usually results from a lack of production and thus are nonregenerative. although mild forms may exist in pregnant sheep and goats and those deficient in vital minerals (e.g., iron, selenium, copper, and zinc), the most common cause of nonregenerative anemia is chronic disease. under these conditions, iron is sequestered in an unusable form in the bone marrow; staining a marrow sample with prussian blue stain reveals large iron stores, differentiating this disease from iron-deficiency anemia. the causes of anemia of chronic disease are numerous and include infectious conditions (e.g., pneumonia, foot rot, and caseous lymphadenitis), malnutrition, and environmental stressors. most anemia does not require treatment. unless loss of rbc mass is rapid and severe, the animal is usually able to compensate to the decreased oxygen-carrying capacity by decreasing activity. it is important to remember in this regard that anemia often first becomes apparent to the manager of a flock or herd when animals appear overly stressed or die during movement or handling. if possible, the cause of the anemia should be addressed. this can involve trying to control internal and external parasites, changing the diet, and treating infectious diseases. maintaining adequate hydration is essential in animals with intravascular hemolysis to avoid hemoglobin-induced renal tubular damage. specialty compounds such as molybdenum salts, such as ammonium molybdate, and sulfur or penicillamine for copper toxicosis and methylene blue ( mg/kg in a % solution in % dextrose or normal saline intravenously) for nitrate toxicity are usually too expensive or difficult to be used on a flock-wide basis but may be useful in valuable individual animals. veterinarians should be aware that methylene blue is no longer approved for use in food-producing animals. animals with severe acute blood loss or hemolysis may benefit from a whole blood transfusion. because transfusion reactions are rare and strong erythrocyte antigens have not been identified in small ruminants (including cervids), almost any donor of the same species is acceptable for a first transfusion. cross-matching can be done to ensure compatibility, which becomes more important if the animal receives more than one transfusion. blood should be withdrawn aseptically from the donor and collected by a bleeding trocar into an open flask or by a catheter into a special collection bag. blood should be mixed at a . : ratio with acid-citrate dextrose, or : with % sodium citrate, or another suitable anticoagulant and administered through a filtered blood administration set. if the jugular vein is not accessible, blood may be infused into the peritoneal cavity, but the slower absorption from that site makes it less effective for treating acute blood loss. the first to minutes of administration should be slow. if no reaction is seen (fever, tenesmus, tachypnea, tachycardia, and shaking), the rate may be increased. transfused erythrocytes may only survive a few days, and therefore, the original cause of the anemia must be addressed. peripheral wbcs include granulocytes (neutrophils, eosinophils, and basophils) and mononuclear cells (lymphocytes and monocytes). immature forms of neutrophils and lymphocytes may be seen during severe inflammatory diseases. abnormalities of the neutrophil line are usually the best cellular evidence of inflammation in small ruminants, and inflammation is almost always a sequela of infection. an increase in neutrophil numbers and their proportional contribution to the total wbc count is usually seen in mild gram positive, subacute, or chronic bacterial infections. animals with more severe disease may exhibit high or normal counts, but a greater proportion of the neutrophils will have toxic changes or be immature forms (band cells, metamyelocytes, or myelocytes). in severe, acute inflammation and many diseases caused by gram negative bacteria, a temporary reduction in neutrophil numbers is observed, often with a concurrent shift toward more toxic or immature forms. if the animal survives the peracute disease, neutropenia should resolve over to days, first through an increase in immature cells, and later through a mature neutrophilic response. another important cause of increased total and relative neutrophil counts is stress (or glucocorticoid administration), which inhibits neutrophil margination and extravasation and thereby increases the number of these cells in the midstream blood. increases in eosinophil counts are usually related to exposure to eukaryotic parasites. decreases are rarely of clinical significance and may be part of the stress response. idiopathic allergic-type reactions also are indicators of pathology but are very rare. increases in basophils are rarely clinically significant. increases in lymphocyte counts often reflect chronic inflammatory disease such as that seen with internal abscesses. in rare cases, lymphocytosis may consist of abnormal, blast-type cells and indicate a lymphoproliferative neoplasm. lymphopenia is an important part of the stress response; nevertheless, the clinician must keep in mind that many diseases stimulate a stress response. therefore, lymphopenia and neutrophilia may represent either stress or inflammation, and an examination of neutrophil morphology and plasma fibrinogen concentrations may be useful in distinguishing the two situations. a high fibrinogen concentration, toxic changes, and high counts of immature neutrophils indicate inflammation under those circumstances. blood monocyte counts also may indicate stress or chronic inflammation. the difficulties in interpreting individual cell count abnormalities highlight the importance of obtaining a differential wbc count and description of cellular morphology in assessing sick sheep and goats. leukogram abnormalities are rarely given specific treatment. it is far more common and useful to use the information from the leukogram to develop a plan to treat the disease responsible for the abnormality. palpation of external lymph nodes is part of the thorough physical examination. lymph nodes that can be found in normal sheep and goats include the submandibular, prescapular, and prefemoral nodes. none of these should be prominent or painful on palpation. additional nodes that may be palpated occasionally in normal animals include the parotid, retropharyngeal, supramammary, perirectal, and popliteal nodes. internal lymph nodes that may be identified during specialized diagnostic procedures include the mediastinal, mesenteric, and other abdominal nodes. enlargement of lymph nodes may be focal, multifocal, or generalized. identification of a single enlarged superficial node does not always rule out a multifocal or generalized disorder because the status of the internal nodes often cannot be determined. enlargement generally indicates either inflammation or neoplasia. inflammatory enlargement is generally related to an associated disease with an infectious component. small ruminants are particularly sensitive to lymph node-based infections (e.g., caseous lymphadenitis), so the search often does not extend beyond aspirating or draining the lymph node itself. neoplastic enlargement almost always results from lymphosarcoma. lymphosarcoma pathogenesis. neoplastic transformation of a member of the lymphocyte cell line leads to unregulated clonal expansion of that cell. the cause of transformation is usually unknown; in rare cases, especially in flock outbreaks in sheep, it can be linked to exposure to the bovine leukemia virus, which has occurred experimentally and as a result of the administration of whole blood anaplasma vaccines. whether the bovine leukemia virus can induce lymphosarcoma in goats and cervids is still unclear. multicentric lymphosarcoma has been reported sporadically in white-tailed deer (odocoileus virginiatus) and other deer, but bovine leukemia virus infection has not been diagnosed in cervids. in one study of neoplastic diseases affecting goats from to , lymphoma was identified as the most common neoplasm, accounting for . % of the assessed tumors. in contrast to other species such as cattle, sheep, and horses, lymphomas in goats are predominantly t-cell lymphomas affecting the mediastinum. a recent study attempted to classify the type of lymphoma affecting goats. using immunohistochemistry (ihc), it was determined that % (n ) of affected goats had t-cell lymphoma and only % (n ) had b-cell lymphoma. proliferation of t or b lymphocytes leads to mass lesions and infiltration of viscera. these changes cause physical obstruction (to breathing, blood flow, urination, defecation, etc.), ulceration of mucosal surfaces (blood loss, bacterial invasion), immune system dysfunction, organ failure, and generalized malaise and cachexia. tissue masses may be internal or visible on external examination. clinical signs. clinical signs in affected animals vary according to the type of lymphoma (t-or b-cell) and the location of the masses. t-cell lymphomas in goats are usually localized in the thoracic cavity and/or neck, suggesting thymic origin or homing. in contrast, b-cell lymphomas tend to have a multicentric distribution. lymphoma in small ruminants usually presents with non-specific signs that can mimic other respiratory or gastrointestinal conditions. slowly progressive weight loss is the most common finding. in some cases, generalized peripheral lymphadenopathy and expansile masses are noted ; at first, they usually are presumed to be caseous lymphadenitis abscesses. progressive chemosis and exophthalmos have been reported in a sheep and a goat with multicentric b-cell lymphoma. , most masses form at the sites of internal or external lymph nodes. in sheep, masses in the brain, skin, joint, and lymphoid tissue have been reported. leukemia is rare. the most common abnormalities are those of chronic disease and cachexia and include nonregenerative anemia and hypoalbuminemia. bone marrow examination may reveal clonal expansion of lymphoid precursor cells. in cervids, lymphadenopathy and multifocal masses affecting the heart, blood vessels, kidney, urinary bladder, and peritoneum have been reported. a more recent report described a subcutaneous maxillary mass in a -year-old captive-born, female whitetailed deer. the mass was diagnosed as focal lymphosarcoma with local metastasis. diagnosis. history and clinical signs are important in the diagnosis of lymphoma in small ruminants. age of affected animals ranges from to years and no gender or breed predisposition has been reported. final diagnosis of affected animals is achieved through necropsy, histopathology, and ihc. lesions seen at necropsy include homogeneous white to tan masses that bulge on the cut surface. they may be small or large. less commonly, diffuse paleness of the reticuloendothelial organs is noted. microscopic examination of these tissues reveals infiltrates of abnormal cells of the lymphocyte line. prevention. avoiding exposure to the bovine leukemia virus and restricting the use of instruments to one animal between cleaning procedures may help prevent the spread of lymphosarcoma. in most animals, however, this neoplasm appears to develop spontaneously. pathogenesis. lambs, kids, and fawns are born with functional lymphocytes that can produce endogenous immunoglobulin. these cells develop the ability to respond to foreign antigens in the fetus during mid to late gestation. because of a lack of in utero exposure, however, basal concentrations of immunoglobulin are very low at birth. these cells therefore are naïve to foreign antigens and unable to develop protective immunity through specific cellmediated and immunoglobulin production. additionally, as with other ruminants, no transplacental passage of maternal immunoglobulin to fetal sheep, goats, and fawns occurs. lambs, kids, and fawns depend exclusively on intestinal absorption of maternally derived colostral antibodies, immune cells (t-lymphocytes), and other immune factors to provide a ready supply of specific immunity and allow opsonization of pathogens for the first months of life. adequate passive transfer requires delivery of a sufficient quantity of good-quality colostrum (immunoglobulin g [igg] concentration in mg/ml) into the gastrointestinal tract, as well as adequate absorption of antibodies (timely) from the colostrum into the blood. however, the amount of maternal colostrum produced by the dam, and its composition, as well as the ability of the newborn to stand and nurse in a timely manner, can be affected by several factors. colostrum igg concentration and volume of production can be influenced by breed, age, nutrition, body condition score (bcs) at parturition, and vaccination status of the dam. the igg concentration in colostrum samples from ewes of different breeds can vary between and mg/ml. one study demonstrated that primiparous ewes with low bcs (, . ) at lambing produced less colostrum compared with multiparous ewes with similar bcs. additionally, ewes with higher bcs (. . ) tended to produce higher volumes of colostrum compared with ewes with lower bcs. another study suggested that undernutrition of ewes during late gestation can affect colostrum quality and immune development and function in newborn lambs. it has been suggested that at least g of total igg should be fed to newborn lambs and kids during the first hours of life to reach adequate transfer of passive immunity. adequate transfer of passive immunity in small ruminant neonates has been suggested as serum igg levels at hours of life of  mg/ml. one study indicated that lambs that nurse low-quality colostrum (igg , mg/ml) had lower serum igg concentrations compared with lambs that that nurse colostrum of higher quality (igg . mg/ml), indicating that the concentration of igg in colostrum is a determining factor for the presentation of failure in the transfer of passive immunity. other factors such as pregnancy toxemia, gastrointestinal parasitism, excess of iodine intake during pregnancy, and inadequate vaccination of the dam can result in poor colostrum synthesis and quality. timely consumption of maternal colostrum during the first hours of life is essential to achieve adequate transfer of passive immunity. in small ruminants, cells of the small intestine are able to internalize and transfer igg into the blood during the first hours of life; however, the absorption efficiency of igg is higher during the first to hours of life. , factors associated with the neonate, such as weakness, inability to stand, and congenital abnormalities, will prevent timely nursing of maternal colostrum and lead to failure of passive transfer (fpt). litter size and body weight (bw) of the kid(s) have also been correlated with inadequate absorption of igg from colostrum. one study demonstrated that litter sizes of three light goat kids (, . kg bw) or more had significantly lower mean serum igg levels at hours of life when compared with litter sizes of one or two heavier kids ( . versus . mg/ml, respectively). this suggests that special attention and monitoring should be paid to multiple fetus gestation as the risk of fpt under these circumstances at kidding is higher; however, the quality of colostrum, amount ingested, and adequacy of absorption are rarely monitored by small ruminant producers in natural or artificial rearing systems. the use of monitoring tools to evaluate colostrum quality and igg absorption is common in modern dairy cattle operations, and these tools are readily available for small ruminant production systems. recent reports have presented the use of %brix in maternal colostrum and neonate serum and its positive correlation with serum total proteins (stps) at hours as effective monitoring tools of fpt in lambs and goat kids. [ ] [ ] [ ] the use of stp has also been used to monitor colostrum deficiency intake in mule deer fawns ; however, adequate values of serum igg for cervid neonates have not been established yet. inadequate colostrum intake and low serum igg at to hours of life have been consistently associated with higher morbidity and mortality rates in lambs, goat kids, and fawns. one study reported that % of lamb mortality between hours and weeks of age can be attributed to fpt. another study suggested that colostrum deficiency and low serum igg in goat kids resulted in higher mortality rates at weeks and of life due to chronic infections with pasteurella multocida and escherichia coli. other reports demonstrated that % of lambs with a serum igg of , mg/ml at hours died before weeks of age compared with only % of the lambs with a serum igg of . mg/ml at hours. in a previous report, mule deer (odocoileus hemionus) fawns with a stp of # g/dl between days and of age developed diarrhea and died before days of age compared with fawns with stp . g/dl. in a more recent report, a -day-old formosan sambar deer (rusa unicolor swinhoei) with a history of colostrum deprivation died due to severe suppurative meningitis caused by e. coli infection. in addition to immunoglobulins, colostrum also contains large quantities of fat-soluble vitamins that do not cross the placenta. the most important of these are vitamins a, d, and e, which are important in bone development and the immune or inflammatory response. neonates that have not ingested enough colostrum are likely to be deficient in these vitamins. diagnosis. history of dam dystocia, inadequate colostrum nursing, complete colostrum deprivation, and signs of undernourishment or sepsis in the first few days after birth are usually a presumptive indication of failure in the transfer of passive immunity. a high prevalence of diarrhea and respiratory disease in neonates should prompt investigation and evaluation of passive transfer of immunity in affected herds or flocks. owners occasionally evaluate lambs or kids for adequate intake by picking up the animal and holding it at ear level, while carefully cradling the head and neck, and then shaking the abdomen to hear milk in the abomasum; however, this is not a reliable indication of adequate transfer of passive immunity. a definitive diagnosis of fpt can be made by direct laboratory measurement (single radial immunodiffusion [srid]) of igg in serum at hours of life. although some practitioners use the value of igg used in dairy calves ( mg/ml), others have suggested an igg value , mg/ml to establish the presence of fpt in small ruminants. numerous semiquantitative methods of estimating igg are available and are easy to use in sheep, goats, and cervids. the most common is the measurement of serum total solids or stp values at hours of life through an optical refractometer. the stp at hours of life in a well-hydrated animal has demonstrated correlation with serum igg in calves, lambs, and goat kids. studies in goat kids indicated that an stp between . and . g/dl was associated with adequate transfer of passive immunity. , another study demonstrated fpt in lambs with stp values , . g/dl at hours of life. a study in mule deer suggested that fawns with an stp # g/dl had inadequate colostrum intake and fpt. recently, the measurement of %brix in maternal colostrum and serum with a digital brix refractometer has become an alternative method to evaluate colostrum quality and fpt in dairy operations. colostrum %brix . % and serum %brix . . % have been associated with adequate transfer of igg in calves and goat kids. other qualitative methods to assess the transfer of passive immunity in large animals include various agglutination (glutaraldehyde), precipitate assays (sodium sulfate), and measurement of g-glutamyl transferase (ggt) in serum. these methods may be relied on to give an overall flock assessment of adequacy of passive transfer, but they are rarely accurate enough to provide definitive information on individual animals. treatment. fpt is not in itself pathologic, but it greatly increases the neonate's susceptibility to infectious diseases. the amount of colostrum absorbed across the gut decreases with time, especially in animals that have been ingesting other proteins (e.g., the casein in milk); it also decreases with illnesses that decrease gastrointestinal function. neonatal small ruminants should receive at least g of igg/kg of bw or ideally g of total mass of igg from a good-quality colostrum source (. mg/ml of igg) during the first hours of life. other authors recommend an intake of to ml of colostrum/kg during the first hours of life. in artificial rearing systems or lamb feedlots, feeding of colostrum every hours until hours of life is recommended. when same species' maternal colostrum is unavailable, goat colostrum or bovine colostrum/colostrum replacers or are a good alternative; however, hemolysis has been reported in lambs receiving cattle colostrum. one study demonstrated that there was no difference in serum igg levels of lambs that received the same volume of sheep or goat colostrum at birth. another study demonstrated that lambs that received ml of a bovine colostrum replacer at birth in addition to ml of stored sheep colostrum at hours of life had higher serum %brix values at hours and had less incidence of disease during the preweaning period compared with lambs that received the same volume of stored sheep colostrum at birth and at hours of life. since igg absorption cannot be extended more than hours after birth, administration of an oral colostrum source is the best treatment in the immediate postpartum period in still-healthy neonates. after the window for immunoglobulin absorption has closed, plasma, serum, or whole blood administered by the iv or intra-peritoneal route is the best way to raise the neonate's blood immunoglobulin concentrations. adult donor plasma contains approximately . to . g of immunoglobulin/dl, so administration of a volume equivalent to % of bw or a dose of to ml/kg has been recommended for the treatment of large animal neonates. if plasma is used instead of colostrum, administration of vitamins a, d, and e also may be beneficial. if colostrum and plasma are unavailable or cost-prohibitive, "closing" the gut as quickly as possible with milk, maintaining high standards of hygiene, and possibly administering prophylactic antibiotics offer the greatest prospects for preventing infectious disease. vaccination of the neonate or the administration of antitoxin hyperimmune serum should not be considered protective but may be of value. prevention. prevention of fpt should be based on the establishment of an adequate colostrum program managing the previously mentioned factors that affect production, quality, and absorption of maternal colostrum components in lambs, goat kids, and fawns. ensuring colostral quality is best done through good nutrition, health care, and vaccination of dam (see chapters and ) . administration of vaccines weeks before parturition, followed in weeks with a booster, provides the highest quantity of protective immunoglobulin in the colostrum. antepartum leakage is rarely the problem in small ruminants that it is in horses and cattle. however, in a flock or herd environment, still-pregnant dams may steal babies from other sheep or goats. to prevent such theft and the resultant loss of colostrum by the "adopted" neonate, owners may choose to keep pregnant animals separate from those that have already delivered. if complete separation is not possible, the dam and her offspring should be allowed to bond with each other in a private pen ("jug" or "crate") for at least hours before being placed back with the flock. clipping excessive wool or mohair from around the perineal area and udder before lambing or kidding, expressing the teats to ensure they are not plugged, and having extra colostrum available when pregnant females are placed in jugs or crates are other good preventive measures. etiology and pathogenesis. uncomplicated diarrhea in lambs, goat kids, and fawns may be caused by infectious agents such as viruses, bacteria, and protozoa. in goat kids and elk calves, metabolic causes of diarrhea have been described. , group b and a rotavirus, enterotoxigenic e. coli k , cryptosporidium parvum, and other cryptosporidium spp. have been commonly identified as causal agents of diarrhea in small ruminant neonates. [ ] [ ] [ ] [ ] with recent advances in diagnostics and metagenomics of the enteric environment of large animals, novel viruses have been identified as potential causal agents of diarrhea in lambs and goat kids. adenovirus, astrovirus, calicivirus, coronavirus, and picornavirus have been identified in feces of diarrheic lambs and goat kids ; however, their role in the pathogenesis of neonatal diarrhea is still uncertain. these organisms differ from the agents of complicated diarrhea in that they do not invade beyond the gut wall or result in systemic toxemia (see chapter ) . additional causes of diarrhea reported in goat kids and elk include lactose intolerance and hypernatremia, respectively. , less frequently, bacteria such as c. perfringens, clostridium difficile, and attaching and effacing e. coli have been associated with complicated diarrhea in small ruminant neonates. , the net result of such an infection is that a large volume of water and electrolytes are lost into the bowel due to malabsorptive, hypersecretory, or hyperosmolar processes. if enough fluid and electrolytes are lost, dehydration and metabolic acidosis arise, inducing systemic clinical signs of depression and weakness in association with diarrhea. in goats, this clinical entity is one component of the floppy kid syndrome. clinical signs. profuse, watery, yellowish-green to brown diarrhea without fever is the hallmark clinical sign. with severe dehydration and acidosis, affected lambs, kids, and fawns become weak and dull and lack appetite. [ ] [ ] [ ] excessive salivation and loss of suckle reflex have also been reported in affected lambs and kids. , mucous membranes become tacky, and skin tenting times are prolonged. shock signs may develop. physical assessment often must take the place of clinicopathologic analysis in affected neonates. mild, nonclinically complicated diarrhea is characterized by profuse diarrhea with minimal systemic signs. the affected animal is bright and alert, with minimal skin tenting, and can stand and eat readily, with a strong suckle reflex. it is less than % dehydrated, with a blood ph of . to . , and bicarbonate deficit is minimal. moderate uncomplicated diarrhea is characterized by profuse diarrhea in a dull but responsive animal. skin tenting is prolonged, but eye luster is normal. the affected animal is able to stand and eat but eats slowly and has a weak suckle reflex. the head typically is held down. it is to % dehydrated, with a blood ph of . to . and a bicarbonate deficit of to meq/l. severe uncomplicated diarrhea is characterized by profuse diarrhea. the affected animal is dull and minimally responsive, with a very long skin tent time and dull, sunken eyes. it can stand only with assistance and prefers to stay in sternal recumbency with its head up. the animal eats very slowly, if at all, and has a minimal suckle reflex. it is to % dehydrated, with a blood ph of . to . and a bicarbonate deficit of meq/l. very severe uncomplicated diarrhea is characterized by profuse diarrhea and profound weakness. the animal's skin remains tented for more than minute, and its eyes are very sunken and dull. it is nonresponsive with no suckle response. it is unable to maintain sternal recumbency, lying on its side instead. the animal is to % dehydrated, with a blood ph of . to . and a bicarbonate deficiency of to meq/l. epidemiology. morbidity and mortality of uncomplicated diarrhea in small ruminants and fawns vary depending on the cause. reports of rotaviral diarrhea in newborn lambs indicate morbidity rates between % and % and mortality rates between and % , ; however, one study reported a % case fatality rate in lambs affected with types b and a rotavirus diarrhea. another study reported mortality rates between % and % in lambs and kids affected with c. parvum diarrhea. most of infectious agents associated with uncomplicated neonatal diarrhea in small ruminants are shed by adult animals and older lambs/kids around stressful events such as lambing/kidding and extreme weather conditions. one study reported that pregnant does shed to times more oocysts during the weeks around kidding compared with other time periods. additionally, poor husbandry/hygiene of lambing/kidding sheds, fecal soiling, flock size (. animals), lambing/kidding season (winter/spring), and the presence of c. perfringens type a in feces have been suggested as potential risk factors for uncomplicated diarrhea in small ruminant neonates. [ ] [ ] [ ] clinical pathology. the leukogram should be normal or show abnormalities compatible with stress. serum biochemical or blood gas analysis may reveal evidence of intestinal malabsorption, electrolyte loss, metabolic acidosis (hypoglycemia, hyponatremia, hypochloremia, hyperkalemia, low bicarbonate, and increased anion gap), and dehydration (hyperalbuminemia and increased blood urea nitrogen [bun] and creatinine). in contrast with the common leukogram and biochemical abnormalities found in calves, lambs, and goat kids with uncomplicated diarrhea, elk calves with diarrhea develop leukocytosis, hyperchloremia, and hypernatremia (serum na . meq/l). additionally, increased anion gap, bun, creatinine, and albumin concentrations have been reported in affected elk calves. a presumptive diagnosis may be based on the characteristic history and clinical signs. response to conservative treatment also is supportive of this diagnosis. identification of the specific causative agent is less important than proper treatment of affected animals; however, feces or intestinal contents from affected animals can be submitted for electron microscopy, reverse-transcription polymerase chain reaction (pcr), and cell culture immunofluorescent assays to identify viruses. [ ] [ ] [ ] additionally, intestinal tissue can be submitted for ihc for rotavirus and c. parvum. , feces of affected animals can also be submitted for enzyme-linked immunosorbent assay (elisa), ziehl-neelsen staining technique, light or fluorescence microscopy, sugar flotation, and auramine or fluorescent antibody staining for the diagnosis of c. parvum infection. fecal culture to determine a bacterial cause is recommended. treatment. the immediate goals of treatment are rehydration, replacement of lost electrolytes, and restoration of acid-base balance as these are usually the leading causes of death in affected neonates. less immediate goals are provision of nutrition and replacement of ongoing losses. the aggressiveness of treatment is dictated by the severity of the condition, as well as economic considerations. . rehydration: calculate the percent dehydration and use to calculate fluid requirements for a -hour period. example: % dehydration in a -kg lamb: dehydration: . kg kg/l . l or ml. maintenance: ml/kg/day . l or ml. total fluids to replace in hours . l or ml fluid loss due to dehydration ( ml in this case) should be replaced during the first hours and the rest can be replaced in the next hours. . replace lost electrolytes: sodium, chloride, and bicarbonate are lost roughly in proportion to extracellular fluid (ecf) in the acute phase of diarrhea ( - days) in untreated animals. potassium tends to be increased in this phase due to the presence of metabolic acidosis and care should be taken when selecting fluids containing potassium to treat affected animals at this time. in chronic cases of diarrhea, and especially in cases where the owner/producer has given oral milk replacer or electrolyte supplements/replacements to affected animals before veterinary evaluation, the serum concentration of sodium, potassium, and bicarbonate might be variable or increased. special care should be taken in these cases when selecting fluids to treat affected animals as the risk of causing hypernatremia is higher. in cases of diarrhea in elk calves, hypernatremia is common, and fluids should be selected accordingly. in the majority of cases, initial replacement of sodium, chloride, and bicarbonate with fluids containing proper composition is recommended. example: assessment suggests a bicarbonate deficit of meq bicarbonate in a -kg, comatose lamb with prolonged skin tenting ( . is the multiplier for ecf in a neonate): . ( meq) kg meq bicarbonate. commercial iv . % sodium bicarbonate solutions contain meq of bicarbonate per milliliter and could be added directly to iv fluids in severely dehydrated and acidotic animals. therefore, the immediate goal is to provide ml of fluid and meq of bicarbonate to this lamb in a formulation that resembles normal ecf. fluids can be given by various routes. selection of route of administration of fluids depends on degree of dehydration, presence or not of a strong suckle reflex, and degree of depression. neonates with advanced degrees of dehydration, depression, and absence of suckle reflex will benefit from iv fluid therapy. in contrast, neonates with mild dehydration and active suckle reflex can be effectively treated with oral electrolytes ; however, if oral fluids have not produced an improvement within to hours, iv treatment should be strongly considered. other routes such as subcutaneous, intra-peritoneal, and intra-osseous can also be used for fluid administration to neonates. • advantages: oral fluids are inexpensive (nonsterile) and easy to give. they are less likely to cause fatal arrhythmias or neurologic disease than iv fluids. • disadvantages: an animal receives a maximum of its gastric volume ( % of bw), and good gastric motility is required. oral fluids may not be well absorbed by a damaged gut. absorption also is slow. intravenous • advantage: this method allows rapid correction of all deficits, even in moribund animals. • disadvantages: it is expensive (sterile), requires venous access, and can rapidly lead to overcorrection. subcutaneous • advantages: this method does not require venous access or good gut motility. • disadvantages: it is expensive (sterile), and the fluids may not be well absorbed in very dehydrated animals. absorption is not as quick as by iv administration. animals should be given only hypotonic or isotonic fluids. intra-peritoneal • advantages: this method does not require venous access or gut motility. fluids are absorbed quickly by this route. • disadvantages: it is expensive (sterile) and can cause peritonitis. isotonic fluids are best used in this route. only a limited volume can be given. many commercial oral electrolyte solutions for neonatal ruminants are available; however, not all of them fulfill the requirements to adequately replace fluids and electrolytes in neonatal ruminants with diarrhea. oral electrolyte solutions must contain enough sodium , provide agents that increase absorption of water (glycine, glucose, and acetate), provide an alkalinizing agent (bicarbonate, propionate, acetate, and citrate; acetate has demonstrated best results), and an energy source (glucose). the amount of carbohydrates might vary and is usually higher in "high-energy" solutions specifically used for severely affected neonates that are not eating and develop negative energy balance. less carbohydrate is needed in less severely affected animals because they are usually eating some and are less likely to have severe negative energy balance. fluids to be avoided include medicated milk replacers and unbuffered saline solutions. iv treatment should be provided with a sterile commercial product. such preparations typically contain to meq/l of base. additional sodium bicarbonate solution or sterile powder can be added to fluid therapy based on the bicarbonate deficit ( meq/ml of . % solution and meq of bicarbonate/g of powder, respectively). the bicarbonate deficit should be over the first hours. after deficits are replaced, the following continued treatments and adjuncts may be considered: . continued administration of fluids (oral rather than iv, if possible) to replace ongoing losses: • oral electrolytes at a volume equal to % of the bw per feeding can be given; the number of feedings can be increased from two (normal) to three to six per day. • iv fluids can be continued at twice the maintenance fluid rate until appetite is restored. • more bicarbonate may be necessary. . consideration of addition of milk to the treatment regimen: • milk or milk replacers should be added to the therapy of neonates with diarrhea. they provide nutrition to the affected neonate, preventing negative energy balance and promoting intestinal healing. • care should be taken to not mix oral electrolyte solutions with milk or milk replacers in the same container as the concentration of sodium and overall osmolarity of the solution can dramatically increase, leading to hypernatremia or other metabolic abnormalities. • milk or milk replacers should be given in small volumes (, % of total requirements) but at a higher frequency (every - hours) to avoid overloading the abomasum and intestine of affected animals. lambs fed milk lose less weight with scours. • free water helps prevent hypernatremia. • milk is a good potassium source (see chapter ). elk deer calves. elk deer calves commonly develop diarrhea with hypernatremia (serum na . meq/l) compared with other large animal neonates, where hyponatremia is more common. therefore, administration of oral electrolyte solutions designed for other ruminants (calves, lambs, and kids) should be avoided in these animals. a dilution ( : or : ) of commercially available bovine calf electrolyte solutions to reduce sodium content is recommended for the treatment of elk calf diarrhea. the use of lactated ringer's solution, which has a low sodium concentration in addition to a very low reduction rate of serum sodium (, . meq/l/hour) has been advocated in the fluid therapy of hypernatremic elk calves with diarrhea. additional therapy. dextrose ( . - %) solutions can be added to the fluid therapy of hypoglycemic animals. the use of nonsteroidal antiinflammatory drugs (nsaids) in neonatal ruminants with diarrhea is controversial due to the risk of renal damage and abomasal ulceration; however, in cases of diarrhea complicated by septicemia or endotoxemia, nsaids should be used to reduce the effects of systemic inflammation. flunixin meglumine at a dose of . to . mg/kg is the only nsaid approved for food animal use. similarly, the use of oral or systemic antibiotics in cases of uncomplicated diarrhea is controversial due to its potential effect on the intestinal microbiota and development of bacterial resistance; however, their use is warranted in the presence of septicemia or endotoxemia in addition to diarrhea. in these cases, b-lactams such as oral amoxicillin or systemic ceftiofur are usually good choices. the effect of mucosal protectants and probiotics in cases of diarrhea is unknown in small ruminant neonates, and their use is left to practitioners based on their own experiences (see appendix ) . prevention. prevention of uncomplicated diarrhea in small ruminant neonates is based primarily on the timely feeding of adequate amounts of good quality maternal colostrum or colostrum replacer (see "failure of passive transfer" section). vaccination of dams with antigens of common infectious agents associated with uncomplicated neonatal diarrhea before parturition has demonstrated to be effective increasing colostrum immunity and prevention of diarrhea in lambs. maintenance of adequate husbandry and hygiene conditions in lambing/kidding sheds or barns is necessary to reduce neonatal exposure to infectious agents normally shed in feces of dams during parturition such as rotavirus and c. parvum. ruling out infectious causes of depression and weakness is difficult, and clinicians often do well to assume that an infectious disease is contributing to clinical signs when making treatment decisions. however, several noninfectious systemic disturbances also can depress neurologic and muscular function. successful treatment often requires identification and correction of each of these disturbances. among the more common abnormalities leading to depression in neonates are hypoxemia, metabolic or respiratory acidosis, hypothermia, hyperthermia, hypoglycemia, dehydration, azotemia, and some electrolyte imbalances. hypothermia and hyperthermia can easily be diagnosed by measuring body temperature with a rectal thermometer. hypothermia is far more common and can result from weakness, shock, and environmental stress. cold, windy weather or tube feeding with cold milk replacer or fluids can lead to a rapid drop in core body temperature, especially in neonates that are small or weak or have been inadequately licked off or were rejected by their dams. strong, vigorous neonates usually are protected by heat produced during muscular activity and are able to seek food and shelter. clinical signs appear when the rectal temperature drops to ° f ( . ° c) or below. protection from wind and cold such as with an individual ewe jug or pen, heat lamps (positioned far enough away so as not to burn the neonate), hot water bottles, blankets, and administration of warm fluids is helpful in treating and preventing hypothermia. shearing the ewe before lambing is of value because it forces the ewe to seek shelter. if this management technique is used, care should be taken to avoid inducing severe hypothermia in the dam. environmental hyperthermia is much less common than fever in neonates. therefore, treatment for infectious diseases in young animals with high temperatures usually is warranted. providing cool shelter with good ventilation, minimizing stressful events, ensuring adequate fluid intake, and shearing the adults are the best defenses against environmental heat stress. hypoglycemia also is easy to diagnose with the aid of an inexpensive, portable glucose meter. lambs and kids typically develop hypoglycemia under the same circumstances as those leading to hypothermia. administering ml/kg of dextrose (approximately . fl oz/lb, or % of bw) in warm milk replacer or ml/kg of % dextrose, by either the iv or oral route (diluted to % dextrose), should provide ample energy to correct hypoglycemia. iv administration may be necessary if gut motility is absent. follow-up treatment may be necessary if the neonate does not regain its appetite. except during severe conditions, normal lambs and kids should be able to maintain normal body core temperature. they should therefore be examined for an underlying disorder if they exhibit signs of hypothermia or hyperthermia. clinicians and owners should not assume that warming and feeding a cold, weak neonate will always correct the problem. hypoxemia is much more difficult to diagnose. portable blood gas meters for arterial analysis and radiography units for thoracic imaging are available but are still not in common use in small ruminant practice. for those reasons, hypoxemia usually is underdiagnosed. hypoxemia can result from prematurity or dysmaturity, infection, depression or weakness (decreased ventilation), meconium aspiration, bullous emphysema, hernias, and other thoracic fluid or tissue masses. it is likely to be a contributing factor in illness and death in most weak neonates younger than days of age. such animals benefit from the provision of supplemental oxygen, either through a nasal insufflation tube or by oxygen tent. in addition to its direct effect on general wellbeing and behavior/ attitude, hypoxemia at birth leads to poor gut function and subsequent poor colostral absorption. many animals that exhibit fpt and subsequent sepsis had a previous bout of hypoxemia. azotemia, metabolic acidosis, and electrolyte imbalances are difficult to diagnose without clinicopathologic analysis. therefore, these problems are best treated in animals showing signs of dehydration with the administration of a balanced, physiologic electrolyte solution. metabolic acidosis usually is accompanied by either obvious evidence of bicarbonate loss (diarrhea) or severe dehydration. however, neither of these conditions is present with floppy kid syndrome. this descriptive title is applied to muscle weakness, anorexia, and depression in kids observed in the first weeks of life. by its strictest definition, floppy kid syndrome refers to metabolic acidosis with a high anion gap without dehydration or any known cause in young kids that were normal at birth. a variety of disorders and conditions have been proposed as the cause of metabolic acidosis without dehydration, including intestinal fermentation of milk in well-fed kids with subsequent absorption of volatile fatty acids, transient neonatal renal tubular acidosis, and lactic acidosis secondary to toxic impairment of cardiovascular function. overgrowth of c. perfringens type a often is suggested as a source of the toxin. with a high anion gap, a pathologic condition that leads to overproduction of an organic acid is more likely than one that leads to bicarbonate loss. the disease can occur in individual animals or in outbreaks; although parity of the dam and number of offspring have not been associated with this metabolic disturbance, aggressively feeding kids are more likely to suffer from milk fermentation or clostridial overgrowth. an infectious etiology appears to be more likely in herds displaying an increased incidence of this metabolic disturbances as the kidding season progresses. the disease also is reported to be more common in meat goats than in dairy goats. the prevalence can vary tremendously from year to year in a single flock or region. a similar disease has been reported in calves and llama crias, and lambs are likely to be susceptible under the right conditions. because blood gas analysis and exclusion of other diseases often are impractical, the term floppy kid syndrome frequently is used by owners to refer to any kid that is weak and does not have an overt, organ-specific sign (e.g., diarrhea). different pathologic processes are grouped together by their common clinical endpoint (as with "thin ewe syndrome"), and the veterinarian is charged with determining the etiology in a specific flock. most possible causes are found in the previous list of conditions that cause weakness and depression in neonates. among these entities, sepsis and hypoxemia are the most important items and therefore must also be considered possible causes of floppy kid syndrome. treatment and prevention of floppy kid syndrome currently follow the same lines as for treatment and prevention of neonatal sepsis or enteritis. spontaneous recovery of animals with floppy kid syndrome may occur. however, in valuable kids, quick assessment of blood chemistry and base deficits will allow requisite correction of electrolyte and blood ph abnormalities with . % sodium bicarbonate. tissue-invading clostridia are large, straight, gram positive rods that are to mm in length. c. perfringens and c. haemolyticum are smaller bacteria, and clostridium novyi, clostridium chauvoei, and clostridium septicum are larger. the bacteria grow best under anaerobic conditions and produce waste gases. clostridia bear spores, which may be the only viable form in the environment (soil and decomposed organic matter). identification of these spores within bacteria on microscopic examination is useful to identify clostridia, but it is not diagnostic of disease. spores in c. perfringens are central and do not affect the shape, whereas most other species have the spore toward one end and appear slightly club shaped. clostridia cause infectious, noncontagious disease. the bacteria inhabit the intestinal tract and are present in the feces of ruminants. small numbers of organisms in their dormant spore form also may reside in tissues such as liver and skeletal muscle. they can be isolated from soil, where most are thought to have short life spans. soil concentrations are highest in locations recently contaminated with ruminant feces, especially crowded, overused facilities such as feedlots and lambing sheds. environmental contaminations are associated with cool, damp times of the year such as late winter and spring. the concentration of organisms and their toxins found in the feces, gut contents, and internal organs of most adult ruminants usually is small. competition and peristalsis prevent overgrowth in the gut, and aerobic conditions prevent overgrowth in other tissues in live animals. however, rapid overgrowth and tissue invasion ensue after death, making rapid postmortem examination essential to ascertain whether clostridial organisms are responsible for the death. pathogenic clostridial organisms all produce heat-labile protein exotoxins. most make a variety of toxins, and the relative contribution of each toxin to the disease state is not known. c. perfringens is a normal commensal of the intestinal tract of clinically healthy large animals, including cervids; however, the number of bacteria and their toxin production within the intestine usually remain low due to peristalsis and normal homeostasis. c. perfringens is classified into five biotypes (a, b, c, d, and e) based on the production of four major exotoxins, namely alpha (cpa), beta (cpb), epsilon (etx), and iota (itx); however, the production of more than different exotoxins in various combinations has been associated with these bacteria, including perfringolysin o (pfo), enterotoxin (cpe), and beta toxin (cpb ). the different biotypes of c. perfringens cause different diseases in relation with the exotoxins they produce. the major effect of the phospholipase/ sphingomyelinase cpa, produced by all c. perfringens biotypes, is cell lysis and hemolysis, and its role on intestinal disease of large animals is not well understood. however, this toxin has been associated with hemolytic disease and hemorrhagic enteritis in large animals; cpb, produced by c. perfringens types b and c, is a trypsinlabile toxin associated with necrotizing enteritis and enterotoxemia in large animal neonates; etx, produced by c. perfringens types d and b, is a trypsin-activated necrotizing toxin associated to vasculitis, edema, and necrosis of the cns and enterotoxemia; and itx, another trypsin-activated necrotizing toxin produced by c. perfringens type e, has also been associated to intestinal disease in small ruminants. , c. perfringens types c and d are considered the most important types in veterinary medicine as they can cause disease in most farm animals. severe clinical disease due to bacteria sporulation and massive toxin production only occurs when the normal intestinal environment and microbial balance are disrupted in affected individuals. decreased peristalsis and poor ruminal and abomasal function have also been proposed as factors that contribute to disease presentation. weather and handling stresses, feed changes, and an overabundance of high-energy feeds such as milk, bakery products, and cereal grains might promote bacteria overgrowth and exotoxin synthesis and release. additionally, other enteric infections that disrupt the mucosal border may increase systemic absorption of toxins and promote severe disease. c. perfringens type a is a normal inhabitant of the intestinal tract of large animals and is ubiquitous in the environment (soil). one study reported c. perfringens type a as the most common isolate among other clostridia from healthy young lambs. c. perfringens type a has been associated with a fatal hemolytic syndrome in younger lambs and cattle but not goats ("yellow lamb disease"), , acute hemorrhagic enteritis and hemolytic enterotoxemia in cattle (hemorrhagic bowel/jejunal syndrome) and goats, , , and intestinal hemorrhage and splenomegaly in farmed deer. , risk factors for infection have not been established; however, high soluble carbohydrate diets and high bcss have been associated with clinical disease. , , this disease occurs most commonly in lambs to months old. under favorable conditions, the organisms proliferate and cause a corresponding increase in alpha toxin production. the alpha toxin (cpa), in synergy with the beta toxin (cpb ), is responsible for hemolytic crisis, vasculitis, and gastrointestinal lesions. the clinical course usually is less than hours. clinical signs. in most cases, sudden death or history of found dead is common. clinical signs observed usually include weakness, depression, fever or hypothermia, icterus, anemia, hemoglobinuria, tachypnea, colic, hemorrhagic diarrhea or absence of feces, and terminal recumbency. , , [ ] [ ] [ ] adult animals also are susceptible to hemolytic disease and vasculitis caused by c. perfringens type a infection. fatal abomasitis and rumenitis in neonates and juveniles also have been blamed on c. perfringens type a, but the rapid postmortem proliferation of the organism makes substantiation of this claim difficult. morbidity in a flock is lower than for many of the other enteric clostridial diseases, but the mortality rate is very high. diagnosis. the most characteristic clinicopathologic change is neutrophilic leukocytosis with a left shift. other evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. laboratory evaluation reveals evidence of intravascular hemolysis. necropsy in sheep, goats, and cervids usually reveals evidence of hemolysis, pallor, jaundice, hemoglobinuria, hyperemic and edematous intestines, splenomegaly, gastrointestinal serosal and mucosal hemorrhage, and multifocal internal petechial hemorrhages. , , [ ] [ ] [ ] the isolation of c. perfringens type a from necropsied animals is not itself diagnostic. definitive diagnosis can be made based on identification of the alpha toxin and the absence of other toxins by elisas or older, live animal assays. more recently, multiplex pcr techniques are replacing immunodiffusion assays for the identification of a specific toxin-producing gene isolate, typing of bacteria, and demonstration of toxins or toxin genes. gut content and intestinal samples collected from freshly dead animals make the most meaningful samples for diagnosis. treatment. administration of high doses (. , iu/kg bid) of penicillin and clostridium antitoxin ( - ml subcutaneously [sc] or orally [po] ) is the mainstay of treatment, although animals may die acutely before therapies can be instituted. prevention. a conditionally licensed toxoid against the clostridial alpha toxin is available for cattle in the united states. a recent report demonstrated that a new vaccine including recombinant cpa, cpb, and etx was effective at inducing protective antibodies to c. perfringens biotypes in cattle, sheep, and goats. this could be an alternative for the prevention of morbidity and mortality caused by c. perfringens type a. prevention efforts should focus on environmental hygiene and avoiding gut conditions favorable for proliferation of the organism (high content of soluble carbohydrates in the diet). because this type appears to survive better in soil than other types, preventing ingestion of soil may be important in preventing disease. c. perfringens types b and c occur in the soil and the animals' housing environment and can be shed by asymptomatic individuals. the reported geographic range of both diseases is limited (type b to the united kingdom and south africa and type c to the united kingdom, australia, and north america), even though infection with c. perfringens type c appears to occur worldwide. these organisms cause very similar diseases called lamb dysentery and hemorrhagic enterotoxemia, respectively. very young lambs and kids ( - days to - weeks of age) are usually affected due to the presence of trypsin inhibitors in colostrum. older animals may become susceptible as a result of overwhelming infection or trypsin inhibition by some soy and sweet potato products or temporary suppression of pancreatic trypsin production (struck in adult sheep). with both diseases, the beta toxin (cpb) is a required pathophysiologic factor, and inactivation of this toxin after maturation of pancreatic trypsinogen secretion is what commonly limits the susceptible population to neonatal animals. the cytolytic and necrotizing effects of the beta toxin (cpb), in synergy with the beta toxin (cpb ), cause necrosis and ulceration of the intestinal mucosa and are translocated into circulation, causing severe toxemia and death. the diseases initially affect lambs and kids younger than days of age, with illness occasionally occurring in older lambs. the incidence of disease in lambs and kids can be around to %, with a case fatality rate of %. high stocking density in lambing areas, cold weather, single-born lambs, and high milk production of dams have been suggested as potential risk factors for type b and c enterotoxemia. because of management practices in young animals and age-related vulnerability, fecal contamination of teats, hands, and equipment that enter the mouths of the neonates (orogastric tubes and nipples) is a major cause of infection. clinical signs. severely affected animals or those at the beginning of an outbreak usually are found dead. less acutely affected animals expel initially yellow, fluid feces that progressively become brownish and/or hemorrhagic. feces may also contain flecks of blood and show splinting of the abdomen, especially when handled, along with signs of colic and feed refusal. the clinical course usually is short, and the disease is almost always fatal. one study reported acute abdominal pain, hemorrhagic diarrhea, and death within hours of experimental oral inoculation of three goat kids with a field strain of c. perfringens type c. dehydration, anemia, and severe weakness are also common clinical signs in affected animals. terminal convulsions and coma occasionally are noted, especially in outbreaks in the united states. c. perfringens type c in older sheep causes the disease known as "struck." affected animals usually are found dead or with signs of toxemia. specific antemortem signs of gastrointestinal disease are rare. specific antemortem signs of gastrointestinal disease are rare. clinical pathology changes observed in these animals include neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. necropsy findings. postmortem examination reveals focal hemorrhagic ulcers (up to . cm in diameter) in the small intestine (mostly in the ileum) with type b infection and diffuse reddening with hemorrhage and necrosis of the abomasum and the entire segments of the intestine with type c infection. type c infections in ruminants can also present with generalized peritonitis, subendocardial and subepicardial hemorrhages, and hemorrhagic lymph nodes. animals that die very rapidly may exhibit minimal or no gross abnormalities of the intestine. a similar syndrome of type c enterotoxemia has been previously reported in a sika deer (cervus nippon). sudden death, severe hemorrhagic gastritis including forestomach and abomasum, and catarrhal enteritis was observed in the affected animal. diagnosis. diagnosis of these diseases is made by identification of characteristic history, clinical signs, postmortem lesions, and positive toxin assays. because the beta toxin is very labile, negative toxin assays are less significant than negative assays for presence of other tissue-invading clostridia. the isolation of c. perfringens type b or c from necropsied animals is not itself diagnostic. immunodiffusion assays or multiplex pcr of intestinal contents for specific isolate and beta toxin (cpb) identification are recommended to obtain final diagnosis (see "diagnosis" in "c. perfringens type a" section). treatment. if the infection is identified early in the disease course, high doses of oral and parenteral penicillin and c. perfringens c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. usually, the condition is not recognized early enough, and animals are found dead or dying. prevention. a beta toxoid is available in the united states and other countries. it usually is packaged with an epsilon toxoid. the best protection is achieved by vaccinating pregnant dams twice, with the second dose administered approximately to weeks before lambing or kidding and annual booster. deer does should receive double the dose of sheep as low antibody responses to clostridia have been reported in these animals. , vaccination of pregnant dams is directed to increase specific colostrum antibodies to protect neonates. juveniles also should be vaccinated twice or three times at months, months, and months. adults, including males, should receive an annual booster. in the face of an outbreak, the lambing area should be moved to a different place. additionally, vaccination of dams and newborns with a beta toxoid and administration of c. perfringens c and d antitoxin can be carried out in the face of an outbreak to reduce morbidity and mortality. c. perfringens type d produces epsilon toxin (etx), which is responsible for causing type d enterotoxemia in sheep, goats, calves, and deer. , other common names for the disease include "overeating disease" or "pulpy kidney disease." the disease has a worldwide distribution and occurs primarily in suckling lambs of to weeks of age, although it has also been reported in weaned lambs up to months of age and adult sheep. the disease is also common in grazing goats and deer. the prevalence of disease has been reported from . to . %, with a % case fatality rate in feedlot lambs. one study on proportional distribution of goatherd mortality in the province of quebec, canada, reported a . % mortality of goats to c. perfringens type d enterotoxemia. the disease is more common in feedlot lambs after they enter the lot. tail docking, castration, and other management interventions are thought to decrease the incidence of this disease by temporarily decreasing appetite. the disease also affects unvaccinated adult sheep, even without any history of stressors or feed changes. sudden changes in the diet are the main predisposing factor in goats. the disease can occur in vaccinated goats, as vaccination has not demonstrated to be completely protective in this species. , c. perfringens type d is normally found in the gastrointestinal tract of healthy ruminants, but the acid environment of the abomasum and continuous peristalsis help to keep numbers of bacteria and levels of toxin production low. however, under specific conditions such as overingestion of high-energy feeds (milk, grain, and lush pasture), excess of fermentable starches in the intestine, and intestinal stasis, the organism proliferates rapidly, producing lethal quantities of epsilon toxin. these conditions are usually triggered in well-conditioned, fast-growing animals that are on a highly nutritious diet. the epsilon toxin, once produced, acts locally, causing increasing gut permeability and widespread tissue damage. epsilon toxin and other exotoxins are then absorbed through the intestinal tract into systemic circulation and transported to the brain, lungs, and kidneys, causing increased endothelial permeability, perivascular edema, and generalized necrosis. , the characteristic increased vascular permeability and perivascular edema in the kidney and brain are responsible for the name of "pulpy kidney disease" and "focal symmetric encephalomalacia." clinical signs. the course of the disease is usually very short ( . - hours), so sudden or spontaneous death is a common clinical sign across affected small ruminant species. , - natural disease caused by c. perfringens type d differs between sheep and goats, possibly because of a difference in relative local and systemic actions of the epsilon toxin, although experimental models have demonstrated that both species develop similar lesions. , , in sheep, systemic actions of the toxin leads to mostly neurological signs such as dullness, depression, ataxia, trembling, stiff limbs, opisthotonus, convulsions, frothy mouth, and rapid death. in goats, actions of the toxin appear to be more localized to the intestinal tract, causing enterocolitis, colic, diarrhea, dehydration, and occasional neurological signs. , necropsy findings. postmortem findings in sheep are characterized by edema of the brain, lungs, and heart in addition to hydropericardium. edema of the kidneys (pulpy kidney lesion) is inconsistent. sheep usually demonstrated minor and inconsistent intestinal changes. other lesions reported in cattle and deer include hemorrhages on the epicardium, thymus, and diaphragm and petechial hemorrhages in the jejunal mucosa. , necropsy lesions reported in goats include pseudomembranous enterocolitis with mucosal ulceration, as well as fibrin, blood clots, and watery contents in the bowel lumen. evidence of systemic toxemia, including multifocal petechial and ecchymotic hemorrhage, proteinaceous exudates in body cavities, pulmonary edema, hydropericardium, and cerebral malacia with perivascular cuffing, have also been reported in goats and affected deer. , , , , clinical pathology. characteristic clinicopathological changes include pronounced hyperglycemia and glucosuria, which are considered a hallmark of c. perfringens d enterotoxemia. additionally, neutrophilic leukocytosis with a left shift and evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. treatment. in general, the course of disease is too acute for the establishment of any treatment. however, as with infections with types b and c, if the disease is identified early in the disease course, high doses of oral and parenteral penicillin in addition to clostridium c and d antitoxin may be of benefit. iv fluids and antiinflammatory agents may be indicated as well. prevention. vaccination of pregnant ewes with two doses of toxoid, with the second dose given to weeks before lambing, and adequate ingestion of colostrum are the best methods of protecting newborn lambs. vaccination of older lambs should occur before exposure to diets rich in carbohydrates (grain-feedlot settings) or lush pastures. in these cases, lambs should be vaccinated twice or three times around , , and months of age. males and adult females that are not part of the breeding program may be vaccinated annually. vaccination has been shown to protect goats from experimental disease, but clinical evidence suggests that well-vaccinated goats are still susceptible to developing clostridial enteritis. the toxoids may not protect against local action of the toxins in the goat, which appears to play a greater role in their disease than it does in the sheep. , , more frequent vaccination (every months) in goats is suggested to increase protection. the adjuvant present in some multivalent clostridial vaccines may cause subcutaneous reactions that may lead to abscess formation. in the face of an outbreak, immediate mass administration of c and d antitoxin ( iu/kg) in addition to vaccination is recommended. nonenteric clostridial infections c. novyi, c. septicum, c. chauvoei, and c. sordelli have been identified as causal agents of severe muscle, liver, and abomasal necrosis in small ruminants and cervid species. , [ ] [ ] [ ] these organisms are usually present in the soil and environment and in the gastrointestinal tract and liver of healthy ruminants. pathogenesis is usually facilitated by trauma of affected tissues, local multiplication of the organism, local and systemic damage by exotoxin production, and ultimately death. , four types of c. novyi have been described, a, b, c, and d. c. novyi type c is considered nontoxigenic and therefore is not associated with disease. c. novyi type a produces alpha toxin and is associated with wound infections and myonecrosis in cases of "bighead" and "malignant edema." c. novyi type b produces alpha and beta toxins and is associated with infectious necrotic hepatitis or "black disease." , the temporal and geographic distributions of black disease resemble those of fascioliasis, with the highest incidence of disease in milder, moister months in many countries. black disease is less common in sheep than in cattle and is rare in goats. , c. novyi type d (c. haemolyticum) produces beta toxin and is associated with bacillary hemoglobinuria (red water disease). c. septicum produces alpha toxin and is associated with malignant edema and necrotic abomasitis (braxy). c. chauvoei produces alpha and beta toxins and is associated with severe myonecrosis observed in blackleg and c. sordelli produces a hemolytic toxin associated with myonecrosis in cases of malignant edema and blackleg. , pathogenesis. spores of the organism shed in feces of carrier animals contaminate the environment and are ingested with feed/ grass and stored within kupffer cells. , liver damage caused by migrating liver fluke larvae (fasciola hepatica, fasciola gigantica, and cysticercus tenuicollis) create perfect ischemic conditions that induce germination of c. novyi type b spores and toxin synthesis and production. , , the alpha toxin is necrotoxic and causes liver necrosis and diffuse damage of the vascular system. the beta toxin is produced in smaller amounts and contributes to vascular damage and systemic toxemia. infective organisms also may be brought into the liver by the flukes. clinical signs. the course of disease from first illness to death is short and never lasts more than a few hours in sheep. therefore, peracute or sudden death is not uncommon in this species. wellnourished adult sheep between and years are more commonly affected. the disease course is a little longer ( - days) in cattle and deer. , affected sheep are debilitated, fail to keep up with the flock, and exhibit generalized weakness, sternal recumbency, separation, and anorexia. tachypnea and tachycardia may be seen; high fever ( - ° f) occurs early in the disease. clinical signs observed in cattle, goats, and deer are similar and may include severe depression, anorexia, abdominal distention, colic, ruminal stasis, and lateral recumbency. , , , , , a report of black disease in a forest reindeer (rangifer tarandus fennicus) described serosanguinous discharge from mucocutaneous orifices (nostrils and anus), periorbital edema, and nystagmus in addition to other clinical signs. necropsy findings. necropsy might be difficult due to rapid autolysis of tissues in affected animals. severe venous congestion usually darkens the underside of the skin of affected animals, giving this disease its common name of "black disease." fluid in the pericardial sac, pleural space, and peritoneal cavity is usually present. endocardial and epicardial hemorrhages are a common finding. the liver is swollen and congested and on its diaphragmatic surface presents pale foci of coagulation necrosis; however, solid organs such as liver and kidneys could be in an advanced state of autolysis. characteristic lesions of black disease in the liver are single or multiple yellow to white areas ( - cm in diameter) of necrosis surrounded by a bright hyperemic zone. a recent report of black disease in a reindeer described moderate amounts of dark red thoracic and pericardial fluid, edema of the lungs and upper respiratory tract, swollen spleen, and several well-circumscribed areas of black discoloration in the liver. diagnosis. the most characteristic clinicopathological change is neutrophilic leukocytosis with a left shift. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen; however, diagnosis of black disease is based on characteristic history (endemic liver fluke areas), clinical signs, and postmortem findings and testing. an impression smear of the margins of the liver might reveal large numbers of gram positive rods, but this is not definitively diagnostic. anaerobic culture of c. novyi from typical liver lesions, in addition to demonstration of the alpha/beta toxins from peritoneal fluid or liver (fresh-refrigerated), through elisa or pcr is required to establish final diagnosis. , the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smear samples or other liver (formalin-fixed) samples have also been described. , treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium ( , - , iu/kg) iv every hours or oxytetracycline mg/kg iv every hours should be initiated. supportive care, including iv fluids, nutritional support, and stress reduction, may be beneficial. in the face of an outbreak, vaccination of the whole herd/flock should be initiated immediately. efforts to control fluke infestation constitute the most effective approach to prevention of this disease. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every months starting around to months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given month before expected fluke exposure may provide additional protection. , deer should be vaccinated in the same fashion as sheep but double the vaccine dose for sheep should be used for these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. , efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis. c. novyi type d (c. haemolyticum) is the etiologic agent associated with red water disease. c. haemolyticum is similar to other clostridial species in its life cycle and appears to thrive on alkaline soils and pastures with standing water. the disease tends to be seasonal occurring at times of high larval fluke migration. similar to c. novyi b, c. haemolyticum colonizes the livers of healthy animals and proliferates after liver damage, including damage caused by migrating flukes (f. hepatica, fascioloides magna, dicrocoelium dendriticum, and c. tenuicollis), liver abscessation (fusobacterium necrophorum or trueperella pyogenes), or damage incurred during liver biopsy. , under ischemic conditions of the liver, spores of c. haemolyticum germinate and produce high amounts of beta toxin. the beta toxin causes localized hepatic necrosis and after reaching circulation induces severe intravascular hemolysis and damage of the capillary endothelium. intravascular hemolysis leads to rapid anemia and death due to anoxia. the disease is seen worldwide and is more commonly reported in sheep than in goats. bacillary hemoglobinuria has been reported in a free-ranging elk calf (cervus elaphus roosevelti) found dead in the southwest of the state of washington, united states. clinical signs. bacillary hemoglobinuria usually affects wellnourished animals older than year of age. , in most cases, the disease is per-acute and sudden dead or found dead is the only sign. in cases where signs are recognized antemortem, affected animals appear weak, depressed, and febrile ( - ° f); blood or blood-tinged froth may be present in the nostrils; rectal bleeding and bloody feces may be present; and severe hemoglobinuria (dark red, port wine-colored urine) is usually observed. , blood appears thin and watery and mucous membranes are pale and icteric. heart and respiratory rates are high and become much higher with any sort of effort or stress. other terminal signs include bloat and the presence of blood in the nostrils, mouth, vagina, and rectum. death occurs within hours to a few days after onset of clinical signs. necropsy findings. gross lesions include jaundice of mucous membranes and tissues and subcutaneous petechial/ecchymotic hemorrhages, edema, and emphysema. marked autolysis of internal organs might prevent identification of typical lesions. dark red urine is present in the bladder. lymph nodes and spleen are congested and hemorrhagic. hemorrhagic abomasitis and enteritis might occur, as well as the presence of hemoglobin-stained transudate in pleural and peritoneal cavities and pericardial sac. pulmonary edema is common. the pathognomonic lesion is the ischemic hepatic infarcts ranging from to cm in diameter with a hyperemic interface with healthy liver tissue. , diagnosis. clinicopathological abnormalities usually include anemia, leukocytosis with mature neutrophilia, and degenerative left shift (immature forms of neutrophils and toxic changes) often is present. , serum biochemical evaluation may reveal increased levels of liver enzymes such as sorbitol dehydrogenase, ggt, aspartate aminotransferase, and increased indirect total serum bilirubin. [ ] [ ] [ ] presumptive diagnosis can be made on history, clinical sigs, clinicopathological abnormalities, and postmortem findings; however, similar to black disease, final diagnosis should be based on anaerobic culture of c. novyi from typical liver lesions in addition to demonstration of the beta toxins from peritoneal fluid or liver (fresh-refrigerated) through elisa or pcr techniques. , , , the use of fluorescent antibody and ihc for the identification of c. novyi on liver impression smears or other liver (formalin-fixed) samples has also been described. , more recently, a pcr assay for the detection of c. novyi type d in cattle has been reported. treatment and prevention. treatment is rarely possible because of the fulminant clinical course of the disease; however, if treatment is attempted, high doses of penicillin g sodium ( , - , iu/kg) iv every hours or oxytetracycline mg/kg iv every hours should be initiated. supportive therapy should include the administration of iv fluids, blood transfusions, and antiinflammatory agents. efforts to control liver flukes and prevent other causes of liver damage are most important. administration of multivalent clostridial vaccines containing c. novyi is highly effective. animals should be vaccinated every months starting around to months of age and before parturition as protective immunity is short lived. in flocks at high risk for developing this disorder, a booster vaccine given month before expected fluke exposure may provide additional protection. deer should be vaccinated in the same fashion as sheep, but double the vaccine dose for sheep should be used as these animals as they do not develop a strong antibody response to commercially available multivalent vaccines. , efforts to eliminate the organism from soil and environment are usually unrewarding but carcasses of animals dying from the disease should be burned, deeply buried, or removed from the premises. pathogenesis and clinical signs. fecal and soil contamination of wounds received during fighting (head-butting) or dehorning (disbudding) leads to proliferation of c. novyi type a in damaged head and neck tissues. accumulation of secreted toxins leads to swelling, edema, serohemorrhagic exudates, and local tissue necrosis. wounds appear and smell gangrenous. systemic toxemia may affect internal organs, leading to the death of the animal. c. sordelli causes identical disease. diagnosis. laboratory analysis may reveal an increase in enzymes of muscle or liver origin as well as neutrophilic leukocytosis with many immature and toxic neutrophils. postmortem findings include local necrosis around the injury site. diagnosis usually is made by characteristic clinical signs and lesions. treatment. wound management (disinfection, debridement) and administration of high doses of penicillin g sodium ( , - , iu/kg) iv every hours are important treatment considerations. prevention. ram management may aid in the prevention of head-butting wounds. vaccination with multivalent clostridial toxoids starting around weaning time ( - months of age) and with annual boosters also may be helpful. in flocks with a high prevalence of this disorder, a booster vaccine given to rams month before the breeding season and to ewes/does before parturition may provide additional protection. pathogenesis. c. septicum is the most important agent in the pathogenesis of malignant edema and braxy. in the case of malignant edema, other tissue-invasive clostridia (c. chauvoei, c. sordelli, and c. perfringens a) have also been associated with this disease, and mixed infections are common. the pathogenesis of infection is often similar to that seen with bighead and blackleg: soil or fecal clostridial invasion of a contaminated wound. in sheep and goats, this disease has been reported following lambing/kidding, after shearing of tail docking. c. septicum can also invade the abomasal lining of lambs, causing severe hemorrhagic, necrotic abomasitis known as braxy. activation of dormant bacteria in previously damaged tissue (myositis/abomasitis) similar to that seen in clostridial necrotic hepatitis also occurs. in both cases (malignant edema and braxy), bacterial toxins precipitate local tissue necrosis and systemic toxemia. the alpha, beta, gamma, and delta toxins produced by c. septicum are lecithinase, deoxyribonuclease, hyaluronidase, and hemolysin, respectively. commonly affected sites of malignant edema include castration, dehorning, and injection sites; the umbilicus; and the postpartum uterus. factors that promote braxy have not been identified, although it usually affects weaned and yearling lambs in the winter after ingestion of frozen feedstuffs implicated as initial causes of abomasitis. , both forms of the disease have worldwide distribution and are described more in sheep than in goats. , clinical signs. malignant edema is characterized by local lesion (wound) or regional pain characterized by swelling and edema that progressively becomes tense and dark (skin). high fever, signs of shock/toxemia, and frothy exudation of the wound are usually present. evidence of subcutaneous gas production is less common in this infection than in blackleg. uterine infection may cause a fetid vaginal discharge. death occurs within hours to a few days after onset of clinical signs. braxy usually causes death before any abnormalities are noted. on rare occasions, signs of sudden onset of illness with high fever, abdominal distention, depression, colic, and recumbency may be seen before death. diagnosis. characteristic clinicopathologic changes include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia (metabolic acidosis, azotemia, and increases in liver and muscle enzymes) also may be seen. examination of a gram-stained smear from the edematous swelling(s) or wound swabs could give an early diagnosis. one study reported the successful use of a pcr assay for the identification of bacteria associated with malignant edema in cattle, sheep, and other ruminants. postmortem changes with malignant edema include dark red, swollen muscle filled with hemorrhagic, proteinaceous exudate and little or no gas. with braxy, the abomasal wall is hemorrhagic and necrotic. both diseases are associated with rapid postmortem decomposition of the carcass. treatment and prevention. wound management and the rapid administration of high doses of penicillin (penicillin g sodium at , - , iu/kg iv every hours) are important in treating malignant edema. local treatment consists of surgical incision of the affected area to provide drainage and irrigation with peroxide. injection of penicillin directly into or in the periphery of the lesions may help. ancillary treatments such as iv fluids, antiinflammatory agents (e.g., flunixin meglumine, mg/kg iv), and nutritional support may be necessary. maintenance of good hygiene during procedures such as lambing, tail docking, shearing, castration, obstetric manipulation, and administering injections is helpful in preventing malignant edema. multivalent clostridial toxoids may provide some protection and should be given annually to animals at risk for the disorder. pathogenesis. several species of clostridial organisms can cause myonecrosis in small ruminants. , , the disease is acute to per-acute, has a short course of duration, and is usually fatal. c. chauvoei, c. septicum, and c. sordelli are commonly involved with clostridial myonecrosis in ruminants. [ ] [ ] [ ] blackleg can be enzootic in some areas or farms because of increased bacterial contamination and occurs more commonly in the warm months of the year. [ ] [ ] [ ] animals between months and years of age can be affected. , c. chauvoei is the most important cause of blackleg. c. sordelli tends to be involved in the myonecrosis of older feedlot animals. these organisms are found in the soil and can gain access to muscles after translocation from the gastrointestinal tract and liver into systemic circulation. additionally, direct inoculation of the organisms by penetrating wounds or intramuscular injections has been suggested. local tissue trauma, wounds, unsanitary procedures (i.e., shearing, tail docking, and castration), umbilical infection (neonates), or vaginal trauma from lambing can create perfect conditions for the germination of clostridial spores inducing rapid toxin synthesis and production. in some cases, bacterial proliferation appears to occur in a site distant from the original wound (i.e., fetal infections after shearing of a ewe and myocardial necrosis in cattle and sheep). bacterial toxins cause severe local tissue necrosis, systemic toxemia, and ultimately death. as with braxy, several other strains of tissue-invasive clostridia can cause this disease and mixed infections are common. clinical signs. clostridial myonecrosis usually progresses rapidly and sudden death or history of found dead is not uncommon. [ ] [ ] [ ] clinical signs in animals who are still alive include local to regional painful, edematous swelling most commonly in the limbs or trunk muscles. skin of the affected area can become discolored and crepitus; however, in affected sheep, subcutaneous edema and gaseous crepitation are uncommon and cannot be felt before death. other signs might include stiff gait, lameness, fever, and signs of shock. in cases where the infection occurred through a wound, there is extensive local damage and malodorous serosanguinous fluid discharge. c. chauvoei also causes uterine infection and severe gangrenous mastitis in postparturient ewes. , in these cases, uterine and mammary infections may cause fetid vaginal and mammary discharge, respectively. death often occurs within to hours after onset of clinical signs. necropsy findings. rapid tissue autolysis is not uncommon in animals that succumb to clostridial myonecrosis. bloodstained fluid and froth can be observed discharging from nostrils and anus. in small ruminants and especially sheep, affected muscle areas are more localized and deeper, brown to black discoloration is present, the subcutaneous edema is not as severe, and, although there is gas present, is not in such large amounts as in cattle. in cases of infection from skin wounds, the area demonstrates subcutaneous edema, swelling, and underlying muscle discoloration. in cases of infection through the urogenital tract, typical lesions are found in the perineal area, vagina, uterus, and fetus. lung congestion, fibrinohemorrhagic pleuritis, pericarditis, myocardial damage, and bloat are also common findings. , diagnosis. it is rarely possible to obtain samples for clinicopathological analysis due to the per-acute course of the disease. if samples can be obtained, common findings include neutrophilic leukocytosis with a left shift. a decrease in wbc and rbc counts also is possible because of the leukocidal and hemolytic effects of the toxins. additional evidence of systemic toxemia-metabolic acidosis, azotemia, and increases in liver and muscle enzymesalso may be seen. presumptive diagnosis can be made from history, characteristic clinical signs, and gross pathology findings; however, aspirates or tissue specimens from affected muscles for direct smear examination, fluorescent antibody testing, or anaerobic culture are required for definitive diagnosis. , a multiplex pcr is available for identification of pathogenic clostridia on fluid and tissue samples. treatment and prevention. aggressive antibiotic therapy (e.g., penicillin g sodium or potassium penicillin at , - , iu/kg iv every hours), in combination with surgical debridement of affected tissues (fasciotomy), and supportive care (nutritional support, iv fluids, and antiinflammatory agents) are important within the treatment plan for clostridial myositis. prognosis for treatment of all types of clostridial myositis cases is usually guarded to poor and depends on the duration and extension of the lesions. maintaining excellent hygiene during invasive procedures such as castration, obstetric manipulation, shearing, tail docking, and administering injections is helpful in preventing blackleg. multivalent clostridial toxoids may provide some protection and should be given to all animals starting at weaning time, before parturition, and annually. , both tetanus and botulism are important diseases in small ruminant medicine. these two diseases are covered elsewhere in this book (see chapters , , , , and ) . older animals are generally more resistant to sepsis than neonates because they have larger amounts of circulating antibodies. however, this resistance can be overwhelmed by aggressive bacteria, or loss of immune function can allow invasion by opportunistic bacteria. malnutrition, parasitism, transport, overcrowding, other diseases, extreme weather conditions, and other stressors are the major causes of immune suppression. sepsis may produce peracute, acute, or chronic disease signs. peracute signs include fever, injected mucous membranes (including the sclera), tachycardia, tachypnea, dyspnea, swollen joints, lameness, splinting of the abdomen, weakness, depression, anorexia, recumbency, seizures, coma, and sudden death. acute signs are similar, except that they persist for a longer period and therefore are more likely to be noticed. chronic signs usually result from the partial clearance of infection after an acute episode, which may be clinical or inapparent. pathogenesis. gram negative bacteria and their toxins gain access to the blood from a site of proliferation or destruction. the most important toxin is endotoxin, a group of lipopolysaccharide molecules that reside within the wall of the bacteria. bacteria or endotoxins incite a systemic inflammatory response, chiefly through activation of host macrophages and stimulation of host cytokine release. these cytokines cause inflammation, produce leukocyte recruitment, increase capillary permeability, induce fever through stimulation of the hypothalamus, and have regional or diffuse vasomotor effects. because the ruminant gut has a plentiful population of gram negative bacteria, it is implicated as the source of most cases of gram negative sepsis. grain overload causes a die-off of the normal gram negative ruminal flora, ulcerative enteric disease allows invasion of bacteria or absorption of their toxins, and ingestion of pathogens provides a suitable place for proliferation and route for invasion of the body. gram negative sepsis caused by opportunistic organisms is best recognized in immunocompromised neonates but also can be seen in stressed or immunocompromised animals of all ages. e. coli is commonly found in fecal material, klebsiella pneumoniae is found in feces and wood products, f. necrophorum lives in the gastrointestinal tract and in soil and invades through compromised gastric mucosa or foot-rot lesions, and pseudomonas aeruginosa is commonly found in water and wash solutions. primary pathogens are most common in adults. although some coliform bacteria may fit into this category, by far, the most important genus is salmonella. sources of salmonella infection are numerous and include carrier animals of the same species, cattle, rodents, birds, other animals, environmental contamination, and possibly feedstuffs. only one serotype of salmonella is specifically adapted to sheep (salmonella abortus ovis), and it is not found in north america. no strain is known to be host-adapted to goats or cervids. therefore, all infections in sheep, goats, and cervids have the potential to spread to and from other species, including humans. serotypes of salmonella that have caused important infections in sheep or goats include salmonella typhimurium, salmonella dublin, and salmonella montevideo. most of these infections lead to bacteremia with mild systemic signs, followed by abortion. s. dublin and s. typhimurium tend to cause more illness in adults because of fibrinonecrotic enteritis. clinical signs. affected animals can exhibit anything, from mild depression with a low-grade fever to shock. common signs include fever, tachycardia, tachypnea, depression with slow or absent eating and drinking, weakness or recumbency, and injection or cyanosis of mucous membranes. organ-specific signs may betray the source or at least the primary location of the infection. fetid discharge may be seen with metritis or abortion; dyspnea and abnormal lung sounds may be seen with pulmonary infection; and bloat, ruminal atony, abdominal distention, and diarrhea may be seen with gastrointestinal infections. diagnosis. the most common abnormality identified on a cbc with peracute gram negative sepsis is panleukopenia. over the course of several days, this condition may resolve, first through an increase in immature neutrophils and later through an increase in mature neutrophils and restoration of lymphocyte counts. very immature cells, severe toxic changes, and persistence of neutropenia suggest a poor prognosis. serum biochemical changes often reflect the severity of the condition. the greater the evidence of shock or tissue damage, the worse the prognosis. metabolic acidosis with a large anion gap and azotemia suggest advanced disease. necropsy findings include diffuse evidence of inflammation, including pulmonary congestion, and polyserositis with body cavity exudates. hemorrhagic pneumonia or fibrinonecrotic enteritis may be seen and reflect the source of bacterial invasion. in all cases, diagnosis is best confirmed by bacteriologic culture of body tissues or fluids. in the live animal, culture of blood, feces, or tracheal fluid yields the best results. when several animals are infected, environmental samples (including feed, water, and bedding) should be tested for the presence of the bacteria. bacteriologic culture of aborted fetuses or placentas frequently yields heavy growth of the organism. prevention. maintaining overall good health and hygiene is the best means of preventing gram negative sepsis. antiendotoxin bacterins are available for cattle in the united states, but their use in small ruminants has been too limited to assess their efficacy. during a flock outbreak, the use of autogenous bacterin may help prevent the spread of disease on a farm. actinobacillus seminis is a gram negative bacillus or coccobacillus that affects primarily the male and female reproductive tracts. infection causes posthitis, epididymitis, and orchitis in rams and metritis and abortion in ewes. other sites of infection, including rare occurrences of chronic sepsis, also are possible. serologic tests are much more useful for identifying infected flocks than infected individuals within flocks. definitive diagnosis depends on bacteriologic culture of the organism and differentiation of it from brucella ovis. the bacillus is common in sheep in some parts of the world but is uncommon in north american sheep and goats. t. pyogenes is best known as an abscess-forming bacterium because of the thick pus formed in response to infection by it and the fibrinous response it elicits. it occasionally also causes sepsis. its association with chronic sepsis lends credence to the belief that trueperella is often a secondary invader that colonizes tissues damaged by another bacterium (see chapter ) . bacillus anthracis is a large, gram positive, anaerobic bacillus that causes anthrax. it forms spores under aerobic conditions (such as on culture plates) but rarely does so when oxygen tensions are low, as in carcasses. the organism affects most mammals, with herbivores being most susceptible. it is usually carried from one area to another by shedding or dying animals and also can multiply in alkaline, nitrogenous soils. periods of heat and intermittent flooding promote overgrowth of the organism. b. anthracis spores may be inhaled or ingested; in rare cases, the bacillus itself may be spread by biting flies. after local replication, the organism gains access to the blood, where it multiples readily. large numbers of the organisms colonize the spleen. b. anthracis secretes a holotoxin made of edema factor), protective antigen, and lethal factor. this toxin impairs phagocytosis, increases capillary permeability, and inhibits clotting. splenic engorgement, generalized edema, circulatory shock, and bleeding diathesis are the most common lesions and signs of anthrax. generalized infection should be considered uniformly fatal. death may occur before or within hours of initial recognition that the animal is sick. prophylactic antibiotic treatment of healthy animals (oxytetracycline mg/kg iv sid) may decrease spread and mortality during outbreaks. the disease is reportable in many areas. local forms of anthrax also occur, most commonly after transmission through a skin wound or fly bite. local heat, pain, swelling, and necrosis are seen first, and the generalized syndrome often follows. bacterial organisms are rarely identified before important treatment decisions must be made. therefore, treatment should follow general principles and have a wide spectrum of efficacy. antimicrobial drugs are the cornerstone of treatment. in meat-or milkproducing animals, the veterinarian must be careful to use drugs within label directions or have a rational plan for extra-label drug use. the issue of extra-label drug use is especially important in small ruminants and cervids because very few pharmaceutical products have been licensed for them in north america. unless a specific organism is suspected (clostridiosis or anaplasmosis), a single antibiotic or combination of antimicrobial drugs to provide a broad spectrum of coverage should be selected. penicillins, macrolides, tetracyclines, and cephalosporins all provide effective coverage against gram positive pathogens. the newer third-generation cephalosporins are effective against many systemic and enteric gram negative pathogens. the gram negative pathogens of the respiratory tract are often sensitive to other classes of antibiotics. macrolides and tetracyclines also are effective against mycoplasma species and rickettsial organisms. nsaids are almost always beneficial in severe infectious conditions because of their antiinflammatory, antipyretic, and antiendotoxic effects. they are likely to be more effective than corticosteroids because they provide benefits without suppressing the immune response. all such drug use should be considered extralabel and administered accordingly with appropriate withdrawal times established. specific antisera are available for some of the clostridial diseases and may be beneficial if given before widespread tissue necrosis has occurred. severely compromised animals should be treated with fluids for shock (see chapter ). the most common zoonotic disease risk posed by exposure to small ruminants is orf, also known as contagious ecthyma in animals (see chapter ) . the disease is caused by an epitheliotropic poxvirus and is transmitted to humans by direct contact with infected animals. skin trauma is a significant risk factor for transmission in both humans and animals. in humans, erythematous macules or papules appear at the site of infection to days following exposure. the infection is generally self-limiting in immunocompetent individuals with complete healing occurring within weeks. brucella melitensis. apart from contagious ecthyma, the greatest risk of zoonotic disease from small ruminants is due to pathogens typically found in the reproductive tract that are transmitted to humans through contact with aborted fetuses, the placenta, or birthing fluids or through the consumption of raw or improperly pasteurized dairy products. b. melitensis is more common in goats than sheep (see chapter ) . swine, cattle, and other ruminants are common hosts. infection in animals usually causes inapparent mammary infection and abortions; infection in humans is characterized by undulant fever, myalgia, and fatigue. coxiella burnetii. c. burnetii is a rickettsial organism that is an important cause of abortion in sheep and goats (see chapter ) . wildlife and farm-raised deer may serve as reservoir hosts for infection in other ruminants and humans. infection is a documented cause of reproductive failure in farmed deer and prolonged shedding of the organism is an important source of environmental contamination. in addition to abortion, newly infected sheep and goats occasionally have mild, transient fevers. c. burnetii is far more important as the cause of q fever in humans, who become infected after inhaling particles, handling contaminated animals, or coming into contact with contaminated body fluids (uterine fluid, milk) from infected animals. infection in humans may be asymptomatic, present with flu-like symptoms, or, in the chronic form, present as granulomatous hepatitis, osteomyelitis, or bacterial endocarditis. chlamydophila spp. chlamydophila abortus (previously chlamydia psittaci) is an obligate intracellular parasite and the cause of enzootic abortion of small ruminants (see chapter ) . chlamydophila pecorum may cause polyarthritis and keratoconjunctivitis (see chapter ) in sheep and goats. transmission between animals and to humans most commonly occurs through direct contact with infected tissues or materials. infection in humans results in an acute febrile syndrome or respiratory symptoms. chlamydial diseases are more commonly reported in sheep than in goats. chlamydial diseases are suspected to cause disease in other species, including deer. recent serologic evaluation of wild ungulates identified multiple species of deer with antibodies against several chlamydial species. the clinical significance of serological infection in these species remains undetermined. francisella tularensis. f. tularensis is more common in sheep than goats. the organism has many hosts, of which the most important are wild rabbits and rodents. it can contaminate water sources. transmission to sheep is usually through biting arthropods that have previously fed on an infected wild mammal. acute or chronic sepsis may be seen, with more widespread and severe disease occurring in sheep with poor immune function. at necropsy, the disease is characterized by military foci of necrosis in the liver, and less commonly in the lymph nodes, spleen, and lungs. most cases in humans result in acute onset of flu-like symptoms a few days after exposure. l. interrogans. pathogenesis. leptospira spp. are spirochete bacteria that live in moist environments. their survival time outside of hosts is usually short, so their most important reservoirs are the kidneys of infected animals, especially rodents. infected animals shed the organisms through urine and most other body fluids. organisms enter new hosts through mucous membranes and skin breaks and cause bacteremia. signs of sepsis range from inapparent to severe, with more severe signs predominating in neonates. intravascular hemolysis may result. in animals that survive the acute stage, infection may localize in sites such as the kidneys, eyes, and fetoplacental unit. abortion may occur a month or more after acute signs first become evident while renal shedding may occur for several months. leptospirosis is zoonotic. in most cases, infections in humans are asymptomatic and selflimiting. however, in approximately % of cases, severe, and potentially fatal, systemic disease may develop, including jaundice, renal failure, and pulmonary hemorrhage. clinical signs acute leptospirosis causes signs of sepsis, including fever, depression, dyspnea, exercise intolerance, weakness, and death (see chapter ) . additionally, many affected animals show signs of intravascular hemolysis such as anemia, icterus, and hemoglobinuria. diagnosis evidence of intravascular hemolysis such as anemia, hyperbilirubinemia, hemoglobinuria, and hemoglobinemia is suggestive of this disease. in chronic infection, non-specific inflammatory changes and azotemia may be seen. animals dying in the acute hemolytic stage are likely to have dark, discolored urine, bladder, and kidneys. spirochetes can be identified on dark-field microscopy of fresh urine or plasma from infected animals and may be cultured with special techniques. in animals with less severe infection, a rise in antibody titers can be used to support a diagnosis of leptospirosis. prevention numerous vaccines are available for sheep. because protection is serotype specific, it is important to vaccinate against common serotypes in the area. leptospira pomona is the most consistent isolate from sheep and goats; leptospira hardjobovis is the predominate serovar in deer. vaccination immunity is thought to be short lived; boosters should be given at least twice a year in endemic areas. vaccination of deer against serovars hardjobovis and pomona has been associated with decreased urine shedding and increased growth rate in young animals. pathogenesis. l. monocytogenes causes disease with similar frequency in sheep and goats (see chapter ) . the organism is a common soil and fecal contaminant. it also proliferates in silage that is not properly acidified and in rotting, woody debris. risk of exposure depends on the feed and environment of the animals. environmental and fecal contamination is a more common source than silage in small ruminants overall because most sheep and goats throughout the world are not fed silage. infection in humans almost always results from ingestion of contaminated food products or unpasteurized milk. clinical signs. nervous system dysfunction and abortion are the most common manifestations of the disease. animals with the brainstem form of the disease display signs reflective of cranial nerve dysfunction, including drooped ears or eyelids, decreased facial sensation, and deviated nasal septum. a head tilt and circling may be present; in advanced cases of the disease, the animal is recumbent. clinical signs are mainly unilateral, occasionally bilateral, according to the nerve nuclei affected. diagnosis. antemortem diagnosis of listeriosis is difficult. a presumptive diagnosis is made based on history, clinical signs, and potential response to treatment. histopathologic identification of microabscesses in the brainstem and culture of the organism from affected tissues can be used to confirm the diagnosis. pathogenesis. p. multocida is a small, gram negative, bipolar, ovoid rod that inhabits the pharynx of healthy ruminants. it can survive in soil and water for varying amounts of time after contamination with ruminant nasal secretions. healthy ruminants shed p. multocida much more frequently than mannheimia haemolytica. disease occurs when bacteria colonize the lower respiratory tract or enter the blood. risk factors for pulmonary and systemic infection include viral or mycoplasmal respiratory diseases, temperature extremes, respiratory tract irritants, transport, overcrowding, changes to higher-energy feeds, and handling stress. these factors are thought to both increase bacterial replication in the airway and suppress mechanisms to clear the infection. pasteurellosis is a major problem in feedlot sheep but less common in small breeding or hobby flocks. pasteurellosis also is a significant disease in certain wild small ruminants such as bighorn sheep. direct spread of the organism between animals occurs with nasal contact, and indirect spread occurs after contact with infected nasal secretions. the organism persists in the environment for longer periods during warm, moist weather. p. multocida produces a polysaccharide capsule that inhibits phagocytosis and an endotoxin that contributes to clinical signs. the major disease caused by p. multocida is pneumonia (see chapter ). however, pasteurella spp. also are capable of entering the blood to cause septicemia in young lambs and hemorrhagic septicemia in adults. occasionally, focal infections such as septic arthritis and mastitis are found. clinical signs. clinical signs of pneumonic and septicemic pasteurellosis include severe depression, bilateral purulent nasal discharge, coughing, diarrhea, anorexia, high fever, and edema of the head, neck, and brisket. the disease course can be short with septicemic pasteurellosis and is usually more insidious with p. multocida pneumonia. pasteurella mastitis is characterized by the bluebag condition or gangrene of the udder. diagnosis. inflammatory changes in the leukogram and hyperfibrinogenemia are the most frequent abnormalities. with severe disease and in the septicemic form, immature neutrophils may predominate over mature cells. inflammation of the intestine and abomasum also may be seen. hemorrhage and fibrin are usually absent or less prominent than in pneumonia caused by m. haemolytica. samples for bacteriologic culture are usually obtained postmortem. blood or tracheal fluid may be obtained before death if the value of the animal warrants it. m. haemolytica is a gram negative rod that is a common commensal inhabitant of the tonsils of young animals. disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lower respiratory tract. clinical signs and diagnosis. the most common syndrome is enzootic pneumonia, which is seen in young lambs and their dams (see chapter ) . hemorrhagic bronchopneumonia is the major lesion and respiratory signs predominate. gangrenous mastitis (bluebag) is seen in some of the dams, presumably after they have been nursed by infected offspring. factors that promote respiratory disease, including viral infections, airborne irritants, high stocking density, and stress, are thought to promote invasion of the lower airway by these bacteria. b. trehalosi is a gram negative rod that is a commensal inhabitant of the upper respiratory tract (see chapter ) . disease is much more frequently described in sheep than in goats and occurs when the organism gains access to the lung or blood. replication occurs in the lung and systemic toxemia or bacteremia resulting in septicemic pasteurellosis. septicemic pasteurellosis is a significant cause of mortality in young lambs and in some farms is the leading cause of death in the age group. clinical signs. septicemic pasteurellosis occurs most commonly in weaned lambs, often following some form of stress such as transport, marketing, or weaning itself. the course of the disease is relatively rapid, and animals may be found dead within hours without showing premonitory clinical signs. when observed, clinical signs include depression, recumbency, and signs of toxemia. diagnosis. septicemic pasteurellosis should be suspected when presented with a dead, recently weaned, sheep with a recent history of stress. diagnosis is best confirmed by typical lesions at necropsy and culture of the organism from bodily tissues. demonstration of b. trehalosi in nasal swabs is of limited value due to the high prevalence of upper respiratory tract colonization in healthy lambs. at necropsy, there may be no evidence of pneumonia, but blood-stained foam can be found in the upper respiratory tract. ulceration of the pharynx and esophagus is commonly present as is subcutaneous hemorrhage of the neck and thorax. prevention. treatment is difficult due to the rapid course of disease. efforts should be made to minimize stressors, particularly during and following weaning, and to manage management factors that may contribute to the disease. vaccination with pasteurella bacterins is rarely effective at controlling natural outbreaks of disease. pathophysiology. abscess-forming bacteria are usually able to survive phagocytosis and thereby avoid destruction by cells of the immune system. alternatively, they invoke such an inflammatory response that the host body "walls off" the entire region with fibrous tissue. abscesses may occur locally, frequently after a wound infection, or at numerous or distant sites from the point of infection. for abscesses to occur at the latter sites, the organism must travel either by way of the blood or within leukocytes. disease characterized by multifocal or internal abscesses usually results from a low-grade, transient event of bacteremia. the best known and most important abscess-forming bacterium in small ruminants is corynebacterium pseudotuberculosis, the gram positive, facultative anaerobic coccobacillus that causes caseous lymphadenitis. infection is usually maintained in a flock by infected animals that spread the organism to others through purulent material draining from open abscesses. the organism is very hardy, so infection can occur through direct contact or indirect contact with contaminated common instruments and facilities. infection is usually introduced into a flock through acquisition of an infected animal, although it also can occur when a naive flock is moved into a contaminated area. horses, cattle, and humans also are minor hosts. infection is thought to occur after ingestion, inhalation, or wound contamination. except for lower respiratory tract invasion, a surface break is thought to be necessary. contaminated shears, tail-docking knives, and emasculators readily spread the organisms through a flock. abscesses can form at the site of invasion or more commonly at the site of the local lymph node. clinical signs. clinical signs of external abscesses include surface swellings and draining lesions. drainage may be intermittent and usually consists of thick, yellow-white purulent material. internal abscesses are more difficult to diagnose. thoracic masses may cause inspiratory dyspnea or occlude venous return to the heart. abdominal lesions may cause tenesmus, stranguria, and occasionally colic. the most common sign of internal abscesses is weight loss with or without intermittent fever. common external sites include the submandibular or retromandibular space and the preinguinal, prefemoral, and supramammary lymph nodes. head and neck lesions are more common in goats, whereas sheep have a more even distribution of cranial and caudal lesions, presumably as a result of shearing wounds. external infections rarely cause clinical illness beyond the draining abscess, although some degree of cachexia may be present. diagnosis. diagnosis is often made by the characteristic lesions with their thick, nonmalodorous pus. bacteriologic culture provides a definitive diagnosis, which may be important for flock management. serologic tests have been developed to identify carrier animals and may be useful if the manager wishes to eliminate infection from the flock. treatment. treatment is often unrewarding: antibiotic sensitivity profiles do not reflect the degree of protection afforded the organisms within the abscesses. long-term treatment with antibiotics and drainage of any compromising masses may lead to some degree of resolution, but internal abscesses are likely to persist. prevention. prevention through the use of vaccines has been attempted. vaccines appear to reduce the severity of the disease but do not completely prevent infection. moreover, live attenuated bacterins lead to de facto infection of all vaccinated animals and therefore should not be used in naïve flocks. other abscess-forming bacteria are most important as differential diagnoses for caseous lymphadenitis. t. pyogenes is another wound contaminant that affects focal areas or regional external lymph nodes. it also commonly colonizes damaged internal tissues such as postpneumonic lungs, postacidotic livers, and damaged feet and heart valves. it is thought to be ubiquitous and poorly invasive in ruminants and therefore does not have the same flock significance as c. pseudotuberculosis. flocks with outbreaks of this infection often have suboptimal management. f. necrophorum causes similar disease and often coinfects with t. pyogenes. it is generally more necrotizing and leads to greater systemic signs of acute illness, including death. f. necrophorum also produces fetid pus, whereas t. pyogenes usually does not. rhodococcus equi is a rare cause of pulmonary abscesses in sheep. numerous small, coalescent, nodular skin abscesses may result from pseudomonas pseudomallei infection (melioidosis). infection usually occurs after the sheep or goat is bitten by an insect that previously fed on an infected rodent. this organism is found in many subtropical regions, including the caribbean, but is not reported in north america. f. necrophorum causes or is associated with a variety of diseases in sheep and is likely to cause many similar diseases in goats. it is best known as a cause of foot rot and hepatic abscesses and appears to be important in lip-leg ulceration. it is an enteric gram negative anaerobe and as such can cause gram negative sepsis after entrance of the bacteria or its toxins into the circulation. f. necrophorum has a poor ability to invade healthy tissue. however, it readily colonizes regions damaged by trauma, persistent moisture, and infection. in addition to endotoxin, the bacterium produces leukocidal and cytolytic toxins that form zones of necrosis around bacterial colonies. this tissue necrosis and the foul-smelling waste gases produced by the bacteria are characteristic of necrobacillosis, or f. necrophorum infection. clinical signs include necrotic, fetid lesions, usually of the mouth or feet, that can cause ingestion or lameness problems. efforts to maintain good hygiene are helpful in preventing fecal contamination. additionally, preventing trauma to foot and mouth tissues through good surface choices and proper pasture drainage is important. pathogenesis. yersinia spp. are gram negative bacteria. yersinia enterocolitica and yersinia pseudotuberculosis both have many mammalian and avian hosts, including humans, and cause clostridial enteritis-like disease in goats. rodent and bird hosts may be important reservoir populations for infections in domestic animals. kids younger than months develop enteritis, bacteremia, and diarrhea that is watery but not bloody. severe toxemia and sudden death can occur. older kids and flocks with chronic exposure tend to have less severe acute disease. instead, chronic diarrhea and weight loss are seen, usually in association with gut wall and abdominal abscesses. sheep, deer, and wild ungulates are rarely affected. clinical signs. signs of enteritis or sepsis predominate in acute disease, whereas signs of wasting are more common in chronic disease. diagnosis. evidence of acute or chronic inflammation is provided by blood work. characteristic necropsy lesions include numerous microabscesses in the gut wall and mesenteric lymph nodes, as well as other evidence of enteritis or sepsis. culture of lesions and demonstration of a rising antibody titer are diagnostic. prevention. avoiding exposure to sources and maintaining overall flock health are helpful in preventing losses due to yersiniosis. pathogenesis. mycobacteria are small, aerobic, straight or curved pleomorphic rods with thick lipid cell walls. they can be stained with acid-fast stains and are usually gram positive. the bacteria live within infected animals of many mammalian species and survive for several years in warm, moist environments. infection occurs after ingestion or inhalation. an identifying characteristic of the mechanism of infection by mycobacteria is the bacteria's ability to survive within macrophages by preventing fusion of phagosomes and lysosomes. the organisms are carried to local lymphatic vessels or lymph nodes, where they form granulomas. as they enlarge, granulomas may develop necrotic or mineralized centers surrounded by macrophages and giant cells. disease can be local, regional, or generalized, depending on the distance the organism is carried from the original site of infection. granulomatous pneumonia, enterocolitis, and lymphadenitis are the most common local and regional forms of the disease. organisms from ruptured granulomas may be spread in contaminated respiratory secretions and feces. mycobacterial infections of all types are uncommon in north american sheep, goats, and cervids, and these species are considered to be relatively resistant to infection. mycobacterium bovis is the most common organism associated with ovine tuberculosis in other countries (see chapter ), but mycobacterium avium is more common in the united states. the most common mycobacterial infection is johne's disease (paratuberculosis) caused by the etiologic agent m. avium subsp. paratuberculosis (see chapter ) . mycobacterium tuberculosis is rare in the united states. mycobacterial infections are reportable in most parts of the united states. some debate is ongoing about human susceptibility to m. avium subsp. paratuberculosis; the other organisms are known to be pathogenic in people. clinical signs. the most common clinical sign is emaciation. diarrhea may be seen terminally in both tuberculosis and paratuberculosis. the disease is insidious, with signs becoming more apparent over several weeks to months. respiratory signs may be seen, especially with infection by m. bovis or m. avium subsp. diagnosis. reports of clinicopathologic abnormalities are rare. hypoalbuminemia and hypoproteinemia are likely to be common with chronic enterocolitis caused by either tuberculosis or paratuberculosis. the most common necropsy lesions seen in tuberculosis are nodular lesions of the lung, liver, lymph nodes, spleen, and intestines. histologic evaluation reveals the nodules to be granulomas with giant cells and acid-fast organisms. frequently, the center of the lesion is necrotic and mineralized. intestinal lesions appear to be more common than pulmonary lesions in goats. the lesions of paratuberculosis are centered around the ileocecocolic junction and the adjacent mesentery. the regions may appear normal or be notably thickened. thickening of bowel or nodular infiltrates of lung or liver may be detected antemortem using imaging modalities, such as ultrasonography or computed tomography. postmortem diagnosis is made by identifying characteristic lesions and culturing the organisms. antemortem diagnosis of tuberculosis is best achieved by observing the reaction to intradermal injection of tuberculin with or without comparative injection of purified protein derivatives of m. bovis and m. avium subsp. paratuberculosis. all tuberculosis testing should be done in accordance with local regulations. antemortem diagnosis of johne's disease can be achieved by fecal culture of the organism, but this test takes several weeks to months to complete and is far less reliable in sheep or goats than cattle, with a sensitivity as low as . . serologic tests (e.g., elisa) appear to be sensitive and specific for johne's disease in animals demonstrating clinical disease rather than preclinical infection. serologic detection of clinical johne's disease in cervids has been shown to be highly sensitive and specific while the sensitivity of fecal culture is low in both sheep and goats. the recommended organism detection method in both species is fecal pcr. fecal or milk pcr can be used on pooled samples for flock identification and to type the organism. prevention. tuberculosis should not be endemic in flocks in the united states because positive animals are quarantined or destroyed. preventing exposure to wild ruminants and other possible sources is crucial. except in goat flocks raised for the production of milk that is to be sold unpasteurized, testing is uncommon, so animals are usually not identified until they develop overt disease. paratuberculosis is much more common and may be maintained in flocks by carrier animals. no effective treatment is available for either disease, nor should any be encouraged because efforts should be concentrated on eliminating infection from the flock or herd. vaccination of sheep is used extensively in australia to control paratuberculosis. prolonged vaccination has been shown to decrease fecal shedding in infected animals over time. pathogenesis. mycoplasma spp. are very small, simple bacteria that parasitize cells of higher species. they are common inhabitants of mucous membranes and can have either a commensal or pathogenic relationship with the host. transmission between animals is most likely through direct or indirect contact with body fluids from infected animals, inhalation of respiratory droplets, and arthropod vectors. common sites for superficial infection include the ocular membranes, lung, mammary gland, and female reproductive tract. the organisms can also enter the blood and cause septicemia, abortion, pleuritis, and polyarthritis. flare-ups often occur during times of crowding and during parturition, when neonates can spread the organisms from the mother's mouth to her udder and in turn become infected by ingesting contaminated milk. the most important mycoplasma species in the united states are mycoplasma conjunctivae, mycoplasma capricolum, and the less pathogenic mycoplasma ovipneumoniae. they are most commonly associated with keratoconjunctivitis, acute or chronic sepsis, and pneumonia, respectively. m. conjunctivae and c. abortus are the most common causes of pinkeye in north american small ruminants. mycoplasma spp. are thought to inhibit tracheal ciliary function and thus may have a role similar to viruses in "shipping fever pneumonia" in facilitating lower respiratory tract invasion by primary bacterial pathogens. many of the major pathogenic serotypes found in other countries (some of which cause severe pleuropneumonia without the participation of another bacteria), including mycoplasma mycoides subsp. mycoides, mycoplasma mycoides subsp. capri, mycoplasma agalactiae, and strain f , are not found in or have been eradicated from north america clinical signs. keratoconjunctivitis, mastitis, exudative vulvovaginitis, fever, cough, dyspnea, exercise intolerance, abortion, lameness, swollen joints, neonatal death, and depression may all be seen with mycoplasma infections. diagnosis. no specific clinical pathologic findings occur with these diseases. mycoplasma infection should be suspected in sheep and goats with severe exudative pleuropneumonia in some parts of the world. mycoplasma can be identified by bacteriologic culture or staining of exudates. examiners must take care in interpreting positive cultures from body surfaces because nonpathogenic mycoplasma are common. prevention. vaccines against mycoplasmal infections are available in some parts of the world, but not in the united states. providing fly control, preventing stress and overcrowding, and isolating sick animals from healthy ones may help prevent the spread of disease. anaplasma ovis, mycoplasma ovis, and babesia spp. a. ovis and m. ovis are small bacteria that lack cells walls and parasitize erythrocytes. these and similar organisms have undergone recent reclassification following molecular analysis. other species of hemotropic mycoplasmas may affect sheep and cervids. the organisms are spread from animal to animal by insect or mechanical vectors. known arthropod vectors for a. ovis include ticks and horseflies; other biting flies may be more important with m. ovis infection. hypodermic needles and equipment used for tail-docking, castrating, or disbudding animals may be important in iatrogenic transmission. after being introduced into a naive host, the organisms proliferate, and the number of red cells infected increases rapidly until an effective immune response begins to weeks later. a similar proliferation of organisms may occur in chronically infected animals after temporary immune suppression. the humoral and cellular immune responses against a. ovis lead to opsonization of parasitized erythrocytes and their removal by cells of the reticuloendothelial system; m. ovis infection is thought to cause more intravascular hemolysis. the result in both cases is hemolytic anemia. the protozoon parasites babesia ovis and babesia motasi have similar life cycles and cause similar diseases, but they have been eradicated and are reportable in the united states. babesia spp. affecting small ruminants are generally less pathogenic than are their bovine counterparts. animals surviving acute hemolytic crisis reduce the parasites to low numbers but rarely clear the infection completely; they serve as sources of infection for other animals. sheep and goats are susceptible to infection by either organism; goats generally appear to be more resistant to the development of severe parasitemia and clinical signs. clinical signs. signs present during hemolytic crises include fever, weakness, pale mucous membranes, and pigmenturia. urine discoloration results from increased amounts of bilirubin in most cases, although hemoglobinuria may be seen in some sheep with m. ovis infection. icterus is usually present only after the acute hemolytic crisis. clinical signs are exacerbated during times of stress, and infection is often first noted when the animals are moved or handled. chronically infected animals may appear clinically normal, may have recrudescence of infection after stress, or may display signs of ill-thrift such as poor body condition and fleece. babesiosis occasionally causes concurrent central neurologic signs. diagnosis. the major clinical laboratory finding is regenerative anemia with detection of the intraerythrocytic bodies. chronically infected sheep often have high counts of nucleated erythrocytes. because m. ovis consumes glucose, hypoglycemia and metabolic acidosis may be detected, especially in blood samples that are not processed immediately. diagnosis is by identification of the organisms on blood smears. special stains are available to make the organisms more visible. postmortem lesions include pallor or icterus of membranes and splenomegaly. some evidence of vasculitis, including edema or exudates in body tissues or cavities, may be seen with m. ovis infection. treatment. mycoplasma spp. and anaplasma spp. are sensitive to tetracycline antibiotics. babesiosis is more difficult to treat. effective drugs include diminazene, pentamidine, and imidocarb dipropionate. supportive care for all blood parasite infections includes whole blood transfusions, nutritional support, and administration of fluids. prevention. prevention in most cases involves maintaining low levels of parasites rather than eliminating them entirely. this method ensures continual stimulation of the immune response, whereas eradication often leaves the animal susceptible to another bout of acute infection. vector control can also be important in management of the disease. pathogenesis. two organisms belonging to the anaplasmataceae family, ehrlichia ovis and anaplasma phagocytophilum, infect ovine wbcs, causing fever, immune suppression, and some organ damage. a. phagocytophilum is the causative agent of tick borne fever in sheep and granulocytic anaplasmosis in horses, dogs, and humans. the organism is transmitted by ticks (ixodes spp.) and maintained in the environment by asymptomatic carrier animals. the distribution and incidence of disease is seasonal with the life cycle of the tick. the organism infects cells of the granulocytic lineage, leading to severe persistent neutropenia and acute lymphopenia. fever occurs to weeks after infection, lasts as long as weeks, and occasionally relapses. chronic infection is common. spleen, lung, liver, and kidney tissue may show some damage because of immune destruction of infected cells, but organ-specific signs are usually the result of secondary infection. secondary bacterial joint infections in lambs infected with a. phagocytophilum develop debilitating lameness known as tick pyemia. e. ovis causes fever (benign ehrlichiosis) to weeks after infection. because of this organism's predilection for mononuclear cells, the degree of immunosuppression and subsequent importance of this disease are much less than for a. phagocytophilum infection. diagnosis. specific diagnosis is best made by identifying darkly stained bodies at the periphery of granulocytic cells, as well as occasional large bodies deep within the cytoplasm of some cells. stained bodies also can be seen on the periphery of mononuclear cells from a blood smear during the acute febrile stage or in tissues during chronic infection. serologic tests are available for detection of anaplasmosis. the available celisa is incapable of distinguishing species of anaplasma and serologic results must be interpreted appropriately, and the species confirmed by pcr. both infections affect sheep and goats (a. phagocytophilum also affects many other ruminants, including white-tailed deer), but neither has been reported in north america. a recent study demonstrated that sheep are capable of being experimentally infected with a human isolate a. phagocytophilum. interestingly, the sheep did not develop clinical disease. such findings suggest that sheep could serve as asymptomatic carriers and potential reservoirs for humans. a. phagocytophilum is widespread in northwestern europe, including the united kingdom, scandinavia, and india, and e. ovis is found mainly in countries bordering the indian ocean. in spite of documented seropositive status of animals, there have been no reports of sheep or goats naturally infected with a. phagocytophilum in the united states developing clinical disease. treatment and prevention. treatment and prevention efforts should focus on reducing vectors and bacterial counts during vector season. both organisms are susceptible to treatment with tetracycline. people and animals can become infected with trypanosome protozoa. the trypanosomes can complete their developmental cycle only in tsetse flies (glossina species). trypanosomes multiply in blood, tissues, and body fluids of their vertebrate hosts and are transmitted between vertebrate hosts in the saliva of blood-sucking flies as they feed. the trypanosome species that are known to infect goats and sheep include trypanosoma congolense, trypanosoma vivax, trypanosoma brucei subsp. brucei, trypanosoma evansi, and trypanosoma simiae. pathogenesis. after entering through the skin, trypanosomes reach the bloodstream by way of the lymphatic system. the parasites multiply, and the prepatent period lasts for to days after infection. the infection is characterized by periods of parasitemia, followed by the absence of parasites. this pattern of infection occurs because of antigenic variation: trypanosomes vary the antigenic nature of their glycoprotein surface coat to evade the host's immune system. this immune system-evasive maneuver prolongs infection and is responsible for chronic disease. some trypanosomes tend to invade extravascular spaces, such as the ocular aqueous humor and cerebrospinal fluid. the pathogenicity of trypanosomes varies with the different host species. trypanosomes may produce a hemolysin early in the course of the disease that causes anemia in the host. later, increased phagocytic activity results in massive erythrocyte destruction. clinical signs. the clinical signs are variable and non-specific and depend on the speed of onset of anemia and the degree of organ impairment. entire herds may be affected. all aspects of production are impaired-fertility, birth weight, lactation, weaning weight, growth, and survival. trypanosomiasis may predispose the animal to the development of other diseases that mask the underlying trypanosome infection. trypanosomiasis may be acute, subacute, or chronic, with chronic infection occurring most commonly. acute disease often causes abortion. dairy goats may show a sudden drop in milk production. depression, anorexia, and a stiff gait may be present. physical examination reveals tachycardia, tachypnea, and a slight fever. hyperemic mucous membranes and excessive lacrimation may be noted. affected animals often become recumbent and anorexic and die within to weeks of onset of clinical signs. if the animal survives, progression to the subacute phase, characterized by listlessness, weight loss, enlargement of superficial lymph nodes, and a dull, dry hair coat, may occur. in such cases, auscultation findings are similar to those in other forms of acute cardiac disease, as well as pale mucous membranes and a pronounced jugular pulse. the animal may linger for several weeks or months, or the chronic form of the disease may develop. affected animals show ill-thrift: dull and dry hair coat, inelastic skin, lethargy, emaciation, peripheral lymphadenopathy, pale mucous membranes, and exercise and stress intolerance. death may occur many months or even years after infection and usually results from congestive heart failure. subclinical trypanosomiasis causes acute episodes when animals are stressed by inadequate nutrition, increased production demands, or concurrent disease. diagnosis. diagnosis is difficult because the parasitemia is intermittent, clinical signs are non-specific, and infection is not always synonymous with disease. a pcr assay is gaining acceptance as the most sensitive diagnostic modality, but not all infected animals exhibit clinical disease. although a tentative diagnosis of pathologic trypanosomiasis can be made on the basis of history, clinical signs, and the presence of appropriate vectors, a definitive diagnosis requires identification of trypanosomes on a fresh blood smear, a giemsa-stained blood smear, or less commonly, a lymph smear. examination of the buffy coat of centrifuged blood with darkfield phase-contrast spore illumination is the most sensitive direct microscopic method and is useful when parasite numbers are low. pathogenic trypanosomes must be distinguished from more ubiquitous, nonpathogenic species particularly common in cattle, such as trypanosoma theileri. repeated blood sampling in individual animals often is necessary, because as noted, parasitemia is intermittent. the diagnosis is supported by evidence of anemia on a cbc. indirect diagnostic methods include an indirect fluorescent antibody test and the elisa. these tests are less helpful for diagnosis of a single clinical case but are useful in assessment for herd infection. both t. congolense and t. brucei readily infect rats and mice, and detection of these pathogens can be used to diagnose the infection indirectly. treatment. treatment consists of the use of trypanocidal agents and supportive care. animals with acute, subacute, and subclinical disease respond better to treatment than those with chronic disease because of the irreversible damage to hematopoiesis associated with chronic infection. with most trypanocides, the therapeutic index is low and varies with the host species. trypanocide efficacy also varies with the species of trypanosome present; resistance to agents is common. some trypanocides are irritating to the skin and may cause severe inflammation at the injection site. in sheep and goats with t. brucei infection, the trypanocide of choice is diminazene aceturate, which should be used at a higher dosage rate ( mg/kg given intramuscularly [im] or sc) than that recommended for cattle. protection after trypanocide use usually lasts to months, depending on the season. animals must be rested before and after treatment. supportive care consists of providing fluids, an environment conducive to rest, good nutrition, and possibly blood transfusions. prevention. vector control, stress and nutrition management, and selection of trypanosome-tolerant breeds of sheep and goats all help control or prevent trypanosomiasis. no vaccine is available. animals can be treated with insecticides (pyrethroids) to prevent bites by tsetse flies and other flies. control is accomplished by strategic use of trypanocides during the peak season. continued parasitologic and clinical surveillance is essential to determine the efficacy of control measures. pathogenesis. sarcocystis spp. are protozoon parasites that have a two-host life cycle. sexual reproduction occurs in the bowel of a carnivore (mainly dogs and wild canids) after the carnivore ingests cysts in the muscles of sheep, goats, and cervids. sporocysts are passed in the carnivore's feces and later ingested by a sheep, goat or cervid. the sporocysts hatch in the ruminant gut and invade the vascular endothelium during three phases of asexual reproduction. after the third phase (approximately to weeks after ingestion), merozoites enter the ruminant's muscle tissue and encyst. clinical signs are uncommon but can occur during the stages of reproduction and muscle invasion of the host. n. caninum has a similar life cycle and causes similar disease, except that it appears more likely to cause abortion and affect the central nervous system. clinical signs. most infections are asymptomatic. however, if a large number of sporocysts are ingested, tissue damage may occur during the intestinal, vascular, and muscle stages of the sarcocystis life cycle. fever, lameness or a stiff gait, reluctance to move, and diarrhea may be seen. central neurologic signs (blindness, changes in mentation, and seizures) may occur if the organisms invade the brain or interrupt blood flow to it. abortion can occur as early as weeks after ingestion. with severe chronic infections, emaciation and anorexia are seen. diagnosis. the most characteristic abnormality is an increase in muscle enzyme activity in the blood. anemia is common and may result from extravascular hemolysis. cerebrospinal fluid may show mild mononuclear pleocytosis or may appear normal. on necropsy, muscles may display pale streaks or macroscopic cysts throughout. other evidence of vasculitis includes hemorrhagic serosal surfaces, body cavity fluids, and lymphadenopathy. microscopic or ultrastructural examination of affected tissues should reveal the presence of organisms. specific antibody tests are available and do not cross-react with t. gondii antibodies. blood antibody titers often peak around the onset of clinical signs and should be markedly higher than baseline values. antibody preparations also are available for identification of organisms in tissue preparations. treatment. sheep infected with sarcocystis species can be treated with salinomycin ( ppm in complete feed), monensin ( . - mg/kg po), or amprolium ( - mg/kg po). drugs such as sulfadiazine or trimethoprim ( - mg/kg im sid), pyrimethamine ( . - mg/kg po sid), and clindamycin have shown some success in treating neospora infections. these treatments are off-label and thus are governed by regulations regarding extra-label drug use. prevention. preventing contamination of feedstuffs with the feces of infected carnivores and preventing ingestion of raw meat by carnivores are most important, but these measures may not be possible in flocks handled with dogs or those living on range land. anticoccidial drugs appear to decrease the chance of clinical disease. pathogenesis. t. gondii is a protozoon parasite with a life cycle very similar to sarcocystis, except that the definitive host is the cat and that a wider range of mammalian and avian species, including humans, appear to be capable of acting as intermediate hosts. sporocysts are infective a few days after passage in cat feces, and most ruminants are infected by eating feed contaminated with cat feces. people can become infected by ingesting raw meat or milk from infected animals. abortion, stillbirth, and neonatal death are the most common forms of clinical disease in sheep and goats, and toxoplasma should be considered one of the most common causes of perinatal losses in small ruminants (see chapter ) . abortion usually occurs during the final month of pregnancy. fever, vasculitis-induced disease, and neurologic disease are less common manifestations. clinical signs. beyond abortion, clinical disease is rare in adults and resembles systemic sarcocystosis. clinical signs include fever, dyspnea, depression, and anorexia. neurologic signs are more common than with sarcocystis infection, especially in lambs and kids infected in utero. diagnosis. no specific laboratory abnormalities are associated with toxoplasmosis. nodular lesions similar to sarcocysts may be seen in various tissues, including the brain. aborted or stillborn fetuses may appear normal except for histologic lesions in the brain, liver, or lung, but more commonly fetuses are macerated. the placenta is usually abnormal, with gross and microscopic evidence of necrosis of the cotyledons. microscopic identification of the organism in body tissues is the most common means of diagnosis. serologic tests also are available. treatment and prevention. drugs similar to those used to treat neospora may be effective against toxoplasma. preventing contamination of feeds with cat feces and preventing ingestion of dead animals by cats are the most important ways of stemming the spread of this organism. both methods are likely to be difficult in most flocks. direct spread from one animal to another is rare. clinical signs. bluetongue disease has two different manifestations-reproductive problems (see chapter ) and acute vasculitis of several organ systems. with vasculitis, a spiked fever often precedes depression, anorexia, and rapid weight loss. leukopenia is present. affected animals may develop edema of the lips, tongue, throat, ears, and brisket. other signs include excessive salivation and hyperemia or cyanosis of the oral mucosa, including the tongue (hence the name bluetongue). affected sheep often produce profuse serous nasal discharge that soon becomes mucopurulent and produces crusts and excoriations around the nose and muzzle. oral lesions progress to petechial hemorrhages, erosions, and ulcers. pulmonary edema is often severe, and pneumonia may develop. skin lesions can progress to localized dermatitis. affected sheep may exhibit stiffness or lameness because of muscular changes and laminitis. cyanosis or hemorrhagic changes of the skin of the coronet can extend into the horny tissue. after recovery, a definite ridge in the horn of the hoof may be present for many months. in severe cases, the hoof sloughs. mortality varies widely. in africa, the virus is much more virulent than in the united states, and mortality ranges from to %. the reproductive or teratogenic form of the disease varies greatly with strain, host, and environmental factors. teratogenic effects include abortions, stillbirths, and weak, live "dummy lambs." congenital defects may include hydranencephaly. diagnosis. in parts of the world where the disease is common, the diagnosis is usually based on clinical signs alone. the virus can be isolated from blood, semen, or tissues (spleen and brain from aborted fetuses). viral isolation from blood obtained during the viremic state is the most definitive means of diagnosis. serologic evaluation involves two types of viral antigen groups called p and p . the former is found in all bluetongue viruses, and the latter determines the serotype. sera are commonly tested with complement fixation, agar gel immunodiffusion (agid), or one of several elisa tests. a competitive elisa is considered the best serologic test for detecting group antibodies to bluetongue virus. a direct fluorescent antibody test is available. molecular tests (e.g., pcr) for bluetongue have recently become available and are extremely sensitive and specific. they can be useful for distinguishing serotypes. other clinicopathologic signs that aid in diagnosis include leukopenia during the early febrile stage of the disease and an increase in serum ck corresponding to the latter phase of muscle stiffness and lameness. treatment. treatment is non-specific and consists of nursing care. because of the reluctance of animals to eat, they should be fed a gruel of alfalfa pellets by stomach tube or encouraged to eat soft feeds and green grass. broad-spectrum antimicrobials are often used to treat secondary pneumonia and dermatitis. animals should be kept on soft bedding with good footing. water and shade should be readily available. nsaids are commonly used. prevention. the culicoides vector is difficult to eliminate, so animals should be kept indoors during periods of peak gnat activity (dusk and early evening). owners should attempt to eliminate gnat breeding grounds such as overflowing watering troughs and shallow septic systems and should limit exposure of sheep to gnats with the use of repellent sprays. modified live vaccines based on local strains and serotypes are available in some parts of the world. some cross-protection among serotypes does occur. the vaccine should be administered at least weeks before breeding season to prevent teratogenic effects. vaccinated breeding rams may have a slight risk of decreased fertility. lambs can be vaccinated in the face of an outbreak. pregnant animals cannot be vaccinated with modified live vaccines. sheep that have recovered from an attack of bluetongue are solidly resistant for months to infection by the same viral strain and to some other viral types. active immunity in sheep requires both humoral and cellular immunity. etiology. epizootic hemorrhagic disease virus (ehdv) is an orbivirus belonging to the family reoviridae. the virus is structurally related to bluetongue virus, and the pathogenesis and clinical signs of disease resulting from these two viral infections are very similar. at least seven distinct serotypes of ehdv are recognized, although formal classification of serotypes has yet to be finalized. only two serotypes (ehdv and ehdv ) have historically circulated throughout north america, and those serotypes are largely considered to be endemic in almost all areas of the united states, with the exception of the northeast and arid areas of the southwest. however, in , ehdv was isolated from surveillance efforts in dead white-tailed deer. since then, ehdv has been increasingly identified from both surveillance samples and clinical cases and is also believed to be endemic in several regions. pathogenesis. epizootic hemorrhagic disease (ehd) is a noncontagious disease that is transmitted by the culicoides biting midges. culicoides sonorensis is the primary vector of ehdv in the united states, although other species are also suspected to transmit the disease based on the geographic distribution of clinical cases, although this has yet to be formally shown. due to the vector-borne route of transmission, peak incidence of the disease is closely associated with peak vector population, namely, in the late summer and fall of the year. although capable of infecting a wide range of wild and domestic ruminants, ehdv is largely a pathogen of wild cervids, particularly white-tailed deer. episodes of clinical disease are less common in mule deer, pronghorn antelope, and bighorn sheep and have lower morbidity and mortality. sheep are only rarely infected with the virus and goats appear to be resistant to the virus. cattle are commonly infected based on seroprevalence surveys, but overt clinical disease is uncommon. as a rule, infection in livestock is usually asymptomatic except for periodic epidemics. the last major ehd epidemic in the united states occurred in and affected a variety of captive and wild ruminant species. in endemic areas, seroprevalence in cervids and other ruminants is high, but clinical disease is not commonly seen. conversely, where seroprevalence is low, introduction of the virus results in widespread infection, where morbidity and mortality can reach % and %, respectively. following transmission of the virus by biting midges, ehdv replicates in the endothelial cells of the lymphatics surrounding the site of the bite. a primary viremia allows for systemic spread of the virus and secondary replication in lymph nodes throughout the body and the spleen. viremia is important for disease propagation and generally lasts no more than weeks following infection, although the virus can occasionally be isolated from deer infected days previously. antibodies to ehdv are first detected to days following infection but are not always capable of completely neutralizing the infection. thus, it is possible to find both neutralizing antibodies and live virus in the same animal. passive antibodies in fawns can be found up to approximately months of age. as in adults, antibodies in fawns may not protect from infection but generally protect from severe clinical signs. clinical signs. clinical disease in white-tailed deer can be peracute, acute, or chronic. the course of the peracute syndrome of diseaseis relatively short, with death often occurring within hours of infection, with or without the presence of clinical signs. when present, clinical signs include severe edema of the head and neck, swelling of the tongue and conjunctiva, anorexia, fever, weakness, and respiratory distress. hemorrhagic diatheses are not present antemortem but may occur after death. in contrast, in the acute form of the disease, the clinical signs of the peracute form are accompanied with bleeding throughout body tissues (figure . a, b) . ulcers may be evident in the oral cavity and throughout the upper gastrointestinal tract, forestomachs, and abomasum. case fatality rates are high for both the peracute and acute forms. deer that recover after several weeks of illness are said to suffer from the chronic form of the disease. signs of previous illness may include breaks or rings in the hoof horn due to interrupted growth and synthesis leading to lameness, sometimes severe. ulceration and scarring of the rumen and gastrointestinal tract may result in loss of body condition despite a seemingly normal appetite and ample nutrition. widespread evidence of vasculitis may be observed histopathologically. diagnosis. the gold standard for ehdv diagnosis is virus isolation. demonstration of neutralizing antibodies to ehdv reference strains is evidence of previous infection but may be of limited value in endemic areas where seroprevalence levels are expected to be high. also, all potentially suspected serotypes must be used when testing the sample, thereby increasing the time and cost involved with the test. continued research and refinement of molecular techniques, including pcr, are ongoing and are attractive due to the short turnaround times and the potential for high throughput of samples. however, it is important to remember that a positive result using molecular techniques does not equate to the presence of infectious virus, and thus, interpretation of results must be done with caution. control. control of ehd is difficult and relies on a combination of disease surveillance, vector control, and potentially, vaccination. eradication of vector-borne diseases from endemic areas is difficult and time-consuming, and thus, disease control is likely more attainable than strict eradication. vector control is more important in the late fall and summer, when populations are at peak levels and viral transmission is more likely. midge-proofed housing and the treatment of animals with pyrethroid insecticides have been attempted but may be logistically challenging and have yet to have been demonstrated efficacious. vaccine availability in north america is limited, but inactivated autogenous vaccines have been developed from isolates obtained from ill or recently diseased animals. autogenous vaccines are tested for purity but not necessarily for efficacy. vaccine usage must be approved by the u.s. department of agriculture prior to administration. etiology. peste des petits ruminants (ppr) is an acute or peracute, febrile, often fatal disease of ruminants caused by a virus in the family paramyxoviridae and genus morbillivirus. sheep are less susceptible than goats and white-tailed deer. cattle are only subclinically infected, and some wild ungulates, as well as camels, appear to suffer the occasional epizootic. the virus (pprv) is serologically related to the virus that causes rinderpest. geographically, the virus is found throughout northern africa, the middle east, and adjacent regions of asia, with possible movement into southern africa and europe noted. pathogenesis. the main route of infection is respiratory, and ppr is spread by airborne droplets. all secretions and excretions of infected animals are contagious throughout the course of the disease, but no carrier state exists. the virus targets lymphoid tissue. lymphocytes are destroyed in germinal centers in lymph nodes, peyer's patches, tonsils, splenic corpuscles, and cecal lymphoid tissue. immunosuppression results from lymphoid destruction. lymphocytes are partially replaced by plasma cells, macrophages, an eosinophilic acellular matrix, and occasionally neutrophils. the epithelial lining of the mouth and digestive tract is highly vulnerable to the pprv. with the loss of the alimentary tract mucosa, weight loss and diarrhea become severe. the incubation period is usually to days, with up to days possible. clinical signs. the clinical disease produced by pprv in sheep and goats closely resembles that of rinderpest, but the course is much more rapid. with the acute form, sheep and goats typically display an abrupt rise in temperature to ° to ° f ( °- ° c). within a few days, infected animals develop nasal and lacrimal discharge, depression, thirst, anorexia, and leukopenia. congestion of the conjunctival and other mucous membranes occurs, followed by serous and mucopurulent exudates. sheep and goats develop oral erosions with necrotic foci, which results in excessive salivation. diarrhea that may be profuse but rarely hemorrhagic develops within to days and is accompanied by abdominal pain, tachypnea, emaciation, and severe dehydration. bronchopneumonia, particularly that caused by pasteurella spp., may be a terminal • fig. . a. the lungs of the adult pen-raised, white-tailed deer, have been retracted to reveal to ecchymoses on the ventral surface of the "ribcage." petechiae and ecchymoses can occur anywhere within the carcass in cases of epizootic hemorrhagic disease (ehd), but common locations are on the epicardium, on the pleural surface the ribs, subcutaneously, and on the surface of the spleen. b. ecchymoses over the surface of the reticulum (bottom right of photo) and the surface of the rumen (left side of photo). in addition to ehd, this deer also had bronchopneumonia (fibrin overlying consolidated lung can be seen in the far right of photo). (courtesy dr. kelley steury, auburn, al.) a b sequela. death usually occurs to days after the onset of fever. pregnant sheep or goats with ppr may abort. diagnosis. a presumptive diagnosis of ppr can be made on the basis of clinical, pathologic, and epizootiologic findings. the diagnosis can be confirmed by isolating the virus from blood or tissues, including lymph nodes, tonsils, spleen, and lung. immunocapture elisa or pcr may be used to detect infection several days before the development of clinical disease. most serologic tests (complement fixation or agid) cannot differentiate between ppr and rinderpest. characteristic postmortem findings include necrotic stomatitis that is generally confined to the inside of the lower lip and adjacent gum, the cheeks near the commissures, and the ventral surface of the free portion of the tongue. abomasal erosions are often present. in the small intestine, peyer's patches are markedly affected, particularly in the first portion of the duodenum and terminal ileum. the large intestine may be severely affected. lesions occurring near the ileocecal valve, at the cecocolic junction, and in the rectum are often described as zebra stripes that indicate areas of congestion along the folds of the mucosa. treatment and prevention. infection with pprv has no specific treatment. mortality can be reduced by supportive care, including the administration of antimicrobial and antiinflammatory agents, as well as nutritional support. in the united states, state and federal veterinarians should be notified if pprv is suspected. methods used to eradicate rinderpest are useful in the eradication and control of ppr. all sick sheep and goats and those exposed should be slaughtered and disposed of by burning, burying, or rendering. the premises should be decontaminated, and the area quarantined. sheep and goats can be protected against ppr by immunization with rinderpest vaccines or by the simultaneous administration of ppr hyperimmune bovine serum and virulent pprv. pathogenesis. louping ill is a tickborne disease caused by a flavivirus. it affects mainly lambs but occasionally also affects other livestock species and infrequently affects deer, camelids, and humans. transmission is most common during tick season, and ixodes ricinus is thought to be the most important infective host. many sheep clear the infection after a few days of fever and viremia, but others develop severe, fatal viral encephalitis. the virus is shed in many secretions, including milk, which is an important source of infection for other animals (and humans). the severity of the disease depends on herd immunity because previous exposure gives long-lasting immunity. colostrum from immune females is protective for the neonate. high antibody titers also appear to shorten the duration and level of viremia and thereby prevent invasion of the central nervous system. naïve flocks may have fatality rates as high as %. clinical signs. high biphasic fever, anorexia, and depression are seen in most infected sheep. lambs may die quickly before illness is noted. some sheep also develop central neurologic signs, including hyperexcitability, muscle tremors, and rigidity. abnormal coordination and muscle activity may cause sheep to move with a bounding gait (hence the name louping ill). diagnosis. the condition has no characteristic gross lesions. microscopic examination of animals with neurologic signs reveals evidence of viral meningoencephalitis. diagnosis is made by history (based on location, signs, and time of year), the identification of characteristic lesions, virus isolation, or fluorescent antibody staining of fresh brain tissue. a demonstrated increase in specific antibody titers in survivors strongly suggests the presence of this infection. prevention. vaccines are available in endemic areas to control infection. vector control during tick season also is important. lambing season should also be timed so that lambs have high colostral antibody protection at the time of exposure to ticks. pathogenesis. foot-and-mouth disease is caused by a highly contagious picornavirus and has been eradicated from the united states. vesicular stomatitis is caused by a rhabdovirus and is intermittently eradicated from the united states. both diseases are highly contagious, nearly indistinguishable from each other clinically, and reportable. foot-and-mouth disease has a broad host range that includes most hoof stock (including pigs but not horses) and several other mammalian species. vesicular stomatitis also affects many species of hoof stock, including both pigs and horses. sheep and goats are relatively less susceptible than cattle, particularly to vesicular stomatitis. the viruses are spread by aerosol and mechanical vectors and primarily colonize skin or mucous membranes. milking machines, flies, birds, and humans all may be important mechanical vectors. vesicular stomatitis tends to remain at the site of infection, and colonization is facilitated by damage to the skin. oral mucous membranes, coronary bands and interdigital skin, and teat-end skin are common sites of lesions. vesicular stomatitis outbreaks in the united states tend to occur in the summer or fall and end with the first killing frost. viremia plays more of a role with foot-and-mouth disease. the virus is present in most body tissues and fluids in infected animals and can be transmitted through milk, meat, bone, and hide products; semen; equipment that pierces the skin; and biting arthropods. it also tends to spread through the circulation from the site of infection to other susceptible tissues, including the sites of vesicular stomatitis, as well as to the nasal cavity, mammary glandular epithelium, and ruminal pillars. the basic lesion for both diseases are the vesicles that form in the oral cavity and on the teats and coronary band. the vesicles quickly rupture and may not be visualized before forming erosions. ruptured vesicles leave deep erosions on the skin or mucous membranes and appear to cause pain. tissue damage and inflammation are often compounded by secondary bacterial infection, which can cause greater morbidity and mortality than the original viral infection. morbidity is related to feed refusal, increased recumbency, and secondary infections of the mouth, udder, and feet. clinical signs. sheep and goats usually develop minor lesions, if any, and are more important in many outbreaks as transport or multiplying hosts than as primary clinical cases. however, identification of lesions should raise suspicion of this disorder. in the worst cases, vesicles, erosions, and ulcers are seen at target sites. they may appear mildly inflamed and erythematous; if they are infected, they may appear severely inflamed with hemorrhage and necrosis. other signs vary according to the location and severity of the lesions. lingual and buccal lesions cause salivation, dysphagia, and feed refusal. foot lesions, which are the most common clinical manifestation in small ruminants, cause lameness and recumbency. teat lesions cause reluctance to be milked or nursed and a decrease in production. fever also may be seen early in the disease, when vesicles are most apparent. the fever then usually abates, and vesicles are replaced by erosions or ulcers. abortion may occur, especially with foot-and-mouth disease, and is probably related to the fever rather than to fetal infection. the disease is usually self-limiting; most animals recover within to weeks. shedding of the virus causing vesicular stomatitis is thought to subside soon after healing of lesions. foot-and-mouth disease virus may be shed for as long as months, and all body secretions and tissues should be considered contagious, including milk, semen, meat, and offal. both viruses have zoonotic potential and cause a disease in humans that resembles mild influenza. the diseases are self-limiting, but people can shed the viruses in sufficient quantities to infect other animals. diagnosis. no characteristic clinicopathologic changes are reported for either virus. gross lesions resemble those seen before death and include vesicular, erosive, and ulcerative lesions of the mouth, feet, and teat ends; foot-and-mouth disease also causes lesions of the mammary gland and ruminal epithelium. microscopic findings include hydropic degeneration of cells of the stratum spinosum of the epidermis without inclusion bodies. secondary bacterial infection may lead to deeper ulcers and complicate identification of the viral etiology of these lesions. myocarditis lesions may be seen with some forms of foot-and-mouth disease. a presumptive diagnosis may be made by identifying characteristic lesions during a season and in an area at risk for one of these infections. in north america, bluetongue should be considered as an important differential diagnosis for ulcerative oral lesions in sheep. a confirmed diagnosis of foot-and-mouth disease is achieved by a combination of virus isolation (from vesicles), ihc, and serology by regulatory officials. identifying the source of infection also is very important. diagnosis of vesicular stomatitis is achieved by complement fixation or fluorescent antibody staining of virus in vesicular fluid or detection of a rise in antibody titers. flocks with either of these diseases in the united states are subject to quarantine and possible destruction (especially for foot-and-mouth disease). prevention. meticulous personal hygiene and avoidance of contact with new animals are important during outbreaks to prevent spread between flocks. vaccines against foot-and-mouth disease are available in many parts of the world, but not in the united states. most nations slaughter or quarantine affected animals. vaccines against vesicular stomatitis are available and are most commonly used if the risk of outbreak is high, but vaccination does not prevent infection or shedding. good hoof and teat care and soft feeds may help prevent spread of the virus by providing a healthy, intact barrier against invasion. pathogenesis. sheep and goat pox are caused by two closely related poxviruses. some strains are infective to both sheep and goats; most are species specific. they are maintained in populations by infected animals, and transmission occurs by aerosol or direct or indirect contact. flies may play an important role as mechanical vectors in some flocks. viruses remain infective in the environment for as long as months. after infection, viremia and inflammation of the oral, nasal, and ocular mucous membranes occur. erythematous papular pox lesions appear a few days later. severity varies according to strain pathogenicity, breed susceptibility, and immune status. mild infections are characterized by lesions concentrated in the non-wooled or hairless regions of the skin. severe infections produce lesions throughout the oral cavity, respiratory tract, and peritoneal cavity. secondary infection is common with the severe form and mortality is high. if the animal survives, lesions heal in to weeks. both diseases have been eradicated from the united states and are reportable. people can develop mild disease on exposure to these viruses. clinical signs. fever, inappetence, conjunctivitis, and upper respiratory signs are seen in the initial stages. pox lesions are visible shortly thereafter. secondary infection can lead to a variety of more serious signs indicative of respiratory disease, sepsis, and shock. diagnosis. characteristic pox lesions are highly suggestive of this disease. microscopic analysis reveals eosinophilic intracytoplasmic inclusion bodies, acantholysis, and pustule formation within the epidermis and occasionally the dermis. viral particles may be seen on ultrastructural examination. gross and microscopic lesions are characteristic with the severe form, but mild disease may produce mild lesions that are difficult to differentiate from other viral diseases that cause oral proliferative or ulcerative lesions. virus can be isolated from blood or tissues (mainly skin) during the acute viremic stage and identified by antibody staining of more chronic lesions. serologic tests are available to detect rising titers in convalescent animals. treatment and prevention. no specific treatment is available for sheep or goat pox. antibacterial drugs may be useful to treat secondary infection. judicious use of insecticides and confinement of affected animals may prevent spread. vaccines are available in some countries, but not in the united states. infected flocks are placed under quarantine or destroyed in regions where the diseases are not endemic. these viruses are difficult to eradicate from flocks because of their environmental persistence and the constant supply of susceptible hosts. caprine arthritis-encephalitis virus (caev) is an enveloped, singlestranded retrovirus in the lentivirus genus. like other retroviruses, caev integrates into the host chromosomal dna before replicating. the virus is able to remain latent or undergo sporadic bouts of productive viral replication. caev is closely related to ovine lentiviruses. clinical signs. clinical disease may be evident in only % of goats from a caev-infected herd at any given time. as many as % of seropositive goats may be clinically normal. caev produces four clinical syndromes: encephalomyelitis, arthritis, interstitial pneumonia, and indurative mastitis. the pattern of disease usually varies with age. arthritis is generally seen in sexually mature goats, whereas encephalomyelitis is generally seen in kids to months old. interstitial pneumonia and indurative mastitis are more common in adult goats. some goats suffer from a wasting disorder characterized by poor body condition and rough hair coat. diagnosis. a presumptive diagnosis of caev can be made on the basis of history and clinical signs suggestive of one or more of the syndromes. in general, elisa tests are better for detecting disease in an individual animal because the sensitivity of the test is higher than that of the agid, whereas the agid is better for herd screening that requires high specificity. with the agid test, false negatives may occur in goats that have not yet seroconverted to recent infection. individual goats may take months or years to seroconvert or may never do so. parturition or advanced stages of disease also may contribute to a false-negative result. false positives may occur in goats younger than days old that have colostral antibodies. for this reason, it is often suggested that kids be at least months old before they are tested. pcr testing has high specificity and sensitivity and can detect infection within a day of exposure. other less commonly used tests include a western blot to detect antibodies and a northern blot to look for mitochondrial rna. because of the limitations in interpreting serologic results, caev-induced disease can only be definitively diagnosed by identification of characteristic lesions from examination of biopsy specimens or postmortem viral isolation. treatment. no specific treatments are available for any of the syndromes associated with caev. young goats suffering from encephalomyelitis may benefit from physical therapy if they are recumbent, and bottle feeding may help maintain hydration and caloric intake. antibiotics may be beneficial to goats affected with interstitial pneumonia or mastitis if secondary bacterial infection is present. generally, the prognosis is poor for the encephalitic form and guarded for the other forms. prevention. prevention of caev is crucial because infection is lifelong. infected colostrum and milk are the most important sources of infection. newborn kids should be prevented from ingesting colostrum from infected does and should instead be fed pasteurized goat's milk or milk from caev-negative goats. all goats in a herd should undergo serologic testing twice yearly; seropositive goats should be segregated or culled to prevent direct contact between infected and uninfected animals. ovine progressive pneumonia (opp) is an ultimately fatal retroviral disease that causes chronic, progressive, debilitating inflammatory conditions of the lungs (united states) and central nervous system (other parts of the world). it also is called maedi-(maeði is icelandic for "shortness of breath") visna (meaning "wasting"). the virus is a member of the lentivirus genus of retroviruses and is closely related to caev. recombination between opp and cae viruses has been observed. the virus primarily affects sheep and rarely goats and has been identified worldwide, except in australia and new zealand. the disease has a long incubation period and protracted clinical course. pathogenesis. only sheep older than years of age are affected by opp virus (oppv). the virus is spread by direct contact, probably in respiratory and salivary secretions, and by excretion in the milk and colostrum. transplacental transfer is of minor importance. virus is excreted by animals that exhibit clinical signs and asymptomatic animals. infection is established in the monocyte and macrophage cell line and spread by these cells to the lungs, lymph nodes, choroid plexus, spleen, bone marrow, mammary gland, and kidneys. like caev, oppv evades the cellular and humoral immune system of the host by incorporation of its provirus in host dna, low-grade replication of virus only when monocytes differentiate into macrophages (restricted replication), and production of antigenic variants that are not neutralized by existing antibodies. continual antigenic stimulation of the host by low-grade replication of oppv results in chronic inflammation and resultant lymphoid proliferation in various target tissues. the virus may prevent b lymphocytes from differentiating into plasma cells in lymph nodes and may thereby impair immunoregulation. seroconversion occurs within to weeks after infection. clinical signs. in the united states, serologic surveys reveal infection rates of between and % but rarely is more than % of a flock lost to oppv. icelandic, texel, border leicester, and finnish landrace appear to be susceptible sheep breeds. more resistant sheep breeds include rambouillet, suffolk, and columbia. various clinical syndromes are associated with oppv and include wasting (thin ewe syndrome), dyspnea occasionally with a dry cough, pneumonia, mastitis ("hard bag"), posterior paresis, arthritis, and vasculitis. in north america, pneumonia and indurative aseptic mastitis are common sequelae of infection. coinfection with the jaagsiekte virus (the cause of pulmonary adenomatosis) worsens respiratory signs. visna, the neurologic form, is more common in goats. over the course of up to a year, subtle signs such as a head tilt or hindlimb weakness progress to gross incoordination, whole body tremors, and rarely more profound cranial nerve signs. diagnosis. a presumptive diagnosis can be made on the basis of clinical signs, poor response to treatment, characteristic postmortem findings, and serologic testing. definitive diagnosis requires pcr or isolation of the virus from wbcs (buffy coat of whole blood sample) or tissues. less expensive and faster serologic tests include agid, elisa, and an indirect immunofluorescence test. the agid test is frequently used as a flock screening test, but the elisa is more sensitive on an individual basis and can detect antibodies earlier in the course of the disease. as with caev, false negatives and false positives are possible. characteristic postmortem lesions include generalized wasting and firm, noncollapsing lung or firm, mottled mammary glands, both with regional lymphadenopathy. microscopic evaluation of those tissues reveals interstitial non-septic, mononuclear cell infiltrates, although these may be complicated by secondary infections. histopathology of nervous tissue reveals meningoleukoencephalitis. treatment. no effective treatment is available for oppv. supportive therapy that includes appropriate husbandry and control of secondary infection with antibiotics may prolong life for a few weeks or months but, ultimately, the disease is fatal. because of the poor prognosis and risk of exposure of naive animals to clinical disease, long-term treatment is not recommended. prevention. the only known method of preventing oppv infection in a flock is to prevent exposure to the virus. management practices that help decrease the incidence of horizontal transmission include disinfection of milking equipment, dehorning instruments, and tail docking and castration tools before use and between animals. contaminated feed and water also are potential routes of infection and should not be shared among infected and uninfected animals. serologic testing and separation or culling of seropositive animals may help reduce infection. although oppv can readily be isolated from ewe colostrum, colostral transmission of oppv has not been definitively established. however, many prevention guidelines recommend that offspring from infected dams be separated from the dam before they nurse and then be fed cow colostrum and artificially reared. quarantine and serologic testing of flock additions before placing them with the current flock and purchase of sheep only from oppv-free flocks are important to prevent the introduction of new infections. because of the potential cross-species spread, all precautions taken for sheep also apply to contact goats. serologic testing should be performed at least annually in a flock until two consecutive negative test results are obtained. border disease virus (bdv) is in the genus pestivirus and family flaviviridae, which also includes the two genotypes of bovine viral diarrhea virus (bvdv) and classical swine fever virus. it rarely causes disease in adults and is most important as a cause of in utero infection of lambs and kids. the condition gets its name from the fact that it was first reported in sheep along the welsh border of the united kingdom. other names such as "hairy shakers" and "fuzzy lamb disease" refer to some of the clinical signs seen in affected newborns. it is important to recognize that although bdv is genetically distinct from the two types of bvdv, sheep and goats also are susceptible to some strains of bvd. pathogenesis. horizontal transmission of bdv occurs through contact with secretions and excretions of body fluids and tissues from infected animals. the virus crosses intact mucous membranes and can spread rapidly through a flock. the major reservoir is the persistently infected sheep or goat. these reservoirs are usually asymptomatic, congenitally infected, and often seronegative animals that shed large quantities of virus. these may be residents of a flock with an ongoing problem or bought in as replacement animals to a naïve flock. some cross-infection from other species is possible, particularly from cattle. adult, immunocompetent sheep rarely show any signs of acute infection. however, if a pregnant ewe or doe is infected, the virus may be transmitted vertically to the embryo or fetus. depending on the stage of gestation, embryonic or fetal infection may have different outcomes ranging from embryonic reabsorption to normal birth. these infections are the most important aspect of border disease. the major organ system targeted by bdv is the fetal central nervous system. the hallmark lesion is hypomyelination, or degeneration of oligodendroglial cells. three factors contribute to this lesion. the first is direct viral damage. the second is viral-induced inhibition of the thyroid gland that causes decreased secretion of thyroid hormones. in the absence of these hormones, a resultant lowered concentration of a specific nucleotide in the central nervous system also contributes to the hypomyelination. the third factor is altered immune function. the virus causes the host to produce a virus-specific delayed hypersensitivity reaction that causes inflammation in the central nervous system. it also causes immunosuppression. death often results from opportunistic conditions such as parasitism, diarrhea, and bronchopneumonia. clinical signs. clinical signs depend on the time during gestation when the fetus or embryo is exposed to the virus. clinical signs also may vary in severity from animal to animal because different fetuses develop competent immune systems at different times. if the fetus or embryo is exposed to the virus within days of conception, it dies and is resorbed or aborted. these losses are not usually noticed by the flock manager. the principal manifestation in the flock is a large number of open ewes and a small lamb crop. infection of the fetus between days and of gestation causes damage to rapidly growing systems such as the skin and nervous, lymphoid, thyroid, and skeletal systems. congenital malformations are seen at birth. lambs have abnormal fleece characteristics (hairy rather than woolly in consistency), small stature, domed heads, shortened legs, and dark pigmentation of the skin, particularly on the dorsal aspect of the neck. the lamb may exhibit tonic-clonic tremors ("hairy shakers") when awake, which may prevent standing or suckling. most of these lambs die within a few days of birth. if they survive, the hair changes disappear in to weeks and the central nervous system signs resolve by weeks. goats infected at this time have similar symptoms except that they rarely exhibit hair coat changes. if kids are infected before day of gestation and are still viable, they may become persistently infected and immunologically compromised. they are small at birth and generally weak. typical outbreaks of border disease cause abortions and birth of weak lambs in the first year as the virus rapidly spreads throughout a susceptible flock and then insignificant losses in the succeeding years as adult sheep develop immunity. however, if new naïve ewes are introduced in the flock, substantial losses may occur in perpetuity. diagnosis. border disease viral antigens can be demonstrated in abomasum, pancreas, kidney, thyroid, skin, and testicle tissues from aborted fetuses and persistently infected animals using fluorescent antibody tests. however, ihc on ear notch samples is not considered as reliable for detecting persistently infected small ruminants as it is for cattle. the virus can be isolated, or viral antigen detected by elisa, from serum, heparinized whole blood, and tissue taken from brain, spinal cord, spleen, and bone marrow from affected lambs. whole blood is better than serum if colostral antibodies are likely to be high; serum is an adequate sample in neonates and juveniles that have not suckled. antibodies to the virus may be quantified by serum neutralization, agid, and complement fixation with hyperimmune bvd antiserum. serologic tests are useful to detect exposure in lategestation (after day ) neonates and unvaccinated animals but may be confounded by colostral antibodies in suckling neonates, previous exposure, and vaccination in older animals. any titer in a presuckling neonate indicates in utero exposure, whereas a serum neutralization titer of : to : suggests infection in adults. the presence of specific antibodies in the cerebral spinal fluid suggests bdv infection. negative presuckling serologic tests do not rule out exposure because persistently infected lambs tend to be immunotolerant to the bdv and therefore are born without an antibody titer. these animals may subsequently develop a titer that is indistinguishable from that of a normal animal. although persistently infected animals do not respond immunologically to the strain of the virus they carry, they may respond to other strains of the virus, including vaccine strains. as with bvd, pcr assays are gaining popularity for the detection of bdv in fluids and tissue samples. these assays appear to be superior to other techniques, except in autolyzed tissues. realtime pcr may also be used to differentiate bdv from bvd and to type isolates. gross postmortem findings include hydranencephaly, porencephaly, microcephaly, cerebellar hypoplasia, abnormal rib curvature, brachygnathia, doming of the frontal bones of the skull, narrowing of the distance between the orbits, shortening the crown-to-rump length, shortening of the diaphyseal length, retention of secondary hair fibers, and abnormal skin pigmentation. the major histopathologic changes include hypomyelination and hypercellularity of the white matter. glial cells appear normal. treatment. no treatment is available for border disease infection. supportive care may include assistance in nursing and standing for affected lambs, provision of good bedding and solid footing, and treatment of secondary opportunistic infection. prevention. control is primarily achieved by eliminating persistently infected carrier animals from the flock and preventing the addition of new carrier animals. this is easiest in a closed flock but especially difficult in small ruminant flocks because of the frequent desire to import new genetics. to identify carriers, virus isolation must be performed on every animal in the flock; carrier animals must be culled. additionally, all unborn animals must be considered potential carriers and should be tested at birth. an alternative solution in hobby flocks is to arrest breeding activity until all animals have been shown to be free of infection. new animals should be quarantined and tested before admission to the flock. herd screening with the ear skin biopsy test using fluorescent antibody staining to detect virus is less expensive and more convenient than the whole blood virus isolation test. the role of vaccination in preventing infection is still unclear. no vaccine against bdv is available, but some reports suggest that bvdv vaccines for cattle may be helpful for sheep at risk. however, these vaccines have proven to be more effective at preventing clinical disease in vaccinated animals than in preventing in utero infection because they do not prevent transient viremia. vaccination decreases viremia and fetal infection but does not eliminate them. therefore, vaccines play a role in decreasing economic loss but do not replace culling of carrier animals as the major method of control. another member of the slow infection group of diseases of small ruminants is scrapie. it is an afebrile, chronic, progressive degenerative disorder of the central nervous system of sheep and occasionally of goats (see chapter ) . scrapie is caused by a prion and, as such, is one of the transmissible spongiform encephalopathies. sheep (and goats and mouflon to a lesser degree) are the natural hosts for scrapie. clinical signs often do not usually appear until animals are years old, and animals as old as years may exhibit clinical disease. both vertical and horizontal transmission have been demonstrated experimentally in sheep and goats. abnormal scrapie protein has been identified in milk, urine, and seminal plasma of sheep up to months prior to the development of clinical signs. also, new evidence from deer with chronic wasting disease, a similar disorder, suggests that infective prions are excreted in the saliva and feces well before the development of clinical signs. these new revelations may help explain horizontal transmission of infection. clinical signs. the onset of scrapie is insidious. initially, sheep show subtle changes in behavior such as mild apprehension, staring or fixed gaze, failure to respond to herding dogs, and boldness around humans. several months later, the animals become intolerant of exercise and develop a clumsy, unsteady gait and floppy ears. later, the sheep develop itchy skin that causes them to rub themselves excessively against firm, immobile objects (origin of the name scrapie). this leads to excoriations and wool damage. there is a general decline in body condition and coordination. diagnosis. histologically, the only consistent lesions are degenerative changes in the central nervous system consisting of bilaterally symmetric vacuolation of the neurons in the brainstem and spinal cord with accompanying spongy degeneration. as a preclinical test, ihc may be performed in lymphoid tissue from the tonsils, third eyelid, or rectoanal mucosa, but none of these methods is foolproof. cwd is discussed in chapters , , and . testing for clinical anaemia caused by haemonchus spp. in goats farmed under resource-poor conditions in south africa using an eye colour chart developed for sheep validation of the fama-cha eye color chart for detecting clinical anemia in sheep and goats on farms in the southern united states validation of the famacha © eye colour chart using sensitivity/ specificity analysis on two south african sheep farms is the famacha chart suitable for every breed? correlations between famacha scores and different traits of mucosa colour in naturally parasite infected sheep breeds rumen bacteria are involved in the onset of onion-induced hemolytic anemia in sheep the role of free radicals in brassicainduced anaemia of sheep: an esr spin trapping study kale poisoning: the brassica anaemia factor hemolytic anemia in sheep fed wild onion (allium validum) copper toxicosis in sheep: a case report chronic copper poisoning in sheep. i. the relationship of methaemoglobinemia to heinz body formation and haemolysis during the terminal crisis chronic copper toxicity of ruminants copper poisoning in a flock of sheep. copper excretion patterns after treatment with molybdenum and sulfur or penicillamine evaluation of mechanisms of leptospiral hemolytic anemia fatal hemolytic anemia attributed to leptospirosis in lambs studies on eperythrozoon ovis-infection in sheep eperythrozoon ovis-a blood parasite of sheep experimental eperythrozoon ovis infection of sheep mycoplasma ovis comb. nov. (formerly eperythrozoon ovis), an epierythrocytic agent of haemolytic anaemia in sheep and goats molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia and concomitant anaplasma ovis infection bovine colostrum as a cause of hemolytic anemia in a lamb heinz body anaemia in lambs with deficiencies of copper or selenium maxillary lymphosarcoma in a white-tailed deer (odocoileus virginianus) large animal internal medicine effect of physical restraint and xylazine sedation on haematological values in red deer (cervus elaphus) seasonal variations in red deer (cervus elaphus) hematology related to antler growth and biometrics measurements the genetic basis and evolution of red blood cell sickling in deer one hundred two tumors in goats lymphoma classification in goats exophthalmos due to multicentric b-cell lymphoma in a goat ocular involvement of multicentric malignant b-cell lymphoma in a ewe. a case report diseases and parasites of white-tailed deer, miscellaneous publication no. . tall timbers research station colostrum composition of santa inês sheep and passive transfer of immunity to lambs effects of maternal undernutrition during late gestation and/or lactation on colostrum synthesis and immunological parameters in the offspring failure in passive transfer of immunoglobulin g to lambs: measurement of immunoglobulin g in ewe colostrums iodine supplementation of the pregnant dam alters intestinal gene expression and immunoglobulin uptake in the newborn lamb short communication: apoptosis regulates passive immune transfer in newborn kids effects of newborn characteristics and length of colostrum feeding period on passive immune transfer in goat kids a field trial evaluating the health and performance of lambs fed a bovine colostrum replacement use of a digital brix refractometer to estimate serum immunoglobulin in goat kids field methods for estimating serum immunoglobulin concentrations in newborn kids colostrum deficiency in mule deer fawns: identification, treatment and influence on neonatal mortality passive transfer of colostral immunoglobulins from ewe to lamb and its influence on neonatal lamb mortality transfer of maternal passive immunity to kids in goat herd suppurative meningitis in a -day-old formosan sambar deer (cervus unicolor swinhoei) caused by escherichia coli factors affecting igg concentration in day-old lambs effects of maternal nutrition on udder development during late pregnancy and on colostrum production in scottish blackface ewes with twin lambs the effect of colostrum source (goat vs. sheep) and timing of the first colostrum feeding ( h vs. h after birth) on body weight and immune status of artificially reared newborn lambs bovine neonatal pancytopenia and anaemia in lambs caused by feeding cow colostrum secondary lactose intolerance in a neonatal goat hypernatremia in neonatal elk calves: cases ( - ) group b rotavirus associated with an outbreak of neonatal lamb diarrhea rotaviruses associated with neonatal lamb diarrhea in two wyoming shed-lambing operations novel group a rotavirus g p[ ] as primary cause of an ovine diarrheic syndrome outbreak in weaned lambs role of enteric pathogens in the aetiology of neonatal diarrhoea in lambs and goat kids in spain enteric viral infections in lambs or kids suspected clostridium difficile-associated hemorrhagic diarrhea in a -week-old elk calf observations and immunohistochemical detection of coronavirus, cryptosporidium parvum and giardia intestinalis in neonatal diarrhoea in lambs and kids giardia duodenalis and cryptosporidium parvum infections in adult goats and their implications for neonatal kids case control study of diarrhoea and faecal soiling in two-to six-month-old lambs comparison of two techniques for diagnosis of cryptosporidiosis in diarrhoeic goat kids and lambs in cyprus fluid therapy in calves passive immunisation of neonatal lambs against infection with enteropathogenic escherichia coli via colostrum of ewes immunised with crude and purified k pili floppy kid syndrome (metabolic acidosis without dehydration in kids clostridium perfringens toxins involved in mammalian veterinary diseases first isolation of clostridium perfringens type e from a goat with diarrhea clostridial enteric diseases of domestic animals isolation and molecular characterization of clostridium perfringens from healthy merino lambs in patagonia region lamb losses associated with clostridium perfringens type a hemorrhagic bowel syndrome in dairy cattle: cases clostridium perfringens type a and beta toxin associated with enterotoxemia in a -week-old goat investigation of a syndrome of sudden death, splenomegaly, and small intestinal hemorrhage in farmed deer gastric mucormycosis in a sika deer (cervus nippon) associated with proliferation of clostridium perfringens the relationship between the presence of helicobacter pylori, clostridium perfringens type a, campylobacter spp, or fungi and fatal abomasal ulcers in unweaned beef calves multiplex pcr method for genotyping clostridium perfringens the effect of clostridium perfringens type c strain cn and its isogenic beta toxin null mutant in goats beta toxin is essential for the intestinal virulence of clostridium perfringens type c disease isolate cn in a rabbit ileal loop model clostridial diseases vaccines for control, prevention and eradication of disease in farmed deer development and application of an oral challenge mouse model for studying clostridium perfringens type d infection enterotoxaemia caused by clostridium perfringens type d in farmed fallow deer rates of diseases and their associated costs in two colorado sheep feedlots ( - ) proportional mortality: a study of goats submitted for necropsy from goat herds in quebec, with a special focus on caseous lymphadenitis the pathology of experimental clostridium perfringens type d enterotoxemia in sheep enterotoxaemia in goats: a review diagnosis of clostridium perfringens intestinal infections in sheep and goats clinico-pathological findings of clostridium perfringens type d enterotoxaemia in goats and its hemolytic activity in different erythrocytes experimental clostridium perfringens type d enterotoxemia in goats clinical signs, treatments, and postmortem lesions in dairy goats with enterotoxemia: cases epsilon toxin is essential for the virulence of clostridium perfringens type d infection in sheep, goats, and mice clinicopathologic features of experimental clostridium perfringens type d enterotoxemia in cattle ulcerative enterocolitis in two goats associated with enterotoxin-and beta toxin-positive clostridium perfringens type d the passive protection of lambs against clostridium perfringens type d with semi-purified hyperimmune serum blackleg in deer bacterial diseases of farmed deer and bison black disease in a forest reindeer bovine vaccines and herd vaccination programs toxigenic clostridia characterization of the catalytic domain of clostridium novyi alpha-toxin first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan clostridium novyi (myonecrosis, black disease, and bacillary hemoglobinuria) and clostridium septicum (braxy) infections first report of infectious necrotic hepatitis (black disease) among nubian goats in sudan liver and biliary system bacillary hemoglobinuria: induction by liver biopsy in naturally and experimentally infected animals bacillary hemoglobinuria in a free-ranging elk calf bacillary hemoglobinuria in dairy cows an outbreak of bacillary haemoglobinuria in sheep in india successful treatment of bacillary hemoglobinuria in japanese black cows acute abomasitis due to clostridium septicum infection in experimental sheep rapid identification and differentiation of pathogenic clostridia in gas gangrene by polymerase chain reaction based on the s- s rdna spacer region suppurative abomasitis associated with clostridium septicum infection clostridial myocarditis in lambs outbreak of clostridial myocarditis in calves clostridial myositis in cattle: bacteriology and gross pathology clostridial vaccination efficacy on stimulating and maintaining an immune response in beef cows and calves failure of clostridium chauvoei vaccines to protect against blackleg prevalence of coxiella burnetti infection in wild and farmed ungulates coxiella burnetii shedding by farmed red deer (cervus elaphus) high prevalence of antibodies against chlamydiaceae and chlamydophila abortus in wild ungulates using two regional seroprevalence of leptospirosis on deer farms in new zealand growth response and shedding of leptospira spp. in urine following vaccination for leptospirosis in young farmed deer corynebacterium pseudotuberculosis paratuberculosis (johne's disease) in cattle and other susceptible species efficacy of a killed vaccine for the control of paratuberculosis in australian sheep flocks detection of a novel reassortant epizootic hemorrhagic disease virus (ehdv) in the usa containing rna segments derived from both exotic (ehdv- ) and endemic (ehdv- ) serotypes the first years ( - ) of epizootic hemorrhagic disease virus serotype in the usa review of the epizootic hemorrhagic disease outbreak in domestic ruminants in the united states peste des petits ruminants demonstration of coinfection with and recombination by caprine arthritis-encephalitis virus and maedi-visna virus in naturally infected goats key: cord- -rrverrsj authors: delano, margaret l.; mischler, scott a.; underwood, wendy j. title: biology and diseases of ruminants: sheep, goats, and cattle date: - - journal: laboratory animal medicine doi: . /b - - / -x sha: doc_id: cord_uid: rrverrsj nan since the first edition of this book, the use of ruminants as research subjects has changed dramatically. formerly, large animals were primarily used for agricultural research or as models of human diseases. over the past decade, ruminants have continued in their traditional agricultural research role but are now extensively used for studies in molecular biology, genetic engi-british stock with egyptian and indian goats. this breed is relatively heat tolerant and produces milk with the highest butterfat (about - %). fiber breeds include the angora and the cashmere. the angora, the source of mohair, originated in turkey. the cashmere breed is found primarily in mountainous areas of central asia. the la mancha, a newer breed of dairy goat first registered in the united states in , has rudimentary ears that are a genetically dominant distinguishing characteristic of the breed. the meat breeds include the boer, sapel, ma tou, kambling, and pygmy. the pygmy goat is small and is sometimes used for both meat and milk. the mubend of uganda and the red sokoto of west africa produce quality skins for fine leather (smith and sherman, ) . most breeds of cattle are classified as "dairy" or "beef"; a few breeds are considered "dual-purpose." common dairy breeds in the united states include holstein-friesian, brown swiss, jersey, ayrshire, guernsey, and milking shorthorn. holsteins have the largest body size, whereas jerseys have the smallest. of breeds in temperate regions, jerseys have been considered to be the most heat tolerant, but holsteins have been found to adapt to warmer climates. there are many beef breeds. the more common in the united states include angus (also called aberdeen-angus), hereford (both polled and horned), and simmental (briggs and briggs, ; schmidt et al., ) . breeds indigenous to other continents, such as the cape buffalo, have been found to have unique innate immune characteristics that protect them from endemic trypanosomiasis (muranjan et al., ) . more detailed information regarding these and other ruminant breeds is available in briggs and briggs ( ) . "rare" or "minor" breeds of sheep, goats, and cattle are studied for their genetic and production characteristics. discussions of these and efforts at conservation are described in detail elsewhere (national research council, ) . several terms are unique to ruminants. in relation to sheep, a ewe is the female, and a ram is the adult intact male. a lamb is the young animal, and ram lamb and ewe lamb are commonly used terms. a wether is a castrated male. the birthing process is referred to as lambing. with respect to goats, a doe or nanny is the female. a buck or billy is the adult intact male. a kid or goatling is a young goat. a young male may be referred to as a buckling, and a young female may be referred to as a doeling. a castrated male in this species is also called a wether. the birthing process is called kidding. with respect to cattle, an adult female is a cow, and an adult male is a bull. a calf is a young animal. a heifer is a female who has not had her first calf. a steer is a castrated male. calving refers to the act of giving birth. ruminants have been used as research models since the inception of the land grant college system, first in production agriculture and now also in basic and applied studies for the anatomic and physiologic sciences and in biomedical research for a variety of purposes. healthy, normal young ruminants serve as models of cardiac transplantation and as preclinical models for evaluation of cardiac assist or prosthetic devices, such as vascular stents and cardiac valves (salerno et al., ) . for many years, ruminants have been useful research subjects for reproductive research, such as research on embryo transfer, artificial insemination, and control of the reproductive cycle (wall et al., ) . several important milestones in gene transfer, cloning, nuclear transfer, and genetic engineering techniques have been developed or demonstrated using these species (ebert et al., ; schnieke, ; cibelli et al., a,b) (see fig. ). one of many proposed uses of genetically engineered ruminants is the production of proteins that will be secreted in the milk and later isolated (ebert et al, ; memon and ebert, ) . healthy sheep and goats are also often used for antibody production (hanly et al., ) . genome mapping developed rapidly during the s; extensive information is available and is increasing for sheep and cattle (broad et al., ; womack, ) . sheep are often selected for studying areas such as ruminant physiology and nutrition. these animals provide obvious bene-fits over the use of cattle in research from the standpoint of size, ease of handling, cost of maintenance, and docile behavior. sheep are also widely used models for basic and applied fetal and reproductive research (buttar, ; rees et al., ; ross and nijland, ) . the species is used for investigating circadian rhythms related to day length (lehman et al., ) , and the interaction between olfactory cues and behavior (kendrick et al., ) . the number and diversity of natural-and induceddisease research models in sheep are great and increasing. natural models include congenital hyperbilirubinemia/hepatic organic anion excretory defect (dubin-johnson syndrome) in the corriedale breed, congenital hyperbilirubinemia/hepatic organic anion uptake defect (gilbert syndrome) in the southdown breed, glucose- -phosphate dehydrogenase deficiency in the dorset breed, gm~ gangliosidosis in the suffolk breed, and pulmonary adenomatosis (jaagsiekte) in many breeds (hegreberg, l a) . induced models include arteriosclerosis, hemorrhagic shock, copper poisoning (wilson's disease), and metabolic toxocosis (hegreberg, lb) . goats are used in a wide variety of agricultural and biomedical disciplines such as immunology, mastitis, nutrition, and parasitology research. vascular researchers select the goat because of the large, readily accessible jugular veins. goats with inherited caprine myotonia congenita ("fainting goats") have been used as a model for human myotonia congenita (thomsen's disease) (kuhn, ) . a line of inbred nubians serves as models for the genetic disease [ -mannosidosis and prenatal therapeutic cell transplantation strategies (lovell et al., ) . (these disorders are discussed in more detail in section iii,b, .) goats are used as a model for osteoporosis research (welch et al., ) . cattle are often used as a source of ruminal fluid for research, teaching, or treatment of other cattle, by placing a permanent fistula in the left abdominal wall to allow sampling of ruminal fluid (dougherty, ) . cattle also serve as models of many infectious diseases, including zoonoses, and several inherited metabolic diseases. this species is useful for the basic and comparative research on the pathogenesis and immunology of inherited and infectious diseases. bovine trichomoniasis, caused by tritrichomonas (trichomonas)fetus, has been identified as a useful model for the human infection by trichomonas vaginalis (corbeil, ) . inherited cardiomyopathies have been found in the holstein-friesian, simmental-red holstein, black spotted friesian, and polled hereford with woolly coat (weil et al., ) . lipofuscinosis has been identified in ayrshires and friesians, and glycogenesis in shorthorns and brahmans. metabolic diseases such as hereditary orotic aciduria and hereditary zinc deficiency have been characterized in holstein-friesian or friesian cattle. holstein cattle also serve as a model for leukocyte adhesion deficiency syndrome (afip, ) . common breeds of normal, healthy ruminants are usually readily available, although seasonality may play a role, as noted below. agricultural sources and reputable farms may be located through land-grant universities or agricultural schools, cooperative extension and -h networks, regional ruminant breeders' associations, and farm bureaus. commercial sources of purposebred animals are found in technical publications and annual listings of research animal vendors. breeds carrying genetic traits of interest, either as animal models or as valuable production characteristics, may be located through literature or internet searches, animal science societies, breed or livestock conservation associations, and information resources such as the armed forces institute of pathology. organizations such as the institute for laboratory animal research (ilar), national center for research resources (ncrr), or the animal welfare information center (awic) may also serve as information sources about the animals needed. purpose-bred research sheep and goats are available from commercial vendors and are usually maintained in registered facilities under federal standards that are also acceptable to research animal accrediting agencies. these commercial animals are frequently described as specific pathogen-free (spf) and housed as biosecure or closed flocks. animal health programs are in place, and health reports or other quality assurance reports are usually available on request. agricultural sources of either small ruminant may be acceptable, but specific research needs may not have been addressed or may not be understood. lambs, kids, and milking goats may be difficult to locate in fall and winter months because most breeds of sheep and goats are seasonal breeders. management practices exist, however, to extend the breeding and milking seasons. most cattle used as animal models in research in the united states are from one of the dairy breeds, usually holstein, because this breed is now the most common. purpose-bred, specific pathogen-free research cattle are not typically available. because of selection and the management of dairy production units, calves and young stock are available year-round. availability of young beef cattle is more seasonal, according to production cycles typically followed by that industry. auction barns or sales are not appropriate sources for research ruminants. many of these animals are culls and will be poor-quality research subjects. they may be in poor body condition and stressed, may be sources of disease, and may contaminate other healthy animals, as well as the research facility. selection of the suppliers should be made only after research needs have been carefully considered. consistently working with and buying directly from as few sources as possible are best. certain types of research (i.e., agricultural nutrition studies) may better be served by selecting animals from local agricultural suppliers rather than commercial vendors located in a different geographical area. the selection of sources for research ruminants includes scrutiny of flock or herd record keeping; health monitoring, vaccination, and preventive medicine programs (including hoof care); production standards and management practices consistent with the industry; management of the breeding flock or herd; sanitation and waste handling programs; vermin and insect control measures (especially for flies and other flying insects); rearing programs for and condition of young stock; the location, health, and condition of the other animals on the premises; intensity of housing; and animal housing facilities. preliminary and periodic visits to the source farms should be conducted. it is important to establish a good relationship with the local attending large-animal veterinarians, who will be valuable resources for current approved therapies and practices. they may need to be oriented on the specific requirements of animal research. creative ways can be used to initiate and foster a good working relationship between the agricultural supplier and the research facility. supplying the vaccines or dewormers required for flock health programs, providing services such as quarterly serological testing or fecal examinations for the herd or flock, and paying a premium (rather than market price) for animals that meet the quality criteria established for the research animals are often helpful. a set of testing standards can be developed based on one high-quality supplier, and then flocks or herds can be "qualified" based on those standards. qualifying entails evaluations utilizing the facility and management aspects mentioned above and testing either a percentage of the herd or flock or the entire herd or flock for a number of infectious agents. the testing regimen itself should be carefully developed and evaluated. once qualified, each source farm should be reevaluated periodically to maintain its status. slaughter checks may be appropriate; otherwise necropsy of sentinel animals may be required. selected animals undergoing screening tests should be quarantined from the rest of flock or herd while awaiting test results. vaccination and deworming regimens can be instituted during these quarantine periods. a second quarantine should occur when animals arrive at the research facility. the animal screening process also depends on the origin of the animal (state, country) and the scientific program. federal and state regulations must be followed. socialization of the animals at the source facility should also be considered in terms of ease of handling and safety for personnel in the confinement of the research lab, barn, or farm. for example, frequently handled calves will be easier to manage, and adult dairy goats that have been acclimated to human contact are preferable. several texts provide information on industry standards for flock and herd management and preventive medicine strategies that can provide helpful orientation to those unfamiliar with these aspects. these references also provide information regarding vaccination products licensed for use in ruminants and typical herd and flock vaccination parasite control schedules ("current veterinary therapy," , "council report," ; "large animal internal medicine," ; smith and sherman, ) when designing a vaccination program during qualification of a source or at the research facility, it is important to evaluate the local disease incidence and the potential for exposure. vaccination programs should be conducted with an awareness of duration of passive immunity and stresses in ruminants' lives (e.g., weaning, grouping, management changes, and shipping) that may impair immunity or increase susceptibility to infectious diseases. it is also prudent to evaluate the cost-effectiveness of vaccination; labor and vaccine expenses may be much higher than the potential animal morbidity or mortality for diseases in a particular locality. not all of the vaccines mentioned subsequently will be necessary in all herds or flocks. vaccination needs for research animals will also depend on the local disease history, intent of the research, the age of the animals needed for research, and the length of time the animals will be housed. typical health screening programs for sheep include q fever (coxiella burnetii); contagious ecthyma; caseous lymphadenitis (corynebacterium pseudotuberculosis); johne's disease (mycobacterium paratuberculosis); ovine progressive pneumonia; internal parasitism such as nasal bots, lungworms, and intes-tinal worms; and external parasitism such as sheep keds. each supplier should be queried about vaccination programs for bluetongue, brucella ovis, campylobacter spp., chlamydia (enzootic abortion of ewes), clostridial diseases, pneumonia complex (parainfluenza , pasteurella haemolytica, and p. multocida), ovine ecthyma, rabies, dichelobacter (bacteroides) nodosus, arcanobacterium pseudotuberculosis, bacillus anthracis, and fusobacterium necrophorum. because of the limited number of biologics approved for small ruminants, products licensed for cattle have been used with success in sheep, and some licensed for sheep are used in goats ("council report," javma, ) . in some cases, approved feed additives, such as coccidiostats, are fed to sheep. the basic screening profile for goats should include q fever (coxiella burnettii), caprine arthritis encephalitis (cae), brucellosis, tuberculosis, and johne's disease (mycobacterium paratuberculosis) . goats may also be tested for caseous lymphadenitis, contagious ecthyma, or mycoplasma as needed. herd vaccination programs may include immunizations against tetanus and other clostridial diseases, chlamydia, campylobacter, contagious ecthyma, caseous lymphadenitis, corynebacterium pseudotuberculosis, and escherichia coli. cattle herds should be screened for johne's disease, brucellosis, tuberculosis, respiratory diseases, internal and external parasitism, and foot conditions such as hairy heel warts and foot rot. determination of the status of the herd with respect to bovine leukemia virus (blv) may be worthwhile. herd programs may include essential or highly recommended vaccines against bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), bovine respiratory syncytial virus (brsv), parainfluenza (pi- ), leptospira pomona, tritrichomonas fetus, rotavirus, coronavirus, campylobacter (vibrio) , pasteurella haemolytica and p. multocida, and brucella abortus. other vaccination programs, dependent on herd status, endemic diseases, or geographic location, may include immunizations against the clostridial diseases, moraxella bovis (pinkeye), fusobacterium necrophorum (foot rot), staphylococcus aureus (mastitis), haemophilus somnus, rabies, tetanus, bacillus anthracis, enterotoxigenic e. coll anaplasma, and other leptospira species. some products considered to have limited efficacy include vaccines against salmonella dublin and s. typhimurium. some autogenous vaccines may be more effective than the commercially available products--for example, the bovine papillomavirus (warts) vaccines. rearing programs for dairy calves differ from those for the smaller ruminants, including the withdrawal of calves from their dams immediately or by hours after birth. in the cattle industry, antibiotics, ionophores (antibiotics that control selected populations of ruminal organisms), coccidiostats, probiotics, and other approved additives may be part of the milk replacers, grain and concentrate formulations, and/or creep feeding regimens. use varies by the segment of the industry, and regulations vary by country. subcutaneous hormonal implants (such as estradiol benzoate and progesterone combined, zeranol, or [~-estradiol) are administered, especially to beef calves destined for market rather than breeding, to promote growth. transportation of the animals from the source to the research facility must be carefully planned, and all applicable livestock travel regulations followed. it is best to have the animals transported in vehicles regularly utilized by the source farm. if commercial haulers are used, then disinfecting trucks, trailers, and associated equipment, such as ramps and chutes, beforehand is particularly important. the loading, footing, and distribution of the animals in the trailers and trucks, as well as environmental conditions during shipping, are important to consider to minimize stress and injury to the animals. sufficient time for acclimation to the facility, pens, handlers, feed, and water must be allowed once at the destination ("livestock handling and transport," ). recent publications address many general considerations as well as specifics about the facilities, husbandry, space requirements, and standard practices for research and production ruminants. institutions, private entities, researchers, and facility staff must also be aware of the recent adoption by the u.s. department of agriculture (usda) of specific guidelines for regulation of farm animals, such as ruminants, that are used in biomedical and other nonagricultural research. the usda animal care policy notes that the "guide for the care and use of agricultural animals in agricultural research and teaching" and the "guide for the care and use of laboratory animals" provide additional information to supplement the existing animal welfare act regulations (cfr, ; fass, ; hays et al., ; nrc, a; usda, ) . in all cases, stress should be considered and minimized in the husbandry and handling of ruminants. animals need to be provided adequate time to adapt to new surroundings. stress decreases feed intake, and the resulting energy, vitamin, and mineral deficiencies will affect the growth and development in younger animals. reproductive soundness and rumen function are affected by transport and similar stresses. standard practices such as weaning, castration, dehorning, vaccinations, deworming and treatments for external parasites, shipping and the associated feed and water deprivation, introduction to a new housing environment and new personnel, and intercurrent disease are all stressors (houpt, ) . animals should be acclimated to the use of halters and leads, temporary restraint devices, and other handling equipment associated with the research program. personnel in the research facility who are unfamiliar with ruminants should be trained in appropriate handling techniques. ap-preciation for ruminant behaviors has grown in recent years, and refined ruminant handling techniques have been published (houpt, ; grandin, ) . when ruminants are confinement-housed, care should be taken to provide adequate but draft-free ventilation. ammonia buildup and other waste gases may induce respiratory problems. in cold weather, if the ceiling, walls, or water pipes condense water, then the ventilation should be increased even at the expense of lower temperatures. even adult goats and younger cattle are quite comfortable in cold, even subfreezing temperatures, if provided with adequate amounts of dry dust-free bedding and draft protection. sheep, because of their wool, are remarkably tolerant to both hot and cold extremes. newborn lambs and recently shorn adults are susceptible to hypothermia, hyperthermia, and sunburn. therefore, in outside housing areas, sheep should be provided with shelters to minimize exposure to sun and inclement weather. animals housed under intensive confinement should be kept clean, and excreta should be removed from the pens or enclosures daily. feed and water equipment should be maintained in sound, clean condition and should be constructed to prevent fecal contamination. waterers should not create a muddy environment in paddocks or pens. there should be sufficient continuous-access waterers placed around the area to prevent competition or fighting. feeders should be constructed to conform to species size and feeding characteristics and to prevent entrapment of head and limbs. pens, other enclosures, passageways, chutes, and floors must be very sturdy to withstand such factors as the frequent cleaning; the strength, weight, and curiosity of all ages of animals; and the investigative and climbing behaviors of goats. chain-link fences are dangerous because goats (as well as some breeds and ages of sheep) are curious and tend to stand on their hind legs against fencing or walls. forelimbs may be caught easily in the mesh. floors in any areas where animals will be housed, led, or herded must ensure secure footing at all times to prevent slipping injuries. all ruminants are social and herding animals. therefore, they should be housed in groups or at least within eyesight and hearing of other animals. singly housed animals should have regular human contact. environmental enrichment should be governed by the experimental protocol or standard operating procedures, and durable play objects should be supplied to those animals that are housed in confinement. calves, in particular, that must be singly housed or that have been recently weaned, need play objects (morrow-tesch, ) . because sheep and goats are sensitive to changes in light cycle (especially reproductive parameters), photoperiod must be taken into account. normally, sheep and goats should be maintained on a cycle comparable to natural conditions. light intensity should be maintained at about lux (ilar, ; fass, ) . light cycles can be manipulated for experimental reasons. the development of the digestive system and the unique function of the rumen are among the most notable comparative anatomic and physiologic characteristics of ruminants. there is a three-compartment forestomach (rumen, reticulum, and omasum) and a true stomach (abomasum). the mature rumen functions as an anaerobic fermentation chamber in which the enzymes, such as cellulase, of the resident bacteria allow the animals to prosper as herbivores. digestion is also aided by other microorganisms, such as protozoa ( - /ml) and bacteria ( - ~ that contribute to rumen fermentation. the result is the production of volatile fatty acids (acetic, propionic, and butyric) . unlike in the monogastrics, fermentative digestion and volatile fatty acid absorption also occur in the large intestines. the main sources of energy for ruminants are volatile fatty acids (vfas) rather than glucose. glucose is formed from propionic acid (or from amino acids) for metabolism in the central nervous system (cns), uterus, and mammary glands. plasma glucose in ruminants is much lower than and is regulated differently from that in nonruminants. the rumen microorganisms also synthesize vitamins, such as b and k, and provide protein that is used by the animals' systems. large amounts of fermentation gases such as co and methane, and small amounts of nitrogen, are naturally eructed (hecker, ; schimdt et al., ) . intestinal immunoglobulin absorption by pinocytosis in the neonates is crucial to the success of passive transfer. this transfer mechanism is functional for approximately the first hr after birth. neonatal ruminants are immunocompetent, however, and this condition is used to advantage for vaccinations against some common diseases of the neonatal and later juvenile periods, such as infectious bovine rhinotracheitis (ibr) vaccine (using modified live virus vaccines) to calves when their dams' colostrum is lacking antibody against this virus. unlike hepatic lipogenesis in humans, lipogenesis in sheep primarily occurs in adipose tissue and the mamrnary gland (hecker, ) . in addition to normal lymph node chains, and as in other ruminants, sheep have small red "nodes" associated with blood vessels. inadvertently named hemal "lymph nodes," they contain numerous red blood cells. sheep have a relatively large pituitary gland, and accessory adrenal medullary tissue may be interspersed throughout the abdominal cavity. three major ovine histocompatability classes have been identified and designated as ovar (ovis aries) classes i, ii, and iii (franz-werner et al., ) . bovines are recognized as having several unique aspects involving their immune systems. the bovine lymphocyte antigen (bola) system ranks after the hu-man (hla) and murine (h- ) systems in terms of depth of knowledge (lewin, ) . cattle are considered free of autoimmune diseases (schook and lamont, ) . the complexity of the immunobiology of the bovine mammary gland is being studied extensively because mastitis is the most prevalent disease in the dairy industry. several innate immune mechanisms and cellular defenses, and their variation throughout lactation, have been described (sordillo et al., ) . hematology and clinical reference texts are available for the ruminant species and include overviews of normal values for age, sex, and breed-specific ranges, as well as discussions regarding the influences on the hemogram of many management, nutritional, geographic, metabolic, physiologic (including lactation), medication, and iatrogenic variables (duncan and prasse, ; jain, ; kaneko et al., ) . these references should be consulted when preparing to include blood collection data in research protocols and when reviewing hematologic findings. in addition, most veterinary diagnostic laboratories have also developed databases for normal ranges for hematologic and clinical chemistry values based on subjects from their service areas, and these may be useful as local and breed references. appropriate control groups must be incorporated into each research plan, however, to establish the normal values (see table i ) for the particular locale, diagnostic facilities, breed, age, sex, and research circumstances. normal hematologic and clinical biochemistry data are presented in tables ii and iii. some general statements apply to most ruminants. most ruminants have fewer neutrophils than lymphocytes. the blood urea nitrogen (bun) values cannot be used as an indicator of renal function because of the metabolism of urea nitrogen by rumen microflora. because of the large volume of rumen water, ruminants can generally go several days without drinking before significant dehydration occurs. erythrocytes may become more fragile during rehydration, resulting in some degree of hemolysis and hemoglobinuria. severe dehydration can occur quickly, however, in animals that are ill. urine ph is generally alkaline in adult ruminants. ruminant erythrocytes are smaller than those in other mammals, and hematocrits tend to be overestimated unless blood samples are centrifuged for longer amounts of time for packing of the cell pellet. increased red-cell fragility is also associated with the smaller erythrocyte. rouleau formation does not occur in cattle but does to a limited extent in sheep and goats. in addition to fetal hemoglobin, sheep are reported to have at least six different hemoglobins (hecker, ) . blood coagulation in sheep is similar to that in humans. (di / , dc / , dp / ) = (di / , dc / , dp / ) = (di / , dc / , dp / ) = permanent dental formula ( / , c / , m / ) = ( / , c / , m / ) = ( / , c / , m / ) = avital sign data for goats are from "large animal internal medicine" ( ) . sheep weight data represent weights of feeder lamb and adult dry ewe (federation of animal science societies [fass], ) . goat weight data are for a large-breed male goat. cattle weight data represent weights of female holstein or guernsey dairy cattle (fass, ) . life span data for sheep and cattle are from brooks et al. ( ) . erythrocytes in pygmy and toggenburg goats tend to be more fragile than erythrocytes from other goat breeds. normal caprine erythrocytes lack central pallor because they are fiat and lack biconcavity. normal caprine erythrocytes may exhibit poikilocytosis. at least five blood groups have been reported in goats: b, c, m, r-o, and x. because transfusion reaction rates may be as high as - %, cross-matching is advisable although not always practical (smith and sherman, ) . blood loss of up to % of the red cell mass at a single time point can be tolerated by healthy goats. blood may safely be obtained in volumes of ml/kg body weight and given in volumes of - ml/kg. in general, aspartate aminotransferase (ast) and lactate dehydrogenase (ldh) are not liverspecific in goats, and alanine aminotransferase (alt; formally serum glutamic-pyruvic transaminase, or sgpt) cannot be used to evaluate hepatic disease in goats. ~,-glutamyltransferase (ggt) and alkaline phosphatase (ap) are associated with biliary stasis, and elevations in ggt are generally associated with hepatic damage. the nutritional needs of ruminants vary considerably according to the species, breed type, different phases of development, the use of the animals, location, and different stresses in their lives. for example, mineral requirements and other nutritional requirements vary even among breeds of cattle. several references are available that describe the varying requirements and are useful for determining the requirements of ruminants consistent with the parameters noted above and the type of feeds available (jurgens, ; "large animal clinical nutrition," ; nrc, nrc, , nrc, , nrc, , b "large animal internal medicine," ) . preformulated commercial feeds, concentrates, and supplements are available specifically for the different species of ruminants. some of these provide complete energy and protein requirements or may be used as supplements for what cannot be provided entirely by pasture, forage, hay, or silage. concentrate mixtures contain salt, minerals, and other elements. concentrates should contain a protein source such as soybean meal, cottonseed meal, or linseed meal. computer programs are also readily available for those who may need to formulate and balance rations. the palatability of feeds should be taken into account. mineral deficiencies and supplementation have been shown to influence several physiologic parameters such as immune function. introduction of young stock should include continuation of the feeding program of the source or gradual transition to appropriate feed for the animals available in the region of the research facility (nrc, ) . good-quality pasture can support ruminants under certain circumstances. lush spring pastures, especially pastures containing alfalfa, can induce bloat, diarrhea, grass tetany, or nitrate poisoning. ruminants not acclimated to lush pasture should be fed good-quality hay and slowly introduced to pasture environments. when ruminants have access to pasture, it is important to be aware of different eating habits. sheep and cattle are grazers. goats are browsers and will readily eat grasses, as well as seeds, nuts, fruit, and woody-stemmed plants. goats, however, can also be selective eaters and will only eat the leafy, more nutritious parts of the plant. therefore, goats have a tendency to "waste" hay. other eating habits should also be considered. finely ground concentrates are not tolerated well by goats; pelleted concentrates are preferred because the goat will pick out large particles in mixes. generally, goats do not prefer "sweet" feeds that contain molasses and do not need supplemental concentrates if a good-quality pasture or hay is fed. when given access to a salt block, goats generally are self-regulating. grass-fed goats and lactating goats may need supplementation with calcium and phosphorus, whereas alfalfa-fed goats do not (bretzlaff et al., ) . horse and sheep feeds may be fed to goats provided that the feed does not contain much molasses (bretzlaff et al., ) . the copper content of horse feed is not excessive for goats, as it is for sheep. pelleted horse feeds with - % fiber and - % protein are good goat rations. goats will consume - % of body weight in dry-matter intake (whereas cattle will usually consume only % of body weight). goats enjoy human contact, and small alfalfa cubes make tasty treats for the goat. rations that have excessive calcium-phosphorus ratios or elevated magnesium levels may induce urinary calculi in male ruminants. these may also occur when forage grasses are high in silicates and oxalates. to increase ovulation rate in does, some producers "flush" females by feeding . - lb concentrate per head per day for several weeks before and after the initiation of the breeding season. thin pregnant dairy goats should be fed lb concentrate per ) . - . ( . ) . - . potassium (k; mmol/l) hp . - . ( . ) . - . ( . ___ . ) . - . ( . adata presented as ranges with mean and standard deviation in parentheses, s, serum; p, plasma; hp, heparinized plasma. clinical biochemistry data from kaneko et al. ( ) . day, with the amount increasing to . lb per head per day during the last weeks of gestation. forage should be fed ad libitum during this time. all newborn ruminants must receive passive immunity from colostrum, the first postpartum milk of a dam that contains concentrated protective maternal antibodies (most as igg ), functional leukocytes, cytokines, vitamins, minerals, and protein. colostrum also has laxative properties. trypsin inhibitors in the colostrum allow the passage of intact antibody molecules, by pinocytosis, through the neonate's gut wall and into the bloodstream during the first few days after birth. the quality of the colostrum is directly related to herd or flock management, vaccination programs, and the dam's overall condition and nutrition throughout gestation and at the time of parturition. ensuring effective colostrum transfer is also dependent on the timing and amount taken by the neonate. most neonatal ruminants can suckle well within hr of birth. those that do so have been shown to have significantly less diarrhea (naylor, ) . neonates weakened by dystocia or hypothermia, for example, should be hand-fed or tube-fed colostrum. if necessary, the dam should be hand-milked and the newborn fed colostrum (for example, - ml for kids) every - hr for the first - days. in typical management situations, dairy calves either are separated from their dams immediately after birth and bottle-fed colostrum, or they remain with their dams for only about hr and suckle fresh colostrum during this time. dairy producers then refrigerate and/or freeze the colostrum that cannot be consumed by the calf during that time and then feed this diluted : with warm water times a day to the calves during the next - days. extra frozen colostrum for emergencies may be obtained from dairy farmers; it is advantageous to obtain colostrum from well-managed herds and from the multiparous cows in the herd (not heifers) in the same geographic locale. holstein calves, for example, should receive a minimum of - liters within hr of birth and then be fed about - % of body weight in colostrum by hr of age. after days, calves are then placed on milk replacers. although young ruminants generally do well receiving their dams' milk, commercially available milk replacers are available and should generally be prepared and fed according to the manufacturer's recommendations. containers used to prepare and feed these replacers should be sanitized daily. the fat content of both calf and lamb milk replacers is excessive; however, calf milk replacers can be used for kids if care is taken not to overfeed. young ruminants can be offered good-quality hay (such as second cutting) to nibble on by week of age. calves may be provided with calf starter, a commercially available concentrate with appropriate levels of energy and protein, fed according to the manufacturer's recommendations at - weeks of age. they can be weaned off milk replacer by - weeks of age. young ruminants ( - months of age) need good-quality forage as well as grain and concentrate supplementation to promote development of the rumen. in farm management situations, forage can be silage, pasture, and hay. in a confinement situation like a research unit, good-quality hay, such as second cutting, is desirable. animals should not be overfed and should be offered a mineral mix free-choice. in contrast to dairy calves, beef calves remain with their mother cows until weaning at months of age. calves tend to suckle many times per day. as they mature, calves are creepfed, with the energy and protein content of the ration determined by the milk production of the dams and by the available forage, such as pasture. several useful references addressing ruminant reproduction in detail are available ("current veterinary therapy: food animal practice," practice," , practice," , "large animal internal medicine," ; "current therapy in large animal theriogenology," ; hafez, ) . sheep are seasonally polyestrous; most breeds will express estrus in the fall (northern hemisphere) and subsequently lamb in the spring. some breeds of sheep may cycle in both the fall and the spring. between seasonal periods of receptivity, the females undergo a long period of sexual quiescence called anestrus. in a research environment, ewes can be artificially stimulated to progress from anestrous to estrous cyclicity by maintaining the females in hr of light and hr of dark for - weeks. puberty is reached at about - months (or earlier) in both rams and ewes; rams will typically reach puberty before their female counterparts. ewes will display signs of estrus for about - hr and will ovulate spontaneously at the end of estrus. the estrous cycle length is - days, with an average of about days. following breeding, the average length of gestation is - days. slightly longer gestations are observed in animals carrying single lambs (singlets), in animals carrying rams, and in certain breeds such as those derived from merinos. prolificacy, or the number of lambs produced per gestation, tends to be dependent on the maturity of the dam (older dams tend to have multiple lambs) and on breed characteristics (some fine-wool breeds have fewer multiple births). the finn and dorset breeds are especially prolific. lambs vary in size at birth from about - lb up to lb. factors that affect birthweight include parental size, number of lambs in the litter (fewer lambs or singlets tend to be larger), age of the ewe (younger ewes have smaller lambs), lamb gender (males tend to be heavier), nutrition, and season or temperature (spring lambs tend to be larger than fall lambs). goats are seasonally polyestrous in temperate regions, so that young are born in favorable times of the year. they are shortday breeders, in that estrus (heat) is brought about by the decreasing light of shorter days. in temperate climates of the northern hemisphere, goats are normally anestrous during the summer and begin cycling in the fall. the actual length of the sexual cycle depends on day length, breed, and nutrition. most dairy goats cycle between august and february or march. nubians often have extended breeding cycles, and the sexual season of some breeds, including the alpine, can be extended by artificial means. the caprine gestation length averages days with a variation of - days. does bear singletons, twins, and triplets, with slightly shorter gestation when the doe is carrying triplets. cows are polyestrous. domestication of cattle has included selection against seasonality of the breeding season, particularly in dairy breeds but to some extent also in the beef breeds. in spite of this, cattle have been found to be still sensitive, in varying manifestations, to photoperiodicity. reproductive physiology in cattle is influenced by many factors. the reproductive programs in source herds and at well-managed facilities will be production-related. extensive coverage of both physiologic basics and specific industry-related criteriamfor retention of a cow as a breeder, for examplenare addressed in detail in texts and references oriented toward herd and production management ("current veterinary therapy," ). gestation in cattle is approximately days, with a range of - days. the length of gestation in cattle is influenced by fetal sex; fetal numbers; age and parity of the cow; breed; genotype of cow, bull, or fetus; nutrition; and local environmental factors. as noted, these factors are also important in sheep and goats. cows usually bear single calves, although twin births do occur. when twins are combinations of male and female calves, the female should be evaluated for freemartinism. ovine estrus detection is usually accomplished by the ram. nonetheless, because artificial insemination is achievable in ewes, clinical signs of estrus are important. typically, ewes in heat will show a mild enlargement of the vulva, with slight increases of mucus secretion. ewes may isolate from the flock and appear anxious. it is often better and clearly more reliable to employ the help of a sterile ram to mark females when they are in standing heat. two mating systems commonly employed include hand mating and group mating. with hand mating, ewes are placed either singly or in small groups with the ram of choice. ewes are removed as serviced. group mating involves placement of a mature ram with approximately - ewes for the entire -week breeding season. in either mating system, it is best to attach a marking harness to the male so that individual ewes can be identified as serviced. this is important so that parturition dates can be calculated. an easy, natural way to estimate pregnancy is by placing sterile teaser rams with the ewes at the end of the breeding season. any animal marked by the ram probably has not conceived. ultrasound scanners are also used for pregnancy detection. the ultrasound transducer is placed against the right abdomen; presence of a fetus is indicated on the machine. claims of % accuracy at weeks postbreeding have been made, although accuracy is generally best beyond days of gestation. interrectal doppler ultrasound probes detect fetal pulses. fetal heart rate is in the range of - beats per minute, whereas maternal heart rates tend to be - beats per minute. accuracy is best beyond days of pregnancy. rectal-abdominal palpation is an inexpensive alternative. a plastic probe is introduced intrarectally into the ewe, which is restrained on her back in a cradle. the plastic probe is then manipulated toward the abdomen while palpating for the fetus with the opposite hand. the age of the doe when she first expresses heat varies with breed. some does will express signs of heat between and months old. however, does should be - months old or at least - lb in weight before being bred. the caprine estrous cycle lasts - days. the duration of estrus is - hr but averages about hr. the estrous cycle can be more erratic in the beginning than in the end of the breeding season (smith, season (with winter delaying), and the level of nutrition (with higher levels hastening puberty). in some cases, the presence of mature cycling cows influences heifer puberty. with adequate nutrition, dairy breeds will reach puberty at - months and beef breeds at - months, and estrous cycles will occur regularly after the pubertal (first) estrus, maturing heifers will often have one or more ovulations before showing overt signs of estrus. only one follicle usually ovulates per estrous cycle (hafez, ) estrus, or standing heat, in cattle averages - hr in length, with a range of - hr ("large animal internal medicine," ) . detection of standing heat is important because it is closely related to the time of ovulation. ovulation occurs approximately - hr after estrus. detection of estrus is usually accomplished by visual observation of vaginal mucous discharge, mounting behavior by other females (i.e., the cow standing to be mounted is the individual in estrus), and receptivity to a bull (willingness to stand). successful visual detection of standing heat is dependent on observation skills of handlers, knowledge of the herd, stresses (e.g., detection decreased in bos taurus during heat stress), barn and yard surfaces (estrus detected better on dirt than on concrete), and maintaining a consistent observation schedule. teaser animals outfitted with marking devices are also used. other methods of detecting estrus include monitoring progesterone levels; glass slide and other evaluations of cervical mucus; change in vaginal ph; and body temperature changes (hafez, ) . estrous cycles are usually days in length, with a range of - days. it is recommended that a heifer deliver her first calf by years of age. after successful conception, progesterone levels in the cow remain elevated for most of the pregnancy, as the result of the ). "standing heat" is usually - hr but can be as short corpus luteum of pregnancy, and they decline only during the as a few hours. signs of estrus in goats include uneasiness, tail switching or "flagging," redness and swelling of the vulva, clear vaginal discharge that becomes white by the end of estrus, vocalization such as continuous bleating, and occasionally riding and standing with other does. a doe that is not in heat will not stand to back pressure or for attempts to hold her tail. does can be induced to show signs of heat by buck exposure and will ovulate within - days after introduction of the buck. goats ovulate during the later part of the estrous cycle, most between - hr after the onset of estrus. nevertheless, goats should be mated once signs of estrus are recognized and every hr until the end of estrus. most goats kid only once a year, although some goats near the equator may kid twice. once bred successfully, a goat will only rarely show signs of heat again. in fact, the first sign of pregnancy is usually a failure to return to heat, so animals should be carefully watched. pregnancy can be affirmed by a variety of means. goats will generally decrease milk production with pregnancy and should have at least a -to -week dry period for the udder to fully involute and prepare for the next milking period. in cattle, age of first estrus is dependent on the breed, the final month. conceptus implantation occurs beginning at about day . if the pregnancy fails before this time, the cow will begin to cycle again between days - , but if the pregnancy ends after day , there may be a delayed return to estrus. realtime ultrasonography can be used to determine pregnancy as early as days after insemination, with embyros seen by days - . fetal gender can also be determined by experienced personnel by this method by about day . detection of pregnancy can be successful by - days after conception by observation of failure to return to estrus or by palpation per rectum (detecting fetal membrane slip by days - and/or amniotic vesicle by days - ). palpation of the fetus is possible by day and placentomes by approximately days - . palpation later in presumed pregnancy will provide information based on differences in size of the two uterine horns, changes in the uterine wall, and fremitus in the miduterine artery. pregnancy can also be determined with reasonable success rates by determining if progesterone levels are elevated at days - after insemination. levels of bovine pregnancy-specific protein b may also be measured; this is produced by trophoblastic cells and is detectable by days - and elevated throughout pregnancy. placentation in sheep, goats, and cattle is epitheliochorial and ft. evaluation of a cow's udder prior to breeding and especotyledonary, in contrast to the diffuse or microcotyledonary cially as parturition approaches is important in order to assure placentas of horses and pigs. the placentomes, the infolded adequate nutrition and success of passive transfer by the functional units of the placenta, are formed as the result of fu-neonate. if the udder is edematous or if mastitis is present, for sion of the villi of the fetal cotyledons projecting into the crypts example, an alternate source of colostrum (such as frozen reof the maternal caruncles (specialized projections of uterine " serves) must made be available. poor udder conformation may mucosa). caruncles of sheep and goats are concave in shape, whereas those of cows are convex. the placentomes are distributed between the pregnant and nonpregant horns of the uterus in sheep, and there are - . in cattle, although the placentomes initially develop around the fetus, they will eventually be distributed to the limit of the chorioallantoic membrane even in the nongravid horn. the placentomes in the nongravid horn will be smaller than in the gravid horn. the total number will be - . the best birthing preparation for all dams is to ensure a proper plane of nutrition (not overnutrition) and adequate exercise. if possible, the dam should be confined to a birthing pasture or sanitized maternity pen a few days prior to parturition. the birthing environment will be very important in the overall health of the dam and offspring; stress minimization and a clean environment will benefit the immune health of both in the short and long term. outdoor parturition in a small birthing pasture has advantages. there is less stress and less intensity of pathogens. indoor maternity pens should be clean, dry, warm, well bedded, well ventilated but draft-free, and well lighted. adequate space per pen minimizes losses of neonates from being stepped and sat on by the dam. management of these pens, especially if concentrated in an area, is important to minimize pathogens to which dam and young are exposed. water troughs or buckets should be elevated or placed outside the pen, because lambs and kids have a tendency to fall or be pushed into them. soiled bedding should be removed from the birthing pen between dams, the area sanitized and allowed to dry, and fresh bedding installed for the next occupant. moving the female immediately before or during parturition may delay the birthing process. in goats, furthermore, in utero death may occur if parturition is unduly delayed. dams should be monitored closely during parturition for dystocias; these may result in loss of young or in young severely weakened from the prolonged birthing process. prior to parturition, ewes should be sheared or crutched. crutching refers to removing wool around the perineal and mammary areas; this minimizes fetal contamination during the birth process. foot trimming can be done at this time as well. the tail and perineal area of the doe should be clipped and cleaned to improve postbirth sanitation. in general, the pregnant doe needs a ft ( . m x . m) area for the birthing process, and area needs to be increased after birthing to allow spacing for kids. each cow should have a minimum pen area of ft x also be problematic; contingency plans should be made to ensure adequate support for the young if they cannot suckle from those udders. inexperienced heifers may react indifferently or aggressively to their offspring and should be monitored more closely than older, multiparous cows with uneventful calving histories. ewes approaching parturition generally isolate themselves from the flock, become restless, stamp their feet, blat, and periodically turn and look at their abdomen. the pelvic region will appear relaxed, and milk will be present in the udder. once hard labor contractions begin, lambs will usually be born quickly. animals that do not appear to be progressing correctly should be examined for dystocia. most cases of fetal malpresentation or malpositioning can be corrected via vagino-uterine manipulation. occasionally cesarean sections will be necessary. sanitation, cleanliness, and adequate lubrication are of utmost importance when performing obstetrical procedures. for about a week before parturition, rectal temperature of the doe will be above normal, or about ~ depending on environmental temperatures. approximately hr prior to birth, rectal temperature will fall to slightly below normal. many large dairy-goat facilities attempt to control the onset of parturition in order to assist birthing. the drug of choice to induce parturition in the goat is prostaglandin f ~ (pgf ~) (ott, ) . on day of gestation, goats given pgf ~ ( . - mg) will deliver kids within - hr. most goats prefer to kid alone and do so unaided. human interaction can actually interfere with normal birthing, especially in young or nervous does. some does may reject kids if extensive human interference occurs. does nearing parturition have an obviously swollen udder and a red, swollen vulva. pelvic ligaments at the base of tail relax. the doe may circle to make a bed, get up and down, look at her tail or sides, push other goats away, and bleat softly. signs of impending parturition include restlessness; vocalization (bleating softly); uneasiness, including getting up and down, pawing, and bedding; and a mucous discharge, leading to a moist tail. eight to hr prior to parturition, the cervix will dilate and the cervical mucous plug will be evident as a tan, smeared substance on the tail and perineum of the dam. kids should present within - hr in either anterior or posterior position. a posterior presentation can be recognized by the presence of upward-pointing feet. most does will rest between fetuses and are best left alone. however, if labor is prolonged more than hr, a vaginal exam is indicated. if the pregnant goat is housed with other goats, then herdmates will express great interest in the dam. unless moved prior to parturition, it is best to leave the dam with the group until after parturition, because removal may delay parturition. goats are not prone to retained placenta. normal kids will be quite active and will quickly attempt to stand and nurse. weak kids should be towel-dried, warmed (via heat lamp, heat pad, or warm water bottle), and assisted to nurse or fed colostrum. the goat is one of the few ungulate species that will exhibit "false pregnancy," or pseudopregnancy. this is a fairly common condition. does may have characteristically distended abdomens and may develop hydrometra and "deliver" large volumes of cloudy fluid at expected due dates. subsequent pregnancies can be normal. goats should be tested for pregnancy by days of age. veterinary use of prostaglandins has been successful in treating this condition. as in other species, parturition in cattle results from a combination of hormonal changes associated with the maturity of the fetus, notably acth (adrenocorticotropic hormone) and subsequent increases in fetal corticosteriods within days of birth. administration of acth to a fetus, or administration to the dam, results in premature birth. pregnancy is extended if fetal pituitary or adrenal glands are removed surgically. the fetal cortisol probably affects placental steroid production, accounting for sharp increases in the estrogens and estrogen precursors. coincident with this, maternal progesterone levels fall. the rising levels of estrogen cause release of maternal pgf ~ and induction of oxytocin receptors. most cows will separate themselves from the rest of the herd. a cow will lift her tail and arch her back when she is within a few hours of delivering the calf, and most cows are recumbent when delivering the calf. typically, the whole birthing process takes about min. the length of labor of cows carrying larger calves also will be longer. nervous heifers will take longer to deliver, and if they are disturbed, their labor may cease. all postparturient animals should be monitored for successful passage of these fetal membranes within hr of birth. veterinary intervention is required if not. cows occasionally eat placentas, which may subsequently obstruct rumen outflow and require surgical correction. for cattle, it is now recommended practice to remove membranes that have passed, in order to prevent ingestion. following lambing, it is critical that the newborns be "processed" so that they will have greatest survival chances. in a well-managed flock, many lambs and ewes will not need much assistance. when assistance is given, the newborn lamb's nose and mouth should be wiped free of secretions; gently swinging the lambs, head down, aids in removal of these fluids. the lamb should be dried off and stimulated through rubbing to aid its breathing. the lamb's navel should be dipped in an iodine solution to prevent subsequent navel infections. and the lamb should be identified by the application of an ear tag or ear notch. it is extremely important that the lamb be supplied with highquality colostrum within the first hr of birth. lambs that are not nursing on their own should be tube-fed with colostrum that has been collected and saved previously (i.e., frozen in ice cube trays) or collected from the mother after parturition. passive transfer can be assessed by measuring serum y-glutamyltransferase (ggt) levels (tessman et al., ) . after the first few days, colostrum changes over to milk. nursing lambs will ingest increasing amounts of milk as they grow. if the ewe cannot produce sufficient milk, the lamb should be "grafted" onto another ewe or fed artificially with a baby bottle. powdered milk replacers are commercially available; the content of ewe milk is much different from that of cow's milk; thus lamb milk replacer should specifically be used. one report notes that - % of lamb deaths occur during the first week of life and up to % occur within the first month. good management of ewes during gestation, care of the lamb at parturition, application of an appropriate vaccination program, and observation and intervention within the first several weeks of a lamb's life will minimize losses (ross, ) . immediately after birth, the placenta and any birthing materials should be removed from the doe's pen. kids do not usually need assistance. if kids are to be raised by the dam, they can be left alone; otherwise, kids should be towel-dried and removed from the dam. kids are cold-sensitive and may require a heat lamp or other source of added warmth in cold weather. navel cords should be dipped in tincture of iodine, and kids should be dehorned and castrated within the first several days of life. to control caprine arthritis encephalitis (cae), kids should be immediately removed from the dam and hand-fed heattreated colostrum. colostrum should be heat-treated for hr at ~ e the first feeding can be up to ml of colostrum. kids should receive a total of ml colostrum within the first - hr of birth. after day , kids can be placed on milk replacer. milk replacers should contain - % fat and - % milkbased protein. by days of age, kids should be consuming approximately . - . liters of milk per day. kids should be introduced to forages as soon as possible and may be weaned by - weeks or - lb body weight. milk that is fed can be reduced by weeks of age by decreasing either the volume fed or the number of feedings. as with other dams, a cow is usually very attentive to her newborn calf, cleaning and softly vocalizing to the neonate. calves typically are standing by hr after birth and are suckling within hr. as noted previously, dairy calves may be removed from the cow even before suckling, and the colostrum milked from the dam and given to the calf. assistance may be required for nervous heifers, after dystocias and in extreme circumstances such as severe cold. cleaning the newborn's nose and mouth, rubbing down the neonate, assuring that the calf does not get chilled, and assuring that it receives adequate colostrum are all important under any of these circumstances. a stressed calf's umbilical may be treated with an iodine or chlorhexidine solution, although some authors note no benefit of navel treatment, specifying that successful transfer of passive immunity and sound sanitary management of birthing area are the most crucial factors in preventing omphalitis (navel ill) (house, ; kersting, ; kasari and roussel, ) . because newborn calves can be deficient in vitamin a and iron, these may be injected to improve disease resistance (wikse and baker, ) . in cases in which the dams' colostrum is known to be deficient in antibodies against common diseases, vaccinations may be administered at day old and followed with boosters at regular intervals. dehorning is performed when horn buds appear. castration is performed between and weeks of age or later. sexing the young in any of the ruminant species is straightforward. the vulva of the female young is located just ventral to the anus. the genitalia of the male include a penis, located along the ventral midline, and a scrotum, located in the inguinal region. the phenomenon of the freemartin, a genetic female born as a twin to a male, is the result of anastomoses between placental circulations of the twin fetuses; the mixing of bloodforming cells and germ cells results in the xx/xy chimeras. this occurs in - % of phenotypic bovine females born as co-twins with males. the female will often have abnormal vulva and clitoris, and the vagina will be a blind end because of the lack of a cervix. sometimes singleton freemartins are born if the male fetus is lost after days' gestation. multiple births are selected for and are common in sheep; the freemartin phenomenon is regarded as rare. twinning is common in goats, and freemartinism occurs in about % of male-female pairs of twins. intersexes are seen in some goat breeds and when polled goats are mated. proof is usually based on evidence of abnormal genital development and reports of abnormal sexual behavior. prior to weaning, it must be established that lambs can nutritionally survive without mother's milk. thus, grain, and later roughage, should be offered to lambs well in advance of the day of weaning so that they can adjust to the feedstuff. to prevent the ewes from ingesting the lamb ration, a "creep" should be set up by building an area adjacent to the ewe-lamb pen and devising a slatted entry for the lambs to enter but not the ewes. therefore, the lambs will be accustomed to the new ration through this creep-feeding process. if lambs and ewes will be pastured later in the spring, it is still beneficial to creep-feed lambs until pasture growth is adequate enough to fulfill the requirements of the growing lambs. lambs that are consuming . - lb of creep feed per day may be weaned. depending on the individual program, lambs may be weaned as early as weeks of age, although - weeks of age is more common. if ewes are of a breed that will cycle twice a year, and if it is expected that they will be rebred, then the lambs must be weaned as early as possible so that lactational anestrus will resolve and ewes will recycle. another factor is the cost of lactation rations for the ewes; if lamb grain is more economical than ewe grain, then lambs should be weaned. about - days prior to weaning, feeding of the lactation ration to the ewes should be discontinued, and only roughage fed. at weaning, the lambs should be removed in the creep, and the ewes removed to an area that is not within sight (and preferably sound) of the lambs. the ewes should be monitored for postweaning mastitis and treated as necessary. ewes that have physical or disease problems or that have not been productive at lambing or feeding their lambs should be culled. the lambs should be monitored to assure that they continue to gain weight and are eating the new ration. kids should be introduced to forages within the first week of life because the natural curiosity of these animals will cause them to investigate sources of feed. kids can be weaned by - weeks or - lb. hand-fed milk should be reduced by weeks of age by reducing the volume fed or by decreasing the number of feedings. dairy calves are now usually removed from their dams immediately after birth. it is less common now to allow the calves to remain with their dams for about hr and suckle fresh colostrum during this time, because their intake will be inadequate. dairy producers refrigerate and/or freeze the colostrum produced during the first hr and feed this, diluted : with warm water, twice a day to the calves during the next - days. holstein calves, for example, should receive a minimum of - liters within hr of birth and then be fed about - % of body weight in colostrum by hr of age. after days, calves are then placed on milk replacers, preformulated powders reconstituted with water that provide complete nutrition. milk replacers are commercially available and should be fed according to manufacturer's recommendations vaccination programs for calves vary with the preventive medicine program for the overall herd. passive immunity provided by colostrum from cows on sound management programs will last until a calf is about - months old; normally vaccinations are not necessary and are contraindicated during those first months. the duration of passive immunity varies considerably among calves, however; some producers choose to begin vaccinating calves at - months of age and continue with monthly booster immunizations until the animals are months old, when passive immunity is no longer a possibility. artificial insemination (ai) in sheep is more difficult than in cattle because sheep are smaller and cannot be reproductively manipulated via the rectum and because the cervix of sheep is more difficult to traverse with the insemination pipette. breeding animals artificially with fresh semen produces pregnancy rates averaging % (not unlike that of cattle); artificial insemination with frozen semen is less successful. several artificial insemination techniques have been used. laparoscopic ai involves the surgical instillation of semen into the uterus through a small abdominal opening. the procedure is successful but is technically involved and costly. cervical ai involves the transvaginal introduction of semen into the cervix. a modification of this technique (transcervical ai) allows for penetration through the cervix into the uterus. this method (called the guelph system for transcervical ai) leads to successful penetration into the uterus in up to % of ewes when performed by an experienced inseminator. artificial insemination is now an integral part of dairy herding; natural insemination as a management practice is relatively rare. technicians performing the ai technique are available through commercial enterprises. dairy production employees are also trained. information regarding the management of the donors and recipients, the storage and handling of the semen, and the skills and record keeping required is covered extensively elsewhere (nebel, ) . because sheep are hormonally similar to other ruminants, estrous synchronization techniques are comparable. progesterone suppresses follicle-stimulating hormone (fsh) secretion, preventing animals from developing follicles and exhibiting estrus. artificial or natural progesterone can be administered in the feed, through parenteral injection, subcuticular implants, and vaginal pessaries. the progesterone is withdrawn in about - days, after which the fsh secretion will initiate the process of follicle development (trower, ) . estrus usually will occur in - hr (average is hr). a natural method of synchronization, often applied to promote flock breeding within a short period of time (and thus parturition will be within a narrow window as well), is the introduction of sterile rams with the ewes before the beginning of the normal fall mating period. pheromones released from males naturally stimulate the females to cycle and to synchronize their heats. it should be noted that introduction of a male during late anestrus will often stimulate ovulation in about days; however, this cycle will generally be without clinical signs of estrus (silent heat). vasectomy of rams is one method of producing sterile "teaser rams." introduction of the buck to a group of does will induce ovulation and may even synchronize does. does that are kept separate from the buck will show signs of estrus, will ovulate within - days, and will have normal pregnancies when introduced to a buck. bucks with horns and intact scent glands are better able to induce ovulation than dehorned bucks, whose scent glands often been removed. control of breeding in the goat has been studied mostly in dairy breeds in order to produce milk throughout the year and to reduce kidding labor. goats in the luteal phase of the estrous cycle, days - , are sensitive to pgf ~ ( . - mg im) and will show estrus in - hr postinjection (bretzlaff, ) . dosing cycling animals twice days apart will synchronize goats, and artificial insemination using this method has resulted in - % conception rates (bretzlaff, ; greyling and van niekerk, ) . programs for timed breeding have been described and involve administering progestogens (bretzlaff, ) . vaginal pessaries of fluorogestone acetate left in place for days in the doe followed by an injection of pregnant mare serum gonadotropin (pmsg) at the time of pessary removal have proven successful. also, when primed by pgf ~, an day regimen of fluorogestone acetate with pmsg given on day has been successful. synchronization of cattle estrous cycles and superovulation are used as management techniques in certain commercial cattle and dairy production settings where estrus synchronization or embryo transfer is advantageous to production and management. the methodology is also used in the research setting for coordinating donors and recipients of embryos or other genetically manipulated tissues for implantation. the options and dosing regimens are described in detail in veterinary clinical texts (wenzel, ; vanderboom et al., ) . in synchronization, the principle is lysis of the existing corpus luteum. the more common practices involve the use of products approved for use in cattle such as pgf ~, one of its analogs, or products containing estradiol valerate. progestogens are also used in conjunction with estradiol valerate. other approaches, involving management techniques combined with pharmacologic interventions, are considered less successful. superovulation regimens involve injections of fsh either alone or with pgf ~ at timed internals. estrus is expected hr after the final injection, and two inseminations are performed at hr intervals after estrus detection. preparation of recipients involves injection of pgf ~ or progestogens with gonadotropins such as pmsg. for greatest success as management tools, these must be combined with a consistent program that provides appropriate nutrition for all cattle involved. synchronization of animals is also influenced by several other factors, however, such as time in the cycle when hormones are administered, response by each individual animal, whether the cow is a dairy or beef animal, parity and maturity of the cows, success of heat detection after the luteolysis, and accurate record keeping. embryo transfer involves the removal of multiple embryos from a superovulated embryo donor and transferring them to synchronized recipients. this method maximizes the genetic potential of the donor animal. the donor animal is hormonally superovulated and inseminated. in sheep, about week after breeding, the embryos are surgically removed from the donor's uterus. in cattle, the procedure is nonsurgical. about % of expected embryos (determined by counting corpora lutea) can be recovered; successful recovery is affected by factors such as age of the donor, reproductive health, and experience of the surgeon or technician. furthermore, not all collected embryos are of transferable quality. recipients are hormonally synchronized with the donor animals. on the day of embryo collection, transferable embryos are implanted into the uterus of the recipient; laparoscopy has been used in the past and is now being replaced by nonsurgical methods. pregnancy rates average about %. if recipients are not available, embryos, like sperm, can be frozen and kept for later transfer. embryo transfer is commonly practiced in cattle as a herd improvement technique and as a research technique for engineered embyros. disease screening programs for all animals involved are important because several pathogens can be transmitted directly or indirectly, such as bovine viral diarrhea virus, bluetongue virus, infectious bovine rhinotracheitis virus, and mycoplasmal species. in sheep flocks and goat herds, as noted, male young are usually castrated by month of age. the elastrator method is the more popular for animals less than week of age. other methods include the emasculatome (crushing) and surgical removal ("knife method"). the distress associated with castration and tail docking in lambs is the subject of debate and has been researched recently (kent et al., ) . as noted, male calves are usually castrated as early as possible and no later than month of age. in some production situations, however, where maximum hormone responsive muscle development and grouping animals together for procedures dictate scheduling, the procedure may be performed on older males. open and closed techniques are used, depending on the age of animals and on veterinary or farm practice. breeding and vasectomized rams and bucks are usually maintained by medium to large production farms. smaller farms often borrow breeding males. breeding males are typically selected by production record, pedigree, and/or breed. vasectomized males are often retired breeders and should be tattooed or identified clearly to avoid any wasted breeding time. the vasectomy technique for both species is comparable (smith and sherman, ) . rams may be housed together for most of the year, whereas bucks are penned separately. because ewes will exhibit only a limited number of estrous cycles before becoming reproductively quiescent, it is critical that the male be capable of successfully breeding the female in an expeditious manner. any defects in the external genitalia, reproductive diseases, or musculoskeletal abnormalities may prevent successful copulatory behaviors. furthermore, it is impor-tant to know the semen quality of the ram as one indicator of fertility. semen can be collected via electroejaculation or by use of a teaser mount. once semen is collected, it should be handled carefully and kept warm to prevent sperm death, leading to improper conclusions about the male. typically, the characteristics usually evaluated as a determinate of sperm quality are volume (normal between . and . ml); motility (% of sperm moving in a forward wave; high quality is associated with motility of approximately %); concentration (sperm count per unit of volume as measured by a hemocytometer; high-quality semen should contain . x sperm per ml); morphology (live versus dead cells, as determined by special stains and the percentage of abnormal-appearing sperm; neither the abnormalities nor the dead sperm should exceed % in high-quality semen). the extensive use of artificial insemination in the dairy cattle industry has minimized the use of bulls on many farms, although a farm may maintain a few bulls for heat detection and for "cleanup" breeding. breeding bulls are maintained in beef production establishments. breeding bulls must be part of the herd vaccination program, with special attention to appropriate timing of immunizations for the commonly transmitted venereal diseases campylobacteriosis and trichomoniasis. tail docking is a relatively recent development in dairy herd management and is practiced in the belief that it will minimize bacterial contamination of the udder and therefore the milk. tails are typically docked to about inches in length. the practice is more popular in certain regions in the united states. to date, there is no published study indicating that this technique provides any distinctive advantage over keeping the tail switch hair clipped short. healthy ruminants have good appetites, chew cud, are alert and curious, have healthy intact coats, move without hindrance, and have clear, bright, clean eyes and cool dry noses. even adult animals, when provided sufficient space, will play. sheep and goats have tidy "pelleted" dark green feces. cattle have pasty, moist, dark green-brown feces. ruminants normally vocalize, and handlers will learn to recognize normal communication among the group or directed at caregivers in contrast to that when animals are stressed. excessive, strained vocalizations are often a sign of stress in cattle. "bruxism," or grinding of the teeth by a ruminant, is usually associated with discomfort or pain. other signs of discomfort, stress, or illness include decreased time spent eating and cud chewing, restlessness, prolonged recumbency with outstretched neck and head, and hunched back when standing. unhealthy ruminants may be thin, may arch their backs or favor a limb, or may have external lumps or swollen joints, an unusual abdominal profile, or rough or dull coats. all ruminants are herd animals to some extent and social individuals; therefore, every effort should be made to allow contact among animals, in terms either of direct contact or of sound, smell, or sight. human contact and handling should be initiated promptly and maintained regularly and consistently throughout the animal's stay in the research facilities. animals should be provided sufficient time to acclimate to handlers and research staff. cattle and sheep can hear at higher frequencies than humans can and may react to sounds not perceived by handlers. knowledge of the peculiarities of sheep behavior will increase the ease of handling and decrease stress-related effects in research. generally, fine-wooled breeds, such as rambouillet, are the most gregarious and are best handled in groups. the meat, or "downs," breeds tend to be less gregarious, and the long-wooled breeds tend to be solitary (ross, ; asia, ) . nonetheless, movement of animals is simplified by proper facility design. sheep have a wide-angle visual field and are easily scared by activities that are taking place behind them. sheep should be moved slowly and gently. to capture individuals within a flock, it is best to confine the flock to a smaller space and use a shepherd's crook or to gently catch the animal in front of the neck/thorax. grabbing the wool can injure the animals, as well as damage the wool and the underlying tissues. sheep move best in chutes that have solid walls, and individual animals will generally follow a lead animal. any escape route will be challenged and, if successfully breached, will disrupt the entire flock movement. sheep movement is also disrupted by contrasts such as light and shadows that impinge on a chute or corral. finally, like most animals, sheep have a flight zone (minimum zone of comfort), the penetration of which will result in sheep scattering. this minimal flight distance can be modified by increasing handling of the animals and working at the edge of the zone, but it should always be considered when working with animals in chutes, pens, or other confined areas. goats exhibit behavioral characteristics that make them quite distinct from other ruminants. their browsing activity makes them quite orally investigative. goats will readily nibble or chew just about anything they come in contact with, so researchers should keep all paperwork and equipment out of reach. a herd of goats will readily chew through wood gates and fencing, especially when confined in areas without alternatives for chewing behavior. goats are also inquisitive, restless, agile jumpers and climbers, and quite mischievous. if maintained in paddocks, strong high fences are essential, as are adequate spaces for exercise or boulders or rock piles for hoof maintenance and recreational climbing. goats are more tolerant of isolation and are more easily acclimated to human contact than sheep are, but goats will confront unfamiliar intruders and make sneezing noises. goats with horns will use them to advantage, and horns may also become entangled in fencing. although less strongly affected by flock behavior, goats are social animals. most goats raised in close human contact are personable and cooperative and can easily be taught to stand for various procedures, including blood collection. an understanding of breed behaviors, sources of stress in cattle, play behaviors, calf behaviors, and dominance determinants will contribute to prevention of injuries to handlers and better health and welfare of the animals. ruminants of all ages, especially cattle of all ages, should be handled with an appreciation of the serious injury to human handlers that may result (houpt, ) . cattle have a wide visual field, as sheep do, and a flight zone that varies in size, according to previous handling experiences (gentle handling and animal tameness make the flight zone smaller) and the circumstances of the moment (grandin, ) . groups of cattle are moved effectively around a facility by utilizing chute systems, with sequences of gates, that minimize chances of animals turning around. dairy cattle have been bred and selected over centuries for their docile, tractable characters and production characteristics. in contrast, beef breeds have not been selected for docility and are generally more difficult to handle and restrain. beef breeds, such as angus, are known for their independent natures and protective maternal instincts. all cattle respond well to feed as a reward for desired behavior. healthy cattle typically are very curious and watchful and are alert to sounds and smells. when not grazing or eating, they hold their heads up. when sleeping, the head and neck may be tucked back. because of ruminant digestive and metabolic needs, much of the day is spent eating or cud chewing. occasionally, adult cows sit upright like dogs. cattle maintained inside tend to be more docile. in addition to forced isolation from other cattle, sources of stress include rough attitudes of handlers and unfamiliar visual patterns, routines, or environments. these stressors may exacerbate signs of systemic illnesses. calves are known for non-nutritive suckling, bar licking, and tongue rolling. non-nutritive suckling behavior is greater in hungry calves and also right after a milk meal. it is best to provide nipples and other clean noninjurious materials for the animals to suck. non-nutritive suckling can be detrimental in group-housed calves because it can result in disease transmission and hair ball formation. environmental enrichment devices have been developed to cope with this behavior. the behavior diminishes as the animals are weaned onto solid food (morrow-tesch, ) . play activity and vocalizations of calves mimic adult dominance behaviors. play activity by young adult cattle is more common in males, can be quite rough, and is often triggered by a change in the environment. dominance behaviors are dependent on direct physical contact among the cattle, and dominance hierarchies are established within a herd. horns, age, and weight have been reported to be the most important determi-nants. aggressive behaviors in cattle may be triggered by newly introduced animals or unfamiliar visual patterns and by feeding when animals are very hungry. aggression is more common among intact adult males. this section focuses primarily on the more common diseases affecting sheep, goats, and cattle in the united states and elsewhere in north america and those that are reportable. for detailed information not included in this limited overview and for diseases of importance internationally, the authors recommend several excellent comprehensive and focused veterinary clinical texts and periodicals that address ruminant diseases, preventive medicine, and individual and flock or herd management. these are listed under "major references" in the reference list at the end of this chapter. recommendations for current drug therapies, both approved and off-label use in ruminants, including withholding prior to slaughter, formularies, and related information can be found in the references noted above and in formularies (hawk and leary, ; plumb, ) . in addition, the food animal residue avoidance databank (farad), accessible on the internet <http://www.farad.org>, should be used as a resource. farad is a food safety project of the u.s. department of agriculture and is an information resource to prevent drug and pesticide residues in food animals and animal products. food; may be anorexic, weak, unthrifty and depressed; and may salivate excessively. diagnosis is made based on clinical signs and is confirmed by culture. epizootiology and transmission. the organism penetrates wounds of the skin, mouth, nose, gastrointestinal tract, testicles, and mammary gland. rough feed material and foreign bodies may play a role in causing abrasions. actino bacillus lignieresii then enters into deeper tissues, where it causes chronic inflammation and abscess formation. lymphatic spread may occur, leading to abscessation of lymph nodes or infection of other organs. necropsy findings. purulent discharges of white-green exudate drain from the tracts that often extend from the area of colonization to the skin surface. exudates will also contain characteristic small white-gray (sulfurlike) granules. the pus is usually nonodorous. differential diagnosis. contagious ecthyma and caseous lymphadenitis are the primary differentials. diseases or injuries causing oral pain and discomfort, such as dental infections, foreign bodies, and trauma, should be considered. treatment. animals should be fed softer feeds. antibiotics such as sulfonamides, tetracyclines, and ampicillin are effective, although high doses and long durations of therapy are required. penicillin is not effective. weekly systemic administration of sodium iodide for several weeks is not as effective as antibiotic therapy. surgical excision and drainage are not recommended. etiology. actinobacillus lignieresii is an aerobic, nonmotile, non-spore-forming, gram-negative rod that is widespread in soil and manure and is found as normal flora of the respiratory, gastrointestinal, and reproductive tracts of ruminants. in sheep and cattle, a. lignieresii causes sporadic, noncontagious, and potentially chronic disease characterized by diffuse abscess and granuloma formation in tissues of the head and occasionally other body organs. this disease, called wooden tongue, has not been documented in goats. clinical signs. skin lesions are common. tongue lesions are more common in cattle than in sheep. lip lesions are more common in sheep. soft-tissue or lymph node swelling accompanied by draining tracts is observed in the head and neck regions, as well as other areas. animals may have difficulty prehending prevention and control. because the organism enters through tissue wounds, especially those associated with oral trauma, feedstuffs should be closely monitored for coarse material and foreign bodies. b. arcanobacterium infection (formerly actinomycosis, or "lumpy jaw") etiology. arcanobacterium (formerly known as actinomyces or corynebacterium) pyogenes and a. bovis are anaerobic, nonmotile, non-spore-forming, gram-positive, pleomorphic rods to coccobacilli. arcanobacterium bovis is a normal part of the ruminant oral microflora and is the organism associated with "lumpy jaw" in cattle; this syndrome is rarely seen in sheep and goats. this organism has also been associated with pharyngitis and mastitis in cattle. clinical signs and diagnosis. arcanobacterium bovis causes mandibular lesions primarily. the mass will be firm, nonpainful, and immovable. draining tracts may develop over time. if teeth roots become involved, painful eating and weight loss are evident. radiographic studies are helpful for determining fistulas. diagnosis is based on clinical signs, and culture is required to confirm arcanobacterium. the prognosis is poor for lumpy jaw. epizootiology and transmission. these organisms are normal flora of the gastrointestinal tracts of ruminants and gain entrance into the tissues through abrasions and penetrating wounds. necropsy. draining lesions with sulfurlike granules (as with actinobacillosis) are frequently observed. ious degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, hematuria, and so on. severe sequelae may include septicemia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, and endocarditis. research complications. young stock affected by omphalophlebitis may be inappropriate subjects because of growth setbacks and physiologic stresses from the infection. affected adult animals will not thrive and, even with therapy, may not be appropriate research subjects. pathogenesis. arcanobacterium pyogenes is known to produce an exotoxin, which may be involved in the pathogenesis. differential diagnosis. actinobacillus lignieresii and caseous lymphadenitis are important differentials for draining tracts. a major differential for omphalophlebitis is an umbilical hernia, which will typically not be painful or infected. there are many differentials for septic joints and polyarthritis: chlamydia spp., mycoplasma spp., streptococci, coliforms, erysipelothrix rhusiopathiae, fusobacterium necrophorum, and salmonella spp. tumors, trauma to the affected area, such as the mandible, and dental disease or oral foreign body should also be considered. prevention and control. arcanobacterium bovis lesions can be prevented or minimized by feeds without coarse or sharp materials. treatment. penicillin or derivatives such as ampicillin or amoxicillin are treatments of choice. sodium iodides (intravenous) and potassium iodides (orally) have been utilized also. extended antibiotic therapy may be necessary. surgical excision is an option. in addition to medications noted above, isoniazid is somewhat effective for a. bovis infections in nonpregnant cattle. research complications. the possibility of long-term infection and long therapy are factors that will diminish the value of affected research animals. omphalophlebitis, omphaloarteritis, omphalitis, and navel ill are terms referring to infection of the umbilicus in young animals. arcanobacterium pyogenes is the most common organism causing omphalophlebitis, an acute localized inflammation and infection of the external umbilicus. most cases occur within the first months of age, and animals are presented with a painful enlargement of the umbilicus. animals may exhibit var- etiology. bacillus anthracis is a nonmotile, capsulated, sporeforming, aerobic, gram-positive bacillus that is found in alkaline soil, contaminated feeds (such as bonemeal), and water. common names for the disease anthrax include woolsorters' disease, splenic fever, charbon, and milzbrand. clinical signs and diagnosis. anthrax is a sporadic but very serious infectious disease of cattle, sheep, and goats characterized by septicemia, hyperthermia, anorexia, depression, listlessness, depression, and tremors. subacute and chronic cases may occur also and are characterized by swelling around the shoulders, ventral neck, and thorax. the incubation period is day to weeks. bloody secretions such as hematuria and bloody diarrhea often occur. abortion and blood-tinged milk may also be noted. the disease is usually fatal, especially in sheep and goats, after - days. death is the result of shock, renal failure, and anoxia. diagnosis is based on the clinical signs of peracute deaths and hemorrhage. stained blood smears may show short, single to chained bacilli. blood may be collected from a superficial vein and submitted for culture. epizootiology and transmission. cattle and sheep tend to be affected more commonly than goats, because of grazing habits. older animals are more vulnerable than younger, and bulls are more vulnerable than cows. although the disease occurs worldwide, and even in cold climates, most cases in the united states occur in the central and western states, and outbreaks usually occur as the result of spore release after abrupt climatic changes such as heavy rainfall after droughts or during warmer, dryer months. spores survive very well in the environment. the anthrax organisms (primarily spores) are generally ingested, sporulate, and replicate in the local tissues. abrasive forages may play a role in infection. transmission via insect bites or through skin abrasions rarely occurs. necropsy. necropsies should not be done around animal pens or pastures, and definitive diagnoses may be made without opening the animals. incomplete rigor mortis, rapid putrefaction, and dark, uncoagulated blood exuding from all body orifices are common findings. blood collected carefully and promptly from peripheral veins of freshly dead animals can be used diagnostically. splenomegaly, cyanosis, epicardial and subcutaneous hemorrhages, and lymphadenopathy are characterisitic of the disease. pathogenesis. the rapidly multiplying organisms enter the lymphatics and bloodstream and result in a severe septicemia and neurotoxicosis. encapsulation protects the organisms from phagocytosis. liberated toxins cause local edema. differential diagnosis. although anthrax should always be considered when an animal healthy the previous day dies acutely, other causes of acute death in ruminants should be considered, e.g., bloat, poisoning, enterotoxemia, malignant edema, blackleg, and black disease. prevention and control. outbreaks must bereported to state officials. anthrax is of particular concern as a bioterrorism agent. any vaccination programs should also be reviewed with regulatory personnel. herds in endemic areas and along waterways are usually vaccinated routinely with the sterne-strain spore vaccine (virulent, nonencapsulated, live). careful hygiene and quarantine practices are crucial during outbreaks. dead animals and contaminated materials should be incinerated or buried deeply. biting insects should be controlled. the disease is zoonotic and a serious public health risk. treatment. treatment of animals in early stages with penicillin and anthrax antitoxin (hyperimmune serum, if available) may be helpful. amoxicillin, erythromycin, oxytetracycline, gentamicin, and fluoroquinolones are also good therapeutic agents. during epidemics, animals should be vaccinated with the sterne vaccine. research complications. natural and experimental anthrax infections are a risk to research personnel; the pathogen may be present in many body fluids and can penetrate intact skin. the organism sporulates when exposed to air, and spores may be inhaled during postmortem examinations. etiology. brucella is a nonmotile, non-spore-forming, nonencapsulated, gram-negative coccobacillus. brucella abortus is one of several brucella species that infects domestic animals but cross-species infections occur rarely. brucella abortus or b. melitensis may cause brucellosis in sheep, cattle, and goats. brucella melitensis (biovar , , or ) is the primary cause of sheep disease (garin-bastuji et al., ) . brucella ovis is more commonly associated with ovine epididymitis or orchitis than abortion. in the united states, clusters of brucellosis are still found in western areas contiguous to yellowstone national park. bang's disease is the common name given to the disease in ruminants. clinical signs and diagnosis. brucella melitensis in the adult ewe is generally asymptomatic and self-limiting within about months. however, because the organism may enter and cause necrosis of the chorionic villi and fetal organs, abortion or stillbirths may occur. abortion usually occurs in the third trimester, after which the ewe will appear to recover. it has been reported that up to % of infected ewes may abort more than once. rams will also be infected and may develop orchitis or pneumonia. the disease caused by b. ovis is manifested by clinical or subclinical infection of the epididymis, leading to epididymal enlargement and testicular atrophy. brucella ovis causes decreased fertility. brucella melitensis is the more common cause of brucellosis in goats. brucella abortus has been shown to infect goats in natural and experimental infections, and b. ovis has also been shown to infect goats experimentally. does infected with b. melitensis will also abort during the third trimester. infections with b. abortus in cattle produce few clinical signs. there may be a brief septicemia during which organisms are phagocytosed by neutrophils and fixed macrophages in lymph nodes. in cows, the organism localizes in supramammary lymph nodes and udders and in the endometrium and placenta of pregnant cows. infection may cause abortions after the fifth month, with resulting retained placentas. permanent infection of the udder is common and results in shedding of organisms in milk. in bulls, the organism may cause unilateral orchitis and epidydimitis and involvement of the secondary sex organs. organisms may be in the semen. in infected herds, lameness may also be a clinical sign. diagnosis of brucellosis can be made by bacterial isolation of the brucella organism from necropsy samples (especially the fetal stomach contents), as well as by supportive serological evidence. many serological tests are available, such as the tube and plate agglutination tests, the card or rose bengal test, the rivanol precipitation test, complement fixation, enzyme-linked immunosorbent assay (elisa), polymerase chain reaction (pcr), and others. test selection is often dependent on state requirements in the united states. epizootiology and transmission. the primary route of transmission of b. abortus is ingestion of the organism from infected tissues and fluids (milk, vaginal and uterine discharges) during and for a few weeks after abortion or parturition; contaminated semen is considered to be a minor source of infection. exposure to the organism may occur via the gastrointestinal tract (contaminated feed or water), the respiratory tract (droplet infection), or the reproductive tract (contaminated semen) and through other mucous membranes such as the conjunctiva. brucella ovis is transmitted in the semen, as well as orally or nasally through contaminated feed and bedding. necropsy findings. a sheep fetus aborted due to brucella will exhibit generalized edema. the liver and spleen will be swollen, and serosal surfaces will be covered with petecchial hemorrhages. peritoneal and pleural cavities often contain serofibrinous exudates. the placenta will be leathery. pathogenesis. ruminants are considered especially susceptible to brucella infection, because of higher levels of erythritol (a sugar alcohol), which is a growth stimulant for the organism. brucella utilizes erythritol preferentially over glucose as an energy source. placentas and male genitalia also contain high levels of erythritol. brucella organisms also evade lysis when phagocytosed by macrophages and neutrophils and survive intracellularly in phagosomes. abortion is the result of placentitis, typically during the third trimester of gestation. brucella ovis enters the host through the mucous membranes, then passes into the lymphatics, causes hyperplasia of reticuloendothelial cells, and is spread to various organs via the blood. the organism localizes in the epididymides, the seminal vesicles, the bulbourethral glands, and the ampullae. orchitis may be a sequelae of the disease. epididymitis can be diagnosed by identifying gross lesions by palpation of the epididymides, by serological evidence of antibodies to b. ovis, and by semen cultures. differential diagnosis. differential diagnoses include all other abortion-causing diseases. many other agents, such as actinobacillus spp., arcanobacterium (actinomyces) pyogenes, eschericia coli, pseudomonas spp., proteus mirabilis, chlamydia, mycoplasma, and others may be associated with ovine epididymitis and orchitis. a clinically and pathologically similar agent, actinobacillus seminis, has been isolated from virgin rams. this organism has morphological and staining characteristics similar to those of b. ovis and complicates the diagnosis (genetzky, ) . prevention and control. the rev vaccine has been recommended for vaccination of ewe lambs in endemic areas, but this vaccine is not used in the united states. separating young rams from potentially infected older males, sanitizing facilities, and vaccinating them with b. ovis bacterin can prevent the disease. over the past years, aggressive federal and state regulatory and cattle herd health programs in the united states have provided control and prevention mechanisms for this pathogen through a combination of serological monitoring of herds, slaughter of diseased animals, herd management, vaccination programs, and monitoring of transported animals. most states are considered brucellosis-free in the cattle populations; thus, procurement of ruminants that have been exposed to this infectious agent will be unlikely. cattle vaccination programs can be very successful when conducted on a herd basis to reduce likelihood of exposure. strain and the recently validated attentuated strain rb are live vaccines and can be used in healthy heifer calves - months old. vaccination for older animals may be done under certain circumstances. vaccination of bull calves is not recommended, because of low likelihood of spread through semen and possibility of vaccination-induced orchitis. the strain vaccine induces long-term cell-mediated immunity, protects a herd from abortions, and protects the majority of a herd from reactors during a screening and culling program. the vaccine will not, however, protect the animals from becoming infected with b. abortus. strain vaccine induces an antibody response in cattle. the rb vaccine does not result in antibody titers and therefore is advantageous because infection with brucella can be determined serologically. the rb vaccine has been designated as the official calfhood bovine brucellosis vaccine in the united states by the u.s. department of agriculture's animal and plant health inspection service (aphis) (stevens et al., ) . brucella vaccine should be administered to unstressed, healthy cattle, with attention to particular side effects of the vaccination material and to prevention of compounding stresses associated with weaning, regrouping, other management changes, and shipping. the rb is regarded as less pathogenic and abortigenic in cattle. clinical signs and diagnosis. ovine vibriosis is a contagious disease that causes abortion, stillbirths, and weak lambs. the organism inhabits the intestines and gallbladder in subclinical carriers. abortion generally occurs in the last trimester, and abortion storms may occur as more susceptible animals, such as maiden ewes, become exposed to the infectious tissues. it is reported that - % of the flock may become infected and up to % of the ewes will die (jensen and swift, ) . some lambs may be born alive but will be weak, and dams will not be able to produce milk. diagnosis is achieved by microscopic identification or isolation of the organism from placenta, fetal abomasal contents, and maternal vaginal discharges. tentative identification of the organism can be made by observing curved ("gull-wing") rods in giemsa-stained or ziehl-neelsen-stained smears from fetal stomach contents, placentomes, or maternal uterine fluids. epizootiology and transmission. campylobacteriosis occurs worldwide. campylobacter spp., such as c. jejuni, normally inhabit ovine gastrointestinal tracts. transmission of the disease occurs through the gastrointestinal tract, followed by shedding, especially associated with aborted tissues and fluids. in abortion storms, considerable contamination of the environment will occur due to placenta, fetuses, and uterine fluids. ewes may have active campylobacter organisms in uterine discharges for several months after abortion. the bacteria will also be shed in feces, and feed and water contamination serve as another source. there is no venereal transmission in the ovine. necropsy. aborted fetuses will be edematous, with accumulation of serosanguinous fluids within the subcutis and muscle tissue fascia. the liver may contain - cm pale foci. placental tissues will be thickened and edematous and will contain serous fluids similar to those of the fetus. the placental cotyledons may appear gray. pathogenesis. the organism enters the bloodstream and causes a short-term bacteremia ( - weeks) prior to the localizing of the bacteria in the chorionic epithelial cells and finally passing into the fetus. should be considered in late gestation ovine abortions. a bacterin is available to prevent the disease. carrier states have been cleared by treating with a combination of antibiotics, including penicillin and oral chlortetra-cycline. aborting ewes should be isolated immediately from the rest of the flock. after an outbreak, ewes will develop immunity lasting - years. treatment. infected animals should be isolated and provided with supportive therapy. prompt decontamination of the area and disposal of the aborted tissues and discharges are important. research complications. losses from abortion may be considerable. campylobacter ssp. are zoonotic agents, and c. fetus subsp, intestinalis may be the cause of "shepherd's scours." ii. clinical signs and diagnosis. preliminary signs of a problem in the herd will be a high percentage of cows returning to estrus after breeding and temporary infertility. this will be particularly apparent in virgin heifers that may return to estrus by days after breeding. long interestrous intervals also serve an indication of a problem. spontaneous abortions will occur in some cases, typically during the fourth to eighth months of gestation. severe endometritis may lead to salpingitis and permanent infertility. demonstration or isolation of the organism, a curved rod with corkscrew motility, is the basis for diagnosis. the vaginal mucous agglutination test is used to survey herds for campylobacteriosis. serology will not be worthwhile, because the infection does not trigger a sufficient antibody response. culture from breeding animals may be difficult because campylobacter will be overgrown by faster-growing species also present in the specimens. epizootiology and transmission. the bacteria is an obligate, ubiquitous organism of the genital tract. transmission is from infected bulls to heifers. older cows develop effective immunity. necropsy findings. necrotizing placentitis, dehydration, and fibrinous serositis will be found grossly. in addition, bronchopneumonia and hepatitis will be seen histologically. pathogenesis. campylobacter organisms grow readily in the genital tract, and infection is established within days of exposure. the resulting endometritis prevents conception or causes embyronic death. differential diagnosis. the primary differential diagnosis for campylobacteriosis is trichomoniasis. other venereal diseases should be considered when infertility problems are noted in a herd. these include brucellosis, mycoplasmosis, ureaplasmosis, infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv), and bovine virus diarrhea (bvd). leptospirosis should also be considered. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. prevention and control. killed bacterin vaccines are available, either as oil adjuvant or as aluminum hydroxide adsorbed. the former is preferred because of duration of immunity but causes granulomas. that vaccine also has specific recommendations regarding administration several months before the breeding season. the latter product is administered closer to the breeding season, and the duration of immunity is not as prolonged. in both cases, boosters should be given after the initial immunization and as part of the regular prebreeding regimen. only one bacterin product is approved for use in bulls. many combination vaccine products contain only the aluminum hydroxide adsorbed product. artificial insemination (ai) is particularly useful at controlling the disease, but bulls used for ai must be part of a screening program for this and other venereal diseases such as trichomoniasis. treatment. cows will usually recover from the infection, and treatment with antibiotics such as penicillin, administered as an intrauterine infusion, improve the chances of returning to breeding condition. etiology. the most common caprine bacterial skin infection is caused by staphylococcus intermedius or s. aureus and is known as staphylococcal dermatitis (smith and sherman, ) . the staphylococcus organisms are cocci and are categorized as primary pathogens or ubiquitous skin commensals of humans and animals. staphylococcus aureus and s. intermedius are classified as primary pathogens and produce coagulase, a virulence factor. clinical signs and diagnosis. small pustular lesions, caused by bacterial infection and inflammation of the hair follicle, occur around the teats and perineum. occasionally, the infection may involve the flanks, underbelly, axilla, inner thigh, and neck. staphylococcal dermatitis may occur secondary to other skin lesions. diagnosis is based on lesions. culture will distinguish s. aureus. pathogenesis. simple boredom may cause rubbing, followed by staphylococcal infection of damaged epidermis. differential diagnosis. the presence of scabs makes contagious ecthyma a differential diagnosis, along with fungal skin infections and nutritional causes of skin disease. treatment. severe infections should be treated with antibiotics based on culture and sensitivity. severe lesions and lesions localized to the underbelly, thighs, and udder benefit by periodic cleaning with an iodophor shampoo and spraying with an antibiotic and an astringent (smith and sherman, ) . h. clostridial diseases i. clostridium perfringens type c infection (enterotoxemia and struck) etiology. clostridium perfringens is an anaerobic, grampositive, nonmotile, spore-forming bacterium that lives in the soil, in contaminated feed, and in gastrointestinal tracts of ruminants. the bacteria is categorized by toxin production. toxins include alpha (hemolytic), beta (necrotizing), delta (cytotoxic and hemoltyic), epsilon, and iota. types of c. perfingens are a, b, c, d, and e. this is a common and economically significant disease of sheep, goats, and cattle. clinical signs and diagnosis. the beta toxin associated with overgrowth of this bacterium results in a fatal hemorrhagic enterocolitis within the first hr of a young ruminant's life. many animals may be found dead, with no clinical presentation. affected animals are acutely anemic, dehydrated, anorexic, restless, and depressed and may display tremors or convulsions as well as abdominal pain. feces may range from loose gray-brown to dark red and malodorous. morbidity and mortality may be nearly %. a similar noncontagious but acutely fatal form of enterotoxemia in adult sheep, called struck, occurs in yearlings and adults. struck is rare in the united states. the disease is also caused by the beta toxin of c. perfringens type c and is often associated with rapid dietary changes or shearing stresses in sheep. although affected animals are usually found dead, clinical signs include uneasiness, depression, and convulsions. mortality is usually less than %. diagnosis is usually based on necropsy findings, although confirmation can be made by culture of the organism. identification of the beta toxin in intestinal contents may be difficult because of instability of the toxin. necropsy findings. necropsy findings include a milk-filled abomasum, and hemorrhage in the distal small intestine and throughout the large intestine. petechial hemorrhages of the serosal surfaces of many organs, especially the thymus, heart, and gastrointestinal tract, will be visible. hydropericardium, hydroperitoneum, and hemorrhagic mesenteric lymph nodes will also be present. pulmonary and brain edema may also be seen. histologically, the gram-positive c. perfringens organisms may be visible in excess numbers along the mucosal surface of the swollen, congested, necrotic intestines. in cases of struck, necropsy findings include congestion and erosions of the mucosa of the gastrointestinal tract, serosal hemorrhages, and serous peritoneal and pericardial fluids. in late stages of the disease and especially if prompt necropsy is not performed, the organism will infiltrate the muscle fascial layers and produce serohemorrhagic and gaseous infiltration of perimysial and epimysial spaces. pathogenesis. hemorrhagic enterotoxemia is an acute, sporadic disease caused by the beta toxin of clostridium perfringens type c. neonates ingest the organism, which then proliferates and attaches to the gastrointestinal microvilli and elaborates primarily the beta toxins. the trypsin inhibitors present in colostrum prevent inactivation of the beta toxin. the toxins injure intestinal epithelial cells and then enter the blood, leading to acute toxemia. the intestinal injury may result in diarrhea, with small amounts of hemorrhage. associated electrolyte and water loss result in dehydration, acidosis, and shock. differential diagnosis. differential diagnoses include other clostridial diseases such as blackleg and black disease, as well as coccidiosis, salmonellosis, anthrax, and acute poisoning. clinical signs in chronic cases in older animals, such as adult goats, include soft stools, weight loss, anorexia, depression, and severe diarrhea, sometimes with mucus and blood. mature affected sheep may be blind and anorectic and may head-press. necropsyfindings. necropsy findings are similar to those seen with c. perfringens type c. additionally, extremely necrotic, soft kidneys ("pulpy kidneys") are usually observed immediately following death. (this phenomenon is in contrast to what is normally associated with later stages of postmortem autolysis.) focal encephalomalacia, and petechial hemorrhages on serosal surfaces of the brain, diaphragm, gastrointestinal tract, and heart are common findings. diagnosis can be made from the typical clinical signs and necropsy findings as well as the observation of glucose in the urine at necropsy. shock, probably through vascular damage. the noncontagious, peracute form of enterotoxemia occurs in suckling, fast-growing animals, either nursing from their dams or on high-protein, high-energy concentrates. the largest, fastest-growing animals generally are predisposed to this condition; for example, lambs, fat ewe lambs, and usually singleton lambs tend to be most susceptible. the hyperglycemia and glucosuria seen in acute cases are due to epsilon toxin effects on liver glycogen metabolism. should be administered to the pregnant animals prior to parturition. an alternative includes administration of an antitoxin to the newborn lambs. the disease may become endemic once it is on the premises. treatment. treatment is difficult and usually unsuccessful. antitoxin may be useful in milder cases, and the antitoxin and toxoid can also be administered during an outbreak. differential diagnosis. tetanus, enterotoxigenic e. coli, botulism, polioencephalomalacia, grain overload, and listeriosis are differentials. prevention and control. vaccination prevents the disease. maternal antibodies last approximately weeks postpartum; thus young animals should be vaccinated at about this time. feeding regimens to young, fast-growing animals and feeding of concentrates to adults should be evaluated carefully. research complications. this disease can be costly in losses of neonates and younger animals. treatment. treatment consists of support (fluids, warmth), antitoxin administration, oral antibiotics, and diet adjustment. toxin that is proteolytically activated by trypsin. this disease caused by c. perfringens tends to be associated with sheep and is of less importance in goats and cattle. clinical signs. the peracute condition in younger animals is characterized by sudden deaths, which are occasionally preceded by neurological signs such as incoordination, opisthotonus, and convulsions. because the disease progresses so rapidly to death (within - hr), clinical signs are rarely observed. hypersalivation, rapid respirations, hyperthermia, convulsions, and opisthotonus have been noted. in acute cases, hyperglycemia and glucosuria are considered almost pathognomonic. etiology. clostridium tetani is a strictly anaerobic, motile, spore-forming, gram-positive rod that persists in soils and manure and within the gastrointestinal tract. at least serotypes of c. tetani exist. clinical signs. infection by c. tetani is characterized by a sporadic, acute, and fatal neuropathy. after an incubation period of days to weeks, the animal exhibits bloat; muscular spasticity; prolapse of the third eyelid; rigidity and extension of the limbs, leading to a stiff gate; an inability to chew; and hyperthermia. erect or drooped ears, retracted lips, drooling, hypersensitivity to external stimuli, and a "sawhorse" stance are frequent signs. the animal may convulse. death occurs within - days, and mortality is nearly %, primarily from respiratory failure. diagnosis is based on clinical signs. musclerelated serum enzymes such as aspartate aminotransferase (ast), creatinine kinase (ck), and lactate dehydrogenase (ldh) might be elevated. (jensen and swift, ) . serum cortisol may also be elevated, and stress hyperglycemia may be evident. permanent lameness may result in survivors. contaminant and is often found as part of the gut microflora of herbivores. the organisms sporulate and persist in the environment. all species of livestock are susceptible, but sheep and goats are more susceptible than cattle. individual cases may occur, or herd outbreaks may follow castration, tail docking, ear tagging, or dehorning. mouth wounds may also be sites of entry. pathogenesis. tetanus, or lockjaw, is caused by the toxins of c. tetani. all serovars produce the same exotoxin, which is a multiunit protein composed of tetanospasmin, which is neurotoxic, and tetanolysin, which is hemolytic. a nonspasmogenic toxin is also produced. contamination of wounds results in anaerobic proliferation of the bacterium and liberation of the tetanospasmin, which diffuses through motor neurons in a retrograde direction to the spinal cord. the toxin inhibits the release of glycine and y-aminobutyric acid from renshaw cells; this resuits in hypertonia and muscular spasms. proliferation of c. tetani in the gut of affected animals may also serve as a source and may produce clinical signs. the uterus is the most common site of infection in postparturient dairy cattle with retained placentas. differential diagnoses. early in the course of the infection, differential diagnoses include bloat, rabies, hypomagnesemic tetany, polioencephalomalacia, white muscle disease, enterotoxemia in lambs, and lead poisoning. polyarthritis of cattle is a differential for the gait changes in that species. necropsy findings. findings are nonspecific except for the inflammatory reaction associated with the wound. because of the low number of organisms necessary to cause neurotoxicosis, isolation of c. tetani from the wound may be difficult. administering tetanus antitoxin (e.g., at least iu in an adult sheep or goat); vaccinating with tetanus toxoid; administering of antibiotics (penicillin, both parenterally [potassium penicillin intravenously and procaine penicillin intramuscularly] and flushed into the cleaned wound), a sedative or tranquilizer (e.g., acepromazine or chlorpromazine) and a muscle relaxant; and keeping the animal in a dark, quiet environment. supportive fluids and glucose must be administered until the animal is capable of feeding. if the animal survives, revaccination should be done days after the previous dose. prevention and control like other ubiquitous clostridial diseases, tetanus is impossible to eradicate. the disease can be controlled and prevented by following good sanitation measures, aseptic surgical procedures, and vaccination programs. tetanus toxoid vaccine is available and very effective for stimulating long-term immunity. tetanus antitoxin can be administered ( iu in lambs) as a preventive or in the face of disease as an adjunct to therapy. both the toxoid and the antitoxin can be administered to an animal at the same time, but they should not be mixed in the syringe, and each should be administered at different sites, with a second toxoid dose administered weeks later. animals should be vaccinated or times during the first year of life. does and ewes should receive booster vaccinations within months of parturition to ensure colostral antibodies. research complications. unprotected, younger ruminants may be affected following routine flock or herd management procedures. contaminated or inadequately managed open wounds or lesions in older animals may provide anaerobic incubation sites. etiology. clostridium novyi, an anaerobic, motile, sporeforming, gram-positive bacteria, is the agent of bighead and black disease. clostridium novyi type d (c. hemolyticum) is the cause of bacillary hemoglobinuria, or "red water." clostridium chauvoei is the causative agent of blackleg. clinical signs. bighead is a disease of rams characterized by edema of the head and neck. the edema may migrate to ventral regions such as the throat. additional clinical signs include swelling of the eyelids and nostrils. most animals will die within - hours. black disease, or infectious necrotic hepatitis, is a peracute, fatal disease associated with c. novyi. it is more common in cattle and sheep but may be seen in goats. the clinical course is - days in cattle and slightly shorter in sheep. otherwise healthy-appearing adult animals are often affected. clinical signs are rarely seen, because of the peracute nature of the disease. occasionally, hyperthermia, tachypnea, inability to keep up with other animals, and recumbency are observed prior to death. bacillary hemoglobinuria is an acute disease seen primarily in cattle and characterized by fever and anorexia, in addition to the hemoglobinemia and hemoglobinuria indicated by the name. animals that survive a few days will develop icterus. mortality may be high. blackleg, a disease similar to bighead, causes necrosis and emphysema of muscle masses, serohemorrhagic fluid accumulation around the infected area, and edema (jackson et al., ) . blackleg is more common in cattle than in sheep. the incubation period is - days and is followed by hyperthermia, muscular stiffness and pain, anorexia, and gangrenous myositis. the clinical course is short, - hr, and untreated animals invariably die. blackleg in cattle can be associated with subcutaneous edema or crepitation; these do not usually occur in sheep. most lesions are associated with muscles of the face, neck, perineum, thigh, and back. epizootiology and transmission. bighead is caused by the toxins of c. novyi, which enters through wounds often associated with horn injuries during fighting. the c. novyi type b organisms produce alpha and beta toxins, and the alpha toxins are mostly responsible for toxemia, tissue necrosis, and subsequent death. clostridium novyi type d is endemic in the western united states. it is hypothesized that the c. chauvoei organisms enter through the gastrointestinal tract. black disease and bacillary hemoglobinuria are associated with concurrent liver disease, often associated with fasciola infections (liver flukes); it is sometimes seen as a sequela to liver biopsies. the diseases are more common in summer months, and fecal contamination of pastures, flooding, and infected carcasses are sources of the organism. birds and wild animals may be vectors of the pathogen. ingested spores are believed to develop in hepatic tissue damaged and anoxic from the fluke migrations. necropsy. diagnosis of black disease is usually based on postmortem lesions. subcutaneous vessels will be engorged with blood, resulting in dried skin with a dark appearance. carcasses putrefy quickly. in addition, hepatomegaly and endocardial hemorrhages are common, and hepatic damage from flukes may be so severe that diagnosis is difficult. blood coagulates slowly in affected animals. pathogenesis. the propagation of the clostridial organisms is self-promoted by the damage caused by the toxins and the increased local anaerobic environment created. clostridium novyi proliferates in the soft tissues of the head and neck, and the resultant clostridial toxin causes increased capillary permeability and the liberation of serous fluids into the tissues. mixed infections with related clostridial organisms may lead to increasing hemorrhage and necrosis in the affected tissues. diagnosis is based on clinical signs. in black disease and bacillary hemoglobinuria disease, the ingested clostridial spores are absorbed, enter the liver, and cause hepatic necrosis. associated toxemia causes subcutaneous vascular dilatation; increased pericardial, pleural, and peritoneal fluid; and endocardial hemorrhages. the toxins produced by c. novyi, identified as beta, eta, and theta, and each having enzymatic or lytic properties or both, also contribute to the hemolytic disease. clostridium chauvoei spores proliferate in traumatized muscle areas damaged by transportation, rough handling, or injury. differential diagnosis. differential diagnoses include other clostridial diseases as well as photosensitization. hemolytic diseases such as babesiosis, leptospirosis, and hemobartonellosis should be included as differentials. treatment. for c. chauvoei infection (blackleg), early treatment with penicillin or tetracycline may be helpful. treatment for black disease is not rewarding even if the animal is found before death. carcasses from bacillary hemoglobinuria losses should be burned, buried deeply, or removed from the premises. prevention and control. vaccinating animals with multivalent clostridial vaccines can prevent these diseases. subcutaneous administration of vaccine material is recommended over intramuscular. vaccinations may be useful in an outbreak. careful handling of ruminants during shipping and transfers will contribute to fewer muscular injuries. for bighead, mature rams penned together should be monitored for lesions, especially during breeding season. control of fascioliasis is very important in prevention and control of black disease and in the optimal timing of vaccinations. etiology. clostridium septicum is the species usually associated with malignant edema, but mixed infections involving other clostridial species such as c. chauvoei, c. novyi, c. sordellii, and c. perfringens may occur. clostridium spp. are motile (c. chauvoei, c. septicum) or nonmotile, anaerobic, spore-forming, gram-positive rods. clinicial signs. malignant edema, or gas gangrene, is an acute and often fatal bacterial disease caused by clostridium spp. the incubation period is approximately - days. the affected area will be warm and will contain gaseous accumulations that can be palpated as crepitation of the subcutaneous tissue around the infected area. regional lymphadenopathy and fever may occur. the animal becomes anorexic, severely depressed, and possibly hyperthermic. edema and crepitation may be noted around the wound; death occurs within hr to days. epizootiology and transmission. the organisms are ubiquitous in the environment and may survive in the soil for years. the disease is especially prevalent in animals that have had recent wounds such as those that have undergone castration, docking, ear notching, shearing, or dystocia. necropsy findings. the tissue necrosis and hemorrhagic serous fluid accumulations resemble those of other clostridial diseases. pathogenesis. in most cases, the clostridial organisms cause a spreading infection through the fascial planes around the area of the injury; vegetative organisms then produce potent exotoxins, which result in necrosis (alpha toxin) and/or hemolysis (beta toxin). furthermore, the toxins enter the bloodstream and central nervous system, resulting in systemic collapse and high mortality. necropsy. spreading, crepitant lesions around wounds are suggestive of malignant edema. affected tissues are inflamed and necrotic. gas and serosanguineous fluids with foul odors infiltrate the tissue planes. large rod-shaped bacteria may be observed on histopathology; confirmation is made through culture and identification. intramuscular inoculation of guinea pigs causes a necrotizing myositis and death. organisms can be cultured from guinea pig tissues. treatment. infected animals can be treated with large doses of penicillin and fenestration of the wound is recommended. prevention and control. proper preparation of surgical sites, correct sanitation of instruments and the housing environment, and attention to postoperative wounds will help prevent this disease. multivalent clostridial vaccines are available. research complications. morbidity or loss of animals from lack of or unsuccessful vaccination and from contaminated surgical sites or wounds may be consequences of this disease. etiology. escherichia coli is a motile, aerobic, gram-negative, non-spore-forming coccobacillus commonly found in the environment and gastrointestinal tracts of ruminants. escherichia coli organisms have three areas of surface antigenic complexes (o, somatic; k, envelope or pili; and h, flagellar), which are used to "group" or classify the serotypes. colibacillosis is the common term for infections in younger animals caused by this bacteria. clinical signs. presentation of e. coli infections vary with the animal's age and the type of e. coli involved. enterotoxigenic e. coli infection causes gastroenteritis and/or septicemia in lambs and calves. colibacillosis generally develops within the first hr of life when newborn animals are exposed to the organism. the enteric infection causes a semifluid, yellow to gray diarrhea. occasionally blood streaking of the feces may be observed. the animal may demonstrate abdominal pain, evidenced by arching of the back and extension of the tail, classically described as "tucked up." hyperthermia is rare. severe acidosis, depression, and recumbancy ensue, and mortality may be as high as %. the septicemic form generally occurs between and weeks of age. animals display an elevated body temperature and show signs suggestive of nervous system involvement such as incoordination, head pressing, circling, and the appearance of blindness. opisthotonos, depression, and death follow. occasionally, swollen, painful joints may be observed with septicemic colibacillosis. blood cultures may be helpful in identifying the septicemic form. in ruminants, e. coli is is a less common cause of cystitis and pyelonephritis. the cystitis is characterized by dysuria and pollakiuria; gross hematuria and pyuria may be present. the infection may or may not be restricted to the bladder; in the later presentation, and in cases of pyelonephritis, a cow will be acutely depressed, have a fever and ruminal stasis, and be anorexic. in chronic cases, animals will be polyuric and undergo weight loss. escherichia coli may also cause in utero disease in cattle, resulting in abortion or weakened offspring. epizootiology and transmission. escherichia coli is one of the most common gram-negative pathogens isolated from ruminant neonates. zeman et al. ( ) classify e. coli infections into four groups: enterotoxigenic, enterohemorrhagic, enteropathogenic, and enteroinvasive. enterotoxigenic e. coli (etec) attach to the enterocytes via pili, produce enterotoxins, and are the primary cause of colibacillosis in animals and humans. fimbrial (pili) antigens associated with ovine disease include k and f . enterohemorrhagic e. coli (ehec) attach and efface the microviuus, produce verotoxins, and occasionally cause disease in humans and animals. enteropathogenic e. coli (epec) colonize and efface the microvillus but do not produce verotoxins. epec are associated with disease in humans and rabbits and cause a secretory diarrhea. enteroinvasive e. coli (eiec) invade the enterocytes of humans and cause a shigella-like disease. overcrowding and poor sanitation contribute significantly to the development of this disease in young animals. the organism will be endemic in a contaminated environment and present on dams' udders. the bacteria rapidly proliferate in the neonates' small intestines. the bacteria and associated toxins cause a secretory diarrhea, resulting in the loss of water and electrolytes. if the bacteria infiltrate the intestinal barrier and enter the blood, septicemia results. diagnosis of the enteric form can be made by observation of clinical signs, including diarrhea and staining of the tail and wool. necropsy findings. swollen, yellow to gray, fluid-filled small and large intestines, swollen and hemorrhagic mesenteric lymph nodes, and generalized tissue dehydration are common. septicemic lambs may have serofibrinous fluid in the peritoneal, thoracic, and pericardial cavities; enlarged joints containing fibrinopurulent exudates; and congested and inflamed meninges. isolation and serotyping of e. coli confirm the diagnosis. elisa and latex agglutination tests are available diagnostic tools. differential diagnosis. differential diagnoses include the enterotoxemias caused by c. perfringens type a, b, or c; campylobacter jejuni; coccidia, rotavirus, coronavirus, salmonella, and cryptosporidia. other contributing causes of abomasal tympany in young ruminants, such as dietary changes, copper deficiency, excessive intervals between feedings of milk replacer, or feeding large volumes should be considered. prevention and control. the best preventive measures are maintenance of proper housing conditions, limiting overcrowding, and frequently sanitizing lambing areas. attention to colostrum feeding techniques and colostral quality are important means of preventing disease. treatment must include intravenous fluid hydration and reestablishment of acid-base and electrolyte abnormalities. treatment. antibiotics such as trimethoprim-sulfadiazine, enrofloxacin, cephalothin, amikacin, and apramycin may be helpful; oral antibiotics are not recommended. vaccines are available for prevention of colibacillosis in cattle. etiology. corynebacterium pseudotuberculosis (previously c. ovis) are nonmotile, non-spore-forming, aerobic, short and curved, gram-positive coccobacilli. caseous lymphadenitis (cla) is such a common, chronic contagious disease of sheep and goats that any presentation of abscessing and draining lymph nodes should be presumed to be this disease until proven otherwise. the disease has been reported occasionally in cattle. clinical signs and diagnosis. abscessation of superficial lymph nodes, such as the superficial cervical, retropharyngeal, subiliacs (prefemoral), mammary, superficial inguinals, and popliteal nodes, and of deep nodes, such as mediastinal and mesenteric lymph nodes, is typical. radiographs may be helpful in identifying affected central nodes. peripheral lymph nodes may erode and drain caseous, "cheesy," yellow-green-tan secretions. the incubation period may be weeks to months. over time, an infected animal may become exercise-intolerant, anorexic, and debilitated. fever, increased respiratory rates, and pneumonia may also be common signs. exotoxin-induced hemolytic crises may occur occasionally. morbidity up to % is common, and morbid animals will often eventually succumb to the disease. diagnosis is based on clinical lesions; elisa serological testing is also available. smears of the exudate or lymph nodes aspirates can be gram-stained. lymph node aspirates may also be sent for culturing. epizootiology and transmission. the organism can survive for months or more in the environment and enters via skin wounds, shearing, fighting, castration, and docking. ingestion and aerosolization (leading to pulmonary abscesses) have been reported as alternative routes of entry. necropsy findings. disseminated superficial abscesses as well as lesions of the mediastinal and mesenteric lymph nodes will be identified. cut surfaces of the affected lymph nodes may appear lamellated. lungs, liver, spleen, and kidneys may also be affected. cranioventral lung consolidation with hemorrhage, fibrin, and edema are seen histologically. pathogenesis. corynebacterium pseudotuberculosis produces an exotoxin (phospholipase d) that damages endothelial and blood cell membranes. this process enhances the organisms' ability to withstand phagocytosis. the infection spreads through the lymphatics to local lymph nodes. the necrotic lymph nodes seed local capillaries and hematogenously and lymphatically spread the organisms to other areas, especially the lungs. differential diagnosis. differentials include pathogens causing lymphadenopathy and abscessation. treatment. antibiotic therapy is not usually helpful. abscesses can be surgically lanced and flushed with iodinecontaining and/or hydrogen peroxide solutions. abscessing lymph nodes can be removed entirely from valuable animals. during warmer months, an insect repellent should be applied to and around healing lesions. all materials used to treat animals should be disposed of properly. because of the contagious nature of the disease, animals with draining and lanced lesions should be isolated from cla-negative animals at least until healed. commercial vaccines are available (piontkowski and shivvers, ) . minimizing contamination of the environment, using proper sanitation methods for facilities and instruments, segregating affected animals, and taking precautions to prevent injuries are all important. research complications. this pathogen is a risk for animals undergoing routine management procedures or invasive research procedures, because of its persistence in the environment, its long clinical incubation period, and its poor response to antibiotics. etiology. corynebacterium renale, c. cystitidis, and c. pilosum are sometimes referred to as the c. renale group. these are piliated and nonmotile gram-positive rods and are distinguished biochemically. corynebacterium renale causes pyelonephritis in cattle, and c. pilosum and c. cystitidis cause posthitis, also known as pizzle rot or sheath rot, in sheep and goats. in many references, all these clinical presentations are attributed to c. renale. clinical signs and diagnosis. acute pyelonephritis is characterized by fever, anorexia, polyuria, hematuria, pyuria, and arched back posture. untreated infections usually become chronic, with weight loss, anorexia, and loss of production in dairy animals. relapses are common, and some infections are severe and fatal. diagnosis of pyelonephritis is based on urinalysis (proteinuria and hematuria) and rectal or vaginal palpation (assessing ureteral enlargement). urine culturing may not be productive. in chronic cases, e. coli and other gram-negatives may be present. posthitis and vulvovaginitis are characteriazed by ulcers, crusting, swelling and pain. the area may have a distinct malodor. necrosis and scarring may be sequelae of more severe infections. fly-strike may also be a complication. diagnosis is based on clinical signs and on investigation of feeding regimens. epizootiology and transmission. ascending urinary tract infections with cystitis, ureteritis, and pyelonephritis are widespread problems, but incidence is relatively low. the vaginitis and posthitis contribute to the venereal transmission, but indirect transmission is possible because the organisms are stable in the environment and present on the wool or scabs shed from affected animals. posthitis occurs in intact and castrated sheep and goats. necropsy findings. pyelonephritis, multifocal kidney abscessation, dilated and thickened ureters, cystitis, and purulent exudate in many sections of the urinary tract are common finding at gross necropsy. of bovine genitourinary tracts. the pilus mediates colonization. conditions such as trauma, urinary tract obstruction, and anatomic anomalies may predispose to infection. in addition, more basic ph urine levels may block some immune defenses. infections ascend through the urinary tract. the bacteria are urease-positive when tested in vitro, and the ammonia produced in vivo during an infection damages mucosal linings, with subsequent inflammation. corynebacterium cystitidis and c. pilosum are normally found around the prepuce of sheep and goats. high-protein diets, resulting in higher urea excretion and more basic urine, are contributing factors. posthitis and vulvovaginitis may develop within a week of change to the more concentrated or richer diet, such as pasture or the addition of high-protein forage. the ammonia produced irritates the preputial and vulvar skin, increasing the vulnerability to infection. differential diagnosis. urolithiasis is a primary consideration for these diseases. contagious ecthyma should be considered for the crusting that is seen with posthitis and vulvovaginitis, although the lesions of contagious ecthyma are more likely to develop around the mouth. ovine viral ulcerative dermatosis is also a differential for the lesions of posthitis and vulvovaginitis. prevention and treatment. because high-protein feed is often associated with posthitis and vulvovaginitis, feeding prac-tices must be reconsidered. clipping long wool and hair also is helpful. treatment. long-term ( weeks) penicillin treatment is effective for pyelonephritis. reduction of dietary protein, clipping and cleaning skin lesions, treating for or preventing fly-strike, and topical antibacterial treatments are effective for posthitis and vulvovaginitis; systemic therapy may be necessary for severe cases. surgical debridement or correction of scarring may also be indicated in severe cases. etiology. erysipelothrix rhusiopathiae is a nonmotile, nonspore-forming, gram-positive rod that resides in alkaline soils. clinical signs. erysipelothrix causes sporadic but chronic polyarthritis in lambs less than months of age. in older goats, erysipelas has been associated with joint infections. epizootiology and transmission. the disease may follow wound inoculation associated with castration, docking, or improper disinfection of the umbilicus. following wound contamination and a -to -day incubation period, the lamb exhibits a fever and stiffness and lameness in one or more limbs. joints, especially the stifle, hock, elbow, and carpus, are tender but not greatly enlarged. necropsy findings. thickened articular capsules, mild increases in normal-appearing joint fluid and erosions of the articular cartilage are usually found. the joint capsule is infiltrated with mononuclear cells, but bacteria are difficult to find. diagnosis is based on clinical signs of polyarthritis, and confirmation is made by culturing the organism from the joints. differential diagnosis. differential diagnoses include polyarthritis caused by chlamydia or other bacteria and stiffness caused by white muscle disease. other bacteria causing septic joints include areanobacterium pyogenes and fusobacterium necrophorum. caprine arthritis encephalitis (cae) should also be considered. prevention and control. proper sanitation and prevention of wound contamination are important in preventing the infection in lambs. screening of goat herds for cae is recommended. therapy. erysipelas is sensitive to penicillin antibiotic m. etiology. dermatophilus congolensis is an aerobic, grampositive, filamentous bacterium with branching hyphae. dermatophilosis is a chronic bacterial skin disease characterized by crustiness and exudates accumulating at the base of the hair or wool fibers (scanlan et al., ) . clinical signs. animals will be painful but will not be pruritic. two forms of the disease exist in sheep: mycotic dermatitis (also known as lumpy wool) and strawberry foot rot. mycotic dermatitis is characterized by crusts and wool matting, with exudates over the back and sides of adult animals and about the face of lambs. strawberry foot rot is rare in the united states but is characterized by crusts and inflammation between the carpi and/or tarsi and the coronary bands. animals will be lame. in goats and cattle, similar clinical signs of crusty, suppurative dermatitis are seen; the disease is often referred to as cutaneous streptothricosis in these species. lesions in younger goats are seen along the tips of the ears and under the tail. diagnosis is based on clinical signs as well as the typical microscopic appearance on stained skin scrapings, cultures, and serology. epizootiology and transmission. the disease occurs worldwide, and the dermatophilus organism is believed to be a saprophyte. transmission occurs by direct or indirect contact and is aggravated by prolonged wet wool or hair associated with inclement weather. biting insects may aid in transmission. necropsy findings. lymphadenopathy as well as liver and splenic changes may be observed. histopathologically, superficial epidermal layers are necrotic and crusted with serum, white blood cells, and wool or hair. dermal layers are hyperemic and edematous and may be infiltrated with mononuclear cells. pathogenesis. lesions typically begin around the muzzle and hooves and the dorsal midline. prevention and control. potash alum and aluminum sulfate have been used as wool dusts in sheep to prevent dermatophilosis. minimizing moist conditions is helpful in controlling and preventing the disease. in addition, controlling external parasites or other factors that cause skin lesions is important. lesions will resolve during dry periods. treatment. animals can be treated with antibiotics such as penicillin and oxytetracycline. treating the animals with povidone-iodine shampoos or chlorhexidine solutions is also useful in clearing the disease. n. etiology. two bacteria, dichelobacter (bacteroides) nodosus and fusobacterium necrophorum, work synergistically in caus-ing contagious foot rot in sheep and goats. other organisms may be involved as secondary invaders. both dichelobacter and fusobacterium are nonmotile, non-spore-forming, anaerobic, gram-negative bacilli. foot rot is a contagious, acute or chronic dermatitis involving the hoof and underlying tissues (bulgin, ) . it is the leading cause of lameness in sheep. at least serotypes of dichelobacter are known. arcanobacterium pyogenes may also contribute to the pathogenicity or to foot abscesses in goats. foot scald, an interdigital dermatitis, is caused primarily by d. nodosus alone. clinical signs. varying degrees of lameness are observed in all ages of animals within - weeks of exposure to the organisms. severely infected animals will show generalized signs of weight loss, decreased productivity, and anorexia associated with an inability to move. the interdigital skin and hooves will be moist, with a distinct necrotic odor. morbidity may reach % in susceptible animals. diagnosis is based on clinical signs. smears and cultures confirm the definitive agents. clinical signs of the milder disease, foot scald, include mild lameness, redness and swelling, and little to no odor. epizootiology and transmission. fusobacterium necrophorum is ubiquitous in soil and manure, in the gastrointestinal tract, and on the skin and hooves of domestic animals. in contrast, dichelobacter contaminates the soil and manure but rarely remains in the environment for more than about weeks. some animals may be chronic carriers. overcrowded, warm, and moist environments are key elements in transmission. outbreaks are likely in the spring season. shipping trailers and contaminated pens or yards should be considered also as likely sources of the bacteria. pathogenesis. both organisms are transmitted to the susceptible animal by direct or indirect contact. the organisms enter the hoof through injuries or through sites where strongyloides papillosus larvae have penetrated. fusobacterium necrophorum initiates the colonization and is followed by d. nodosus. the latter attaches and releases proteases; these cause necrosis of the epidermal layers and separation of the hoof from the underlying dermis. the pathogenicity of the serotypes of d. nodosus is correlated with the production of these proteases and numbers of pili. additionally, f. necrophorum causes a severe, damaging inflammatory reaction. differential diagnosis. foot abscesses, tetanus, selenium/ vitamin e deficiencies, copper deficiency, strawberry foot rot, bluetongue virus infection (manifested with myopathy and coronitis), and trauma are among the many differentials that must be considered. treatment. affected animals are best treated by manually trimming the necrotic debris from the hooves, followed by application of local antibiotics and foot wraps. systemic antibiotics such as penicillin, oxytetracycline, and erythromycin may be used. goats have improved dramatically when given a single dose of penicillin ( , u/kg) (smith and sherman, ) . footbaths containing % zinc sulfate, % copper sulfate, or % formalin (not legal in all states) can be used for treatment as well as for prevention of the disease. affected animals should be separated from the flock. vaccination has been shown to be effective as part of the treatment regimen. some breeds of sheep and some breeds and lines of goats are resistant to infection. individual sheep may recover without treatment or are resistant to infection. epizootiology and transmission. cases may be sporadic, or epizootics may occur. bos taurus dairy breeds and animals with wide interdigital spaces are more commonly affected. the factors here are comparable to those present in foot rot of smaller ruminants. necropsy findings. findings at necropsy include dermatitis and necrosis of the skin and subcutaneous tissues. although necropsy would rarely be performed, secondary osteomyelitis may be noted in severe cases by sectioning limbs. prevention and control. prevention and control programs involve scrutiny of herd and flock management; quarantine of incoming animals; vaccination; segregation of affected animals; careful and regular hoof trimming; discarding trimmings from known or suspected infected hooves; maintaining animals in good body condition; avoiding muddy pens and holding areas; and culling individuals with chronic and nonresponsive infections. dichelobacter nodosus bacterins are commercially available; cross protection between serotypes varies. biannual vaccinination in wet areas may be essential. some breeds may develop vaccination site lumps. footbaths of % zinc sulfate, % formalin (where allowed by state regulations), or % copper sulfate are also considered very effective preventive measures. goats are less sensitive than sheep to the copper in the footbaths. treating and controlling foot rot is costly in terms of time, initial handling and treatments and their follow-up, housing space, and medications. etiology. interdigital necrobacillosis of cattle is caused by the synergistic infection of traumatized interdigital tissues by fusobacterium necrophorum and bacteroides melaninogenicus. like f. necrophorum, b. melaninogenicus is a nonmotile, anaerobic, gram-negative bacterium. dichelobacter nodosus, the agent of interdigital dermatitis, may be present in some cases. this is a common cause of lameness in cattle. clinical signs. clinical signs include mild to moderate lameness of sudden onset. hindlimbs are more commonly affected, and cattle will often flex the pastern and bear weight only on the toe. the interdigital space will be swollen, as will be the coronet and bulb areas. characteristic malodors will be noted, but there will be little purulent discharge. in more severe cases, animals will have elevated body temperature and loss of appetite. the les~ons progress to fissures with necrosis until healing occurs. the diagnosis is by the odor and appearance. anaerobic culturing confirms the organisms involved. pathogenesis. the bacteria enter through the skin of the interdigital area after trauma to the interdigital skin, from hardened mud, or from softening of the skin due to, for example, constant wet conditions in pens. colonization leads to cellulitis. in addition, f. necrophorum releases a leukocidal exotoxin that reduces phagocytosis and causes the necrosis, whereas the tissues and tendons are damaged by the proteases and collagenases produced by b. melaninogenicus. zinc deficiency may play a role in the pathogenesis in some situations. differential diagnoses. the most common differentials for sudden lameness include hairy heel warts and subsolar abcesses. bluetongue virus should also be considered. grain engorgement and secondary infection from cracks caused by selenium toxicosis should also be considered. the exotic footand-mouth disease virus would be considered in areas where that pathogen is found. prevention and control. as with foot rot in smaller ruminants, management of the area and herd are important. paddocks and pens should be kept dry, well drained, and free of material that will damage feet. footbaths and chlortetracycline in the feed have been shown to control incidence. affected animals should be segregated during treatment. chronically affected or severely lame animals should be culled. new cattle should be quarantined and evaluated. ing within a week include cleaning the feet and trimming necrotic tissue; parenteral antimicrobials, such as oxytetracycline or procaine penicillin, or sulfonomethazine in the drinking water or tetracyclines in feed; and footbaths (such as % zinc sulfate, . % formalin, or % copper sulfate) twice a day. in severe cases, more aggressive therapy such as bandaging the feet or wiring the digits together may be needed. animals can recover without treatment but will be lame for several weeks. acquired immunity is reported to be poor. research complications are comparable to those noted for foot rot in smaller ruminants. fusobacterium necrophorum is also associated with foot abscesses, the infection of the deeper structures of the foot, in sheep and goats. only one claw of the affected hoof may be involved. the animals will be three-legged lame, and the affected hoof will be hot. pockets of purulent material may be in the heel or toe. etiology. bacteria such as fusobacterium spp., bacteroides spp., and dichelobacter nodosus have been isolated from bovine heel lesions. spirochete-like organisms have also been shown in the lesions of cows with papillomatous digital dermatitis (pdd), in the united states and europe; these have culturing requirements similar to those of treponema species. treatment. antibiotic and antiseptic regimens have been used successfully for this problem. antibiotics include parenteral cephalosporins and pencillins, as well as topical tetracyclines with bandaging. antiseptic or antibiotic solutions in footbaths include tetracyclines, zinc sulfate, lincomycin, spectinomycin, copper sulfate, and formalin. the footbaths must be well maintained, minimizing contamination by feces and other materials. tandem arrangements, such as the cleaning footbaths and then the medicated footbaths, and preventing dilution from precipitation are useful. other treatments such as surgical debridement, cryotherapy, and caustic topical solutions have been successful. research complications. infectious, contagious ppd is one of the major causes of lameness among heifers and dairy cattle and is a costly problem to treat. the outbreaks are generally worse in younger animals in chronically infected herds. the immune response is not well understood, and it may be temporary in older animals. clinical signs. all lesions occur on the haired, digital skin. one or all feet may be affected. most lesions occur on the plantar surface of the hindfoot (near the heel bulbs and/or extending from the interdigital space), but the palmar and dorsal aspect of the interdigital spaces may also be involved. progression of lesions, typically over - weeks, includes erect hairs, loss of hair, and thickening skin. moist plaques begin as red and remain red or turn gray or black. exudate or blood may be present on the plaque. plaques enlarge and "hairs" protrude from the roughened surface. lesioned areas are painful when touched. the lesions may or may not be malodorous. epizootiology and transmission. facility conditions and herd management are considered contributing factors. the following have been examined as contributing factors: nutrition, particularly zinc deficiency; poorly drained, low-oxygen, organic material underfoot; poor ventilation; rough flooring; damp and dirty bedding areas; and overcrowding. these interdigital lesions occur commonly in young stock and in dairy facilities throughout the world. the disease is seen only in cattle. pathogenesis. the organisms noted above, combined with poor facility and herd management, are critical in the pathogenesis. differential diagnosis. differentials for lameness will include sole abscesses, laminitis, and trauma. prevention and control. each facility and management condition noted above should be addressed in conjunction with appropriate antibiotic and/or antiseptic treatment regimens. all equipment used for hoof trimming must be cleaned and disinfected after every use. trucks and trailers should also be sanitized between groups of animals. etiology. haemophilus somnus is a pleomorphic, nonencapsulated, gram-negative bacterium. diseases caused by this organism include thromboembolic meningoencephalitis (teme), septicemia, arthritis, and reproductive failures due to genital tract infections in males and females. haemophilus somnus is a also major contributor to the bovine respiratory disease complex. haemophilus spp. have been associated with respiratory disease in sheep and goats. clinical signs. the neurologic presentation may be preceded by - weeks of dry, harsh coughing. neurologic signs include depression, ataxia, falling, conscious proprioceptive deficits; signs such as head tilt from otitis interna or otitis media, opisthotonus, and convulsions may be seen as the brain stem is affected. high fever, extreme morbidity, and death within hr may occur. respiratory tract infections are usually part of the complex with infectious bovine rhinotracheitis virus, bovine respiratory syncytial virus, bovine viral diarrhea virus, parainfluenza , mycoplasma, and pasteurella, and the synergism among these contributes to the signs of bovine respiratory disease complex (brdc). in acute neurologic as well as chronic pneumonic infections, polyarthritis may develop. abortion, vulvitis, vaginitis, endometritis, placentitis, and failure to conceive are manifestations of reproductive tract disease. in all cases, asymptomatic infections may also occur. diagnosis based on culture findings is difficult because h. somnus is part of the normal nasopharyngeal flora. paired serum samples are recommended; single titers in some animals seem to be high because of passive immunity, previous vaccination, or previous exposure. in cases of abortion, other causes should be eliminated from consideration. because the organism is considered part of the normal flora of cattle and can be isolated from numerous tissues, the distinction between the normal flora and the status of chronic carrier is not clear. outbreaks are associated with younger cattle in feedlots in western united states, but stresses of travel and coinfection with other respiratory pathogens are involved in some cases. adult cattle have also been affected. vaccination for viral respiratory pathogens may increase susceptibility. transmission is by respiratory and genital tract secretions. the organism does not persist in the environment. times of stress to the cattle is worthwhile. killed whole-cell bacterins are commercially available; these have been shown to be effective in controlling the respiratory disease presentation. control of other clinical aspects of the h. somnus disease by these bacterins has not been well described. treatment. rapid treatment at the first signs of neurologic disease is important in an outbreak. haemophilus somnus is susceptible to several antibiotics, such as oxytetracycline and penicillin, and these are often used in sequence until the cattle are recovered. necropsy findings. pathognomonic central nervous system lesions include multifocal red-brown foci of necrosis and inflammation on and within the brain and the meninges. many thrombi with bacterial colonies will be seen in these affected areas. ocular lesions may also be seen, including conjunctivitis, retinal hemorrhages, and edema. usually animals with neurological disease will not have respiratory tract lesions. the respiratory tract lesions include bronchopneumonia and suppurative pleuritis. when combined with pasteurella infection, the pathology becomes more severe. aborted fetuses will not show lesions, but necrotizing placentitis will be evident histologically. pathogenesis. inhalation of contaminated respiratory secretions from carrier animals is the primary means of transmission. the anatomical location of bacterial residence within the carriers has not been identified. after gaining access by way of the respiratory tract, the bacteria proliferate, and a bacteremia develops. the bacteria are phagocytosed by neutrophils but are not killed. the thrombosis formation is due to the adherence by the nonphagocytosed organisms to vascular endothelial cells, degeneration and desquamation of these cells, and exposure of subendothelial collagen, with subsequent initiation of the intrinsic coagulation pathway. antigen-antibody complex formation, resulting in vasculitis, is also correlated with high levels of agglutinating antibodies. other pathogens associated with neurological disease and respiratory disease such as pasteurella hemolytica, p. multocida, and p. aeruginosa. in smaller ruminants, corynebacterium pseudotuberculosis should be considered. prevention and control. stressed animals or those exposed to known carriers can be treated prophylactically with tetracycline administered parenterally or orally (in the feed or water). the late-stage polyarthritis is resistant to antibiotic therapy, because of failure of the antibiotic to reach the site of infection. planning vaccination programs carefully will decrease chances of outbreaks. for example, avoiding vaccinating animals for infectious bovine rhinotrachetitis and bovine viral diarrhea during clinical signs. leptospirosis is a contagious but uncommon disease in sheep and goats. the disease may cause abortion, anemia, hemoglobinuria, and icterus and is often associated with a concurrent fever. after a -to -day incubation period, the organism enters the bloodstream and causes bacteremia, fever, and red-cell hemolysis. leptospiremia may last up to days. immune stimulation is apparently rapid, and antibodies are detectable at the end of the first week of infection; crossserovar protection does not occur. during active bacteremia, hemolysis may result in hemoglobin levels of % below normal. hyperthermia, hemoglobinuria, icterus, and anemia may be observed during this phase, and ewes in late gestation may abort. abortion usually occurs only once. mortality rates of above % have been reported in infected ewes and lambs (jensen and swift, ) . subclinical infection is more common in nonpregnant and nonlactating animals. sheep infected with leptospirosis may display a hemolytic crisis associated with igm acting as a cold-reacting hemagglutinin. acute and chronic infections in cattle are more common than infections in sheep and goats. acute forms in cattle display signs similar to those in sheep. acute infection in calves may progress to meningitis and death. lactating cows will have severe drops in production. chronic cases may lead to abortion, with retained placenta, and weakened calves or animals that carry the infection. infertility may also be a sequela. epizootiology and transmission. leptospires are a large genus, and leptospirosis is a complicated disease to prevent, treat, and control. the organism survives well in the environment, especially in moist, warm, stagnant water. cattle, swine, and other domestic and wild animals are potential carriers of serovars common to particular regions. wild animals often serve as maintenance hosts, but domestic livestock may be reservoirs also. organisms are shed in urine, in uterine discharges, and through milk. animals become carriers when they are infected with a host-adapted serovar; sporadic clinical disease is more commonly associated with exposure to a non-hostadapted serovar (heath and johnson, ) . infection may occur via oral ingestion of contaminated feed and water, via placental fluids, or through the mucous membranes of the susceptible animal. placental or venereal transmission may occur. as the organisms are cleared from the bloodstream, they chronically infect the renal convoluted tubules and the reproductive tract (and occasionally the cerebrospinal fluid or vitreous humor). chronically infected animals may shed the organism in the urine for days or longer. necropsy. diagnosis is confirmed by identification of leptospires in fetal tissues. the leptospires are visible in silver-or fluorescent antibody-stained sections of liver or kidney. leptospires may also be seen under dark-field or phase-contrast microscopy of fetal stomach contents. fetal and maternal serology, and diagnostic tests such as the microscopic agglutination test, are useful; interpretation is complicated because of cross reaction of antibodies to many serovars. differential diagnosis. more than one serovar may cause infection in one animal, and each serovar should be considered as a separate pathogen. because of the associated anemia, differential diagnoses should include copper toxicity and parasites, in addition to other abortifacient diseases. prevention and control. polyvalent vaccines, tailored to common serovars regionally, are available and effective for preventing leptospirosis in cattle. immunity is serovar specific. because serological titers tend to diminish rapidly ( - days in sheep [jensen and swift, ] ), frequent vaccination may be necessary. other prevention measures such as species-specific housing, control of wild rodents, and proper sanitation should be instituted. treatment. antibiotic treatment is aimed at treating ill animals and trying to clear the carrier state. treatment methods for acute leptospirosis include oxytetracycline for - days. addition of oxytetracycline or chlortetracycline to the feed for week may be helpful. these antibiotics are considered best for removal of the carrier state of some serovars. vaccination and antibiotic therapy can be combined in an outbreak. research complications. leptospirosis is zoonotic and may be associated with flulike symptoms, meningitis, or hepatorenal failure in humans. etiology. listeria monocytogenes is a pleomorphic, motile, non-spore-forming, [ -hemolytic, gram-positive bacillus that inhabits the soil for long periods of time and has been often found in fermented feedstuffs such as spoiled silage. of the known serovars, several produce clinical signs in ruminants. listeria ivanovii (associated with abortions in sheep) is serovar . clinical signs. listeriosis is an acute, sporadic, noncontagious disease associated with neurological signs or abortions in sheep and other ruminants. the overall case rate is low. the disease may present as an isolated case or with multiple animals affected. three forms of disease are described: encephalitis, placentitis with abortion, and septicemia with hepatitis and pneumonia. the encephalitic form is most common in sheep; septicemic forms may occur in neonatal lambs (scarratt, ) . clinically, the encephalitic form begins with depression, anorexia, and mild hyperthermia after an incubation period of - weeks. as the disease progresses, animals exhibit nasal discharges and conjunctivitis and begin to walk in circles, as if disoriented. facial paralytic lesions, including drooping of an ear or eyelid, dilation of a nostril, or strabismus occur unilaterally on the affected side as the result of dysfunction of some or all the cranial nerves v-xii. the neck will by flexed away from the affected side. facial muscle twitching, protrusion of the tongue, dysphagia, hypersalivation, and nasal discharges may be noted. the hypersalivation may lead to metabolic acidosis in advanced cases in cattle. anorexia, prostration, coma, and death follow. the placental form usually results in last-trimester abortions in ewes and does, which typically survive this form of the disease. the affected females may be asymptomatic or may show severe clinical signs such as fever and depression, with subsequent retained placenta or endometritis. abortion usually occurs within weeks of listeria infection. in cattle, abortion occurs during the last months of gestation and has been induced experimentally - days after exposure. cows present with the range of clinical signs seen in smaller-ruminant dams. there is no long-term effect on the fertility of affected dams. epizootiology and transmission. the organism is transmitted by oral ingestion of contaminated feeds and water or possibly by inhalation. by the oral route, the organism enters through breaks in the oral cavity and ascends to the brain stem by way of nerves. when severe outbreaks occur, feedstuffs should be assessed for spoilage. listeria organisms can be shed by asymptomatic carriers, especially at the end of pregnancy and at lambing. diagnosis and necropsy findings. diagnosis is usually made from clinical signs. culture confirms the diagnosis (cold enrichment at ~ is preferable but not essential for isolation). impression smears will show the pleomorphic gram-positive characterisitics of the pathogen. tissue fluorescent antibody techniques may also be utilized. gross lesions are not observed with the encephalitic form. microscopic lesions include thrombosis, neutrophilic or mononuclear foci in areas of inflammation, and neuritis. the pons, medulla, and anterior spinal cord are primarily affected in the encephalitic form. microabscesses of the midbrain are characteristic of listeria encephalitis in sheep. aborted fetuses that are intact may show fibrinous polyserositis, with excessive serous fluids; small, necrotic foci of the liver; and small abomasal erosions. necrotic lesions of the fetal spleen and lungs may also be seen. in goats, listeria-induced neurological lesions occur only in the brain stem. placentitis, focal bronchopneumonia, hepatitis, splenitis, and nephritis may be seen with other forms. pathogenesis. with the encephalitic form, the organism penetrates mucosal abrasions and enters the trigeminal or hypoglossal nerves. the listeria organisms then migrate along the nerves and associated lymphatics to the brain stem (medulla and pons). in the septicemic form, the organism penetrates tissues of the gastrointestinal tract and enters the bloodstream, to be distributed to the liver, spleen, lungs, kidneys, and placenta. after infection, organisms are shed in all body secretions (infected milk is an important risk factor for zoonosis). a toxin produced by listeria monocytogenes is correlated with pathogenicity, but the mechanism of the pathogenesis of this molecule has not been elucidated. differential diagnoses. rabies, bacterial meningitis, brain abscess, lead toxicity, and otitis media must be considered as differentials. in sheep, the differentials include organisms that cause abortion, and neurological signs, such as enterotoxemia due to clostridium perfringens type d. in goats, the major differentials include caprine arthritis encephalitis viral infection and chlamydial and mycoplasmal infections. in both species, scrapie is a differential. in cattle, aberrant parasite migration or hemophilus somnus infection must also be considered. prevention and control. affected dams should be segregated and treated. other animals in the group may be treated with oxytetracycline as needed. aborted tissues should be removed immediately. proper storage of fermented feeds minimizes this source of contamination. when silage spoils, the ph increases, producing a suitable growth environment for the organism. commercial vaccines are not available in the united states. treatment. affected animals can be treated aggressively with penicillin, ampicillin, oxytetracycline, or erythromycin. exceptionally high levels of penicillin are required for treating affected cattle. severely affected animals should receive appropriate fluid support and other nursing care. treatment is less successful, and mortality is especially high in sheep. recovered animals tend to resist reinfection. research complications. in addition to the loss of fetal animals, stress to the dams, and risks to other animals, any aborted tissue by a ruminant should be regarded as a potential zoonotic risk. listeria can cause mild to severe flulike symptoms in humans and may be a particular risk for pregnant women and for older or immune-compromised individuals. listeriosis in humans is a reportable disease. etiology. lyme disease is caused by the spirochete borrelia burgdorferi. clinical signs and diagnosis. reports in ruminants indicate seroconversion to b. burgdorferi, but there are few definitive correlations to the arthritis that is present. diagnosis requires culturing from the affected joints and diagnostic elimination of other causes of lameness and arthritis. epizootiology and transmission. the organism is present throughout much of the northern hemisphere and has been reported in many mammals and also in birds. ticks of the ixodes ricinus complex are the major vectors of the spirochete and must be attached for hr for successful transmission. pathogenesis. the ixodes ticks have three life stages: larval, nymphal, and adult. feeding occurs once during each stage, and wild animals are the source of blood meals. the larval stages feed from rodents, such as the white-footed deer mouse, peromyscus leucopus, from which they acquire the spirochete. the nymphal stage is that which usually infects other animals. the adult ticks are usually found on deer. differential diagnosis. seroconversion to b. burgdorferi does not necessarily confirm the cause of arthritis. other causes of arthritis and lameness in ruminants include trauma, caprine arthritis encephalitis virus, mycoplasma spp., chlamydia psittaci, erysipelothrix spp., arcanobacterium pyogenes, brucella spp., and rickets. prevention and control. control of the tick vector is the most important factor in preventing the possibility of exposure or disease. treatment. antibiotic therapy, with tetracycline, penicillin, amoxicillin, and cephalosporins, is used for diagnosed or suspected lyme arthritis. research complications. lyme disease is zoonotic, and the lxodes ticks transmit the disease to humans. v. mastitis i. ovine mastitis mastitis in ewes may be acute, subclinical, or chronic. acute mastitis often results in anorexia, fever, abnormal milk, and swelling of the mammary gland. pasteurella haemolytica is the most common cause of acute mastitis. additional isolates may include, in order of prevalence, staphylococcus aureus, actinomyces (corynebacterium) spp., and histophilus ovis. escherichia coli and pseudomonas aeruginosa have also been found to cause acute mastitis. as many as six serotypes of pasteurella haemolytica have been isolated from the mammary glands of mastitic ewes. furthermore, intramammary inoculation of these organisms isolated from ovine and bovine pulmonary lesions has resulted in clinical mastitis in ewes (watkins.and jones, ) . subclinical mastitis is detected only indirectly, by counting somatic cells. the most common isolate from ewes with subclinical mastitis is coagulase-negative staphylococci. other isolates include actinomyces bovis, streptococcus uberis, s. dysgalactiae, micrococcus spp., bacillus spp., and fecal streptococci. most of these organisms are commonly found in the environment. diffuse chronic mastitis, or hardbag, results from interstitial accumulations of lymphocytes in the udder. both glands are usually affected, but no inflammation is present. serological evidence suggests that diffuse chronic mastitis is caused by the retrovirus that causes ovine progressive pneumonia (opp or maedi/visna virus). other bacterial agents or mycoplasma have not usually been isolated from udders with this type of mastitis. acute mastitis occurs in approximately % of lactating ewes annually, and it usually occurs either soon after lambing or when lambs are - months old (lasgard and vaabenoe, ) . subclinical mastitis occurs in - % of lactating ewes (kirk and glenn, ) . subclinical mastitis is more common in ewes from high-milk-producing breeds. skin or teat lesions and dermatitis increase the prevalence of disease. acute mastitis can be diagnosed in ewes with associated systemic signs of disease by physical examination of the udder and inspection of the milk. subclinical mastitis is often suggested by somatic cell counts elevated above x cells/ml. when high somatic cell counts are identified, subclinical mastitis can be diagnosed by milk culture. the california mastitis test may also be helpful as an indicator of mastitis. manual palpation of a hard, indurated udder as well as serological testing for the maedi/visna virus is helpful in confirming the diagnosis of diffuse chronic mastitis. treatment for acute bacterial mastitis should include aggressive application of broad-spectrum antibiotics (intramammary and systemic) and supportive therapy such as fluids and anti-inflammatory drugs. it is may be helpful to milk out the infected ud-der frequently; oxytocin injections preceding milking will improve gland evacuation. because somatic cell counting is often not routinely performed, treatment of subclinical mastitis is seldom done. there is currently no treatment available for diffuse chronic mastitis. ii. caprine mastitis lactating goats are subject to inflammation of mammary gland, or mastitis. the primary causative organisms are staphylococcus epidermidis and other coagulasenegative staphylococcus spp. clinical signs of mastitis include abnormal coloration or composition of milk, mammary gland redness, heat and pain, enlargement of the mammary gland, discoloration of the mammary gland, and systemic signs of septicemia. large abscesses may be present in the affected gland. staphylococcus aureus is also associated with caprine mastitis, and toxemia may be part of the clinical picture. this organism produces a necrotizing alpha toxin that can result in gangrenous mastitis. caprine mastitis may be clinical or subclinical, and the first indication of mastitis may be weak, depressed, or thin kids. diagnosis is based on careful culture of mastitic milk. treatment includes frequent stripping, intramammary antibiotics, and nonsteroidal anti-inflammatory drugs. oxytocin ( - u) may help milk letdown for frequent strippings. bovine mastitis products can be used in the goat; however, care should be taken not to insert the mastitis tube tip fully, because damage to the protective keratin layer lining the teat canal may occur. in severe acute systemic cases, steroids, fluids, and systemic antibiotics may be necessary. other less common causes of mastitis in goats include streptococcus spp. (s. agalactiae, s. dysgalactiae, s. uberis, and zooepidemicus). gram-negative causes of caprine mastitis include escherichia coli, klebsiella pneumoniae, pasteurella spp., pseudomonas, and proteus mirabilis. corynebacterium pseudotuberculosis can cause mammary gland abscessation, whereas mycoplasma mycoides may cause agalactia and systemic disease. "hard udder" can be caused by caprine arthritis encephalitis virus (caev). brucellosis and listeriosis can cause a subclinical interstitial mastitis (smith and sherman, ) . iii. bovine mastitis mastitis is the disease of greatest economic importance for the dairy cattle industry. the majority of the impact will be on the production and overall health of the cows, but low-incidence herds also diminish the risk of calves' ingesting or being exposed to pathogens. the most common bovine mastitis pathogens include staphylococcus aureus and streptococcus agalactiae, s. dysgalactiae, and s. uberis; coliform agents such as escherichia coli, enterobacter aerogenes, serratia marcescens, and klebsiella pneumoniae; mycoplasmal species such as mycoplasma bovis, m. bovigenitalium, m. californicum, m. canadensis, and m. alkalescens; and salmonella spp. such as s. typhimurium, s. newport, s. enteritidis, s. dublin, and s. muenster. many of these agents such as staphylococcus spp., salmonella spp., and the coliforms can cause both acute and chronic mastitis, as well as severe systemic disease, including fever and anorexia. these must be regarded as herd and environmental pathogens in terms of treatment and prevention. the pathogenesis of staphylococcal infections is comparable to that in goats. staphylococcus agalactiae can be cleared from udders because it does not invade other tissues, is an obligate resident of the glands, and is susceptible to penicillin. in contrast, s. uberis and s. dysgalactiae are environmental organisms and can be highly resistant to pencillin. mycoplasma bovis is the more common of the mycoplasmal pathogens and can cause severe infections. transmission of the mycoplasmas is not well defined but may be related to their presence in other organ systems. treatments for mycoplasmal mastitis are not successful; culling is recommended. there are many interrelated factors associated with prevention and control of mastitis in a herd, including herd health and dry cow management, order of animals milked, milking procedures, milking equipment, condition of the teats, and the condition of the environment. management of the overall herd includes aspects such as vaccination programs, nutrition, isolation of incoming animals, and quarantine and treatment of or culling diseased individuals. culturing or testing newly freshened cows and monitoring the bulk milk tank serve as indicators of subclinical mastitis. herd management will diminish teat lesions. bacterin vaccines are available for preventing and controlling coliform mastitis and s. aureus mastitis. at the time of dry-off, all cows must be treated by intramammary route. some infections can be successfully cleared during this time. younger, disease-free animals should be milked first; any animals with diagnosed problems should be milked after the rest of the herd and/or segregated during treatment. milkers' hands easily serve as a means of pathogen transmission, and wearing rubber gloves is recommended. teat and udder cleaning practices include washing and drying with single-service paper or cloth towels or pre-and postmilking dipping. milking equipment must be maintained to provide proper vacuum levels and pumping rates, and liners should be the appropriate size. facilities that provide clean and dry areas for the animals to rest, feed, and move will diminish teat injuries and reduce exposures to mastitis pathogens. in that regard, inorganic bedding such as clean sand harbors few pathogens in contrast to shavings and sawdust. w. etiology. moraxella bovis, a gram-negative coccobacillus, is the most common cause of infectious bovine keratoconjunctivitis (ibk) in cattle. this organism is not a cause of keratoconjunctivitis in sheep and goats. the disease includes conjunctivitis and ulcerative keratitis. the pathogenic m. bovis strain is piliated, and at least seven serotypes exist. clinical signs. lacrimation, photophobia, and blepharospasm are seen initially. conjunctival injection and chemosis develop within a day of exposure, and then keratitis with corneal edema and ulcers. anterior uveitis may be a sequela within a few days, and thicker mucopurulent ocular discharge may be seen. corneal vascularization begins by days after onset. reepithelialization of the corneal ulcers occurs by - weeks after onset. diagnosis is usually based on clinical signs, but culturing is helpful and fluoroscein staining is useful for demonstrating corneal ulceration. epizootiology and transmission. the disease is more severe in younger cattle. the clinical signs of ibk tend to be more severe in cattle that are also infected with infectious bovine rhinotracheitis (ibr) virus or those that have been vaccinated recently with modified live ibr vaccine. the bacteria are shed in nasal secretions and cattle with no clinical symptoms may be carriers. transmission is by fomites, flies, aerosols, and direct contact. incidence in winter months is very low. nonhemolytic strains are associated with the winter epidemics, and hemolytic strains are associated with summer epidemics. necropsy findings. necropsy is not typically performed on these cases. corneal edema, ulceration, hypopyon, and uveitis would be noted, depending on the stage of infection. pathogenesis. the pili ofm. bovis bind to receptors of corneal epithelium. the virulent strains of the bacteria then release the enzymes that damage the corneal epithelial cells. other factors contributing to infection include ultraviolet light and trauma from dust and plant materials. differential diagnoses. infectious bovine rhinotrachetitis virus causes conjunctivitis, but the central corneal ulceration that is characteristic of ibk is not seen with m. bovis infections. mycoplasma, listeria, branhamella (neisseria) , and adenovirus may be cultured from affected bovine eyes but none has been shown to produce the corneal lesions when inoculated into susceptible animals. prevention and control. cattle should not be immunized intranasally with modified live infectious bovine rhinotracheitis vaccine during ibk outbreaks; this will likely exacerbate the infection. new animals should be quarantined and treated prophylactically before introduction to herds. the available vaccines, containing. m. bovis pili or killed m. bovis, help decrease incidence and severity of disease; these preparations are not completely effective, because the m. bovis strain may not be homologous to that used for the vaccine preparation. other preventive measures include % permethrin-impregnated bilateral ear tags, pour-on avermectins, or dust bags or face rubbers containing insecticide (such as % coumaphos) to control flies throughout the season and premises; mowing of high pasture grass to minimize ocular trauma; provision of shade; control of dust and sources of other mechanical trauma; and segregation of animals by age. treatment. cattle can recover without treatment, but younger animals should be treated as soon as the infection is detected. antibiotic treatments include topical, subconjunctival administration and intramuscular dosing. several standard topical antibiotics have been shown to be effective, including oxytetracycline, gentamicin, and triple antibiotic combinations. these should be administered twice per day. subconjunctival injections of antibiotics, such as penicillin g, provide higher corneal levels of drug; these are typically administered only once or twice in severe cases. intramuscular doses of long-acting oxytetracycline, given on alternate days, are effective in larger herds, and doses hr apart eliminate carriers. third-eyelid flaps, temporary tarsorrhaphy, or eye patches may be useful in certain cases. epizootiology and transmission. although m. bovis can be killed by sunlight, it otherwise survives a long time in the environment and in cattle feces. animals acquire the infection from the environment or from other animals via aerosols, from contaminated feed and water, and from secretions such as milk, semen, genital discharges, urine, and feces. clinically normal animals may serve as carriers. the bacilli stimulate an initial neutrophilic tissue response. neutrophils become necrotic and are phagocytosed by macrophages, forming giant epithelioid cells called langhans' giant cells. an outer lymphocytic zone is formed, and fibrotic encapsulation creates the classical caseous nodules. vascular erosion and hematogenous migration of the organisms may lead to lesions throughout the body. necropsy findings. yellow primary tubercles (granulomas) with central areas of caseous necrosis and calcification are present in the lungs. caseous nodules are also associated with gastrointestinal organs and mesenteric lymph nodes. research complications. this pathogen does present a complication due to the carrier status of some animals, the likelihood of herd outbreaks, the severity of disease in younger animals, and the morbidity, possible progression to uveitis, and time and treatment costs associated with infections. the overall condition of the cattle will be affected for several weeks, and permanent visual impairment or loss, as well as ocular disfigurement, may occur. mycobacterium bovis infection (tuberculosis) etiology. mycobacteria are aerobic, nonmotile, non-sporeforming, acid-fast pleomorphic bacteria. most cases of tuberculosis in sheep are related to mycobacterium bovis or m. avium. cases in goats have been attributed to m. bovis, m. avium, or m. tuberculosis. mycobacterium bovis, or the bovine tubercle bacillus, is the cause in cattle but has been isolated from many domestic and wild mammals. other agents of mammalian tuberculosis include m. microti and m. africanum. clinical signs. tuberculosis is a sporadic, chronic, contagious disease of ruminants and is zoonotic. the infection is often asymptomatic later in the illness, and it may be diagnosed only at necropsy. the respiratory system (m. bovis) or the digestive system (m. avium) is the primary site of infection; other tissues such as mammary tissue and reproductive tract may be infrequently involved. locations of the characteristic tubercles will determine whether clinical signs are seen. respiratory signs may include dyspnea, coughing, and pneumonia. digestive tract signs include diarrhea, bloat, or constipation; diarrhea is most common. lymphadenopathy occurs in advanced cases. fever and generalized disease may be seen after calving. infected goats lose weight and develop a persistent cough. prevention and control. significant progress has been made in eradication programs in the united states during the past several decades, but during the s, infected animals continued to be found in domestic cattle herds and particularly in captive deer herds in hunting preserves. the intradermal tuberculin test, using purified protein derivative (ppd), is usually used as a diagnostic indicator in live animals. this test should be performed annually on bovine and caprine dairy herds (and bison herds); the official tests are the caudal fold, comparative cervical, and single cervical tests. notification to state officials is required following identification of intradermal-positive animals. great care must be exercised in any handling of tissue or necropsies of reactors, and state animal health officials should be consulted regarding disposal of materials and cleaning of premises following depopulation of positive animals. no treatment is recommended, and treatment is usually not allowed, because of the zoonotic potential, chronicity of the disease, and the treatment costs. slaughter is preferred, to prevent potential transmission to humans. paratuberculosis, or johne 's disease (mycobacterium paratube rculo sis) etiology. mycobacterium paratuberculosis, the causative agent of johne's disease, is a fastidious, non-spore-forming, acid-fast, gram-positive rod. the organism is actually a subspecies of m. avium, but m. paratuberculosis does not produce the siderophore mycobactin (an iron-binding molecule) of m. avium. clinical signs and diagnosis. johne's disease is a chronic, contagious, granulomatous disease of adult ruminants and is characterized by unthriftiness, weight loss, and intermittent diarrhea. in sheep and goats, chronic wasting is usually seen, occasionally with pasty feces or diarrhea. in cattle, chronic diarrhea and rapid weight loss are the most common clinical signs of the disease. usually older adult animals are infected, but over time in an infected herd, younger animals will become infected when sufficient doses of organisms are ingested. although clinical signs are nonspecific, johne's disease should be considered if the affected diarrheic animals have a good appetite and are on a good anthelmintic program. the disease is diagnosed based on clinical signs and laboratory analyses, although none of the tests is more than % sensitive. in addition, the sensitivity of the serological tests differs between species. the standard is the fecal culture that takes - weeks. theenzyme-linked immunosorbent assay (elisa) is now considered the most reliable serological test, but false negatives do occur. other serological tests such as agar gel immunodiffusion (agid) and complement fixation are useful. herd screening may be done using the agid or elisa serological tests. identification of the organism on culture, or the presence of acid-fast organisms on mucosal or mesenteric lymph node smears or from rectal biopsies, helps confirm the diagnosis. some animals serologically negative for johne's disease, however, have been found to be positive on fecal culture. commercial agid tests approved for use in cattle may be useful in diagnosing johne's disease in sheep (dubash et al., ) . serological tests cross-react with other species of mycobacterium, especially m. avium. epizootiology and transmission. the organism is prevalent in the environment and is transmitted to young animals by direct or indirect contact. although vertical transmission has been reported, the organism more commonly enters the gastrointestinal tract and penetrates the mucosa of the distal small intestine, primarily the ileum. chronic carriers may intermittently shed the organisms. parasite that grows only in macrophages of infected animals. nursing infected dams are a primary source of infection of neonates. if the organism is not cleared, it proliferates slowly in the tissue, leading to inflammatory reactions that progress through neutrophilic to mononuclear stages. the organism may penetrate the lymphatics and proliferate in mesenteric lymph nodes. after an incubation period of a year or more, some of the carriers will progress to clinical disease manifested by fibrotic and hyperplastic changes in the ileum, leading to the classic thickening in the region. gut changes result in intermittent diarrhea, with subsequent dehydration, electrolyte imbalances, and malnutrition, although this clinical sign is more common in cattle than in sheep or goats. necropsy and diagnosis. the ileum from infected cattle is grossly thickened; this is not seen in sheep and goats. ileal and ileocecal lymph nodes provide the best samples for histology and acid-fast staining. differential diagnosis. diseases causing chronic wasting and poor coat and body condition of all ruminants should be considered. these include chronic salmonellosis, peritonitis, severe parasitism, winter dysentery, and pyelonephritis. deer can be infected, and the lesions can be confused with those of tuberculosis. prevention and control. prevention is the most effective method to manage this pathogen. efforts should be focused on eliminating the disease through test and slaughter. neonates should not be reared by infected dams. some states have johne's disease eradication programs. facilities and pastures where animals testing positive for johne' disease were maintained should be thoroughly cleaned and kept vacant for a year after culling. other considerations. mycobacterium paratuberculosis is being investigated as a factor in the development of crohn's disease in humans. etiology. the most common organism causing infection of the umbilicus is arcanobacterium (formerly actinomyces, corynebacterium) pyogenes; other bacteria may be present. arcanobacterium spp. are anaerobic, nonmotile, non-sporeforming, gram-positive, pleomorphic rods to coccobacilli. other environmental contaminants are also associated with this disease, such as escherichia coli, enterococcus spp., proteus, streptococcus spp., and staplylococcus spp. clinical signs and diagnosis. navel ill is an acute localized inflammation and infection of the external umbilicus. animals present with fever and painful enlargement of the umbilicus. animals may exhibit various degrees of depression and anorexia, and purulent discharges may be seen draining from the umbilicus. involvement of the urachus is usually followed by cystitis and associated signs of dysuria, stranguria, and hematuria. other common severe sequelae include septicemia, pneumonia, peritonitis, septic arthritis (joint ill), meningitis, osteomyelitis, uveitis, endocarditis, and diarrhea. neonates, and most cases occur within the first months of age. cleanliness of the birthing and housing environment and successful transfer of passive immunity are important factors in the occurrence of the disease. dystocia resulting in weak neonates can be a factor predisposing to the development of the disease. navel ill is diagnosed by typical clinical signs. the presence of microabscesses and palpation of the umbilical area for firm intra-abdominal structures extending from the umbilicus are abnormal. assessment of colostral immunoglobulin transfer may contribute to determination of the prognosis. navel ill should always be considered for young ruminants with fever of unknown origin during the first week of life and for slightly older lambs, kids, or calves that are not thriving. arthrocentesis of affected joints and culture of the fluid for identification of the pathogen are also diagnostic options and essential for effective antimicrobial selection. differential diagnosis. the major differential is an umbilical hernia, which will typically not be painful or infected and can often be reduced. mycoplasmal arthritis is a differential in kids. in the past, erysipelothrix rhusopathiae was a common navel ill pathogen in sheep. treatment. omphalitis can be treated with a to day course of broad-spectrum antibiotics such as ampicillin, amoxicillin, penicillin, ceftiofur, florfenicol, and erythromycin. if an isolated abscess is palpable, it should be surgically opened and repeatedly flushed with iodine solutions. surgical reduction of the infected umbilicus is indicated if intra-abdominal structures are involved. the prognosis for recovery is good if systemic involvement has not occurred. prevention and control. the disease is best prevented and controlled by providing clean birthing environments, ensuring adequate colostral immunity, thoroughly dipping the umbilicus of newborns in tincture of iodine or strong iodine solution (lugol's), monitoring for dystocias, and maintaining young growing animals in noncontaminated environments. may invade the bloodstream, causing disseminated septicemia. clinically, the lambs may exhibit nasal discharge of mucopurulent to hemorrhagic exudate, hyperthermia, coughing, dyspnea, anorexia, and depression. with the respiratory form, auscultation of the thorax suggests dullness and consolidation of anteroventral lobes; this will be confirmed by radiographs. the disease is diagnosed by clinical signs, blood cultures from septicemic animals, blood smears showing bipolar organisms, and history of predisposing stressors. in cultures, p. hemolytica is distinguished from p. multocida by hemolysis on blood agar; only p. multocida produces indole. epizootiology and transmission. the organism is ubiquitous in the environment and in the respiratory tracts of these animals. younger ruminants, between and months of age, are especially prone to infection during times of stress, such as weaning, transportation, dietary changes, weather changes, and overcrowding. the pneumonic form appears as a complex associated with concurrent infections such as parainfluenza , adenovirus type , respiratory syncytial virus, mycoplasmas, chlamydia, pasteurella multocida and bordetella parapertussis (martin, ; brogden et al., ) . the organism is transmitted between animals by direct and indirect contact, through inhalation or ingestion. necropsy findings. necropsy lesions include areas of necrosis and hemorrhage in the small intestines and multifocal mm lesions distributed on the surfaces of the lungs and liver. with the pneumonic form, serofibrinous exudates fill the alveoli; ventral lung lobes are consolidated and are congested and purple-gray in color. fibrinous pleuritis, pericarditis, and hematogenously induced arthritis also may be evident.. the disease can be costly to treat, and the toll taken on young animals due to the consequences of systemic infection may detract from their research value. etiology. pasteurella hemolytica and p. multocida are aerobic, nonmotile, non-spore-forming, bipolar, gram-negative rods. biotype a serotypes are associated with pneumonia and septicemia in all ruminants (ellis, ) . serotype of p. hemolytica is considered a major cause of pulmonary lesions of bovine bronchopneumonia and fibrinous bronchopneumonia. clinical signs. pasteurellosis is an acute bacterial disease characterized by bronchopneumonia, septicemia, and sudden death. the organism invades the mucosa of the gastrointestinal tract or respiratory tract and causes localized areas of necrosis, hemorrhage, and thrombosis. the lungs and liver are frequent areas of formation of microabscesses. acute rhinitis or pharyngitis often precedes the respiratory form. the organism also pathogenesis. a leukotoxin is considered to be a key factor in the pathogenesis of the p. hemolytica infection. macrophages and neutrophils are lysed by the toxin as they arrive at the lung, and the enzymes released by the neutrophils cause additional damage to the tissue. treatment. treatment may include the use of antibiotics such as penicillin, ampicillin, tylosin, sulfonamides, or oxytetracycline. newer antibiotics, such as ceftiofur, tilmicosin, spectinomycin, and florfenicol, are very effective and approved for use in cattle. in outbreaks, cultures from fresh necropsies are helpful for determining sensitivities useful for the remaining group. prevention and control. the incidence of disease can be decreased by minimizing the degree of stress; by improving management, such as nutrition and control of parasitism; and, in cattle and sheep, by vaccinating for viral respiratory infections such as parainfluenza. early pasteurella hemolytica bacterin vaccines for use in cattle are not considered effective, but newer products based on immunizing against the leukotoxin and some bacterial capsule surface antigens are effective. pasteurella multocida bacterins and live streptomycin-dependent mutant vaccines are available. in young animals, passive immunity is protective. preventive measures also include maintaining good ventilation in enclosures and barns. new animals to the flock or herds should be quarantined for at least weeks before introduction. etiology. salmonella typhimurium is a motile, aerobic to facultatively anaerobic, non-spore-forming, gram-negative bacillus and is the organism associated with enteric disease and some abortions in ruminants. it is a common inhabitant of the gastrointestinal tract of ruminants. current nomenclature categorizes s. typhimurium as a serovar within the species s. enteritidis (the other two species are s. typhi and s. choleraesuis). salmonella typhimurium, s. dublin, and s. newport are the common species seen in bovine cases. salmonella typhimurium, s. dublin, s. anatum, and s. montevideo are seen in ovine and caprine cases, although a host-adapted species has not been identified in the goat. ovine abortions due to various salmonella species are not reported in the united states but are enzootic in other countries. salmonella serotypes have been associated with aborted fetuses in all ruminant species. clinical signs and diagnosis. salmonellosis causes acute gastroenteritis, dysentery, and septicemia (anderson and blanchard, ) . clinically, the animals become anorexic and hyperthermic. diarrhea or dysentery develops; feces may contain mucus and/or blood and have a putrid odor. animals become severely depressed and weak, losing a high percentage of their body weight. animals may die in - days because of dehydration associated with dysenteric fluid loss, septicemia, shock, and acidosis. morbidity may be %, and mortality may be high. septicemia may result in subsequent meningitis, polyarthritis, and pneumonia. chronically infected animals may have intermittent diarrhea. in goats, salmonellosis may be recognized as diarrhea and septicemia in neonates, as enteritis in preweaned kids and mature goats, and, rarely, as abortion. adult cases may be sporadic, with intermittent bouts of diarrhea, subacute or even chronic. morbidity and mortality will be highest in neonates, and some may simply be found dead. the older animals generally tend to fare better during the disease. abdominal distension with profuse yellow feces is common. kids become severely depressed, anorexic, febrile (with temperatures as high as ~ dehydrated, acidotic, recumbent, and comatose. salmonella abortions may occur throughout gestation. there may not be any other clinical signs, or abortion may be seen with diarrhea, fever, and vulvar discharges. hemorrhage, placental necrosis, and edema will be present. metritis and placental retention may occur. some mortality of dams may occur. diagnosis is based on clinical signs and can be confirmed by culturing fresh feces or at necropsy. because of intermittent shedding of organisms, culture may be difficult; repeated cultures are recommended. leukopenia and a degenerative shift to the left are not uncommon hematological findings. epizootiology and transmission. stresses associated with recent shipping, overcrowding, and inclement weather may predispose the animal to enteric infection. birds and rodents may be natural reservoirs of salmonella in external housing environments. transmission is fecal-oral. after ingestion, the organisms may proliferate throughout the gastrointestinal tract and may penetrate the mucosa of the intestines, invade the peyer's patches and lymphatics, and migrate to the spleen, liver, and other organs. animals that survive may become chronic carriers and shedders of the organisms, and this has been demonstrated experimentally (arora, ) . fecal-oral transmission is also associated with salmonella abortion; veneral transmission has not been reported. necropsy findings and diagnosis. animals will have noticeable perineal staining. intestines (particularly the ileum, cecum, and colon) may contain mucoid feces with or without hemorrhages. petechial hemorrhages and areas of necrosis may be noticed on the surface of the liver, heart, and mesenteric lymph nodes. the wall of the intestines, gallbladder, and mesenteric lymph nodes will be edematous, and a pseudodiphtheritic membrane lining the distal small intestines and colon may be observed. this membrane is not normally seen in the goat (smith and sherman, ) . splenomegaly may be present. aborted fetuses will often be autolysed. placentitis, placental necrosis, and hemorrhage are commonly seen. serologic evidence of recent infection can be demonstrated in the dam. salmonella can be isolated from the aborted tissues. pathogenesis. after ingestion, the organism proliferates in the intestine. damage to the intestines and the resulting diarrhea are due to the bacterial production of cytoxin and endotoxin. although the salmonella organisms will be taken up by phagocytic cells involved in the inflammatory response, they survive and multiply further. septicemia is a common sequela, with the bacteria localizing throughout the body. in latently infected animals, it is often shed from the gallbladder and mesenteric lymph nodes. younger animals may be susceptible because of immature immunity and intestinal flora and higher intestinal ph. carriers may develop clinical disease when stressed. differential diagnoses. in young animals, differentials include other enteropathogens: escherichia coli, rotavirus and coronavirus, clostridia, cryptosporidia, and other coccidial forms. these pathogens may also be present in the affected animals. differentials in adults include bovine viral diarrheas and winter dysentery in cattle and parasitemia and enterotoxemia in all ruminants. prevention and control. affected animals should be isolated during herd outbreaks. samples for culture should include herdmates, water and feed sources, recently arrived livestock (other species), and area wildlife, including birds and rodents. repeated cultures, culling of animals, intensive cleaning, and disinfection of facilities are all important during outbreaks. the bacteria survive for about a week in moist cow manure. vaccination using the commercially available killed bacterin or autologous bacterins may be useful in outbreaks involving pregnant cattle, although the j- bacterin is now considered better. treatment. nursing care includes rehydration and correction of acid-base abnormalities. antibiotic therapy may be useful in cases with septicemia, but it is controversial because it may induce carrier animals. gentamicin, trimethoprim-sulfadiazine, ampicillin, enrofloxacin, and amikacin antibiotics may be successful. negative, rod-shaped bacterium. type a is more virulent than type b. clinical signs. although tularemia is a disease of livestock, pets, and wild animals, sheep are most commonly affected. the disease is characterized by hyperthermia, muscular stiffness, and lymphadenopathy. infected animals move stiffly, are depressed, and are hyperthermic. anemia and diarrhea may develop, and infected lymph nodes enlarge and may ulcerate. mortality may reach %. animals that recover will have immunity of long duration. epizootiology and transmission. the disease is most commonly transmitted by ticks or biting flies. the wood tick, dermacentor andersoni, is an important vector in transmitting the disease in the western united states, and, as natural hosts, wild rodents and rabbits tend to be reservoirs of the pathogen. research complications. salmonellosis is zoonotic, and some serotypes of the organism have caused fatalities even in immunocompetent humans. attempts should be made to identify and cull carrier animals. pathogenesis. the organisms, entering the tick bite wound, move via lymphatics to lymph nodes and subsequently to the bloodstream, where they cause septicemia. the organisms can also be transmitted orally through contaminated water. etiology. spirochete-like organisms are associated with this disease; it is now recognized that the agent is not a chlamydial organism. the disease has been reported only in the foothills bordering the central valley of california. necropsy findings. ticks may also be present on the carcasses. suppurative, necrotic lymph nodes are typical. lungs will be congested and edematous. diagnosis is confirmed by prompt culturing of the organism from lymph nodes, spleen, or liver where granulomatous lesions form; p. tularensis does not survive for long periods in carcasses. serological findings may also be helpful. clinical signs. cows that become infected with the causative agent before months of gestation abort or give birth to weak calves without any clinical sign of infection. cows infected after months of gestation give birth to normal calves. affected cows rarely abort in subsequent pregnancies. the tick vector is ornitho- necropsy. fetuses show several pathological changes, including enlargement of the cervical lymph nodes, spleen, and liver. the calf's thymus will be small, and histologically there will be losses of thymic cortical lymphocytes. histologic changes in lymph nodes and spleen include vasculitis, necrosis, and histiocytosis. treatment. chlortetracycline treatment has been effective in controlling this disease. etiology. tularemia is caused by pasteurella (francisella) tularensis a nonmotile, non-spore-forming, aerobic, gram-control and prevention. eliminating the tick vectors can prevent tularemia. animals should be provided with fresh water frequently. the organism can survive in freezing conditions and in water and mud for long periods of time. caretakers, veterinarians, and researchers should take special precautions before handling the tissues of infected sheep, because this is a method of zoonotic spread. research complications. the disease is zoonotic, and transmission to people may result from tick bites or from handling contaminated tissues. although not a major disease of concern in sheep, researchers using potentially infected animals from western range states of the united states should be aware of it. the organism is antigenically related to brucella spp. etiology. yersiniosis is caused by infections with yersinia enterocolitica, a gram-negative, aerobic, and facultative anaerobe of the family enterobacteriaceae. there are serotypes reported for y. enterocolitica. yersinia pseudotuberculosis infections have also been seen in ruminants. enteric infections predominate in the diseases caused by these bacteria. clinical signs and diagnosis. clinical disease may be seen rarely in many groups of ruminants. goats of - months old suffer from the enteric form of the disease, which is characterized by sudden death or the acute onset of watery diarrhea lasting or more days. spontaneous abortions and weak neonates are also clinical manifestations of infection. lactating does may have mastitis that becomes chronically hemorrhagic. bacteremia results in internal abscesses, abortion, and acute deaths. yersinia pseudotuberculosis has been associated with laboratory goat epizootics (obwolo, ) . diarrhea in pastured sheep, stressed by other factors, has also been reported. diagnosis is based on culture and serology. epizootiology and transmission. the bacteria are carried by wild birds and rodents, and transmission is by ingestion of contaminated feed and water. research complications. yersinia is zoonotic. prevention and control. control measure are not well defined, because the epidemiology of the disease is poorly understood (smith and sherman, ) . tissues from affected goats must be handled and disposed of properly. areas housing affected goats must be thoroughly sanitized. treatment. in case of an abortion storm, treatment of goats with tetracycline has been useful. other broad-spectrum antibiotics may also be useful. clinical signs. contagious caprine pleuropneumonia is characterized by severe dyspnea, nasal discharge, cough, and fever (mcmartin et al., ) . infections with other mycoplasma species also have similar clinical signs. septicemia without respiratory involvement may also be a presentation. epizootiology and transmission. this disease is highly contagious, with high morbidity and mortality. transmission is by aerosols. mycoplasma mycoides subsp, mycoides has become a serious cause of morbidity and mortality of goat kids in the united states. necropsy. large amounts of pale straw-colored fluid and fibrinous pneumonia and pleurisy are typical. some lung consolidation may be present. meningitis, fibrinous pericarditis, and fibrinopurulent arthritis may also be found. diagnosis is usually made at necropsy by culture of the organism from lungs and other internal organs. differential dagnosis. in the united states, the principal differential for m. mycoides subsp, mycoides is caprine arthritis encephalitis. treatment. tylosin and oxytetracycline are effective. some infections are slow to resolve. prevention and control. vaccines are available in some areas. infected herds are quarantined. new goats should be quarantined before introduction to the herd. research complications. the worldwide distribution of the f biotype, as well as the aerosol transmission and high mor-bidity and mortality characteristics of mycoplasmal infectious, make these infections economically important diseases. considerable attention is presently given to this genus as a source of morbidity and mortality in goats. iv. mycoplasma conjunctivae (mycoplasmal keratoconjunctivitis) etiology. mycoplasma conjunctivae causes infectious conjunctivitis, or pinkeye, in sheep and goats with associated hyperemia, edema, lacrimation, and corneal lesions. mycoplasma mycoides subsp, mycoides, m. agalactiae, m. arginini, and acholeplasma oculusi have also been associated with keratoconjunctivitis in these species. respiratory disease and other infections, such as mastitis, may also be observed. clinical signs and diagnosis. all ages of animals may be affected. initially, lacrimation, conjunctival vessel injection, and then keratitis and neovascularization are seen. sometimes uveitis is evident. although the presentation is usually unilateral, bilateral involvement is possible. recurring infections are common. culturing provides the better diagnostic information, and cultures will be positive even after clinical signs have diminished. ily between animals by direct contact. animals can become reinfected, and carrier animals may be a factor in outbreaks. necropsy. it is unlikely that animals would die or be euthanized and undergo necropsy for this problem. conjunctival scrapings would include neutrophils during earlier stages and lymphocytes during later stages. epithelial cell cytoplasm should be examined for organisms. differential diagnosis. the primary differential in sheep and goats is chlamydia, as well as branhamella, rickettsia (colesiota) conjunctivae, and infectious bovine rhinotracheitis in goats only. it is important to consider these differentials if arthritis, pneumonia, or mastitis is present in the group or the individual. treatment. animals do recover spontaneously within about weeks. tetracycline ointments and powders are also used. third-eyelid flaps may be necessary if corneal ulceration develops. prevention and control. new animals should be quarantined and, if necessary treated, before introduction to the flock or herd. etiology. eperythrozoonosis is a rare, sporadic, noncontagious, blood-borne disease in ruminants worldwide caused by the rickettsial agent eperythrozoon. host-specific species of importance are e. ovis, the causative species in sheep and goats, and e. wenyoni, e. tegnodes, and e. tuomii, the causative agents in cattle. although the disease is of minor importance, it can cause severe anemia and debilitation in affected animals. haemobartonella bovis is also rare, and is usually found only in association with other rickettsial diseases. clinical signs and diagnosis. the disease is more severe in sheep. following an incubation period of - weeks, infected animals exhibit episodic hyperthermia, weakness, and anemia. losses may be greater in younger lambs. cattle are usually latently infected but may have swollen and tender teats and legs. fever, anemia, and depression will be present if the cattle are stressed by another systemic disease. diagnosis is based on clinical evidence of anemia and is confirmed by observing the rickettsiae on the surface of red blood cells in a blood smear. epizootiology and transmission. the rickettsial organisms are transmitted typically to young sheep by biting insects, ticks, contaminated needles or blood-contaminated surgical instruments. necropsyfindings. necropsy findings include splenic enlargement and tissue icterus. has resulted in transient hyperthermia, mild respiratory disease, and mastitis. abortions, stillbirths, and births of weak lambs are also seen. epizootiology and transmission. coxiella burnetii is extremely resistant to environmental changes as well as to disinfectants; persistence in the environment for a year or longer is possible. the organism is associated with either a free-living or an arthropod-borne cycle. coxiella burnetii is found in a variety of tick species, such as ixodid or argasid, where it replicates and is excreted in the feces. once introduced into a mammal, coxiella may be maintained without a tick intermediate. the organism is especially concentrated in placental tissues, replicates in trophoblasts, and will be in reproductive fluids. additionally, the organism is shed in milk, urine, feces, and oronasal secretions. necropsy findings. no specific lesion will be seen in aborted or stillborn fetuses, but necrotizing placentitis will be a finding in cases of abortion. the placenta will contain white chalky plaques and a red-brown exudate. the disease can be diagnosed by identifying the rickettsial organisms in smears of placental secretions. the organism has been found in the placentas of clinically normal animals. the organism stains red with modified ziehl-neelsen and macchiavello stains and purple with giemsa stain. pathogenesis. the organism invades and destroys red blood cells. it is believed that intravascular hemolysis and erythrophagocytosis contribute to the macrocytic anemia. as with other red blood cell parasites, splenectomy aggravates the disease. differential diagnosis. because of the organisms' similarity to chlamydia, confirmation must be made by culture techniques, immunofluorescent procedures, elisa, and complement fixation tests. differential diagnosis. clontridium novyi type d, babesiosis, and leptospirosis are the primary differentials. prevention and control. following strict sanitation practices for surgical procedures and controlling external parasites prevent the disease. treatment. treatment is not usually recommended, but oxytetracycline has been used. sheep will develop immunity if supported nutritionally during the disease. research complications. splenectomized animals are the experimental models used to study these diseases. ii. q fever, or query fever (coxiella burnetii) etiology. coxiella burnetii is a small, gram-negative, obligate intracellular rickettsial organism that causes query fever and is regarded as a major cause of late abortion in sheep. clinical signs. infection of ruminants with c. burnetii is usually asymptomatic. experimental inoculation in other mammals treatment. coxiella can be treated with oxytetracyclines. a vaccine is not commercially available. prevention and control. any aborting animals should be segregated from other animals, and other pregnant animals should be treated prophylactically with tetracycline. serologic screening of ruminant sources should be performed routinely. barrier housing, a review of ventilation exhaust, and defined handling procedures are often required. all placentas and all aborted tissues should be handled and disposed of carefully. q fever has been reported in many mammalian species, including cats. research complications. coxiella burnetii-free animals are particularly important in studies involving fetuses and placentation. because of its zoonotic potential, c. burnetii presents a unique problem in the animal research facility environment. a single organism has been shown to cause disease. some of the greatest concerns are the risk to immunocompromised individuals, pregnant women, and other animals, and the presence of carrier animals or those that may shed the organism in placentas, for example. etiology. the ruminant adenoviruses are dna viruses that cause respiratory and reproductive tract diseases. nine antigenic types of the bovine adenovirus have been identified, with type associated with respiratory disease. two of the ovine and two of the caprine antigenic types have been identified. clinical signs. signs of infection range from subclinical to severe, including pneumonia, enteritis, conjunctivitis, keratoconjunctivitis, weak calf syndrome, and abortion. respiratory tract and intestinal tract diseases may be concurrent. infections caused by this virus are often found associated with other viral and bacterial infections. epizootiology and transmission. the virus is believed to be widespread, but prevalence and characteristics of infection have not been characterized. transmission of adenoviruses in other species (e.g., canine) is by aerosols or fecal-oral routes. necropsy findings. lesions found after experimental infections include atelectasis, edema, and consolidation of the lungs. etiology. the bluetongue virus is an rna virus in the orbivirus genus and reoviridae family. five serotypes ( , , , , and ) have been identified in the united states, where it is seen mostly in western states. bluetongue is an acute arthropodborne viral disease of ruminants, characterized by stomatitis, depression, coronary band lesions, and congenital abnormalities (bulgin, ) . clinical signs and diagnosis. sheep are the most likely to show clinical signs. clinical disease is less common in goats and cattle. early in the infection, animals will spike a fever and will develop hyperemia and congestion of tissues of the mouth, lips, and ears. the virus name, bluetongue, is associated with the typical cyanotic membranes. the fever may subside, but tissue lesions erode, causing ulcers. increased salivary discharges and anorexia are often related to ulcers of the dental pad, lips, gums, and tongue, although salivation and lacrimation may precede apparent ulceration. chorioretinitis and conjunctivitis are also common signs in cattle and sheep. lameness may be observed associated with coronitis and is evident in the rear legs. skin lesions such as drying and cracking of the nose, alopecia, and mammary glands are also observed. secondary bacterial pneumonia may also occur. animals may also develop severe diarrhea and become recumbent. sudden deaths due to cardiomyopathy may occur at any time during the disease. hematologically, animals will be leukopenic. the course of the disease is about weeks, and mortality may reach %. if animals are pregnant, the virus crosses the placenta and causes central nervous system lesions. abortions may occur at any stage of gestation in cattle. prolonged gestation may result from cerebellar hypoplasia and lack of normal sequence to induce parturition. cerebellar hypoplasia will also be present in young born of the infected dams, as well as hydrocephalus, cataracts, gingival hyperplasia, or arthrogryposis. diagnosis is suspected with the characteristic clinical signs and exposure to viral vectors. virus isolation is the best diagnostic approach if blood is collected during the febrile stage of the disease or brains from aborted fetuses. fluorescent antibody tests, elisa, virus neutralization tests, pcr, and agar gel immunodiffusion (agid) tests are also used to confirm the diagnosis. necropsy findings. at necropsy, erosive lesions may be observed around the mouth, tongue, palate, esophagus, and pillars of the rumen. ulceration or hyperemia of the coronary bands may also be seen. many of the internal organs will contain petechial and ecchymotic hemorrhages of the surfaces, and hemorrhage may be seen at the base of the pulmonary artery. pathogenesis. the virus multiplies in the hemocoel and salivary glands of the fly and is excreted in transmissible form in the insect's saliva. after entering the host, the virus causes prolonged viremia. the incubation period is - days. the virus migrates to and attacks the vascular endothelium. the resulting vasculitis accounts for the lesions of the skin, mouth, tongue, esophagus, and rumen and the edema often found in many tissues. ballooning degeneration of affected tissues, followed by necrosis and ulceration, occurs. the effects on fetuses appear to be due to generalized infections of developing organs. differential diagnosis. differentials include other infectious vesicular diseases such as foot-and-mouth disease, contagious ecthyma, bovine viral diarrhea virus-mucosal disease, infectious bovine rhinotracheitis, bovine papular stomatitis, and malignant catarrhal fever. rinderpest is a differential in countries where it is endemic. photosensitization should be considered. foot rot is a differential for the lameness and coronitis. differentials for the manifestations such as arthrogryposis include border disease virus and genetic predispositions of some breeds such as charolais cattle and merino sheep. prevention and control. cellular and humoral immunity are necessary for protection from infection. the bluetongue virus is insidious because the genome is capable of reassortment, and some vaccines will not have the antigenic components represented in the local infection. in addition, there is little to no cross protection between strains. modified live vaccines are available in some parts of the united states but should not be used in pregnant animals. vaccinating lambs and rams in an outbreak is worthwhile, for example, but vaccinating lategestation ewes may cause birth defects or abortions. congenital defects are more common from vaccine use than from naturally occurring infection. minimizing exposure to the vector in endemic areas will decrease the incidence of the disease. treatment. supportive care and nursing care are helpful, including gruels or softer feeds, easily accessed water, and shaded resting places. nonsteroidal anti-inflammatory drugs are often administered. for the cases of secondary bacterial pneumonia and some cases of bluetongue conjunctivitis, antibiotics may be administered. research complications. this is a reportable disease because clinical signs resemble foot-and-mouth disease and other exotic vesicular diseases. etiology. bovine lymphosarcoma refers to lymphoproliferative diseases in young cattle that are not associated with bovine leukemia virus (blv) infection, and those in older cattle that are associated with b lv. b lv is a b lymphocyte-associated retrovirus (johnson and kaneene, a,b,c) . clinical signs. forms of bovine lymphosarcoma that are not associated with blv infection are calf, or juvenile; thymic, or adolescent (animals months to years old); and cutaneous (any age). the calf form is rare and characterized by generalized lymphadenopathy. onset may be sudden, and the disease is usually fatal within a few weeks. signs include lymphadenopathy, anemia, weight loss, and weakness. some animals may be paralyzed because of spinal cord compression from subperiosteal infiltration of neoplastic cells. the adolescent form is also rare, the course rapid, and the prognosis poor. the disease is seen most often in beef breeds such as hereford cattle and is characterized by space-occupying masses in the neck or thorax. these masses are also often present in the brisket. secondary effects of the masses are loss of condition, dysphagia, rumen tympany, and fatal bloat. the cutaneous presentation has a longer course and may wax and wane. the masses are found at the anus, vulva, escutcheon, shoulder, and flank; they are painful when palpated, raised, and often ulcerated. the animals are anemic, and neoplastic involvement may affect cardiac function. generalized or limited lymphadenopathy may be apparent. only the adult, or enzootic, form of bovine lymphosarcoma is associated with blv infection. many animals do not develop any malignancies or clinical signs of infection and simply remain permanently infected. some cows manifest disease only during the periparturient period. malignant lymphoma is the more common, whereas leukosis, due to b-lymphocyte proliferation, is rare. clinical signs are loss of condition and a drop in production of dairy cattle, anorexia, diarrhea, ataxia, paresis, and other signs dependent on the location of the neoplastic tissue. tumors are associated with lymphoid tissues. common sites also include the abomasum, spinal canal, and uterus. cardiac tumors develop at the right atrial or left ventricular myocardium, and associated beat and rate abnormalities may be auscultated. the common ocular manifestation of the disease is exophthalmos due to retrobulbar masses. many internal organs may be involved, and tumors may be palpable per rectum. secondary infections will be due to immunosuppression and the weakened state of the animal. sheep have acquired blv infection naturally and have been used as experimental models; in both situations, this species is susceptible to tumor and leukemia development. goats seroconvert but do not develop the clinical syndromes. diagnosis is based on the animal's age, clinical signs, serology, hematology findings according to the form, aspirates or biopsies of masses, and necropsy findings. kits are available for running agid, for which the blv antigens gp- and gp- are used; antibodies may be detected within weeks after exposure and may also help in predicting disease in clinically normal cattle. elisa and pcr diagnostic aids will also be helpful. worldwide. it is estimated that at least % of the cattle in the united states are infected with blv. as few as % of these animals develop lymphosarcoma, but the adult form of the disease described here is the most common bovine neoplastic disease in the united states. larger herds tend to have higher rates. genetic predisposition may be involved; in addition to the presence of blv, the type of bovine lymphocyte antigen (bola) may be correlated to resistance or susceptibility and to the course of the disease. transmission is believed to be by inhalation of blv in secretions; in colostrum; horizontally by contaminated equipment not sanitized between cattle; and by rectum (e.g., mucosal irritation during per-rectum exams or procedures). natural-service bulls may transmit the infection to cows. cows infected with blv may transmit the infection to their calves in utero. tabanid and other flies also serve as vectors, but these represent a minor means of transmission. necropsy findings. neoplastic infiltration of many organs and tissues are found in the calf form and the cutaneous forms. tumors may be local or widely distributed in the enzootic form. definitive diagnosis of neoplastic tissue specimens is by histology. pathogenesis. as with other retroviruses, the blv integrates viral dna into host target cell dna by means of the reverse transcriptase enzyme, creating a provirus. epizootiology and transmission. the virus is reported to be widespread. occurrence is often seasonal, and biting insects may be vectors. transmission with successful infection requires deep penetration of the skin. transmission may be by contaminated milkers' hands, contaminated equipment, and other fomites. differential diagnosis. differential diagnoses include other diseases that cause lesions on teats such as pseudocowpox, papillomatosis, and vesicular stomatitis. other vesicular diseases may be considered, but other more severe clinical signs might be associated with those. there is no vaccine for this disease. development and maintenance of a blv-free herd, or controlling infection within a herd, requires financial and programmatic commitments: blv-positive and blv-negative animals maintained separately; serologic testing (such as at least every months) and separating positive animals; and washing and then disinfecting instruments, needles (or using sterile singleuse products), and equipment for ear tagging and dehorning and other such equipment between animals. a fresh rectal exam sleeve and lubricant should be used for each animal examined. otherwise serologically positive cows may have undetectable antibodies during the periparturient period. embryo transfer recipients should be negative, and the virus will not be transferred by the embryonic stage. calves should be fed colostrum from serologically negative cows. treatment. treatment regimens of corticosteroids and cancer chemotherapeutic agents provide only short-term improvement. in cases where ova, embryos, or semen need to be collected, supportive care for the affected animals is essential. research complications. the united states and several countries, some in europe, have official programs for eradication of enzootic bovine leukosis. prevention and control. established milking hygiene practices are important control measures: having milkers wash their hands with germicidal solutions or wear gloves, cleaning equipment between animals, and separating affected animals. treatment. there is no treatment, and affected animals should be separated from the herd and milked last. lesions can be cleaned and treated with topical antibacterials. etiology. the bovine viral diarrhea virus (bvdv) is a pestivirus of the flaviviridae family. the flaviviridae include hog cholera virus and border disease virus of sheep. the virus contains a single strand of positive-sense rna. a broad range of disease and immune effects is produced by b vdv only in cattle. in addition, this virus is important in the etiology of bovine pneumonias. bovine viral diarrhea/mucosal disease (bvd/md) is one of the most important viral diseases and one of the most complex diseases of cattle. strains of bvdv are characterized as cytopathic (cp) and noncytopathic (ncp), based on cell-culture growth characteristics. the virus has also been categorized as type and type isolates. heterologous strains exist that may confound even sound vaccination programs. etiology. bovine herpesvirus causes bovine herpes mammillitis, a widespread disease characterized by teat and udder lesions, as well as oral and skin lesions. clinical signs and diagnosis. lesions begin suddenly with teat swelling; the tissue will be edematous and tender when touched. the udder lesions may extend to the perineum. the lesions progress to vesicles, then to ulcers; these may take weeks to heal. lesions rarely may also develop focally around the mouth and generally on the skin of the udder. secondary mastitis may occur, because of bacteria associated with the scabs. diagnosis is by clinical signs and serologically. clinical signs and diagnosis. signs of bvdv infections may be subclinical but also include abortions, congenital abnormalities, reduced fertility, persistent infection (pi) with gradual debilitation, and acute and fatal disease. the presence of antibodies, whether from passive transfer or immunizations, does not necessarily guarantee protection from the various forms of the disease. an acute form of the disease, caused by type bvdv, occurs in cattle without sufficient immunity. after an incubation period of - days, clinical signs include fever, anorexia, oculonasal discharge, oral erosions (including on the hard palate), diarrhea, and decreased milk production. the disease course may be shorter with hemorrhagic syndrome and death within days. clinical signs of b vdv in calves also include severe enteritis and pneumonia. when susceptible cows are infected in utero from gestational be found extending throughout the gastrointestinal tract to the days - , or gestational cows are vaccinated with a modi-cecum. the respiratory tract lesions will often be complicated fled live vaccine, abortion or stillbirth result. congenital defects caused by bvdv during gestational days - include impaired immunity (thymic atrophy), cerebellar hypoplasia, ocular defects, alopecia or hypotrichosis, dysmyelinogenesis, hydranencephaly, hydrocephalus, and intrauterine growth retardation. typical signs of cerebellar dysfunction will be evident in calves, such as wide-based stance, weakness, opisthotonus, hyperflexion, hypermetria, nystagmus, or strabismus. some severely affected calves will not be able to stand. ophthalmic effects include retinal degeneration and microphthalmia. fetuses can also be infected in utero, normal at birth, immunotolerant to the virus, and persistently infected (pi). the term mucosal disease is commonly associated with this form of the infection. many pi animals do not survive to maturity, however, and many have weakened immune systems. the pi animals are important because they shed virus and will probably show the clinical signs of mucosal disease (md) caused by a cp b vdv strain derived from an ncp b vdv strain. these md clinical signs include fever, anorexia, and profuse diarrhea that may include blood and fibrin casts, and oral and pharyngeal erosions, as well as erosion at the interdigital spaces and on the teats and vulva. many other associated clinical signs include anemia, bloat, lameness, or corneal opacities and discharges. secondary effects of hemorrhage and dehydration also contribute to the morbidity and mortality. animals that do not succumb to the disease will be chronically unthrifty, debilitated, and infection-prone. diagnosis in affected calves is based on herd health history, clinical signs, and antibodies to b vdv in precolostral serum. viral culturing from blood may be useful. in older animals, oral lesions, serology, detection of viral antigen, and virus isolation contribute to the diagnosis. leukopenia, and especially lymphopenia, are seen. serology must be interpreted with the awareness of the possibility of pi immunotolerant animals. vaccination against the disease carries its own set of side effects and potential problems, especially when using modified live vaccines, whether against cp or ncp strains. the condition of the animals is also a variable. epizootiology and transmission. bvdv is present throughout the world. transmission occurs easily by direct contact between cattle, from feed contaminated with secretions or feces, and by aborted fetuses and placentas. pi females transmit the virus to their fetuses. semen also is a source of virus. necropsy findings. in affected calves, histopathologic findings include necrosis of external germinal cells, focal hemorrhages, and folial edema. later in the disease, large cavities develop in the cerebellum, and atrophy of the cerebellar folia and thin neuropil are evident. older calves may have areas of intestinal necrosis. in cases where oral erosions occur, erosions will by secondary bacterial pneumonia. when the hemorrhagic syndrome develops, petechiation and mucosal bleeding will be present. pathogenesis. the cp and ncp strains are thought to be related mutations of the bvdv; the cp short-lived isolates are believed to arise from the ncp strains. the ncp strains are those present in the pi animals, and the strains are maintained in cattle populations. cp and ncp isolates vary in virulence, and classification of these types is based on viral surface proteins. considerable antigenic variation also exists between strains and types. other viral infections, such as bovine respiratory syncytial virus and infectious bovine rhinotracheitis, may also be present in the same animals. the pathology caused by b vdv is due to its ability to infect epithelial cells and impair the functioning of immune cell populations through out the bovine system. in type bvdv hemorrhagic syndrome, death results from viral-induced thrombocytopenia. in fetuses, the virus infects developing germinal cells of the cerebellum. the purkinje's cells in the granular layer are killed, and necrosis and inflammation follow. the immune effects are the result of the virus's interfering with neutrophil and macrophage functions and of lymphocyte blastogenesis. all of these predispose the affected animals to bacterial infections with pasteurella haemolytica. b vdv damages dividing cells in fetal organ systems, resulting in abortions and congenital effects. differential diagnosis. many differentials must be considered for the clinical manifestations of b vdv infections. differentials for enteritis of calves include viral infections, cryptosporidia, escherichia coli, salmonella, and coccidia. salmonella, winter dysentery, johne's disease, intestinal parasites, malignant catarrhal fever (mcf), and copper deficiency are differentials for the diarrhea seen in the disease in adult animals. respiratory tract pathogens such as bovine respiratory syncytial virus, pasteurella, haemophilus, and mycoplasma must be considered for the respiratory tract manifestations. oral lesions are also produced by mcf, vesicular stomatitis, bluetongue, and papular stomatitis. infectious bovine herpesvirus , leptospirosis, brucellosis, trichomoniasis, and mycosis should be considered in cases of abortion. prevention and control. combined with sound management in a typical cattle herd, vaccination is the best way to prevent b vdv and should be integrated into the herd health program, timed appropriately preceding breeding, gestation, or stressful events. vaccine preparations for b vdv are modified live virus (mlv) or killed virus. each has advantages and disadvantages. the former induces rapid immunity (within week) after a single dose, provides longer duration of immunity against sev-eral strains, and induces serum neutralizing antibodies. mlv vaccines are not recommended for use in pregnant cattle, may induce mucosal disease, and may be immunosuppressive at the time of vaccination. the immunosuppression is detrimental if cattle are concurrently exposed to field-strain virus because it will facilitate infection and possible clinical disease. the mlv strains may cross the placenta, resulting in fetal infections. the killed vaccines are safer in pregnant animals but require booster doses after the initial immunization, may need to be given - times per year, and do not induce cell-mediated immunity. passive immunity may protect most calves for up to - months of age. subsequent vaccination with mlv may provide lifelong immunity, but this is not guaranteed. annual boosters are recommended to protect against vaccine breaks. the virus persists in the environment for weeks and is susceptible to the disfectants chlorhexidine, hypochlorite, iodophors, and aldehydes. maintenance of a closed herd to prevent any possibility of the introduction of the virus is difficult. isolation of new animals, avoidance of the purchase of pregnant cows, scrutiny of records from source farms, use of semen tested bulls, minimization of stress, testing of embryo-recipient cows, and maintainenance of populations of ruminants (smaller or wild species) separately on the premises will minimize viral exposure. other management strategies may require a program for testing and culling pi cattle. this can be expensive but may be a worthwhile investment to remove the virus shedders from a herd. no specific treatment is available. supportive care and treatment with antibiotics to prevent secondary infection are recommended. animals that survive the infection should be evaluated a month after recovery to determine their status as pi or virus-free. etiology. cache valley virus (cvv), of the arbovirus genus of the bunyaviridae family, is a cause of congenital defects in lambs. cvv infection in fetal and newborn lambs include arthrogryposis, microencephaly, hydranencephaly, porencephaly, cerebellar hypoplasia, and micromyelia. stillbirths and mummified fetuses are seen. lambs will be born weak and will act abnormally. diagnosis is by evidence of seroconversion in precolostral blood samples or fetal fluids, as the result of in utero infection. western united states, although it has been isolated in a few midwestern states. although considered a disease of sheep, virus has been isolated from cattle and from wild ruminants and antibodies found in white-tailed deer. transmission is by arthropods during the first trimester of pregnancy. etiology. caprine arthritis encephalitis virus (caev) occurs worldwide, with a high prevalence in the united states. caprine arthritis encephalitis (cae) is considered the most important viral disease of goats. the caev is in the lentivirus genus of the retroviridae family. it causes chronic arthritis in adults and encephalitis in young. caev is in the same viral genus as the ovine progressive pneumonia virus (oppv). clinical signs and diagnosis. the most common presentation in goats is an insidious, progressive arthritis in animals months of age and older. animals become stiff, have difficulty getting up, and may be clinically lame in one or both forelimbs. carpal joints are so swollen and painful that the animal prefers to eat, drink, and walk on its "knees." in dairy goats, milk production decreases, and udders may become firmer. this retrovirus also causes neurological clinical signs in young kids - months old. kids may be bright and alert, afebrile, and able to eat normally even when recumbent. some kids may initially show unilateral weakness in a rear limb, which progresses to hemiplegia or tetraplegia. mild to severe lower motor neuron deficits may be noted, but spinal reflexes are intact. clinical signs may also include head tilt, blindness, ataxia, and facial nerve paralysis. older animals in the group may experience interstitial pneumonia or chronic arthritis. the pneumonia is similar to the pneumonia in sheep caused by oppv; the course is gradual but progressive, and animals will eventually lose weight and have respiratory distress. some animals in a herd may not develop any clinical signs. diagnosis is based on clinical signs, postmortem lesions, and positive serology for viral antibodies to caev. an agar gel immunodiffusion (agid) test identifies antibodies to the virus and is used for diagnosis. kids acquire an anti-caev antibody in colostrum, and this passive immunity may be interpreted as indicative of infection with the virus. the antibody does not prevent viral transmission. ep&ootiology and transmission. the virus is prevalent in most industrialized countries. the common means of transmission, from adults to kids, is in the colostrum and milk in spite of the presence of anti-caev antibody in the colostrum. transmission may occur among adult goats by contact. intrauterine transmission is believed to be rare. transmission to sheep has occurred only experimentally; there is no documented case of natural transmission. necropsy findings. necropsy and histopathology reveal a striking synovial hyperplasia of the joints with infiltrates of lymphocytes, macrophages, and plasma cells. other histologic lesions include demyelination in the brain and spinal cord, with multifocal invasion of lymphocytes, macrophages, and plasma cells. in severe cases of mastitis, the udder may appear to be composed of lymphoid tissue. tem, resulting in the formation of non-neutralizing antibody to viral core proteins and envelope proteins. immune complex formation in synovial, mammary gland, and neurological tissue is thought to result in the clinical changes observed. most commonly, the carpal joint is affected, followed by the stifle, hock, and hip. the infection is lifelong. differential diagnosis. the differential diagnosis for the neurologic form of caev should include copper deficiency, enzootic pneumonia, white muscle disease, listeriosis, and spinal cord disease or injury. the differential diagnosis for caev arthritis should include chlamydia and mycoplasma. prevention and control. herds can be screened for cae by testing serologically, using an agid or an enzyme-linked immunosorbent assay (elisa) test. the elisa is purported to be more sensitive, whereas the agid is more specific. individual animals show great variation in development of antibody. because cae is highly prevalent in the united states, and because seronegative animals can shed organisms in the milk, retesting herds at least annually may be necessary. recently, an immunoprecipitation test for cae has been developed that has high sensitivity and specificity. control measures include management practices such as test and cull, prevention of milk transmission, and isolation of affected animals. parturition must be monitored, and kids must be removed immediately and fed heat-treated colostrum ( ~ for hr). caev-negative goats should be separated from caevpositive goats. treatment. there is no treatment for caev. is also referred to as bovine herpesvirus (bhv- ) and is an alphaherpesvirus. ibrv causes or contributes to several bovine syndromes, including respiratory and reproductive tract diseases. it is one of the primary pathogens in the bovine respiratory disease complex. strains include bhv-i. (associated with respiratory disease), bhv . (associated with respiratory and genital diseases), and bhv . (associated with neurological diseases), which has been reclassified as bovine herpesvirus . clude conjunctivitis, rhinotracheitis, pustular vulvovaginitis, balanoposthitis, abortion, encephalomyelitis, and mastitis. the respiratory form is known as infectious bovine rhinotracheitis, and clinical signs may range from mild to severe, the latter particularly when there are additional respiratory viral infections or secondary bacterial infections. the mortality rate in more mature cattle is low, however, unless there is secondary bacterial pneumonia. fever, anorexia, restlessness, hyperemia of the muzzle, gray pustules on the muzzle (that later form plaques), nasal discharge (that may progress from serous to mucopurulent), hyperpnea, coughing, salivation, conjunctivitis with excessive epiphora, and decreased production in dairy animals are typical signs. open-mouth breathing may be seen if the larynx or nasopharygneal areas are blocked by mucopurulent discharges. neonatal calves may develop respiratory as well as general systemic disease. in these cases, in addition to the symptoms already noted, the soft palate may become necrotic, and gastrointestinal tract ulceration occurs. young calves are most susceptible to the encephalitic form; signs include dull attitude, head pressing, vocalizations, nystagmus, head tilt, blindness, convulsions, and coma, as well as some signs, such as discharges, seen with respiratory tract presentations. this form is usually fatal within days. abortion may occur simultaneously with the conjunctival or respiratory tract diseases, when the respiratory infection appears to be mild, or may be delayed by as much as months after the respiratory tract disease signs. infectious pustular vulvovaginitis is most commonly seen in dairy cows, and clinical signs may be mild and not noticed. otherwise, signs are fever, depression, anorexia, swelling of the vulvar labia, vulvar discharge, and vestibular mucosa reddened by pustules. the cow will often carry her tail elevated away from these lesions. these soon coalesce, and a fibrous membrane covers the ulcerated area. if uncomplicated, the infection lasts about - days, and lesions heal in weeks. younger infected bulls may develop balanoposthitis with edema, swelling, and pain such that the animals will not service cows. epizootiology and transmission. ibrv is widely distributed throughout the world, and adult animals are the reservoirs of infection. the disease is more common in intensive calf-rearing situations and in grouped or stressed cattle. transmission is primarily by secretions, such as nasal, during and after clinical signs of disease. modified live vaccines are capable of causing latent infections. necropsy findings. fibrinonecrotic rhinotracheitis is considered pathognomic for ibrv respiratory tract infections. there will be adherent necrotic lesions in the respiratory, ocular, and reproductive mucosa. when there are secondary bacterial infections, such as pasteurella bronchopneumonia, findings will include congested tracheal mucosa and petechial and ecchymotic hemorrhages in that tissue. lesions from the encephalitic form include lymphocytic meningoencephalitis and will be found throughout the gray matter (neuronal degeneration, perivascular cuffing) and white matter (myelitis, demyelination). intranuclear inclusion bodies are not a common finding with this herpesvirus. pathogenesis. in the encephalitic form, the virus first grows in nasal mucosa and produces plaques. these resolve within days, and the encephalitis develops after the virus spreads centripetally to the brain stem by the trigeminal nerve dendrites. latent infections are also established in neural tissue. differential diagnosis. the severe oral erosions seen with bvdv infections are rare with infectious bovine rhinotracheitis-infectious pustular vulvovaginitis (ibr-ipv). the conjunctivitis of ibr may initially be mistaken for that of a moraxella bovis (pinkeye) infection; the ibr will be peripheral, and there will not be corneal ulceration. bovine viral diarrhea virus and ibrv are the most common viral causes of bovine abortion. differentials for balanoposthitis include trauma from service. vated, attenuated, modified live, and genetically altered preparations. some are in combination with parainfluenza (pi- ) virus. the mlv preparations are administered intranasally; these are advantageous in calves for inducing mucosal immunity even when serologic passive immunity is already present and adequate. some newer vaccines, with gene deletion, allow for serologic differentiation between antibody responses from infection or immunization. bulls with the venereal form of the infection will transmit the virus in semen; intranasal vaccine may be used to provide some immunity. treatment. uncomplicated mild infections will resolve over a few weeks; palliative treatments, such as cleaning ocular discharges and supplying softened food, are helpful in recovery. antibiotics are usually administered because of the high likelihood of secondary bacterial pneumonia. the encephalitic animals may need to be treated with anticonvulsants. etiology. parainfluenza , an rna virus of the family paramyxoviridae, causes mild respiratory disease of ruminants when it is the sole pathogen. the viral infection often predisposes the respiratory system to severe disease associated with concurrent viral or bacterial pathogens. viral strains are reported to vary in virulence. serotypes seen in the smaller ruminants are distinct from those isolated from cattle. clinical signs and diagnosis. infections ranging from asymptomatic to mild signs of upper respiratory tract disease are associated with this virus by itself; infections are almost never fatal. clinical signs include ocular and nasal discharges, cough, fever, and increased respiratory rate and breath sounds. in pregnant animals, exposure to pi- can result in abortions. clinical signs become apparent or more severe when additional viral pathogens are present, such as bovine viral diarrhea virus, or a secondary bacterial infection, such as pasteurella haemolytica infection, is involved. greater morbidity and mortality will be sequelae of the bacterial infections. viral isolation or direct immunofluorescence antibody (ifa) from nasal swabs can be used for definitive diagnosis. presently it is assumed that the virus is widespread in goats, but firm evidence is lacking. for an infection of pi- only, findings will be negligible. some congestion of respiratory mucosa, swelling of respiratory tract-associated lymph nodes, and mild pneumonitis may be noted grossly and histologically. intranuclear and intracytoplasmic inclusion bodies may be present in the mucosal epithelial cells. findings will be similar but not as severe as those caused by bovine respiratory syncytial virus. immunohistochemistry may also be used. pathogenesis. pi- infects the epithelial mucosa of the respiratory tract; however, the disease is often asymptomatic when uncomplicated. differential diagnosis. differentials, particularly in cattle, include infections with other respiratory tract viruses of ruminants: ibrv, bvdv, bovine respiratory syncytial virus, and type bovine adenovirus. prevention and control. immunization, management, and nutrition are important for this respiratory pathogen, as for others. in cattle, modified live vaccines for intramuscular (im), subcutaneous (sc), or intranasal (in) administration are available. the im and sc routes provide immune protection within week after administration but will not provide protection in the presence of passively acquired antibodies. it is contraindicated for pregnant animals because it will cause abortion. the in route immunizes in the presence of passively acquired antibodies, provides immunity within days of administration, and stimulates the production of interferon. other vaccine formulations, about which less information is reported, include inactivated or chemically altered live-virus preparations; both are administered im, and followup immunizations are needed within weeks. booster vaccinations are recommended for all preparations within - months after the initial immunization. all presently marketed vaccine products come in combination with other bovine respiratory viruses as multivaccine products. the humoral immunity protects against pi- abortions. there is no approved pi- vaccine for sheep and goats. the use of the cattle formulation in these smaller ruminants is not recommended. sound management of housing, sanitation, nutrition, and preventive medicine programs are all equally important components in prevention and control. treatment. uncomplicated disease is not treated. etiology. the respiratory syncytial viruses are pneumoviruses of the paramyxoviridae family and are common causes of severe disease in ruminants, especially calves and yearling cattle. two serotypes of the bovine respiratory syncytial virus (brsv) have been described for cattle; these may be similar or identical to the virus seen in sheep and goats. clinicalfindings and diagnosis. infections may be subclinical or develop into severe illness. clinical signs include fever, hyperpnea, spontaneous or easily induced cough, nasal discharge, and conjunctivitis. interstitial pneumonia usually develops, and harsh respiratory sounds are evident on auscultation. development of emphysema indicates a poor prognosis, and death may occur in the severe cases of the viral infection. secondary bacterial pneumonia, especially with pasteurella haemolytica, with morbidity and mortality, is also a common sequela. abortions have been assciated with brsv outbreaks. diagnosis is based on virus isolation and serology (acute and convalescent). nasal swabs for virus isolation should be taken when animals have fever and before onset of respiratory disease. prevention and control. vaccination should be part of the standard health program, and all animals should be vaccinated regularly. vaccinations should be administered within - months of stressful events, such as weaning, shipping, and introduction to new surroundings. currently available vaccines include an inactivated preparation and a modified live virus preparation administered intramuscularly or subcutaneously; immunity develops well in yearling animals, and colostral antibodies develop when cows are vaccinated during late gestation. passive immunity from colostrum provides at least partial protection to calves in herds where disease is prevalent. but this immunity suppresses the mucosal iga response and serum antibody responses. the basis for successful immune protection is the mucosal memory iga, but this is difficult to achieve with present vaccine formulations. the virus is easily inactivated in the environment. preventive measures in preweaning animals should include preconditioning to minimize weaning stress. treatment. recovery can be spontaneous; however, antibiotics and supportive therapy are useful to prevent or control secondary bacterial pneumonia. in severe cases, antihistamines and corticosteriods may also be necessary. use of vaccine during natural infection is not productive and may result in severe disease. etiology. ulcerative dermatosis is a contagious disease of sheep only. it is caused by a poxvirus similar to but distinct from the causative agent of contagious ecthyma ("current veterinary therapy," ). epizootiology and transmission. these viruses are considered ubiquitous in domestic cattle and are transmitted by aerosols. teroventral lung lobes. edema and emphysema are present. as the name indicates, syncytia, which may have inclusions, form in areas of the lungs infected with the virus. necrotizing bronchiolitis, bronchiolitis obliterans, and hyaline membrane formation will be evident microscopically. crusts associated with the skin and mucous membranes of the genitalia, face, and feet (bulgin, ) . genital lesions are much more common than the facial or coronal lesions. discomfort may be associated with the lesions. paraphimosis occasionally occurs. these lesions are painful; during breeding season, animals will avoid coitus. morbidity is low to moderate, and mortality negligible if the flock is otherwise healthy. diagnosis is based on clinical signs. pathogenesis. the severe form of the disease, which often follows a mild preliminary infection, is thought to be caused by immune-mediated factors during the process of infection in the lung. virulence may vary greatly among viral strains. united states, ulcerative dermatosis is transmitted through direct contact with abraded skin of the prepuce, vulva, face, and feet. necropsy findings. necropsy would rarely be necessary to diagnose an outbreak in a healthy flock. findings will be similar to those described for contagious ecthyma. when no contact with cattle has occurred. persistently infected animals, such as lambs, are shedding reservoirs of the virus in urine, feces, and saliva throughout their lives. pathogenesis. following an incubation period of - days, the virus replicates in the epidermal cells and leads to necrosis and pustule formation. pustules rapidly break, forming weeping ulcers. the ulcers scab over and eventually form a fibrotic scar. the disease usually resolves in - weeks. rarely, the disease will persist for many months to more than a year. differential diagnosis. the main differential is contagious ecthyma, which is grossly and histopathologically associated with epithelial hyperplasia. this is also a feature of ulcerative dermatosis. imals, especially males, should not be used for breeding. treatment. affected animals should be separated from the rest of the flock. treatment is supportive, including antiseptic ointments and astringents. research complications. breeding and maintenance of the flocks' condition, because of the pain associated with eating, will be compromised during an outbreak. etiology. border disease, also known as hairy shaker disease (or "fuzzies" in the southwestern united states), is a disease of sheep caused by a virus closely related to the bovine viral diarrhea virus (bvdv), a pestivirus of the togaviridae family. goats are also affected. the virus causes few pathogenic effects in cattle. clinical signs and diagnosis. border disease in ewes causes early embryonic death, abortion of macerated or mummified fetuses, or birth of lambs with developmental abnormalities. lambs infected in utero that survive until parturition may be born weak and often exhibit a number of congenital defects such as tremor, hirsutism (sometimes darkly pigmented over the shoulders and head), hypothyroidism, central nervous system defects, and joint abnormalities, including arthrogryposis. later, survivors may be more susceptible to diseases and may develop persistent, sometimes fatal, diarrhea. the virus infection produces similar clinical manifestations in goats, except that the hair changes are not seen. diagnosis includes the typical signs described above, as well as serological evidence of viral infection. virus isolation confirms the diagnosis. wide, and reports of disease are sporadic. disease has occurred necropsy findings. lesions include placentitis, and characteristic joint and hair-coat changes in the fetus. histologically, axonal swelling, neuronal vacuolation, dysmyelination, and focal microgliosis are observed in central nervous system structures. pathogenesis. the virus entering the ewe via the gastrointestinal or respiratory tracts penetrates the mucous membranes and causes maternal and fetal viremia. infection during the first days of gestation causes embryonic death. in lambs infected between and days, the virus activates follicular development, diminishes the myelination of neurons, and causes dysfunction of the thyroid gland. infection after days of gestation results in lambs that are born persistently infected. infected lambs have high perinatal mortality; survivors have diminished signs over time but, as noted, continue to shed the virus. prevention and control. border disease can be prevented by vaccinating breeding ewes with killed-bvdv vaccine. congenitally affected lambs should be maintained separately and disposed of as soon as humanely possible. new animals to the flock should be screened serologically. if cattle are housed nearby, vaccination programs for bvdv should be maintained. treatment. there is no treatment other than supportive care for affected animals. etiology. contagious ecthyma, also known as contagious pustular dermatitis, sore mouth, or off, is an acute dermatitis of sheep and goats caused by a parapoxvirus. this disease occurs worldwide and is zoonotic. naturally occurring disease has also been reported in other species such as musk ox and reindeer. other parapoxviruses infect the mucous membranes and skin of cattle, causing the diseases bovine pustular dermatitis and pseudocowpox. clinical signs and diagnosis. the disease is characterized by the presence of papules, vesicles, or pustules and subsequently scabs of the skin of the face, genitals of both sexes, and coronary bands of the feet. lesions develop most frequently at mucocutaneous junctions and are found most commonly at the commissures of the mouth. off is usually found in young animals less than year of age. younger lambs and kids will have difficulty nursing and become weak. lesions may also develop on udders of nursing dams, which may resist suckling by offspring to nurse, leading to secondary mastitis. the scabs may appear nodular and raised above the surface of the surrounding skin. morbidity in a susceptible group of animals may exceed %. mortality is low, but the course of the disease may last up to weeks. diagnosis is based on characteristic lesions. biopsies may reveal eosinophilic cytoplasmic inclusions and proliferative lesions under the skin. electron microscopy will reveal the virus itself. disease is confirmed by virus isolation. epizootiology and transmission. all ages of sheep and goats are susceptible. seasonal occurrences immediately after lambing and after entry into a feedlot are common; stress likely plays a role in susceptibility to this viral disease. older animals develop immunity that usually prevents reinfection for at least or more years. resistant animals may be present in some flocks or herds. the virus is very resistant to environmental conditions and may contaminate small-ruminant facilities, pens, feedlots, and the like for many years as the result of scabs that have been shed from infected animals. transmission occurs through superficial lesions such as punctures from grass awns, scrapes, shearing, and other common injuries. necropsy findings. necropsy findings include ballooning degeneration of epidermal and dermal layers, edema, granulomatous inflammation, vesiculation, and cellular hyperplasia. secondary bacterial infection may also be evident. pathogenesis. the virus is typical of the poxviridae, resembling sheep poxvirus (not found in the united states) and vaccinia virus and replicating in the cytoplasm of epithelial cells. following an incubation period of - days, papules and vesicles develop around the margins of the lips, nostrils, eyelids, gums, tongue, or teats; skin of the genitalia; or coronary band of the feet. the vesicles form pustules that rupture and finally scab over. virus should be considered in both sheep and goats. an important differential in goats is staphylococcal dermatitis. prevention and control. individuals handling infected animals should be advised of precautions beforehand, should wear gloves, and should separate work clothing and other personal protective equipment. clippers, ear tagging devices, and other similar equipment should always be cleaned and disinfected after each use. colostral antibodies may not be protective. vaccinating lambs and kids with commercial vaccine best prevents the disease. dried scabs from previous outbreaks may also be used by rubbing the material into scarified skin on the inner thigh or axilla. animals newly introduced to infected premises should be vaccinated upon arrival. precautions must be taken when vaccinating animals, because the vaccine may induce orf in the animal handlers; it is not recommended to vaccinate animals in flocks already free of the disease. affected dairy goats should be milked last, using disposable towels for cleaning teat ends. treatment. affected animals should be isolated and provided supportive care, especially tube feeding for young animals whose mouths are too sore to nurse. treatment should also address secondary bacterial infections of the orf lesions, including systemic antibiotics for more severe infections. treatment for myiasis may also be necessary. the viral infection is self-limiting, with recovery in about weeks. research complications. carrier animals may be a factor in flock or herd outbreaks. contagious ecthyma is a zoonotic disease, and human-to-human transmission can also occur. the virus typically enters through abrasions on the hands and results in a large (several centimeters) nodule that is described as being extremely painful and lasting for as many as weeks. lesions heal without scarring. etiology. foot-and-mouth disease (fmd) is caused by the foot-and-mouth disease virus, a picornavirus in the aphthovirus genus. the disease is also referred to as aftosa or aphthous fever. seven immunologically distinct types of the virus have been identified, with subtypes within those . epidemics of the disease have occurred worldwide. north and central america have been free of the virus since the mid- s. this is a reportable disease in the united states; clinical signs are very similar to other vesicular diseases. cattle (and swine) are primarily affected, but disease can occur in sheep and is usually subclinical in goats. clinical signs and diagnosis. in addition to vesicle formation around and in the mouth, hooves, and teats, fever, anorexia, weakness, and salivation occur. vesicles may be as large as cm, rupture after days, and subsequently erode. secondary bacterial infections often occur at the erosions. anorexia is likely due to the pain associated with the oral lesions. high morbidity and low mortality, except for the high mortality in young cattle, are typical. diagnosis must be based on elisa, virus neutralization, fluorescent antibody tests, and complement fixation. epizootiology and transmission. domestic and wild ruminants and several other species, such as swine, rats, bears, and llamas are hosts. asymptomatic goats can serve as virus reservoirs for more susceptible cohoused species such as cattle. greater mortality occurs in younger animals. the united states, great britain, canada, japan, new zealand, and australia are fmd-free, whereas the disease is endemic in most of south america, parts of europe, and throughout asia and africa. the virus is very contagious and is spread primarily by the inhalation of aerosols, which can be carried over long distances. transmission may also occur by fomites, such as shoes, clothing, and equipment. human hands, soiled bedding, and animal products such as frozen or partially cooked meat and meat products, hides, semen, and pasteurized milk also serve as sources of virus. necropsy findings. vesicles, erosions, and ulcers are present in the oral cavity as well as on the rumen pillars and mammary alveolar epithelium. myocardial and skeletal muscle degeneration (zenker's) is most common (and accounts for the greater mortality) in younger animals. histological findings include lack of inclusion bodies. vesicular lesions include intracellular and extracellular edema, cellular degeneration, and separation of the basal epithelium. replicates in the pharynx and digestive tract in the cells of the stratum spinosum, and viremia and spread of virus to many tissues occur before clinical signs develop. virus shedding begins about hr before clinical signs are apparent. vesicles result from the separation of the superficial epithelium from the basal epithelium. fluid fills the basal epithelium, and erosions develop when the epithelium sloughs. persistent infection also occurs, and virus can be found for months or years in the pharnyx; the mechanisms for the persistence are not known. differential diagnosis. vesicular stomatitis is the principal differential. other differentials include contagious ecthyma (orf), rinderpest, bluetongue, malignant catarrhal fever, bovine papular stomatitis, bovine herpes mammillitis, and infectious bovine rhinotracheitis virus infection. products from endemic areas is regulated. quarantine and slaughter are practiced in outbreaks in endemic areas. quarantine and vaccination are also used in endemic areas, but vaccines must be type-specific and repeated or times per year to be effective and will provide only partial protection. autogenous vaccines are best in an outbreak. passive immunity protects calves for up to months after birth. the virus is inactivated by extremes of ph, sunlight, high temperatures, sodium hydroxide, sodium carbonate, and acetic acid. treatment. nursing care and antibiotic therapy to minimize secondary reactions help with recovery. humoral immunity is considered the more important immune mechanism, with cellmediated immunity of less importance. research complications. rare cases in humans have been reported. importation into the united states of animal products from endemic areas is prohibited. etiology. malignant catarrhal fever (mcf) is a severe disease primarily of cattle. the agents of mcf are viruses of the gammaherpesvirinae subfamily. alcelaphine herpesvirus and and ovine herpesvirus are known strains. the alcelaphine strains are seen in africa. the ovine strain is seen in north america. the alcelaphine and ovine strains differ in incubation times and duration of illness. disease may occur sporadically or as outbreaks. clinical signs and diagnosis. signs range from subclinical to recrudescing latent infections to the lethal disease seen in susceptible species, such as cattle. sudden death may also occur in cattle. presentations of the disease may be categorized as alimentary, encephalitis, or skin forms; all three may occur in an animal. corneal edema starting at the limbus and progressing centripetally is a nearly pathognomonic sign; photophobia, severe keratoconjunctivitis, and ocular involvement may follow. other signs include prolonged fever, oral mucosal erosions, salivation, lacrimation, purulent nasal discharge, encephalitis, and pronounced lymphadenopathy. as the disease progresses, cattle may shed horns and hooves. in north america, cattle will also have severe diarrhea. the course of the disease may extend to week. recovery is usually prolonged, and some permanent debilitation may occur. the disease is fatal in severely affected individuals. history of exposure, as well as the clinical signs and lesions, contributes to the diagnosis. serology, pcr-based assays, viral isolation, and cell-culture assays, such as cytopathic effects on thyroid cell cultures, are also used. because of the susceptibility of rabbits, inoculation of this species may be used. in less severe outbreaks or individual animal disease, definitive diagnosis may never be made. necropsy. gross findings at necropsy include necrotic and ulcerated nasal and oral mucosa; thickened, edematous, ulcerated, and hemorrhagic areas of the intestinal tract; swollen, friable, and hemorrhagic lymph nodes and other lymphatic tissues; and erosion of affected mucosal surfaces. lymph nodes should be submitted for histological examination. histological findings include nonsuppurative vasculitis and encephalitis; large numbers of lymphocytes and lymphoblasts will be present without evidence of virus. pathogenesis. the incubation period may be up to months. vascular endothelium and all epithelial surfaces will be affected. the virus is believed to cause proliferation of cytotoxic t lymphocytes with natural killer cell activities, and the resulting lesions are due to an autoimmune type of phenomenon. differential diagnoses. the differentials for this disease are bovine viral diarrhea/mucosal disease, bovine respiratory disease complex, infectious bovine rhinotracheitis, bluetongue, vesicular stomatitis, and foot-and-mouth disease. causes of encephalitis, such as bovine spongiform encephalopathy and rabies, should be considered. in africa, rinderpest is also a differential. other differentials are arsenic toxicity and chlorinated naphthalene toxicity. in north america, sheep, as well as cattle that have been either exposed or that have survived the disease, are reservoirs for outbreaks in other cattle. if there is concern regarding presence of the virus, animals should be screened serologically; once an animal has been infected, it remains infected indefinitely. lambs can be free of the infection if removed from the flock at weaning. the virus is very fragile outside of host's cells and will not survive in the environment for more than a few hours. lobes; and hematological findings indicate anemia and leukocytosis. the rare neurological signs include flexion of fetlock and pastern joints, tremors of facial muscles, progressive paresis and paralysis, depression, and prostration. death occurs in weeks to months. the disease can be serologically diagnosed with agar gel immunodiffusion (agid) tests, virus isolation, serum neutralization, complement fixation, and enzyme-linked immunosorbent assay (elisa) tests. sixty-eight percent of sheep in some states have been infected with the virus (radostits et al., ) . it is transmitted horizontally via inhalation of aerosolized virus particles and vertically between the infected dam and fetus. in addition, transmission through the milk or colostrum is considered common (knowles, ) . necropsy findings. lesions are observed in lungs, mammary glands, joints, and the brain. pulmonary adhesions, ventral lung lobe consolidation, bronchial lymph node enlargement, mastitis, and degenerative arthritis are visualized grossly. meningeal edema, thickening of the choroid plexus, and foci of leukoencephalomalacia are seen in the central nervous system (cns). histologically, interalveolar septal thickening, lymphoid hyperplasia, histiocyte and fibrocyte proliferation, and squamous epithelial changes are seen in the lungs. meningitis, lymphoid hyperplasia, demyelination, and glial fibrosis are seen in the cns. affected and any exposed animals should be isolated from healthy animals. there is no specific treatment for mcf; supportive treatment may improve recovery rates. corticosteroids may be useful. etiology. an rna virus in the lentivirus group of the retroviridae family causes ovine progressive pneumonia (opp), or maedi/visna. maedi refers to the progressive pneumonia presentation of the disease; visna refers to the central nervous system disease, which is reported predominantly in iceland. visna has been reported in goats but may have been due to caprine arthritis encephalitis infection. clinical signs and diagnosis. opp is a viral disease of adult sheep characterized by weakness, unthriftiness, weight loss, and pneumonia (pepin et al., ; de la concha bermejillo, ) . clinically, animals exhibit signs of progressive pulmonary disease after an extremely long incubation period of up to years. respiratory rate and dyspnea gradually increase as the disease progresses. the animal continues to eat throughout the disease; however, animals progressively lose weight and become weak. additionally, mastitis is a common clinical feature. thoracic auscultation reveals consolidation of ventral lung pathogenesis. the virus has a predilection for the lungs, mediastinal lymph nodes, udder, spleen, joints, and rarely the brain. after initial infection, the virus integrates into the dna of mature monocytes and persists as a provirus. later in the animal's life, infected monocytes mature as lung (and other tissue) macrophages and establish active infection. the virus induces lymphoproliferative disease, histiocyte and fibrocyte proliferation in the alveolar septa, and squamous metaplasia. pulmonary alveolar and vascular changes impinge on oxygen and carbon dioxide exchange and lead to serious hypoxia and pulmonary hypertension. secondary bacterial pneumonia may contribute to the animal's death. pulmonary adenomatosis is the differ-prevention and control. isolating or removing infected animals can prevent the disease. facilities and equipment should also be disinfected. ii. proliferative stomatitis (bovine papular stomatitis) etiology. a parapoxvirus is the causative agent of bovine papular stomatitis. this virus is considered to be closely related to the parapoxvirus that causes contagious ecthyma and pseudocowpox. it is also a zoonotic disease. the disease is not considered of major consequence, but high morbidity and mortality may be seen in severe outbreaks. in addition, lesions are comparable in appearance to those seen with vesicular stomatitis, bovine viral diarrhea virus, and foot-and-mouth disease. the disease occurs worldwide. clinical signs and diagnosis. raised red papules or erosions or shallow ulcers on the muzzle, nose, oral mucosa (including the hard palate), esophagus, and rumen of younger cattle are the most common findings. in some outbreaks, the papules will be associated with ulcerative esophagitis, salivation, diarrhea, and subsequent weight loss. lesions persist or may come and go over a span of several months. morbidity among herds may be %. mortalities are rare. bovine papular stomatitis is associated with "rat tail" in feedlot cattle. animals continue to eat and usually do not show a fever. no lesion is seen on the feet. the infection may also be asymptomatic. diagnosis is based on clinical signs, histological findings, and viral isolation. epizootiology and transmission. cattle less than year of age are most commonly affected, and disease is rare in older cattle. transmission is by animal-to-animal contact. necropsy findings. raised papules may be found around the muzzle and mouth and involve the mucosa of the esophagus and rumen. histologically, epithelial cells will show hydropic degeneration and hyperplasia of the lamina propria. eosinophilic inclusions will be in the cytoplasm of infected epithelial cells. pathogenesis. following exposure to the virus, erythematous macules most commonly appear on the nares, followed by the mouth. these become raised papules within a day, regressing after days to weeks; the lesions that remain will be persistent yellow, red, or brown spots. some infections may recur or persist, with animals showing lesions intermittently or continuously over several months. differential diagnosis. pseudocowpox, vesicular stomatitis, foot-and-mouth disease, and bovine viral diarrhea virus infection are the differentials for this disease. the differential for the "rat tail" clinical sign is sarcocystis infection. there is no vaccine available for bovine papular stomatitis. because of the similarity of this virus to the parapoxvirus of contagious ecthyma, it is important to be aware of the persistence in the environment and susceptibility of younger cattle. vaccination using the local strain, and the skin scarification technique for off, have been protective. handlers should wear gloves and protective clothing. treatment. cattle usually will not require extensive nursing care, but lesions with secondary bacterial infections should be treated with antibiotics. their hands at sites of contact with lesions of cattle. iii. pseudocowpox etiology. pseudocowpox is a worldwide cattle disease caused by a parapoxvirus related to the causative agents of contagious ecthyma and bovine papular stomatitis (see sections iii,a, ,m and iii,a, ,q,ii). lesions are confined to the teats. this is also a zoonotic disease. clinical signs and diagnosis. minor lesions are usually confined to the teats. these are distinctive because of the ring-or horseshoe-shaped scab that develops after days. additional lesions sometimes develop on the udder, the medial aspect of the thighs, and the scrotum. the teat lesions may predispose to mastitis. etiology. pulmonary adenomatosis is a rare but progressive wasting disease of sheep, with worldwide distribution. pulmonary adenomatosis is caused by a type d retrovirus antigenically related to the mason-pfizer monkey virus. jaagsiekte was the designation when the disease was described originally in south africa. progressive respiratory signs such as dyspnea, rapid respiration, and wasting. the disease is diagnosed by these chronic clinical signs and histology. epizootiology and transmission. the disease is transmitted by aerosols. body fluids of viremic animals, such as milk, blood, saliva, tears, semen, and bronchial secretions, will contain the virus or cells carrying the virus. necropsy. the adenomas and adenocarcinomas will be small firm lesions distributed throughout the lungs. the adenocarcinomas metastasize to regional lymph nodes. pathogenesis. as with ovine progressive pneumonia (opp), the incubation period is up to years long. adenocarcinomatous lesions arising from type ii alveolar epithelial cells may be discrete or confluent and involve all lung lobes. with or is a differential diagnosis for opp. etiology. cutaneous papillomatosis is a very common disease in cattle and is much less common among sheep and goats. the disease is a viral-induced proliferation of the epithelium of the neck, face, back, and legs. these tumors are caused by a papillomavirus (dna virus) of the papovaviridae family, and the viruses are host-specific and often body site-specific. most are benign, although some forms in cattle and one form in goats can become malignant. in cattle, the site specificity of the papillomavirus strains are particularly well recognized. designations of the currently recognized bovine papillomavirus (bpv) types are bpv- through bpv- . clinical signs and diagnosis. the papillomas may last up to months and are seen more frequently in younger animals. lesions have typical wart appearances and may be single or multiple, small ( mm) or very large ( mm). the infections will generally be benign, but pain will be evident when warts develop on occlusal surfaces or within the gastrointestinal tract. in addition, when infections are severe, weight loss may occur. when warts occur on teats, secondary mastitis may develop. in cattle, bpv- and bpv- cause fibropapillomas on teats and penises or on head, neck, and dewlap, respectively. bpv- causes flat warts that occur in all body locations, b pv- causes warts in the gastrointestinal tract, and b pv- causes small white warts (called rice-grain warts) on teats. warts caused by bpv- and bpv- do not regress spontaneously. prognosis in cattle is poor only when papillomatosis involves more than % of the body surface. in sheep, warts are the verrucous type. the disease is of little consequence unless the warts develop in an area that causes dis-comfort or incapacitation such as between the digits, on the lips, or over the joints. in adult sheep, warts may transform to squamous cell carcinoma. in goats, the disease is rare, and the warts are also of the verrucous type and occasionally may develop into squamous cell carcinoma. warts on goat udders tend to be persistent. diagnosis is made by observing the typical proliferative lesions. epizootiology and transmission. older animals are less sensitive to papillomatosis than young animals, although immunosupressed animals of any age may develop warts as the result of harbored latent infections. the virus is transmitted by direct and indirect (fomite) contact, entering through surface wounds and sites such as tattoos. pathogenesis. the incubation period ranges from to months. the virus induces epidermal and fibrous tissue proliferation, often described as cauliflower-like skin tumors. the disease is generally self-limiting. differential diagnosis. in sheep and goats, differentials include contagious ecthyma, ulcerative dermatosis, strawberry foot rot, and sheep and goat pox. for cattle) or autogenous vaccines must be used with a recognition that papovavirus strains are host-specific and that immunity from infection or vaccination is viral-type-specific. autogenous vaccines are generally considered more effective. some vaccine preparations are effective at prevention but not treatment of outbreaks. viricidal products are recommended for disinfection of contaminated environments. minimizing cutaneous injuries and sanitizing equipment (tattoo devices, dehorners, ear taggers, etc.) in a virucidal solution between uses are also recommended preventive and control measures. halters, brushes, and other items may also be sources of virus. treatment. warts will often spontaneously resolve as immunity develops. in severe cases or with flockwide or herdwide problems, affected animals should be isolated from nonaffected animals, and premises disinfected. warts can be surgically excised and autogenous vaccines can be made and administered to help prevent disease spread. cryosurgery with liquid nitrogen or dry ice has also proven to be successful for wart removal. topical agents such as podophyllin (various formulations) and dimethyl sulfoxide may be applied to individual lesions once daily until regression. etiology. pseudorabies is an acute encephalitic disease caused by a neurotropic alphaherpesvirus, the porcine herpesvirus . one serotype is recognized, but strain differences exist. the disease has worldwide distribution. it is a primarily a clinical dis-ease of cattle, with less frequent reports (but no less severe clinical manifestations) in sheep and goats. during the rapid course of this usually fatal disease. at the site of virus inoculation or in other locations, abrasions, swelling, intense pruritus, and alopecia are seen. pruritus will not be asymmetric. animals will also become hyperthermic and will vocalize frantically. other neurological signs range from hoof stamping, kicking at the pruritic area, salivation, tongue chewing, head pressing and circling, to paresthesia or hyperesthesia, ataxia, and conscious proprioceptive deficits. nystagmus and strabismus are also seen. animals will be fearful or depressed, and aggression is sometimes seen. recumbency and coma precede death. diagnostic evidence includes clinical findings; virus isolation from nasal or pharyngeal secretions or postmortem tissues; and histological findings at necropsy. serology of affected animals is not productive, because of the rapid course. if swine are housed nearby, or if swine were transported in the same vehicles as affected animals, serological evaluations are worthwhile from those animals. epizootiology and transmission. swine are the primary hosts for pseudorabies virus, but they are usually asymptomatic and serve as reservoirs for the virus. the infection can remain latent in the trigeminal ganglion of pigs and recrudesce during stressful conditions. other animals are dead-end hosts. the unprotected virus will survive only a few weeks in the environment but may remain viable in meat (including carcasses) or saliva and will survive outside the host, in favorable conditions, in the summer for several weeks and the winter for several months. transmission is by oral, intranasal, intradermal, or subcutaneous introduction of the virus. when the virus is inhaled, the clinical signs of pruritus are less likely to be seen. transmission can also be by inadvertent exposure (e.g., contaminated syringes) of ruminants to the modified live vaccines developed for use in swine. spread between infected ruminants is a less likely means of transmission, because of the relatively short period of virus shedding. transport vehicles used for swine may also be sources of the virus. raccoons are believed to be vectors of the virus. horses are resistant to infection. there is no pathognomonic gross lesion. definitive histologic findings include severe, focal, nonsuppurative encephalitis and myelitis. eosinophilic intranuclear inclusion bodies (cowdry type a) may be present in some affected neurons. methods such as immunofluorescence and immunoperoxidase staining can be used to show presence of the porcine herpesvirus . pathogenesis. the incubation period is - hr and duration of the illness is - hr. the longest duration is seen in animals with pruritus around the head. differential diagnoses. differentials for the neurologic signs of pseudorabies infection include rabies, polioencephalomalacia, salt poisoning, meningitis, lead poisoning, hypomagnesemia, and enterotoxemia. those for the intense pruritus include psoroptic mange and scrapie in sheep, sarcoptic mange, and pediculosis. prevention and control. pseudorabies is a reportable disease in the united states, where a nationwide eradication program exists; states are rated regarding status. effective disinfectants include sodium hypochlorite ( % solution), formalin, peracetic acid, tamed iodines, and quaternary ammonium compounds. five minutes of contact time is required, and then surfaces must be rinsed. other disinfectant methods for viral killing include hr of formaldehyde fumigation, or min of ultraviolet light. transport vehicles should be cleaned and disinfected between species. serological screening for pseudorabies of swine housed near ruminants is essential. there is no treatment, and most affected ani-research complications. swine housed close to research ruminants should be serologically screened prior to purchase, and all transport vehicles should be cleaned and disinfected between loads of large animals. humans have been reported to seroconvert. the porcine herpesvirus shares antigens with the infectious bovine rhinotracheitis virus. etiology. rabies is a sporadic but fatal, acute viral disease affecting the central nervous system. the rabies virus is a neurotropic rna virus of the lyssavirus genus and the rhabdoviridae family. sheep, goats, and cattle are susceptible. the zoonotic potential of this virus must be kept in mind at all times when handling moribund animals with neurological signs characteristic of the disease. rabies is endemic in many areas of the world and within areas of the unites states. this is a reportable disease in north america. clinical findings and diagnosis. animals generally progress through three phases: prodromal, excitatory, and paralytic. many signs in the different species during these stages are nonspecific, and forms of the disease are also referred to as dumb or furious. during the short prodromal phase, animals are hyperthermic and apprehensive. animals progress to the excitatory phase, during which they refuse to eat or drink and are active and aggressive. repeated vocalizations, tenesmus, sexual excitement, and salivation occur during this phase. the final paralytic stage, with recumbency and death, occurs over several hours to days. this paralytic stage is common in cattle, and animals may simply be found dead. the clinical course is usually - days. diagnosis is based on clinical signs, with a progressive and fatal course. confirmation presently is made with the fluorescent antibody technique on brain tissue. epizootiology and transmission. the rabies virus is transmitted via a bite wound inflicted by a rabid animal. cats, dogs, raccoons, skunks, foxes, wild canids, and bats are the common disease vectors in north america. virus is also transmitted in milk and aerosols. necropsy findings. few lesions are seen at necropsy. many secondary lesions from manic behaviors during the course of disease may be evident. histological findings will include nonsuppurative encephalitis. negri bodies in the cytoplasm of neurons of the hippocampus and in purkinje's cells are pathognomonic histologic findings. pathogenesis. after exposure, the incubation period is variable, from weeks to several months, depending on the distance that the virus has to travel to reach the central nervous system. the rabies virus proliferates locally, gains access to neurons by attaching to acetylcholine receptors, via a viral surface glycoprotein, migrates along sensory nerves to the spinal cord and brain, and then descends via cranial nerves (trigeminal, facial, olfactory, glossopharyngeal) to oral and nasal cavity structures (i.e., salivary glands). the fatal outcome is currently believed to be multifactorial, related to anorexia, respiratory paralysis, and effects on the pituitary. differential diagnosis. rabies should be included on the differential list when clinical signs of neurologic disease are evident. other differentials for ruminants include herpesvirus encephalitis, thromboemobolic meningoencephalitis, nervous ketosis, grass tetany, and nervous cocciodiosis. prevention and control. vaccines approved for use cattle and sheep are commercially available and contain inactivated virus; there is not one available in the united states for goats. ruminants in endemic areas, such as the east coast of the united states, should be routinely vaccinated. any animals housed outside that may be exposed to rabid animals should be vaccinated. vaccination programs generally begin at months of age, with a booster at year of age and then annual or triennial boosters. awareness of the current rabies case reports for the region and wildlife reservoirs, however, is important. monitoring for and exclusion of wildlife from large-animal facilities are worthwhile preventive measures. the virus is fragile and unstable outside of a host animal. research complications. aerosolized virus is infective. personal protective equipment, including gloves, face mask, and eye shields, must be worn by individuals handling animals that are manifesting neurological disease signs. bovine spongiform encephalopathy, a transmissible spongiform encephalopathy (tse), is not known to occur in the united states, where since it has been listed as a reportable disease. the profound impact of this disease on the cattle industry in great britain during the past two decades is well known. the disease may be caused by a scrapielike (prion) agent. it is believed that the source of infection for cattle was feedstuff derived from sheep meat and bonemeal that had been inadequately treated during processing. the incubation period of years, the lack of detectable host immune response, the debilitating and progressive neurological illness, and the pathology localized to the central nervous system are characteristics of the disease, and are is comparable to the characteristics of other tse diseases such as scrapie, which affects sheep and goats. in addition, the infectious agent is extremely resistant to dessication and disinfectants. confirmation of disease is by histological examination of brain tissue collected at necropsy; the vacuolation that occurs during the disease will be symmetrical and in the gray matter of the brain stem. molecular biology techniques, such as western blots and immunohistochemistry, may also be used to identify the presence of the prion protein. differentials include many infectious or toxic agents that affect the bovine nervous and musculoskeletal systems, such as rabies, listeriosis, and lead poisoning. metabolic disorders such as ketosis, milk fever, and grass tetany are also differentials. there is no vaccine or treatment. prevention focuses on import regulations and not feeding ruminant protein to ruminants; recent usda regulations prohibit feeding any mammalian proteins to ruminants. etiology. scrapie is a sporadic, slow, neurodegenerative disease caused by a prion. scrapie is a reportable disease. it is much more common in sheep than in goats. the disease is similar to transmissible mink encephalopathy, kuru, creutzfeldt-jakob disease, and bovine spongiform encephalopathy (mad cow disease). prions are nonantigenic, replicating protein agents. clinical signs and diagnosis. during early clinical stages, animals are excitable and hard to control. tremors of head and neck muscles, as well as uncoordinated movements and unusual "bunny-hopping" gaits are observed. in advanced stages of the disease, animals experience severe pruritus and will self-mutilate while rubbing on fences, trees, and other objects. blindness and abortion may also be seen. morbidity may reach % within a flock. most animals invariably die within - weeks; some animals may survive months. in goats, the disease is also fatal. pruritus is generally less severe but may be localized. a wide range of clinical signs have also been noted in goats, including listlessness, stiffness or restlessness, or behavioral changes such as irritability, hunched posture, twitching, and erect tail and ears. as with sheep, the disease gradually progresses to anorexia and debilitation. diagnosis can be made by clinical signs and histopathological lesions. a newer diagnostic test in live animals is based on sampling from the third eyelid. tests for genetic resistance or susceptibility require a tube of edta blood and are reasonably priced. epizootiology and transmission. the suffolk breed of sheep tends to be especially susceptible. scrapie has also been reported in several other breeds, including cheviot, dorset, hampshire, corriedale, shropshire, merino, and rambouillet. it is believed that there is hereditary susceptibility in these breeds. targhees tend to be resistant. genomic research indicates there are two chromosomsal sites governing this trait; these sites are referred to codons (q, r, or h genes can be present) and (a or v genes can be present). of the five genes, r genes appear to confer immunity to clinical scrapie in suffolks in the united states. affected suffolks in the united states that have been tested have been aa qq. the disease is also enzootic is many other countries. the disease tends to affect newborns and young animals; however, because the incubation period tends to range from to years, adult animals display signs of the disease. scrapie is transmitted horizontally by direct or indirect contact; nasal secretions or placentas serve as sources of the infectious agent. vertical transmission is questioned, and transplacental transmission is considered unlikely. necropsy findings. at necropsy, no gross lesion is observed. histopathologically, neuronal vacuolization, astrogliosis, and spongiform degeneration are visualized in the brain stem, the spinal cord, and especially the thalamus. inflammatory lesions are not seen. pathogenesis. replication of the prions probably occurs first in lymphoid tissues throughout the host's body and then progresses to neural tissue. differential diagnosis. in sheep and goats, depending on the speed of onset, differentials for the pruritus include ectoparasites, pseudorabies, and photosensitization. prevention and control. if the disease diagnosed in a flock, quarantine and slaughter, followed by strict sanitation, are usually required. the u.s. department of agriculture has approved the use of % sodium hydroxide as the only disinfectant for sanitation of scrapie-infected premises. prions are highly resistant to physicochemical means of disinfection. artificial insemination or embryo transfer has been shown to decrease the spread of scrapie (linnabary et al., ) . research complications. as noted, this is a reportable disease. stringent regulations exist in the united states regarding importation of small ruminants from scrapie-infected countries. etiology. vesicular stomatitis (vs) is caused by the vesicular stomatitis virus (vsv), a member of the rhabdoviridae. three serotypes are recognized: new jersey, indiana, and isfahan. the new jersey and indiana strains cause sporadic disease in cattle in the united states. the disease is rare in sheep. clinical signs and diagnosis. adult cattle are most likely to develop vs. fever and development of vesicles on the oral mucous membranes are the initial clinical signs. lesions on the teats and interdigital spaces also develop. the vesicles progress quickly to ulcers and erosions. the animal's tongue may be severely involved. anorexia and salivation are common. weight loss and decreased milk production are noticeable. morbidity will be high in an outbreak, but mortality will be low to nonexistent. diagnostic work should be initiated as soon as possible to distinguish this from foot-and-mouth disease. diagnosis is based on analysis of fluid, serum, or membranes associated with the vesicles. virus isolation, enzyme-linked immunosorbent assay (elisa), competitive elisa (celisa), complement fixation, and serum neutralization are used for diagnosis. epizootiology and transmission. this disease occurs in several other mammalian species, including swine, horses, and wild ruminants. vsv is an enveloped virus and survives well in different environmental conditions, including in soil, extremes of ph, and low temperatures. outbreaks of vs occur sporadically in the united states, but it is not understood how or in what species the virus survives between these outbreaks. incidence of disease decreases during colder seasons. equipment, such as milking machines, contaminated by secretions is a mechanical vector, as are human hands. transmission may also be from contaminated water and feed. transmission is also believed to occur by insects (blackflies, sand flies, and culicoides) that may simply be mechanical vectors. it is believed that carrier animals do not occur in this disease. necropsy. it is rare for animals to be necropsied as the result of this disease. typical vesicular lesion histology is seen, with ballooning degeneration and edema. there is no inclusion body formation. pathogenesis. lesions often begin within hr after exposure. the virus invades oral epithelium. injuries or trauma in any area typically affected, such as mouth, teats, or interdigital areas, will increase the likelihood of lesions developing there. animals will develop a long-term immunity; this immunity can be overwhelmed, however, by a large dose of the virus. differential diagnosis. foot-and-mouth disease lesions are identical to vs lesions. other differentials in cattle include bovine viral diarrhea, malignant catarrhal fever, contagious ecthyma, photosensitization, trauma, and caustic agents. prevention and control. quarantine and restrictions on shipping infected animals or animals from the premises housing affected animals are required in an outbreak. vaccines are available for use in outbreaks and have decreased the severity of lesions. phenolics, quaternaries, and halogens are effective for inactivating and disinfecting equipment and facilities. treatment. affected animals should be segregated from the rest of the herd and provided with separate water and softened feed. these animals should be cared for after unaffected animals. any feed or water contaminated by these animals should not be used for other animals; contaminated equipment should be disinfected. topical or systemic antibiotics control secondary bacterial infections. cases of mastitis secondary to teat lesions must be treated as necessary. any abrasive materials that could cause further trauma to the animals should be removed. research complications. animals developing vesicular lesions must be reported promptly to eliminate the possibility of an outbreak of foot-and-mouth disease. personal protective equipment, especially gloves, should be worn when handling any animals with vesicular lesions. vsv causes a flulike illness in humans. x. viral diarrhea diseases i. ovine. rotavirus, of the family reoviridae, induces an acute, transient diarrhea in lambs within the first few weeks of life. four antigenic groups (a-d) have been identified by differences in capsid antigens vp and vp . primarily group a, but also groups b and c, have been isolated from sheep. the disease is characterized by yellow, semifluid to watery diarrhea occurring - days after infection. the disease can progress to dehydration, anorexia and weight loss, acidosis, depression, and occasionally death. the virus is ingested with contaminated feed and water and selectively infects and destroys the enterocytes at the tips of the small intestinal villi. the villi are replaced with immature cells that lack sufficient digestive enzymes; osmotic diarrhea results. virus may remain in the environment for several months. the disease is diagnosed by virus isolation, electron microscopy of feces, fecal fluorescent antibody, fecal elisa tests (marketed tests generally detect group a rotavirus), and fecal latex agglutination tests. rotavirus diarrhea is treated by supportive therapy, including maintaining hydration, electrolyte, and acid-base balance. a rotavirus vaccine is available for cattle; because of cross-species immunity, oral administration of high-quality bovine colostrum from vaccinated cows to infected sheep may be helpful ("current veterinary therapy," ). coronavirus, of the family coronaviridae, produces a more severe, long-lasting disease when compared with rotavirus. clinical signs are similar to above, although the incubation period tends to be shorter ( - hr), and animals exhibit less anorexia than those with rotavirus. additionally, mild respiratory disease may be noted (janke, ) . like rotavirus, coronavirus also destroys enterocytes of the villus tips. the virus can be visualized with electron microscopy. treatment is supportive; close consideration of hydration and acid-base status is essential. bovine vaccines are available. ii. caprine. rotavirus, coronavirus, and adenoviruses affect neonatal goats; however, little has been documented on the pathology and significance of these agents in this age group. it appears that bacteria play a more important role in neonatal kid diarrheal diseases then in neonatal calf diarrheas. iii. bovine. rotaviruses, coronaviruses, parvoviruses, and bovine viral diarrhea virus (bvdv) are associated with diarrheal disease in calves. each pathogen multiplies within and destroys the intestinal epithelial cells, resulting in villous atrophy and clinical signs of diarrhea (soft to watery feces), dehydration, and abdominal pain. these viral infections may be complicated by parasitic infections (e.g., cryptosporidium, eimeria) or bacterial infections (e.g., escherichia coli, salmonella, campylobacter). treatment is aimed at correcting dehydration, electrolyte imbalances, and acidosis; cessation of milk replacers and administration of fluid therapy intravenously and by stomach tube may be necessary, depending on the presence of suckle reflex and the condition of the animals. diagnosis is by immunoassays available for some viruses, viral culture, exclusion or identification of presence of other pathogens (by culture or fecal exams), and microscopic examination of necropsy specimens. prevention focuses on calves suckling good-quality colostrum; other recommendations for calf care are in section ii,b, . combination vaccine products are available for immunizing dams against rotavirus, coronavirus, and enterotoxigenic e. coli. additional supportive care for calves includes providing calves with sufficient energy and vitamins until milk intake can resume. rotaviruses of serogroup a are the most common type in neonatal calves; -to -day old calves are typically affected, but younger and older animals may also be affected. the small intestine is the site of infection. antirotavirus antibody is present in colostrum, and onset of rotavirus diarrhea coincides with the decline of this local protection. transmission is likely from other affected calves and asymptomatic adult carriers. the diarrhea is typically a distinctive yellow. colitis with tenesmus, mucus, and blood may be seen. this virus may be zoonotic. coronaviruses are commonly associated with disease in calves during the first month of life, and they infect small-and large-intestinal epithelial cells. the virus infection may extend to mild pneumonia. transmission is by infected calves and also by asymptomatic adult cattle, including dams excreting virus at the time of parturition. calves that appear to have recovered continue to shed virus for several weeks. parvovirus infections are usually associated with neonatal calves. b vdv infections also are seen in neonates and also affect many systems and produce other clinical signs and syndromes that are described in section iii,a, ,e. iv. winter dysentery. winter dysentery is an acute, winterseasonal, epizootic diarrheal disease of adult cattle, although it has been reported in -month-old calves. the etiology has not yet been defined, but a viral pathogen is suspected. coronavirus-like viral particles have been isolated from cattle feces, either the same as or similar to the coronavirus of calf diarrhea. outbreaks typically last a few weeks, and first-lactation or younger cattle are affected first, with waves of illness moving through a herd. individual cows are ill for only a few days. the incubation period is estimated at - days. the outbreaks of disease are often seen in herds throughout the local area. clinical signs include explosive diarrhea, anorexia, depression, and decreased production. the diarrhea has a distinctive musty, sweet odor and is light brown and bubbly, but some blood streaks or clots may be mixed in with the feces. animals will become dehydrated quickly but are thirsty. respiratory symptoms such as nasolacrimal discharges and coughing may develop. recovery is generally spontaneous. mortalities are rare. diagnosis is based on characteristic patterns of clinical signs, and elimination of diarrheas caused by parasites such as coccidia, bacterial organisms such as salmonella or mycobacterium paratuberculosis, and viruses such as b vdv. pathology is present in the colonic mucosa, and necrosis is present in the crypts. etiology. chlamydia psittaci is a nonmotile, obligate, intracytoplasmic, gram-negative bacterium. clinical signs. enzootic abortion in sheep and goats is a contagious disease characterized by hyperthermia and late abortion or by birth of stillborn or weak lambs or kids (rodolakis et al., ) . the only presenting clinical sign may be serosanguineous vulvar discharges. other animals may present with arthritis or pneumonia. infection of animals prior to about days of gestation results in abortion, stillbirths, or birth of weak lambs. infection after days results in potentially normal births, but the dams or offspring may be latently infected. latently infected animals that were infected during their dry period may abort during the next pregnancy. ewes or does generally only abort once, and thus recovered animals will be immune to future infections. and specific antigens associated with the cell surface. the group antigen is common among all chlamydia; the specific antigen is common to related subgroups. two subgroups are recognized, one that causes eae and one that causes polyarthritis and conjunctivitis. the disease is transmitted by direct contact with infectious secretions such as placental, fetal, and uterine fluids or by indirect contact with contaminated feed and water. necropsy. placental lesions include intercotyledonary plaques and necrosis and cotyledonary hemorrhages. histopathological evidence of leukocytic infiltration, edema, and necrosis is found throughout the placentome. fetal lesions include giant-cell accumulation in mesenteric lymph nodes and lymphohistiocytic proliferations around the blood vessels within the liver. diagnosis is based on clinical signs and laboratory (serological or histopathological) identification of the organism. impression smears in placental tissues stained with giemsa, gimenez, or modified ziehl-neelsen can provide preliminary indications of the causative agent. immunofluorescence, enzyme-linked immunosorbent assay (elisa), and polymerase chain reaction (pcr) methods also aid in diagnosis. differential diagnosis. q fever will be the major differential for late-term abortion and necrotizing placentitis. campylobacter and toxoplasma should also be considered for late-term abortion. treatment. animals may respond to treatment with oxytetracycline. abortions are prevented through administration of a commercial vaccine, but the vaccine will not eliminate infections. this is a sheep vaccine and should be administered before breeding and annually to at least the young females entering the breeding herd or flock. research complications. in addition to losses or compromise of research animals, pregnant women should not handle aborted tissues. etiology. chlamydia psittaci is a nonmotile, obligate intracellular, gram-negative bacterium. chlamydial polyarthritis is an acute, contagious disease characterized by fever, lameness (bulgin, ) , and conjunctivitis (see section iii,a, ,c) in growing and nursing lambs. clinical signs. clinically, animals will appear lame on one or all legs and in major joints, including the scapulohumeral, humeroradioulnar, coxofemoral, femorotibial, and tibiotarsal joints. lambs may be anorexic and febrile. animals frequently also exhibit concurrent conjunctivitis. the disease usually resolves in approximately weeks. joint inflammation usually resolves without causing chronic articular changes. epizootiology and transmission. the disease is transmitted to susceptible animals by direct contact as well as by contaminated feed and water. the organism penetrates the gastrointestinal tract and migrates to joints and synovial membranes as well as to the conjunctiva. the organism causes acute inflammation and associated fibrinopurulent exudates. necropsy findings. lesions are found in joints, tendon sheaths, conjunctiva, and lungs. pathological sites will be edematous and hyperemic, with fibrinous exudates but without articular changes. lesions will be infiltrated with mononuclear cells. lung lesions include atelectasis and alveolar inspissation. diagnosis is based on clinical signs. synovial taps and subsequent smears may allow the identification of chlamydial inclusion bodies. treatment. animals respond to treatment with parenteral oxytetracycline. etiology. chlamydia psittaci, a nonmotile, obligate intracellular, gram-negative bacterium, is the most common cause of infectious keratoconjunctivitis in sheep. chlamydia and mycoplasma are considered to be the most common causes of this disease in goats. chlamydial conjunctivitis is not a disease of cattle. clinical signs. infectious keratoconjunctivitis is an acute, contagious disease characterized in earlier stages by conjunctival hyperemia, epiphora, and edema and in later stages by, corneal edema, ulceration, and opacity. perforation may result from the ulceration. animals will be photophobic. in less severe cases, corneal healing associated with fibrosis and neovascularization occurs in - days. lymphoid tissues associated with the conjunctiva and nictitating membrane may enlarge and prolapse the eyelids. morbidity may reach - %. bilateral and symmetrical infections characterize most outbreaks. relapses may occur. other concurrent systemic infections may be seen, such as polyarthritis or abortion in sheep and polyarthritis, mastitis, and uterine infections in goats. epizootiology and transmission. direct contact, and mechanical vectors such as flies easily spread the organism. necropsy. if the chlamydial or mycoplasmal agents are suspected, diagnostic laboratories should be contacted for recommendations regarding sampling. conjunctival smears are also useful. pathogenesis. the pathogen penetrates the conjunctival epithelium and replicates in the cytoplasm by forming initial and elementary bodies. the infection moves from cell to cell and causes an acute inflammation and resultant purulent exudate. the chlamydial organism may penetrate the bloodstream and migrate to the opposite eye or joints, leading to arthritis. diagnosis is suggested by the clinical signs. cytoplasmic inclusions observed on conjunctival scrapings and immunofluorescent techniques help confirm the diagnosis. differential diagnosis. nonchlamydial keratoconjunctivitis also occurs in sheep and goats. the primary agents involved include mycoplasma conjunctiva, m. agalactiae in goats, and branhamella (neisseria) ovis. a less common differential for sheep and cattle is listeria monocytogenes. other differentials include eye worms, trauma, and foreign bodies such as windblown materials (pollen, dust) and poor-quality hay; these latter irritants and stress may predispose the animals' eyes to the infectious agents. should be minimized whenever possible. quarantine of new animals and treatment, if necessary, before introduction into the flock or herd are important measures. shade should be provided for all animals. treatment. the infections can be self-limiting in - weeks without treatment. treatment consists of topical application of tetracycline ophthalmic ointments. systemic or oral oxytetracycline treatments have been used with the topical treatment. atropine may be added to the treatment regimen when uveitis is present. shade should be provided. a. protozoa i. anaplasmosis etiology. anaplasmosis is an infectious, hemolytic, noncontagious, transmissible disease of cattle caused by the protozoan anaplasma marginale. anaplasma is a member of the anaplasmatacae family within the order rickettsiales. in sheep and goats, the disease is caused by a. ovis and is an uncommon cause of hemolytic disease. anaplasmosis has not been reported in goats in the united states. some controversy exists regarding the classification. most recently it is classified as a protozoal disease because of similarities to babesiosis. it has also been classified as a rickettsial pathogen. this summary addresses the disease in cattle with limited reference to a. ovis infections, but there are many similarities to the disease in cattle. clinical signs and diagnosis. acute anemia is the predominant sign in anaplasmosis, and fever coincides with parasitemia. weakness, pallor, lethargy, dehydration, and anorexia are the result of the anemia. four disease stagesnincubation, developmental, convalescent, and carriermare recognized. the incubation stage may be long, - weeks, and is characterized by a rise in body temperature as the infection moves to the next stage. most clinical signs occur during the -to -day developmental stage, with hemolytic anemia being common. death is most likely to occur at this stage or at the beginning of the convalescent stage. death may also occur from anoxia, because of the animal's inability to handle any exertion or stress, especially if treatment is initiated when severe anemia exists. reticulocytosis characterizes the convalescent stage, which may continue for many weeks. morbidity is high, and mortality is low. the carrier stage is defined as the time in the convalescent stage when the animal host becomes a reservoir of the disease, and anaplasma organisms and any parasitemia are not discernible. common serologic tests are the complement fixation test and the rapid card test. these become positive after the incubation phase and do not distinguish between the later three stages of disease. definitive diagnosis is made by clinical and necropsy findings. staining of thin blood smears with wright's or giemsa stain allows detection of basophilic, spherical a. marginale bodies near the red blood cell peripheries. evidence will most likely be found before a hemolytic episode. a negative finding should not eliminate the pathogen from consideration. epizootiology and transmission. the disease is common in cattle in the southern and western united states. anaplasma organisms are spread biologically or mechanically. mechanical transmission occurs when infected red blood cells are passed from one host to another on the mouthparts of seasonal biting flies. sometimes mosquitoes or instruments such as dehorners or hypodermic needles may facilitate transfer of infected red cells from one animal to another. biological transmission occurs when the tick stage of the organism is passed by dermacentor andersoni and d. occidentalis ticks. the carrier stage covers the time when discernible anaplasma organisms can be found on host blood smears. recovered animals serve as immune carriers and disease reservoirs. necropsy. pale tissues and watery, thin blood are typical findings. splenomegaly, hepatomegaly, and gallbladder distension are common findings. pathogenesis. the parasites infect the host's red blood cells, and acute hemolysis occurs during the parasites' developmental stage. the four stages of the parasite's life cycle are described above because these are closely linked to the clinical stages. differential diagnosis. the clinical disease closely resembles the protozoal disease babesiosis. whole organism) programs are not entirely effective, and vaccine should not be administered to pregnant cows. neonatal isoerythrolysis may occur because of the antierythrocyte antibodies stimulated by one vaccine product. vaccinated animals can still become infected and become carriers. the cattle vaccine has shown no efficacy in smaller ruminants, and there is no a. ovis vaccine. identifying carriers serologically and treating with tetracycline during and/or after vector seasons may be an option. removing carriers to a separate herd is also an approach. interstate movement of infected animals is regulated. treatment. oxytetracycline, administered once, helps reduce the severity of the infection during the developmental stage. other tetracycline treatment programs have been described to help control carriers. ii. babesiosis (red water, texas cattle fever, cattle tick fever) etiology. babesia bovis and ba. bigemina are protozoa that cause subclinical infections or disease in cattle. these are intraerythrocytic parasites. babesia bovis is regarded as the more virulent of the two organisms. this disease is not seen in the smaller ruminants in the united states. clinical signs and diagnosis. the more common presentation is liver and kidney failure due to hemolysis with icterus, hemoglobinuria, and fever. hemoglobinuria indicates a poor prognosis. acute encephalitis is a less common presentation and begins acutely with fever, ataxia, depression, deficits in conscious proprioception, mania, convulsions, and coma. the encephalitic form generally also has a poor prognosis. sudden death may occur. thin blood smears stained with giemsa will show babesia trophozoites at some stages of the disease, but lack of these cannot be interpreted as a negative. the trophozoites occur in a variety of shapes, such as piriform, round, or rod. complement fixation, immunofluorescent antibody, and enzyme immunoassay are the most favored of the available serologic tests. babesiosis is present on several continents, including the americas. in addition to domestic cattle, some wild ruminants, such as white-tailed deer and american buffalo, are also susceptible. bos indicus breeds have resistance to the disease and the tick vectors. innate resistance factors have been found in all calves. if infected, these animals will not show many signs of disease during the first year of life and will become carriers. stress can cause disease development. prevention and control. offspring of immune carriers resist infection up to months of age because of passive immunity. vector control and attention to hygiene are essential, such as between-animal rinsing in disinfectant of mechanical vectors such as dehorners. there is no entirely effective means, however, to prevent and control the disease. vaccination (killed necropsy findings. signs of acute hemolytic crisis are the most common findings, including hepatomegaly, splenomegaly, dark and distended gallbladder, pale tissues, thin blood, scattered hemorrhages, and petechiation. animals dying after a longer course of disease will be emaciated and icteric, with thin blood, pale kidneys, and enlarged liver. pathogenesis. the protozoon is transmitted by the cattle fever ticks boophilus annulatus, b. microplus, and b. decoloratus; these one-host ticks acquire the protozoon from infected animals. it is passed transovarially, and both nymph and adult ticks may transmit to other cattle. only b. ovis is transmitted by the larval stage. clinical signs develop about weeks after tick infestations or mechanical transmission but may develop sooner with the mechanical transmission. hemolysis is due to intracellular reproduction of the parasites and occurs intra-and extravascularly. in addition to the release of merozoites, proteolytic enzymes are also released, and these contribute to the clinical metabolic acidosis and anoxia. the development of the encephalitis form is believed to be the result of direct invasion of the central nervous system, disseminated intravascular coagulation, capillary thrombosis by the parasites and infarction, and/or tissue anoxia. differential diagnosis. in addition to anaplasmosis, other differentials for the hemolytic form of the disease are leptospirosis, chronic copper toxicity, and bacillary hemoglobinuria. several differentials in the united states for the encephalitic presentation include rabies, nervous system coccidiosis, polioencephalomalacia, lead poisoning, infectious bovine rhinotracheitis, salt poisoning, and chlorinated hydrocarbon toxicity. prevention and control. control or eradication of ticks and cleaning of equipment to prevent mechanical transmission, as noted in section iii,a, ,a,i, are important preventive measures. some vaccination approaches have been effective, but a commercial product is not available. treatment. supportive care is indicated, including blood transfusions, fluids, and antibiotics. medications such as diminazene diaceturate, phenamidine diisethionate, imidocarb diprionate, or amicarbalide diisethionate are most commonly used. treatment outcomes will be either elimination of the parasite or development of a chronic carrier state immune to further disease. research complications. this is a reportable disease in the united states. iii. coccidiosis etiology. coccidiosis is an important acute and chronic protozoal disease of ruminants. in young ruminants, it is characterized primarily by hemorrhagic diarrhea. adult ruminants may carry and shed the protozoa, but they rarely display clinical signs. intensive rearing and housing conditions and stress increase the severity of the disease in all age groups. coccidia are protozoal organisms of the phylum apicomplexa, members of which are obligatory intracellular parasites. there are at least reported species of coccidia in sheep, of which several are considered pathogenic: eimeria ashata, e. crandallis, and e. ovinoidalis (schillhorn van veen, ). at least species of eimeria have been recognized in the goat (foreyt, ) . eimeria ninakohlyakimovae, e. arloingi, and e. christenseni are regarded as the most pathogenic. eimeria bovis and e. zuernii (highly pathogenic), and e. auburnensis and e. alabamensis (moderately pathogenic), are among the species known to infect cattle. eimeria zuernii is more commonly seen in older cattle and is the agent of "winter coccidiosis." clinical signs and diagnosis. hemorrhagic diarrhea develops days to weeks after infection. fecal staining of the tail and perineum will be present. animals will frequently display tenesmus; rectal prolapses may also develop. anorexia, weight loss, dehydration, anemia, fever (infrequently), depression, and weakness may also be seen in all ruminants. the diarrhea is watery and malodorous and will contain variable amounts of blood and fibrinous, necrotic tissues. the intestinal hemorrhage may subsequently lead to anemia and hypoproteinemia. depending on the predilection of the coccidial species for small and/or large intestines, malabsorption of nutrients or water may occur, and electrolyte imbalances may be severe. concurrent disease with other enteropathogens may also be part of the clinical picture. in sheep, secondary bacterial infection with organisms such as fusobacterium necrophorum may ensue. young goats may die peracutely or suffer severe anemia from blood loss into the bowel. older goats may lose the pelleted form of feces. cattle may have explosive diarrhea and develop anal paralysis. the disease is usually diagnosed by history and clinical signs. numerous oocysts will frequently be observed in fresh fecal flotation (salt or sugar solution) samples as the diarrhea begins. laboratory results are usually reported as number of oocysts per gram of feces. coccidia seen on routine fecal evaluations reflect shedding, possibly of nonpathogenic species, without necessarily being indicative of impending or resolving mild disease. epkzootiology and transmission. as noted, coccidiosis is a common disease in young ruminants. in goats, young animals aged weeks to months are primarily affected, but isolated outbreaks in adults may occur after stressful conditions such as transportation or diet changes. coccidia are host-specific and also host cell-specific. the disease is transmitted via ingestion of sporulated oocysts. coccidial oocysts remain viable for long periods of time when in moist, shady conditions. necropsy. necropsies provide information on specific locations and severity of lesions that correlate with the species involved. ileitis, typhlitis, and colitis with associated necrosis and hemorrhage will be observed. mucosal scrapings will frequently yield oocysts. various coccidial stages associated with schizogony or gametogony may be observed in histopathological sections of the intestines. fibrin and cellular infiltrates will be found in the lamina propria. pathogenesis. this parasite has a complex life cycle in which sexual and asexual reproduction occurs in gastrointestinal enterocytes (speer, ) . the severity of the disease is correlated primarily with the number of ingested oocysts. specifics of life cycles vary with the species, and those characteristics contribute to the pathogenicity. in most cases, the disease is well established by the time clinical signs are seen. oocysts must undergo sporulation over a -to -day period in the environment. after ingestion of the sporulated oocysts, sporozoites are released and penetrate the intestinal mucosa and form schizonts. schizonts initially undergo replication by fission to form merozoites and eventually undergo sexual reproduction, forming new oocysts. the organisms cause edema and hyperemia; penetration into the lamina propria may lead to necrosis of capillaries and hemorrhage. differential diagnosis. differential diagnoses include the many enteropathogens associated with acute diarrhea in young ruminants: cryptosporidia, colibacilli, salmonella, enterotoxins, yersinia, viruses, and other intestinal parasites such as helminths. in cattle, for example, bovine viral diarrhea virus and helminthiasis caused by ostergia must be considered. management factors, such as dietary-induced diarrheas, are also differentials. in older animals, differentials in addition to stress are malnutrition, grain engorgement, and other intestinal parasitisms. prevention and control. good management practices will help prevent the disease. oocysts are resistant to disinfectants but are susceptible to dry or freezing conditions. proper sanitation of animal housing and minimizing overcrowding are essential. coccidiostats added to the feed and water are helpful in preventing the disease in areas of high exposure. treatment. affected animals should be isolated. on an individual basis, treatment should also include provision of a dry, warm environment, fluids, electrolytes (orally or intravenously), antibiotics (to prevent bacterial invasion and septicemia), and administration of coccidiostats. coccidiostats are preferred to coccidiocidals because the former allow immunity to develop. although many coccidial infections tend to be self-limiting, sulfonamides and amprolium may be used to aid in the treatment of disease. other anticoccidial drugs include decoquinate, lasalocid, and monensin; labels should be checked for specific approval in a species or specific indications. animals treated with amprolium should be monitored for development of secondary polioencephalomalacia. pen mates of affected animals should be considered exposed and should be treated to control early stages of infection. mechanisms of immunity have not been well defined but appear to be correlated with the particular coccidial species and their characteristics (for example, the extent of intracellular penetration). immunity may result when low numbers are ingested and there is only mild disease. immunity also may develop after more severe infections. iv. cryptosporidiosis etiology. cryptosporidium organisms are a very common cause of diarrhea in young ruminants. four cryptosporidium species have been described in vertebrates: c. baileyi and c. meleagridis in birds and c. parvum and c. muris in mammals. cryptosporidium parvum is the species affecting sheep (rings and rings, ) . debate continues regarding whether there are definite host-specific variants. clinical signs and diagnosis. cryptosporidiosis is characterized by protracted, watery diarrhea and debilitation. the diarrhea may last only - days or may be persistent and fatal. the diarrhea is watery and yellow, and blood, mucus, bile, and undigested milk may also be present. infected animals will display tenesmus, anorexia and weight loss, dehydration, and depression. in relapsing cases, animals become cachectic. overall, morbidity will be high, and mortality variable. mucosal scrapings or fixed stained tissue sections may be useful in diagnosis. the disease is also diagnosed by detecting the oocysts in iodine-stained feces or in tissues stained with periodic acid-schiff stain or methenamine silver. cryptosporidium also stains red on acid-fast stains such as kinyoun or ziehl-neelsen. fecal flotations should be performed without sugar solutions or with sugar solutions at specific gravity of . (foryet, ) . fecal immunofluorescent antibody (ifa) techniques have also been described. epizootiology and transmission. younger ruminants are commonly affected: lambs, kids (especially kids between the ages of and days old), and calves less than days old. like other coccidians, cryptosporidium is transmitted via the fecal-oral route. in addition to local contamination, water supplies have also been sources of the infecting oocysts. the oocysts are extremely resistant to desiccation in the environment and may survive in the soil and manure for many months. necropsy findings. the lesions caused by cryptosporidium are nonspecific. animals will be emaciated. moderate enteritis and hyperplasia of the crypt epithelial cells with villous atrophy as well as villous fusion, primarily in the lower small intestines, will be present. cecal and colonic mucosae may sometimes be involved. gastrointestinal smears may be made at necropsy and stained as described above. pathogenesis. although cryptosporidium infections are clinically similar to eimeria infections (moore, ) , cryptosporidium, in contrast to eimeria, invades just under the surface but does not invade the cytoplasm of enterocytes. there is no intermediate host. the oocysts are half the size of eimeria oocysts and are shed sporulated; they are, therefore, immediately infective. within - days of exposure, diarrhea and oocyst shedding occur. the diarrhea is the result of malabsorption and, in younger animals, intraluminal milk fermentation. autoinfection within the lumen of the intestines may also occur and result in persistent infections. in addition, several other pathogens may be involved, such as concurrent coronavirus and rotavirus infections in calves. environmental stressors such as cold weather increase mortality. intensive housing arrangements increase morbidity and mortality. differential diagnosis. other causes of diarrhea in younger ruminants include rotavirus, coronavirus, and other enteric viral infections; enterotoxigenic escherichia coli; clostridium; other coccidial pathogens; and dietary causes (inappropriate use of milk replacers). in addition, these other agents may also be causing illness in the affected animals and may complicate the diagnosis and the treatment picture. eimeria is more likely to cause diarrhea in calves and lambs at - weeks of age. giardia organisms may be seen in fecal preparations from young ruminants but are not considered to play a significant role in enteric disease. blood. animals exhibit fever, dehydration, and depression. chronic cases may result in a "poor doer" syndrome with weight loss and unthriftiness. giardia can be diagnosed by identifying the motile piriform trophozoites in fresh fecal mounts. oval cysts can be floated with zinc sulfate solution ( %). standard solutions tend to be too hyperosmotic and to distort the cysts. newer enzyme-linked immunosorbent assay (elisa) and ifa tests are sensitive and specific. epizootiology and transmission. giardia infection may occur at any age, but young animals are predisposed. chronic oocyst shedding is common. transmission of the cyst stage is fecaloral. wild animals may serve as reservoirs. necropsy findings. gross lesions may not be evident. villous atrophy and cuboidal enterocytes may be evident histologically. prevention and control. precautions should be taken when handling infected animals. affected animals must be removed and isolated as soon as possible. animal housing areas should be disinfected with undiluted commercial bleach or % ammonia. formalin ( %) fumigation has proven successful (foryet, ) . after being cleaned, areas should be allowed to dry thoroughly and should remain unpopulated for a period of time. because enteric disease often is multifactorial, other pathogens should also be considered, and management and husbandry should be examined. no known drug treatment is available. the disease is generally self-limiting, so symptomatic, supportive therapy aimed at rehydrating, correcting electrolyte and acid-base balance, and providing energy is often effective. supplementation with vitamin a may be helpful. age resistance begins to develop when the animals are about month old. research complications. cryptosporidiosis is a zoonotic disease. it is easily spread from calves to humans, for example, even as the result of simply handling clothing soiled by calf diarrhea. adult immunocompetent humans are reported to experience watery diarrhea, cramping, flatulence, and headache. the disease can be life-threatening in immunocompromised individuals. v. giardiasis etiology. giardia lamblia (also called g. intestinalis and g. duodenalis) is a flagellate protozoon. giardiasis is a worldwide protozoal-induced diarrheal disease of mammals and some birds (kirkpatrick, ), but it not considered to be a significant pathogen in ruminants. clinical signs and diagnosis. diarrhea may be continuous or intermittent, is pasty to watery, is yellow, and may contain pathogenesis. following ingestion, each giardia cyst releases four trophozoites, which attach to the enterocytes of the duodenum and proximal jejunum and subsequently divide by binary fission or encyst. the organism causes little intestinal pathology, and the cause of diarrhea is unknown but is thought to be related to disruption of digestive enzyme function, leading to malabsorption. disturbances in intestinal motility may also occur (rings and rings, ) . prevention and control. intensive housing and warm environments should be minimized. cysts can survive in the environment for long periods of time but are susceptible to desiccation. effective disinfectants include quaternary ammonium compounds, bleach-water solution ( : or : ), steam, or boiling water. after cleaning, areas should be left empty and allowed to dry completely. treatment. giardia has been successfully treated with oral metronidazole. benzimidazole anthelmintics are also effective, but these are not approved for use in animals for this purpose. should be taken when handling infected animals. etiology. neosporosis is a common, worldwide cause of bovine abortion caused by the protozoal species neospora caninum. abortions have also been reported in sheep and goats. neonatal disease is seen in lambs, kids, and calves. until , these infections were misdiagnosed as caused by toxoplasma gondii. some similarities exist between the life cycles and pathogeneses of both organisms. clinical signs and diagnosis. abortion is the only clinical sign seen in adult cattle and occurs sporadically, endemically, or as abortion storms. bovine abortions occur between the third and seventh month of gestation; fetal age at abortion correlates with the parity of the dam as well as with pattern of abortion in the herd. although cows that abort tend to be culled after the first or second abortion, repeated n. caninum-caused abortions will occur progressively later in gestation (up to about months) and within a shorter time frame in the same cow (thurmond and hietala, ) . although infections in adults are asymptomatic other than the abortions, decreased milk production has been noted in congenitally infected cows. many neospora-infected calves will be born asymptomatic. weakness will be evident in some infected calves, but this resolves. rare clinical signs include exophthalmos or asymmetric eyes, weight loss, ataxia, hyperflexion or hyperextension of all limbs, decreased patellar reflexes, and loss of conscious proprioception. some fetal deaths will occur, and resorption, mummification, autolysis, or stillbirth will follow. immunohistochemistry and histopathology of fetal tissue are the most efficient and reliable means of establishing a postmortem diagnosis. serology (ifa and elisa) is useful, including precolostral levels in weak neonates, but this indicates only exposure. titers of dams will not be elevated at the time of abortion; fetal serology is influenced by the stage of gestation and course of infection. earlier and rapid infections are less likely to yield antibodies against neospora. none of the currently available tests is predictive of disease. epizootiology and transmission. the parasite is now acknowledged to be widespread in dairy and cattle herds. the life cycle of n. caninum is complex, and many aspects remain to be clarified. the definitive host is the dog (mcallister et al., ) . placental or aborted tissues are the most likely sources of infection for the definitive host and play a minor role in transmission to the intermediate hosts. the many intermediate hosts include ruminants, deer, and horses. transplacental transmission is the major mode of transmission in dairy cattle and is the means by which a herd's infection is perpetuated. a less significant mode of transmission is by ingestion of oocysts, which sporulate in the environment or in the intermediate host's body. reactivation in a chronically infected animal's body is the result of rupture of tissue cysts in neural tissue. seropositive immunity does not protect a cow from future abortions. many seropositive cows and calves will never abort or show clinical signs, respectively. some immunological cross-reactivity may exist among neospora, cryptosporidia, and coccidium. necropsy findings. aborted fetuses will usually be autolysed. in those from which tissue can be recovered, tissue cysts are most commonly found in the brain. spinal cord is also useful. histological lesions include mild to moderate gliosis, nonsuppurative encephalitis, and perivascular infiltration by mixed mononuclear cells. pathogenesis. as with toxoplasma, cell death is the result of intracellular multiplication of neospora tachyzoites. neospora undergoes sexual replication in the dog's intestinal tract, and oocysts are shed in the feces. the intermediate hosts develop nonclinical systemic infections, with tachyzoites in several organs, and parasites then localize and become encysted in particular tissues, especially the brain. infections of this type are latent and lifelong. except when immunocompromised, most cattle do not usually develop clinical signs and do not have fetal loss. fetuses become infected, leading to fetal death, mid-gestation abortions, or live calves with latent infections or congenital brain disease. it usually takes - weeks for a fetus to die and to be expelled. many aspects of the role of the maternal immune response and pregnancy-associated immunodeficiency in the patterns of neospora abortions remain to be elucidated. differential diagnosis. even when there is a herd history of confirmed neospora abortions, leptospirosis, bovine viral diarrhea virus (bvdv), infectious bovine rhinotracheitis virus (ibrv), salmonellosis, and campylobacteriosis should be considered. bvdv in particular should be considered for abortion storms. differentials for weak calves are b vdv, perinatal hypoxia following dystocia (immediate postpartum time), bluetongue virus, toxoplasma, exposure to teratogens, or congenital defects. prevention and control. the primary preventive measure is preventing contact with contaminated feces. oocysts will not survive dry environments or extremes of temperature. dog populations should be controlled, and dogs and other canids should not have access to placentas or aborted fetuses. dogs should also be restricted from feed bunks and other feed storage areas. preventive culling is not economically practical for most producers. a vaccine recently became available. if embryo transfer is practiced, recipients should be screened serologically before use. laxis. there is no known treatment or immunoprophy- clinical signs and diagnosis. clinical signs of sarcocystosis infection are seen in cattle during the stage when the parasite encysts in soft tissues. often the infections are asymptomatic. fever, anemia, ataxia, symmetric lameness, tremors, tail-switch hair loss, excessive salivation, diarrhea, and weight loss are clinical signs. abortions in cattle occur during the second trimester and in smaller ruminants days after ingestion of the sporulated oocysts. definitive diagnosis is based on finding merozoites and meronts in neural tissue lesions. clinical hematology results include decreased hematocrit, decreased serum protein, and prolonged prothrombin times. sarcocystis-specific igg will increase dramatically by - weeks after infection. there is no cross-reaction between sarcocystis and toxoplasma. epizootiology and transmission. infection rates among cattle in the united states are estimated to be very high. transmission is by ingestion of feed and water contaminated by feces of the definitive hosts. dogs are the definitive hosts for the species that infect the smaller ruminants. cats, dogs, and primates (including humans when s. hominis is involved) are the definitive hosts for the species that infect cattle. necropsy. aborted fetuses may be autolysed. lesions in neural tissues, including meningoencephalomyelitis, focal malacia, perivascular cuffing, neuronal degeneration, and gliosis, are most marked in the cerebellum and midbrain. lesions may be found in other tissues, such as lymphadenopathy, and hemorrhages may be found in muscles and on serous surfaces. cysts in cardiac and skeletal muscles are common incidental findings during necropsies. pathogenesis. ingestion of muscle flesh from an infected ruminant results in sarcocystis cysts' being broken down in the carnivore's digestive system, release of bradyzoites, infection of intestinal mucosal cells by the bradyzoites, differentiation into sexual stages, fusion of the male and female gametes to form oocysts, and shedding as sporocysts by the definitive hosts. the sporocysts are eaten by the ruminant and penetrate the bowel walls; several stages of development occur in endothelial cells of arteries. merozoites are the form that enters soft tissues, such as muscle, and subsequently encysts. prevention and control. feed supplies of ruminants must be protected from fecal contamination by domestic and wild carnivores. these animals should be controlled and must also not have access to carcasses. in larger production situations, monensin may be fed as a prophylactic measure. treatment. monensin fed during incubation is prophylactic, but the efficacy in clinically affected cattle is not known. etiology. toxoplasmosis is caused by the obligate intracellular protozoon toxoplasma gondii, a coccidial parasite of the family eimeridae. cats are the only definitive hosts, and several warm-blooded animals, including ruminants, have been shown to be intermediate hosts. the disease is a major cause of abortion in sheep and goats and less common in cattle. clinical signs and diagnosis. clinical signs depend on the organ or tissue parasitized. toxoplasmosis is typically associated with placentitis, abortion, stillbirths, or birth of weak young (underwood and rook, ; buxton, ) . it has also been shown to cause pneumonia and nonsuppurative encephalitis. the enteritis at the early stage of infection may be fatal in some hosts. hydrocephalus does not occur in animals as it does in human fetal toxoplasma infections. rare clinical presentations in ruminants include retinitis and chorioretinitis; these are usually asymptomatic. infection of the ewe during the first trimester usually leads to fetal resorption, during the second trimester leads to abortion, and during the third trimester leads to birth of weak to normal lambs with subsequent high perinatal mortality. congenitally infected lambs may display encephalitic signs of circling, incoordination, muscular paresis, and prostration. in sheep, weak young will develop normally if they survive the first week after birth. infected adult sheep show no systemic illness. infected adult goats, however, may die. diagnosis may be difficult, and biological, serological, and histological methods are helpful. serological tests are the most readily available. complement fixation and the sabin-feldman antibody test may assist in diagnosis. antibodies found in fetuses are indicative of congenital infection and are typically detectable days after infection; fetal thoracic fluid is especially useful in demonstrating serological evidence of exposure. biological methods, such as tissue culture or inoculation of mice with maternal body fluids, or with postmortem or necropsy tissues, are more time-consuming and expensive. epizootiology and transmission. this protozoon is considered ubiquitous. fifty percent ( %) of adult western sheep and % of feedlot lambs have positive hemagglutination titers ( : or higher) (jensen and swift, ) . transmission among the definitive host is by ingestion of tissue cysts. necropsy findings. at necropsy, placental cotyledons contain multiple small white areas that are sites of necrosis, edema, and calcification. fetal brains may show nonspecific lesions such as coagulative necrosis, nonsuppurative encephalomyelitis, pneumonia, myocarditis, and hepatitis. histologically, granulomas with toxoplasma organisms may be seen in the retina, myocardium, liver, kidney, brain, and other tissues. impression smears of these tissues, stained appropriately (e.g., with giemsa), provide a rapid means of diagnosis. identification of the organism in tissue sections (especially of the heart and the brain) also confirms the findings. toxoplasma gondii is crescent-shaped, with a clearly visible nuclei, and will be found within macrophages. pathogenesis. the protozoon has three infectious stages: the tachyzoite, the bradyzoite, and the sporozoite within the oocyst. the definitive hosts, felids, become infected by ingesting cyst stages in mammalian tissues, by ingesting oocysts in feces, and by transplacental transfer. ingested zoites invade epithelial cells and eventually undergo sexual reproduction, resulting in new oocysts, which the cats will shed in the feces. cats rarely show clinical signs of infection. one cat can shed millions of oocysts in gm of feces, but the asymptomatic shedding takes place for only a few weeks in its life. oocysts sporulate in cat feces after day. ruminants are intermediate hosts of toxoplasmosis and become infected by ingesting sporulated oocyst-contaminated water or feed. as in the definitive host, the ingested sporozoite invades epithelial cells within the intestine but also further invades the bloodstream and is transported throughout the host. the organism migrates to tissues such as the brain, liver, muscles, and placenta. placental infection develops about days after ingestion of the oocysts. the damage caused by an infection is due to multiplication within cells. toxoplasma does not produce any toxin. campylobacter, chlamydia, and q fever. prevention and control. feline populations on source farms should be controlled. eliminating contamination of feed and water with cat feces is the best preventive measure. sporulated oocysts can survive in soil and other places for long periods of time and are resistant to desiccation and freezing. vaccines for abortion prevention in sheep are available in new zealand and europe. treatment. toxoplasmosis treatment is ineffective, although feeding monensin during pregnancy may be helpful (underwood and rook, ) . (monensin is not approved for this use in the unites states.) weak lambs that survive the first week after birth will mature normally and will not deliver toxoplasmainfected young. research complications. because toxoplasmosis is zoonotic, precautions must be taken when handling tissues from any abortions or neurological cases. infections in immunocompromised humans have been fatal. etiology. trichomoniasis is an insidious venereal disease of cattle caused by tritrichomonas (also referred to as trichomonas) fetus, a large, pear-shaped, flagellated protozoon. the organism is an obligate parasite of the reproductive tract, and it requires a microaerophilic environment to establish chronic infections. in the united states, it is now primarily a disease seen in western beef herds. there are many similarities between trichomoniasis and campylobacteriosis; both diseases cause herd infertility problems. clinical signs and diagnosis. clinical signs include infertility manifested by high nonpregnancy rates as well as periodic py-ometras and abortions during the first half of gestation. often the problem is not recognized until herd pregnancy checks indicate many "open," delayed-estrus, late-bred cows, or cows with postcoital pyometras. the abortion rate varies from % to %, and placentas will be expelled or retained. tritrichomonas fetus also causes mild salpingitis but this does not result in permanent damage. other than these manifestations, infection with t. fetus causes no systemic signs. diagnosis is based on patterns of infertility and pyometras. for example, pyometras in postcoital heifers or cows are suggestive of this pathogen. diagnostic methods include identifying or culturing the trichomonads from preputial smegma, cervicovaginal mucus, uterine exudates, placental fluids, or abomasal contents of aborted fetuses. other nonpathogenic protozoa from fecal contamination may be present in the sample. the trichomonad has three anterior flagellae, one posterior flagella, and an undulating membrane; it travels in fluids with a characteristic jerky movement. culturing must be done on specific media, such as diamond's or modified pastridge. real exposure from breeding bulls or cows or, in some cases, contaminated breeding equipment. necropsy findings. nonspecific lesions, such as pyogranulomatous bronchopneumonia of fetuses and placentitis, may be seen in aborted material; some cases will have no gross lesions. histologically, trichomonads may be visible in the fetal lung lesions and the placenta; those tissues are also the most useful for culturing. pathogenesis. tritrichomonas fetus colonizes the female reproductive tract, and subsequent clinical manifestations may be related to the size of the initial infecting dose. tritrichomonas fetus does not interfere with conception. embryonic death occurs within the first months of infection. affected cows will clear the infection over a span of months and maintain immunity for about months. infections in younger bulls are transient; apparently organisms are cleared by the bulls' immune systems and are dependent on exposure to infected females. older bulls become chronic carriers, probably because of the ability of t. fetus to colonize deeper epithelial crypts of the prepuce and penis. differential diagnosis. campylobacteriosis is the other primary differential for reduced reproductive efficiency of a herd. other venereal diseases should be considered when infertility problems are noted in a herd: brucellosis, mycoplasmosis, ureaplasmosis, and infectious pustular vulvovaginitis. in addition, management factors such as nutrition and age of heifers at introduction to the herd should be considered. heifers, cows, and breeding bulls are vaccinated subcutaneously twice at to week intervals, with the booster dose administered weeks before breeding season starts. similar timing is recommended for administration of the annual booster; a long, anamnestic response does not occur. bulls used for artificial insemination (ai) are screened routinely for t. fetus (and campylobacter) . ai reduces but does not eliminate the disease. the use of younger, vaccinated bulls is recommcmded in all circumstances. new animals should be tested before introduction to the herd. control measures also include culling affected cows or else removing them from the breeding herd for months to rest and clear the infection. culling chronically infected bulls is strongly recommended. treatment. imidazole compounds have been effective, but the use of these is not permitted in food animals in the united states. therapeutic immunizations are worthwhile when a positive diagnosis has been made. these will not curtail fetal losses but will shorten the convalescence of the affected cows and improve immunity of breeding bulls. research complications. trichomoniasis should be considered whenever natural service is used and fertility problems are encountered. nematodes are important ruminant pathogens that cause acute, chronic, subclinical, and clinical disease in adults and adolescents. the major helminths may cause gastroenteritis associated with intestinal hemorrhage and malnutrition. nematodiasis is associated with grazing exposure to infective larvae; animals procured for research may have had exposure to these helminths. mixed infections of these parasites are common. generally, older animals develop resistance to some of the species; thus, animals between about months and years of age are most susceptible to infection. because of the parasites' effects on the animals' physiology, infection in these younger animals is a major contributor to a cycle of poor nutrition and digestion, compromised immune responses, and impaired growth and development. diagnosis is primarily based on fecal flotation techniques; however, because many of these nematodes have similar-appearing ova, hatching the ova and identifying the larvae are often required (baermann technique). a number of anthelmintics can be used to interrupt nematode life cycles. see zajac and moore ( ) and pugh et al. ( ) for comprehensive reviews of treatment and control of nematodiasis. i. haemonchus contortus, h. placei (barber's pole worm, large stomach worm) . haemonchus contortus is the most important internal parasite of sheep and goats, and the brief description here focuses on the disease in the smaller ruminants. haemonchus contortus and h. placei infections do occur in younger cattle and are similar to the disease in sheep. haemonchus is extremely pathogenic, and the adults feed by sucking blood from the mucosa of the abomasum. severe anemia may lead to death. weight loss, decreased milk production, poor wool growth, and intermandibular and cervical edema due to hypoproteinemia ("bottle jaw") are also common clinical signs. diarrhea is not seen in all cases but may sometimes be severe or chronic. the life cycle is direct. under optimal conditions, a complete life cycle, from ingestion of larvae to eggs passed in the feces, occurs in weeks. embryonated eggs may develop into infective larvae within a week. hypobiotic (arrested) larvae may exist for several months in animal tissues, serving as a reservoir for future pasture contamination. periparturient increases in egg shedding by ewes contribute to large numbers of eggs spread on spring pastures ("spring rise"). resistance to common anthelmintics has developed; currently ivermectin or benzimidazole products are used, with a minimum of dosings given - weeks apart. levamisole is also used. in severe cases, animals may benefit from blood transfusions and iron supplementation. because animals may easily acquire infective larvae from ingestion of contaminated feed and from contaminated pastures, general facility sanitation and pasture management and rotation are important preventive and control measures. haemonchus contortus is susceptible to destruction by freezing temperatures and dry conditions. ii. ostertagia (teladorsagia) circumcincta (medium stomach worm). ostertagia circumcincta is also highly pathogenic for sheep and goats and, like haemonchus, attaches to the abomasal mucosa and ingests blood. the life cycle is comparable to that of haemonchus, including the phenomenon of hypobiosis. larvae are especially resistant to cool temperatures, however, and will overwinter on pastures. larvae-induced hyperplasia of abomasal epithelial glands results in a change of gastric ph from about . to near . , leading to decreased digestive enzyme activity and malnutrition. clinical syndromes are categorized as type or type . the former type is associated with infections acquired in fall or spring and is seen in younger animals. the latter type is associated with emergence of the arrested larvae during spring or fall. clinical signs include anemia, weight loss, decreased milk production, and unthriftiness. diarrhea is usually seen in type only; the symptoms of type are comparable to those of haemonchus infections. anthelmintic drug therapy is comparable to that for haemonchus, and drug resistance is also a problem with ostertagia. iii. ostertagia ostertagi (cattle stomach worm). ostertagia ostertagi is the most pathogenic and most costly of the cattle nematodes. ostertagia leptospicularis and o. bisonis also cause disease. the life cycle is direct, and egg shedding by the cattle may occur within - weeks of ingestion of infective larvae. hypobiosis is also a characteristic of o. ostertagi. in the initial steps of infection, the normal processes of the abomasum are profoundly disrupted and cells are destroyed as the larvae develop within and emerge from the glands. moroccan leather appearance is the term to describe the result of cellular hyperplasia and loss of cell differentiation. cycles of infection and morbidity depend on geographic location, climate, and production cycles. type cattle ostertagiasis is associated with ingestion of large numbers of infective larvae, occurs in animals less than years old, and causes diarrhea and anorexia. type ostertagiasis occurs in cattle - years old and older adults, is the result of the emergence and development of hypobiotic larvae, and in addition to signs seen with type , hypoproteinemia with development of submandibular edema, fever, and anemia is a clinical sign. treatment options include ivermectin, fenbendazole, and levamisole; all are effective against the arrested larvae. ostertagia is susceptible to desiccation but is resistant to freezing. iv. trichostrongylus vitrinus, t. axei, t. colubriformis (hair worms) . trichostrongylus species favor cooler conditions, and some larvae may overwinter. although the different species may affect different segments of the gastrointestinal tract, the nematode attaches to the mucosa and affects secretion and/or absorption. trichostrongylus vitrinus and t. colubriformis infect the small intestine of sheep and goats. trichostrongylus axei infects the abomasum of cattle, sheep, and goats and causes increases in abomasal ph similar to those seen with ostertagia. mucosal hyperplasia is not seen. the prepatent period is about weeks. affected animals display unthriftiness, anorexia, decreased milk production, weight loss, diarrhea, and dehydration. these worms show intermediate resistance to freezing temperatures and dry conditions. v. nematodirus spathiger, n. battus (thread-necked worms vii. strongyloides papillosus. strongyloides papillosus is a small-intestinal parasite of sheep and cattle. strongyloides has a different life cycle from that of many nematodes. the eggs, expelled in the feces, are larvated, and when they hatch, they form both free-living males and females or parasitic females only. the parasitic females may enter the gastrointestinal tract through oral ingestion, such as in milk during nursing, or through direct penetration of the skin. penetrating larvae enter the bloodstream and are transported to the lungs, where they penetrate the alveoli, are coughed up, and then swallowed to ultimately enter the gastrointestinal tract. adult females may reproduce in the small intestines by parthenogenesis. clinical signs associated with strongyloides include weight loss, diarrhea, unthriftiness, and dermatitis in cases where large numbers migrate through the skin. the current broad-spectrum anthelmintics are effective against strongyloides. strongyloides, bunostomum infection may involve oral ingestion or direct penetration of the skin (followed by tracheal migration and swallowing). the larvae mature in the small intestines and suck blood. larvae are susceptible to desiccation and freezing. heavy infection with bunostomum may result in anemia, diarrhea, intestinal hemorrhage, edema, and weight loss. ix. oesophagostomum columbianum, o. venulosum (nodule worms) . oesophagostomum spp. primarily infect the large intestine and occasionally the distal small intestine, causing nodule worm disease, or simply gut. oesophagostomum columbianum and o. venulosum infect sheep and cattle. these nematodes may affect sheep from months to years of age, and the prepatent period is about weeks. larvae are highly sensitive to freezing and desiccation and rarely overwinter. larvae penetrate the large-intestinal mucosa but occasionally move into the deeper areas of the intestinal wall near the serosa. the resultant inflammatory reaction may lead to the formation of a caseous nodule that may mineralize over time. intestinal lesions may accelerate peristalsis, leading to diarrhea, or may inhibit peristalsis (later stages), resulting in constipation. clinical signs include weakness, unthriftiness, alternating episodes of diarrhea and constipation, and severe weight loss. nodular lesions are typical at necropsy. x. chabertia ovis (large-mouth bowel worm). chabertia ovis is a minor colon parasite of sheep, goats, and cattle and is seen primarily in sheep. signs of infection are not usually seen in cattle. prepatent periods are up to days. heavy infection, which may result from as few as worms located at the proximal end of the colon, may lead to hemorrhagic mucoid diarrhea, weight loss, weakness, colitis, and mild anemia. xi. trichuris (whipworms). trichuris spp. are mildly pathogenic nematodes and are usually attached to the cecal mucosa. trichuris has a rather long prepatent period, extending from to months. the oval eggs are double-operculated and survive well in pasture environmental extremes. the adult worms also have a characterisitic morphology, with one thicker end appearing as a whip handle. the nematodes cause a minor cecitis and will feed on blood. clinical infection is rare and results in diarrhea with mucus and blood. treatment and prevention methods are similar to those for other nematodes. xii. dictyocaulus (lungworms). dictyocaulus spp., or lungworms, are nematodes that cause varying clinical signs in ruminants. in sheep, dictyocaulus filaria, protostrongylus rufescens, and muellerius capillaris cause disease; dictyocaulus is the most pathogenic. goats are infected by the same species as sheep, but infections are uncommon. dictyocaulus viviparus is the only lungworm found in cattle, causing "fog fever." infections with these parasites in the united states tend to be associated with cooler, moister climates. lungworms induce a severe parasitic bronchitis (known as husk, or verminous pneumonia) in sheep between approximately and months of age. sheep infected with any of the lungworm species may display coughing, dyspnea, nasal discharge, weight loss, unthriftiness, and occasionally fever. coughing and dyspnea are symptoms in goats. diagnosis is suggested by persistent coughing and nasal discharge and is confirmed by identifying larvae in the feces or adults in pathological samples. the baermann technique, involving prompt examination of room-temperature feces, is usually used; zinc sulfate flotation is also used. dictyocaulus has a direct life cycle. the adult worms reside in the large bronchi. dictyocaulus produces embryonated eggs that are coughed up and swallowed; the eggs then hatch in the intestines, and larvae are expelled in the feces. the expelled larvae are infectious in about - days and, after ingestion, penetrate the intestinal mucosa and move through the lymphatics and blood into the lungs, where they develop into adults in about weeks. dictyocaulus filaria causes an especially severe bronchitis in sheep. protostrongylus inhabits smaller bronchioles. muellerius is of minor pathogenicity. protostrongylus and muellerius require the snail or slug as an intermediate host. infection occurs through ingestion of infected snails; infections are less likely than those caused by the direct ingestion of dictyocaulus larvae. immunity wanes over a year. viral and bacterial respiratory tract infections may be associated with the parasitic infection. more severe illness is seen after infections with cooperia and ostertagia, because of a synergism between the nematodes even if the cattle are not currently infected with those parasites. hypobiosis (arrested development of immature worms in lung tissue) is associated with dictyocaulus infections; cattle will be silent carriers, showing no clinical signs and serving as a means for the infection to survive over winter or a dry season. pastures can be heavily contaminated during the next grazing season. necropsy lesions include bronchiolitis and bronchitis, atelectasis, and hyperplasia of peribronchiolar lymphoid tissue. nematodes frequently reside in the bronchi of the diaphragmatic lung lobes and are frequently enmeshed with frothy exudate. prevention and control of the disease involve appropriate pasture management. elimination of intermediate hosts is important in sheep and goat pastures. in a laboratory setting, animals may be procured that are already harboring the disease. infected animals can be treated with anthelmintics such as ivermectin or levamisole. muellerius tends to be resistant to levamisole. there is no anthelmintic currently approved for goats, but fenbendazole, administered weeks apart, has been effective for all three tapeworms are rarely of clinical or economic importance. in younger animals, heavy infections result in potbellies, constipation or mild diarrhea, poor growth, rough coat, and anemia. moniezia expansa, and less commonly moniezia benedini, inhabit the small intestines of grazing ruminants. moniezia expansa has the widest distribution of the tapeworm species in north america. soil mites (galumna spp. and oribatula spp.) contribute to the life cycle as intermediate hosts, a period that lasts up to weeks. cysticercoids released from the mites are grazed, pass into the small intestines, and mature. no clinical or pathological sign is usually observed with moniezia infection; diagnosis is made by observing the characteristic triangularshaped eggs in fecal flotation examinations. infection is treated with cestocides. thysanosoma actinoides, or the fringed tapeworm, is a cestode that resides in the duodenum, bile duct, and pancreatic duct of sheep and cattle raised primarily west of the mississippi river in the united states. thysanosoma is of the family anoplocephalidae. the life cycle is indirect, and the intermediate host is the psocid louse. larval forms, or cysticercoids, are ingested by grazing animals, and the prepatent period is several months. typically, no clinical signs are observed with thysanosoma infection; nonetheless, liver damage, resulting in liver condemnation at slaughter, occurs. necropsy lesions include bile and/or ductal hyperplasia and fibrosis. thysanosoma is diagnosed premortem by identifying the gravid segments in the feces. ii. abdominal or visceral cysticercosis. abdominal or visceral cysticercosis is an occasional finding at slaughter. the socalled bladder worms typically affect the liver or peritoneal cavity and are the larval form of taenia hydatigena, the common tapeworm of the dog family. taenia hydatigena resides in the small intestines of canids, and its gravid segments, oncospheres, contaminate feed and water sources. after ingestion, the larvae penetrate the intestinal mucosa, are transported via the bloodstream to the liver, and cause migration tracts throughout the liver parenchyma. the larvae may leave the liver and migrate into the peritoneal cavity, where they attach and develop over the next - months into small fluid-filled bladders. the life cycle is completed only after these bladders are ingested by a carnivore, thus completing the maturation of the adult tapeworms. although larval migration may cause nonspecific signs such as anorexia, hyperthermia, and weight loss, affected animals are usually asymptomatic. at necropsy, the bladder worms will be observed attached to the peritoneal or organ surfaces. migration tracts may result in fibrosis and inflammation. diagnosis is usually made at necropsy. because of the migration through the liver, fasciola hepatica is a differential diagnosis. minimizing exposure to canine feces-contaminated feeds and water effectively interrupts the life cycle. research animals may have been exposed prior to purchase. echinococcosis, like cysticercosis, is an occasional finding at slaughter or necropsy. the hydatid cyst is the larval intermediate of the adult tapeworm echinococcus granulosus, which resides in the small intestines of dogs and wild canids. embryonated ova are expelled in the feces of the primary host and are ingested by herbivores, swine, and potentially humans. the eggs hatch in the gastrointestinal tract, and the oncospheres penetrate the mucosal lining, enter the bloodstream, and are transported to various organs such as the liver and lungs. the cystic structure develops and potentially ruptures, forming new cystic structures. clinically, echinococcosis presents minimal clinical signs; unthriftiness or pneumonic lesions may be associated with infected organs. cysts are typically observed at necropsy. prevention should be aimed at decreasing fecal contamination of feed and water by canids. additionally, tapeworm-infected dogs can be treated with standard tapeworm therapies. treatment of infected ruminants is uncommon. iv. gid. coenuris cerebralis, the larval form of the canid tapeworm taenia (multiceps) multiceps, is the causative agent of the rare condition called gid. the disease occurs in ruminants as well as many other mammalian species. the larval parasite, ingested from fecal-contaminated food and water, invades the brain and spinal cord and develops as a bladder worm that causes pressure necrosis of the nervous tissues. the resultant signs of hyperesthesia, meningitis, paresis, paralysis, ataxia, and convulsions are observed. diagnosis is usually made at necropsy. eliminating transfer from the canid hosts prevents the disease. the cercariae leave the intermediate host, swim to grassy vegetation, lose their tail, and become a cystlike metacercaria. the metacercariae may remain in a dormant stage on the grass for months or longer until ingested by a ruminant. the ingested metacercariae penetrate the small-intestinal wall and migrate through the abdominal cavity to the liver. there they locate in a bile duct, mature, and remain for up to years. acute liver fluke disease is related to the damage caused by the migration of immature flukes. migratory flukes may lead to liver inflammation, hemorrhage, necrosis, and fibrosis. fascioloides magna infections in sheep and goats can be fatal as the result of just one fluke tunneling through hepatic tissue. in cattle, infections are often asymptomatic because of the host's encapsulation of the parasite. liver fluke damage may predispose to invasion by anaerobic clostridium species such as c. novyi that could lead to fatal black disease or bacillary hemoglobinuria. chronic disease may result from fluke-induced physical damage to the bile ducts and cholangiohepatitis. blood loss into the bile may lead to anemia and hypoproteinemia. liver damage also is evidenced by increases in liver enzymes such as y-glutamyl transpeptidase (ggt). persistent eosinophilia is also seen with liver fluke disease. other clinical signs of liver fluke disease include anorexia, weight loss, unthriftiness, edema, and ascites. at necropsy, livers will be pale and friable and may have distinct migration tunnels along the serosal surfaces. bile ducts will be enlarged, and areas of fibrosis will be evident. diagnosis can be made from clinical signs and postmortem mites cause a chronic dermatitis. the principal symptom of these infections is intense pruritus. in addition, papules, crusts, alopecia, and secondary dermatitis are seen. anemia, disruption of reproductive cycles, and increased susceptibility to other diseases may also occur. mites are rare in ruminants in the united states, but infections of sarcoptes and psorergates mange must be reported to animal health officials. ruminants in poorly managed facilities are generally the most susceptible to infection, and infections are more frequent during winter months. diagnosis is based on signs, examination of skin scrapings, and response to therapy. no effective treatment for demodectic mange in large animals has been found. the differential for mite infestations is pediculosis. several genera of mites may affect sheep. these have been eradicated from flocks in the united states or are very rare and include psoroptes ovis (common scabies), sarcoptes scabiei (head scabies, barn itch), psorergates ovis (sheep itch mite), chorioptes ovis (foot scabies, tail mange), and demodex ovis (follicular mange). goats can also be infected by sarcoptic, chorioptic, and psoroptic mange. the scabies mite sarcoptes rupicaprae invades epidermal tissue and causes focal pruritic areas around the head and neck. the chorioptic mite, either chorioptes bovis or c. caprae, does not invade epidermal tissue but rather feeds on dead skin tissue. the chorioptic mite prefers distal limbs, the udder, and the scrotum and can be a significant cause of pruri-tus. the psoroptic mite psoroptes cuniculi commonly occurs in the ear canal and causes head shaking and scratching. repeated treatments of lime sulfur, amitraz, or ivermectin may be effective (smith and sherman, ) . goats are also susceptible to demodectic mange caused by demodex caprae. adult mites invade hair follicles and sebaceous glands. pustules may develop with secondary bacterial infection. psoroptes bovis continues to be present in cattle in the united states, although it has been eradicated from sheep. chorioptes bovis typically infects lower hindlimbs, perineum, tail, and scrotum but can become generalized. the sarcoptic mange mite s. scabei can survive off the host, so fomite transmission is a factor. the mange usually begins around the head but then spreads. this parasite can be transmitted to humans. demodex bovis infects cattle; nodules on the face and neck are typical. demodex bovis infections may resolve without treatment. lindane, coumaphos, malathion, and lime sulfur are used to treat psoroptes and psorergates. ivermectin is effective against sarcoptes and is approved for use in cattle. lice that infect ruminants are of the orders mallophaga, biting or chewing lice, and anoplura, sucking lice. these are wingless insects. members of the mallophaga are colored yellow to red; members of the anoplura are blue gray. lice produce a seasonal (winter-to-spring), chronic dermatitis. in sheep, biting lice include damalinia (bovicola) ovis (sheep body louse). sucking lice that infect sheep include linognathus ovillus (blue body louse) and l. pedalis (sheep foot louse). in goats, biting lice infection are caused by d. caprae (goat biting louse), d. limbatus (angora goat biting louse), and d. crassipes. suckir/g louse infections in goats are caused by l. stenopis and l. africanus. damalinia bovis is the cattle biting louse. sucking lice include l. vituli, solenopotes capillatus, haematopinus eurysternus, and h. quadripertusus. pruritus is the most common sign and often results in alopecia and excoriation. the host's rubbing and grooming may not correlate with the extent of infestation. hairballs can result from overgrooming in cattle. in severe cases, the organisms can lead to anemia, weight loss, and damaged wool in sheep and damaged pelts in other ruminants. young animals with severe infestations of sucking lice may become anemic or even die. pregnant animals with heavy infestations may abort. in sheep infected with the foot louse, lameness may result. lice are generally species-specific. those infecting ruminants are usually smaller than mm. goats may serve as a source of infection for sheep by harboring damalinia ovis. transmission is primarily by direct contact between animals. transmission can also occur by attachment to flies or by fomites. some animals are identified as carriers and seem to be particularly susceptible to infestations. biting or chewing lice inhabit the host's face, lower legs, and flanks and feed on epidermal debris and sebaceous secretions. sucking lice inhabit the host's neck, back, and body region and feed on blood. lice eggs or nits are attached to hairs near the skin. three nymphal stages, or instars, occur between egg and adult, and the growth cycle takes about month for all species. lice cannot survive for more than a few days off the host. all ruminant mite infestations are differentials for the clinical signs seen with pediculosis. animals that are carriers should be culled, because these individuals may perpetuate the infection in the group. lice are effectively treated with a variety of insecticides, including coumaphos, dichlorvos, crotoxyphos, avermectin, and pyrethroids. label directions should be read and adhered to, including withdrawal times. products should not be used on female dairy animals. treatments must be repeated at least twice at intervals appropriate for nit hatches (about every days) because nits will not be killed. fall treatments are useful in managing the infections. systemic treatments in cattle are contraindicated when there may be concurrent larvae of cattle grubs (hypoderma lineatum and h. bovis). back rubbers with insecticides, capitalizing on self-treatment, are useful for cattle. sustained-release insecticide-containing ear tags are approved for use in cattle. etiology. ruminants are susceptible to many species of ixodidae (hard-shell ticks) and argasidae (softshell ticks). many diseases, including anaplasmosis, babesiosis, and q fever are transmitted by ticks. clinical signs and diagnosis. tick infestations are associated with decreased productivity, loss of blood and blood proteins, transmission of diseases, debilitation, and even death. feeding sites on the host vary with the tick species. ticks are associated with an acute paralytic syndrome called tick paralysis. this disease is characterized by ascending paralysis and may lead to death if the tick is not removed before the paralysis reaches the respiratory muscles. diagnosis is based on identification of the species. epizootiology and transmission. ticks are not as host-specific as lice. ticks are classified as one-host, two-host, or three-host; this refers to whether they drop off the host between larval and nymphal stages to molt. pathogenesis of tick infestations. patterns of feeding on the host differ between argasidae and ixodidae. the former feed repeatedly, whereas the latter feed once during each life stage. pathogenesis of tick paralysis. following a tick-feeding period of - days, the tick salivary toxin travels hematogenously to the myoneural junctions and spinal cord and inhibits nerve transmission. removal of the ticks reverses the syndrome unless paralysis has migrated anteriorly to the respiratory centers of the medulla. in these cases, death due to respiratory failure occurs. insecticides. ticks can be treated using systemic or topical h. other parasites i. nasal bots (nasal myiasis, head grubs). nasal myiasis causes a chronic rhinitis and sinusitis. the disease is caused by the larval forms of the botfly oestrus ovis. the botfly deposits eggs around the nostrils of sheep. the ova hatch, and the larvae migrate throughout the nasal cavity and sinuses, feeding on mucus and debris. in - months, the larvae complete their growing phase, migrate back to the nasal cavity, and are sneezed out. the mature larvae penetrate the soil and pupate for - . months and emerge as botflies. clinically, early in the disease course, animals display unique behaviors such as stamping, snorting, sneezing, and rubbing their noses against each other or objects. hypersensitivity to the larvae occurs (dorchies et al., ) . later, mucopurulent nasal discharges associated with the larval-induced inflammation of mucosal linings will be observed. at necropsy, larvae will be observed in the nasal cavity or sinuses. mild inflammatory reactions, mucosal thickening, and exudates will accompany the larvae. the disease is diagnosed by observing the behaviors or identifying organisms at necropsy. up to % of a flock will potentially be infected; treatment should be employed on the rest of the flock. ivermectins and other insecticides will eliminate the larvae; but treatment should be done in the early fall, when larvae are small. fly repellents may be helpful at preventing additional infections. ii. screwworm flies. cochliomyia hominivorax (callitroga americana) is the the screwworm that causes occasional disease in the southwestern united states along the mexico border. eradication programs have been pursued, and the disease is reportable. large greenish flies lay large numbers of white eggs as shinglelike layers at the edges of open wounds (including docking and castration sites), soiled skin, or abrasions. eggs hatch within hr. larvae are obligate parasites of living tissue, and the cycle is perpetuated because the increasingly large wound continues to be attractive to the next generation of flies. larvae eventually drop off, pupate best in hot climates, and hatch in weeks. large cavities in parasitized tissue are formed, and lesions are characterized by malodor, large volumes of brown exudate, and necrosis. single animals or entire herds may be affected. treatment is intensive, with dressings and larvicidal applications. if there is no intervention, the host succumbs to secondary infections and fluid loss. effective current control regimens include subcutaneous injection of ivermectin and programs that release sterile male flies. iii. sheep keds ("sheep ticks"). in sheep and goats, sheep keds produce a chronic irritation and dermatitis with associated pruritus. the disease is caused by melophagus ovinus, which is a fiat, brown, blood-sucking, wingless fly; the term sheep tick is incorrectly used. the adult fly lives entirely on the skin of sheep. females mate and produce - larvae following a gestation of about - days. the larvae attach to the wool or hair and then pupate for about weeks. the adult female feeds on blood and lives for - months; the life cycle is completed in about - weeks. infection is highest in fall and winter. pruritus develops around the neck, sides, abdomen, and rump. in severe cases, anemia may occur. keds can transmit bluetongue virus. keds are diagnosed by gross or microscopic identification. ivermectin or other insecticides are useful treatment agents. portant, other immune mechanisms are not well understood. immunity may not be of long duration. recovery is enhanced by correcting nutritional deficiencies and improving housing and ventilation problems. a number of topical treatments, such as - % lime-sulfur solution, % captan, iodophors, thiabendazole, and . % sodium hypochlorite, can be used. in severe cases, systemic therapy with griseofulvin may be successful. prevention and control. the animals' environment and overall physical condition should be reassessed with particular attention to ventilation, crowding, sanitation, and nutrition. pens should be thoroughly cleaned and disinfected. research complications. ringworm is a zoonotic disease. etiology. dermatophytosis, or infection of the keratinized layers of skin, is caused mostly by species of the genera trichophyton and microsporum. the primary causes in sheep are t. mentagrophytes and t. verrucosum. in goats, the agents are t. mentagrophytes, m. canis, m. gypseum, t. verrucosum, t. schoenleinii, and epidermophyton floccosum. in cattle, t. verrucosum is the primary causative agent. dermatophytosis is a common fungal infection of the epidermis of cattle and is less common in sheep and goats. clinical signs and diagnosis. multiple, gray, crusty, circumscribed, hyperkeratotic lesions are characteristic of infection. lesions will vary in size. in all ruminants, lesions will be around the head, neck, and ears. in goats and cattle, lesions will extend down the neck, and in cattle, lesions develop particularly around the eyes and on the thorax. cattle lesions are unique in the marked crustiness, which progressively appears wartlike. hair shafts become brittle and break off. intense pruritus is often associated with the alopecic lesions. the disease can be diagnosed by microscopic identification of hyphae and conidia on the hairs following skin scraping and % potassium hydroxide digestion. dermatophyte test media (dtm) cultures are the most reliable means to diagnose the fungus. broken hairs from the periphery of the lesion are the best sources of the fungus. epizootiology and transmission. younger animals are more susceptible, and factors such as crowding, indoor housing, warm and humid conditions, and poor nutrition are also important. transmission is by direct contact or by contact with contaminated fomites, such as equipment, fencing, or feed bunks. pathogenesis. incubation can be as long as weeks. the organisms invade and multiply in hair shafts. treatment. spontaneous recovery occurs in all species in - months. although cell-mediated immunity is considered im- inverted eyelids are a common inherited disorder of lambs and kids of most breeds. generally, the lower eyelid is affected and turns inward, causing various degrees of trauma to the conjunctiva and cornea. young animals will display tearing, blepharospasm, and photophobia initially. if the disorder is left uncorrected, corneal ulcers, perforating ulcers, uveitis, and blindness may occur. placing a suture or a surgical staple in the lower eyelid and the cheek, effectively anchoring the lid in an everted position, successfully treats the condition. the procedure likely results in the formation of some degree of scar tissue within the lower lid, because when the suture eventually is removed, the condition rarely returns. other treatments include the injection of a "bleb" of penicillin in the lid, regular manual correction over a -day period early in the animal's life, and application of ophthalmic ointments, powders, and solutions. boric acid or % argyrol solutions have been used as treatments. because of the genetic predisposition, prevention of the condition requires removal of maternal or paternal carriers. [ -mannosidosis is an autosomal recessive lysosomal storage disease of goats. the disease affects kids of the nubian breed and is identified by intention tremors and difficulty or inability of newborns to stand. cells of affected animals are vacuolated because of a lack of lysosomal hydroxylase, which results in accumulation of oligosaccharides. newborn kids are unable to rise, and they have characteristic flexion of the carpal joint and hyperextension of the pastern joint. kids are born deaf and with musculoskeletal deformities such as domed skull, small narrow muzzle, small palpebral fissures, enophthalmos, and depressed nasal bridge (smith and sherman, ) . carrier adults can be identified by plasma measurements of [ -mannosidase activity. caprine congenital myotonia is an inherited autosomal dominant disease that affects voluntary striated skeletal muscles. goats with this disease are commonly known as fainting goats. "fainting" is actually transient spasms of skeletal musculature brought about by visual, tactile, or auditory stimuli (smith and sherman, ) . muscle fiber membranes appear to have fewer chloride channels than normal, resulting in decreased chloride conduction across the membrane, with subsequent increased membrane excitability and repetitive firing (smith and sherman, ) . contractions of skeletal muscle are sustained for up to min. kids exhibit the condition by weeks of age, and males appear to exhibit more severe clinical signs than females (smith and sherman, ) . electromyographic studies produce an audible "dive-bomber" sound characteristic of hyperexcitable cell membranes (smith and sherman, ) . i. congenital erythropoietic porphyria. congenital erythropoietic porphyria (cep) is an autosomal recessive disease of cattle seen primarily in holsteins, herefords, and shorthorns. the disease also occurs in limousin cattle, humans, and some other species. in the homozygous recessive animal, symptoms of the disease may vary from mild to severe and occur at different times of the year and in different ages of animals. a reddish brown discoloration of teeth and bones is a characteristic of the disease, as is discolored urine, general weakness and failure to thrive, photosensitization, and photophobia. bones are more fragile compared with bones of normal animals. a regenerative anemia occurs as the result of the shortened life span of erythrocytes, due to accumulations of porphyrins. the genetic defect is associated with low activity of an essential enzyme, uroporphyrinogen iii synthase, in the porphyrin-heme synthesis pathway in erythrocytic tissue. the ranges in the presentation of the disease are believed to be related to varying cycles of porphyrin synthesis. porphyrins are excreted in varying amounts in the urine and the discoloration fluoresces under a wood's lamp. diagnosis is based on these clinical and visible signs of porphyria; skin biopsy provides definitive diagnosis. heterozygotes may have milder symptoms. many other genetic defects, in all major organ systems, have been described in numerous breeds of cattle and are described in detail elsewhere ("large animal internal medicine," ) . in many cases, the genetic basis has been clarified, and associated defects also noted. many defects are reported in particular breeds, but as crossbreeding increases and new breeds are developed, these traits are appearing in these animals. the bovine genome continues to be further characterized, and more linkage maps and gene locations are forthcoming (womack, ) . some bovine genetic defects are also regarded as models of genetic disease, such as leukocyte adhesion deficiency of holstein cattle. some of the more commonly reported defects include syndactyly in holsteins and other breeds and polydactyly in simmentals; lysosomal storage diseases such as a-mannosidosis in some beef breeds; enzyme deficiencies such as citrullinemia in holsteins; and progressive degenerative myeloencephalopathy ("weaver") in brown swiss. ii. goiter of sheep. a defect in the synthesis of thyroid hormone has been identified in merino sheep (radostits et al., ) . lambs born with the defect have enlargement of the thyroid gland, a silky appearance to the wool, and a high degree of mortality. edema, bowing of the legs, and facial abnormalities have also been noted in animals with this disorder. immaturity of the lungs at birth causes neonatal respiratory distress and resuits in dyspnea and respiratory failure. spider lamb syndrome is an inherited, often lethal, musculoskeletal disorder primarily occurring in suffolk and hampshire breeds. severely affected lambs die shortly after birth. animals that survive the perinatal period develop angular limb deformities, scoliosis, and facial deformities. with time, affected animals become debilitated, exhibit joint pain, and develop neurological problems associated with the spinal abnormalities. radiologically, secondary ossification centers--especially the physis, subchondral areas, and cuboidal bonesmare affected. abnormal endochondral ossification leads to excess cartilage formation, notably apparent in the elbows. lambs will typically display abnormally long limbs, medial deviation of the carpus and tarsus, flattening of the sternum, scoliosis/kyphosis of the vertebrae, and a rounded nose. muscle atrophy is common. diagnosis can be based on typical clinical signs, which are similar to those seen with marfan syndrome in humans (rook et al., ) . long-term survival is rare; treatment is unsuccessful. i. abomasal and duodenal ulcers. abomasal and duodenal ulcers occur more frequently in calves and adult cattle than in sheep and goats. like rumenitis, abomasal and duodenal ulcers may be associated with lactic acidosis. concurrent disease, such as salmonellosis, bluetongue, or overuse of anti-inflammatory drugs, or recent shipping or environmental stresses may also lead to ulcer formation. copper deficiency, dietary changes, mycotic infections, clostridium perfringens abomasitis, and abomasal bezoars are associated with this disease in calves. in older adult cattle, abomasal lymphosarcoma may be the underlying condition. gastric acid hypersecretion in conjunction with insufficient gastric mucous secretion will physically destroy the gastric epithelium. deep ulceration may cause serious hemorrhage and/or perforation with peritonitis. chronic hemorrhage may lead to anemia. although ulcers are often asymptomatic in calves, perforation with peritonitis is more common than hemorrhage. dark feces or melena and abdominal pain may be observed. arched back, restlessness, kicking at the abdomen, bruxism, and anorexia are common signs of abdominal pain. fecal occult blood is as an easy diagnostic test. treatment includes gastrointestinal protectants and histamine antagonists. anemia may be symptomatically treated with parenteral iron injections and anabolic steroids. preventive measures in cattle herds include ensuring optimal passive immunity for calves, minimizing stress to calves, and striving for a herd free of bovine leukosis virus. ii. abomasal emptying defect. abomasal emptying defect of sheep is a sporadic syndrome associated with abomasal distension and weight loss. suffolks tend to be especially predisposed, although the disease has been diagnosed in hampshires, columbias, and corriedales. the mechanism of the disease is unknown. affected animals will exhibit a gradual weight loss with a history of normal appetites. feces will continue to be normal. ventral abdominal distension associated with abomasal accumulation of feedstuffs will be apparent in many of the animals. diagnosis is primarily based on history and clinical signs. elevations in rumen chloride concentrations (> meq/liter) are commonly found. radiography or ultrasonography may be helpful at identifying the distended abomasum. abomasal emptying defect is usually eventually fatal. medical treatment with metoclopramide and mineral oil may be helpful in early disease. iii. abomasal displacement. displaced abomasum (da) is a sporadic disorder usually associated with multiparous -to year-old dairy cows in early lactation, but the condition can occur even in young calves. displacement to the right (rda) may be further complicated by torsion (rta), a surgical emergency. left displacement (lda) is more common than rda. clinical signs include anorexia, lack of cud chewing, decreased frequency of ruminal contractions, shallow respirations, increased heart rate, treading, and decreased milk production. diagnosis is based on characteristic areas of tympanic resonance during auscultation-percussion of the lateral to lateral-ventral abdomen ("pings"), ruminal displacement palpated per rectum, and clinical signs. cow-side clinical chemistry findings include hypoglycemia and ketonuria; more extensive evaluations will often indicate moderate to severe electrolyte and acid-base abnormalities. da occurs because of gas accumulation within the viscus, and the abomasum "floats" up from its normal ventral location to the lateral abdominal wall. no exact cause of da has been identified, but it is commonly associated with stress; high levels of concentrate in the diet, leading to forestomach atony; and many disorders, including lack of regular exercise, mastitis, hypocalcemia, retained placenta, metritis, or twins. factors such as body size and conformation indicate the possibility of genetic predisposition. treatments include surgical and nonsurgical techniques for lda; the former has a better chance of per-manent correction. emergency surgery is necessary for rta; the disorder is fatal within hr. recurrence is rare after surgical correction. electrolyte and acid-base imbalances are likely in severe cases and especially with rta. prevention includes reducing stress, taking greater care in the introduction and feeding of concentrates, and reducing incidence of predisposing diseases noted above (rohrbach et al., ) . fat cow syndrome is seen in peri-or postparturient overconditioned or obese multiparous dairy cows. factors in the development of the condition include negative energy balance related to the normal decreased dry matter intake as parturition approaches; hormonal changes associated with parturition; and concurrent diseases of parturition that decrease feed intake and increase energy needs. the possible concurrent diseases include metritis, retained fetal membranes, mastitis, parturient paresis, and displaced abomasum. signs are nonspecific and include depression, anorexia, and weakness. prognosis is usually guarded. diagnosis is based on herd management, the animal's condition, ketonuria, and clinical signs. in prepartum cattle and in lactating cows, blood levels of nonesterified fatty acids (nefa) greater than ~teq/liter and - ~teq/liter, respectively, are abnormal (gerloff and herdt, ) . triglyceride analysis of liver biposy specimens are useful. in affected cows, body fat is mobilized, in the form of nefa in response to the energy demands. hepatic lipidosis occurs rapidly as the nefa are converted into hepatic triglycerides. the ability of the liver to extract the albumin-bound nefa from the blood is better than that of other tissues that need and can also use nefa as an energy source. treatment for any concurrent diseases must be pursued aggressively, as well as measures to increase and stabilize blood glucose, decrease nefa production, and increase forestomach digestion to improve production of normally metabolized volatile fatty acids. therapeutic measures include intravenous glucose drips, insulin (nph or lente) injections every hr, and transfaunation of ruminal fluid from a normal cow. prevention includes minimizing stress to lategestation cows. dry and lactating cows should be maintained separately; their energy, protein, and dry matter requirements are very different. cows with prolonged lactation or delayed breeding should be managed to prevent weight gain. i. bloat. bloat or tympanites refers to an excessive accumulation of gas in the rumen. the condition most frequently occurs in animals that have been recently fed abundant quantities of succulent forages or grains. bloat is classified into two broad categories: frothy bloat and free-gas bloat. frothy bloat is associated with ingestion of feeds that produce a stable froth that is not easily expelled from the rumen. fermentation gases such as co , ch , and minor gases such as n , , h , and h s incorporate into the froth, overdistend the rumen, and eventu-ally compromise respiration by limiting diaphragm movement. the froth is often derived from a combination of salivary mucoproteins, protozoal or bacterial proteins, and proteins, pectins, saponins, or hemicellulose associated with ingested leaves or grain. typical foodstuffs that cause frothy bloat include green legumes, leguminous hay (alfalfa, clover), or grain (especially barley, corn, and soybean meal). free-gas bloat is less related to feeds ingested; rather, it is caused by rumen atony or by physical or pathological problems that prevent normal gas eructation. some examples of causes of free-gas bloat are esophageal obstructions (foreign bodies, tumors, abscesses, and enlarged cervical or thoracic lymph nodes), vagal nerve paralysis or injury, and central nervous system conditions that affect eructation reflexes. clinically, the animal will exhibit rumen distension, and tympany will be observed in the left paralumbar fossa. additional signs may include colic-like pain of the abdomen and dyspnea. passage of a stomach tube helps to differentiate between free-gas bloat and frothy bloat; and with free-gas bloat, expulsion of gas through the stomach tube aids in treatment of the disorder. once rumen distension is alleviated with free-gas bloat, the underlying cause must be investigated to prevent recurrence. frothy bloat is more difficult to treat, because the foam blocks the stomach tube. addition of mineral oil, household detergents, or antifermentative compounds via the tube may help break down the surface tension, allowing the gas to be expelled. in acute, life-threatening cases of bloat, treatment should be aimed at alleviating rumen distension by placing a trocar or surgical rumenotomy into the rumen via the paralumbar fossa. limiting the consumption of feedstuffs prone to induce bloat can prevent the disease. additionally, poloxalene or monensin will decrease the incidence of frothy bloat. ii. lactic acidosis. lactic acidosis, or rumen acidosis, is an acute metabolic disease caused by engorgement of grains or other highly fermentable carbohydrate sources. the disease is most frequently related to a rapid change in diet from one containing high roughage to one containing excessive carbohydrates. diet components that predispose to acidosis include common feed grains; feedstuffs such as sugar beets, molasses, and potatoes; by-products such as brewer's grains; and bakery products. biochemically, ingestion of large amounts of the carbohydrate-rich diet causes the normally gram-negative rumen bacterial populations to shift to gram-positive streptococcus and lactobacillus species. the gram-positive organisms efficiently convert the starches to lactic acid. the lactic acid acidifies the rumen contents, leading to rumen mucosal inflammation, and increases the osmolality of rumen fluids, leading to sequestration of fluids and osmotic attraction of plasma and tissue fluid to the rumen. lactic acid-induced rumenitis predisposes the animal to ulcers, to liver abscesses from "absorbed" bacterial pathogens, to laminitis from absorbed toxins, and to polioencephalomalacia from the inability of the new rumen bacterial populations to produce sufficient thiamine needed to maintain normal nervous system function. clinically, animals will become anorexic, depressed, and weak within - days after the initial insult. incoordination, ataxia, dehydration, hemoconcentration, rapid pulse and respiration, diarrhea, abdominal pain, and lameness will also be noted rumen distension and an acetone-like odor to the breath, milk, or urine may also be observed. diagnosis is based on history and clinical signs. blood, urine, or milk ketones can be detected (moore and ishler, ) . additionally, rumen ph, which is normally above . , will drop to less than . and in severe cases may achieve levels as low as . . similarly, urine ph will become acidic, blood ph will drop below . , and hematocrit will appear to increase due to the relative hemoconcentration. necropsy findings will be determined by secondary conditions. the primary lactic acidosis will cause swelling and necrosis of rumen papillae and abomasal hemorrhages and ulcers. treatment must be applied early in the syndrome. in early hours of severe carbohydrate engorgement, rumenotomy and evacuation of the contents are appropriate. the t patient should be given mineral oil and antlfermentatlves to prevent the continued conversion of starches to acids and the absorption of metabolic products. bicarbonate or other antacids like magnesium carbonate or magnesium hydroxide introduced into the rumen will aid in adjusting rumen ph. furthermore, animals can be given oral tetracycline or penicillin, which will decrease the gram-positive bacterial population. iii. rumen parakeratosis. parakeratosis is a degenerative condition of the rumen mucosa that leads to keratinization of the papillary epithelium excessive and continuous feeding of diets low in roughage causes the mucosal changes generally, this condition is seen in feedlot lambs and steers that are fed an all-grain diet. clinically, animals may exhibit only poor rates of gain, due to changes in the absorptive capacity of the injured mucosa. at necropsy, papillae will be thickened and rough. they will frequently be dark in color, and multiple papillae will clump together. abscessation may be observed. histopathologically, papilla surfaces will have hyperkeratinization of the squamous epithelium. chronic laminitis may be observed. however, diagnosis of parakeratosis is generally made at necropsy. feeding adequate roughage, such as stemmy hay, will prevent the disease. antibiotics may be administered to prevent secondary liver abscess formation. iv. rumenitis. rumenitis is an acute or chronic inflammation of the rumen, which occurs most commonly as a sequela to lactic acidosis in addition to concentrate feeding, inadequate roughage in the diet is also associated with this disorder rumenitis may occur with contagious ecthyma infection or following ingestion of poisons or other irritants. because rumenitis is often associated with lactic acidosis, it tends to occur in feedlot animals. the inflamed ruminal epithelium becomes necrotic and sloughs, creating ulcers. endogenous rumen bacteria such as fusobacterium necrophorum may invade the ulcers, penetrate the circulatory system, and induce abscesses of the liver. clinically, the animals will appear depressed and anorexic. rumen motility will be decreased, and animals will lose weight. the disease may resolve in a week to days; mortality may reach %. necropsy lesions include rumen inflammation and ulcers in the anteroventral sac. granulation tissue and scarring may be observed following healing. rumenitis is not typically diagnosed clinically; thus, specific treatment is not commonly done. the disease can be prevented by minimizing the incidence of lactic acidosis. etiology. traumatic reticulitis-reticuloperitonitis is a disease of cattle related to their exploratory tendencies and ingestion of many different, nonvegetative materials. the disease is rarely seen in smaller ruminants. clinical signs. clinical signs range from asymptomatic to severe, depending on the penetration and damage by the foreign object after settling in the animal's forestomach. many signs during the early, acute stages will be nonspecific, ranging from arched back, listlessness, anorexia, fever, decrease in production, ketosis, regurgitation, decrease or cessation of ruminal contractions, bloat, tachypnea, tachycardia, and grunts when urinating, defecating, or being forced to move. the prognosis is poor when peritonitis becomes diffuse. sudden death can occur if the heart, coronary vessels, or other large vessels are punctured by the migrating object. epizootiology and transmission. this is a noncontagious disease. the occurrence is directly related to sharp or metallic indigestible items in the feed or environment that the cattle mouth and swallow. necropsy findings. in severe cases, necropsy findings include extensive inflammation throughout the cranial abdomen, malodorous peritoneal fluid accumulations, and lesions at the reticular sites of migration of the foreign objects. cardiac puncture will be present in those animals succumbing to sudden death. pathogenesis. consumed objects initially settle in the rumen but are dumped into the reticulum during the digestive process, and normal contraction may eventually lead to puncture of the reticular wall. this sets off a localized inflammation or a localized or more generalized peritonitis. the inflammation may also temporarily or permanently affect innervation of local tissues and organs. further damage may result from migration and penetration of the diaphragm, pericardium, and heart. diagnosis is based on clinical signs, knowledge of herd management techniques in terms of placement of forestomach magnets, and reflection of acute or chronic infection on the hemogram. radiographs and abdominocentesis may be useful. differential diagnosis. differentials include abomasal ulcers, hepatic ulcers, neoplasia (such as lymphosarcoma, usually in older animals, or intestinal carcinoma), laminitis, and cor pulmonale. infectious diseases that are differentials include systemic leptospirosis and internal parasitism. diseases causing sudden death may need to be considered. prevention and control. this problem can be prevented entirely by elimination of sharp objects in cattle feed and in the housing and pasture environments. adequately sized magnets placed in feed handling equipment and forestomach magnets (placed per os with a bailing gun in young stock at - months of age) are also significant prevention measures. treatment. provision of a forestomach magnet, confinement, and nursing care, including antibiotics, are the initial treatments. in severe cases, rumenotomy may be considered. etiology. pregnancy toxemia is a primary metabolic disease of ewes and does in advanced pregnancy. beef heifers are susceptible to protein energy malnutrition (pem) syndrome, which is also referred to as pregnancy toxemia. clinical signs. in sheep, this disease is characterized by hypoglycemia, ketonemia, ketonuria, weakness, and blindness. hypoglycemic and ketotic ewes begin to wander aimlessly and to move away from the flock. they become anorexic and act uncoordinated, frequently leaning against objects. advanced signs may include blindness, muscle tremors, teeth grinding, convulsions, and coma. body temperature, heart rate, respiratory rate, and rumen motility continue normally. up to % of infected ewes may die from the disease. the course of the disease may last up to a week. in goats, the disease usually occurs in the last weeks of gestation, especially in does carrying triplets. pregnancy toxemia should be considered with any goat showing signs of illness in late gestation. the doe may separate herself from the herd, stagger, or circle and may appear blind. appetite is poor, and tremors may be evident. a rapid metabolic acidosis results in subsequent recumbency. urinalysis will readily reveal ketonuria. if fetal death occurs, acute toxemia and death of the doe may result. in beef heifers, weight loss and thin body condition, weakness and inability to stand, and depression are clinical signs. some cows develop diarrhea. because the catabolic state is often so advanced, most affected heifers die even if treated. pregnancy toxemia is diagnosed by evidence of typical clinical signs. sodium nitroprusside tablets or ketosis dipsticks may be used to identify ketones in the urine or plasma of ewes and does. blood glucose levels found to be below mg/dl and ketonuria are good diagnostic indicators. in cattle, ketonuria is not a typical finding; hypocalcemia and anemia may be present. that are obese or bearing twins or triplets. the disease develops during the last weeks of pregnancy. pem most frequently occurs in heifers during the final trimester of pregnancy. necropsy findings. at necropsy, affected ewes will often have multiple fetuses, which may have died and decomposed. the liver will be enlarged, yellow, and friable, with fatty degeneration. the adrenal gland may also be enlarged. in cattle, heifers will be very thin, and in addition to a fatty liver, signs of concurrent diseases may be present. pathogenesis. rapid fetal growth, a decline in maternal nutrition, and a voluntary decrease in food intake in overfat ewes result in an inadequate supply of glucose needed for both maternal and fetal tissues. the ewe develops a severe hypoglycemia in early stages of the disease. the ruminant absorbs little dietary glucose; rather, it produces and absorbs volatile fatty acids (acetic, propionic, and butyric acids) from consumed feedstuffs. propionic acid is absorbed and selectively converted to glucose through gluconeogenesis. when the animal is in a state of negative energy balance, it hydrolyzes fats to glycerol and fatty acids. glycerol is converted to glucose while the fatty acids are metabolized for energy. the oxidation of fatty acids in the face of declining oxaloacetate levels (required for normal krebs cycle function) results in the formation of ketone bodies (acetone, acetoacetic acid, and [ -hydroxybutyric acid), thus causing the condition ketoacidosis. heifer cattle have high energy requirements for completing normal body growth and supporting a pregnancy. additional energy requirements are needed during pregnancy for winter conditions and during concurrent diseases. marginal diets and poor-quality forage will place the cows in a negative energy balance. differential diagnosis. hypocalcemia is a common differential diagnosis. in cattle, differentials include chronic or untreated diseases such as johne's disease, lymphosarcoma, parasitism, and chronic respiratory diseases. prevention and control. pregnancy toxemia can be prevented by providing adequate nutrition during late gestation and by maintaining animals in appropriate nonfat condition during pregnancy. in late pregnancy, the dietary energy and protein should be increased . - times the maintenance level. pem can be prevented by maintaining appropriate body condition earlier in pregnancy and supplying good-quality forage for the last trimester. treatment. in sheep, because the morbidity may be as high as %, treatment should be directed at the flock rather than the in-dividual. treating the individual is usually unsuccessful. oral administration of ml of propylene glycol or % glucose twice a day, anabolic steroids, and high doses of adrenocorticosteroids may be helpful. if ewes are still responsive and not severely acidotic or in renal failure, cesarean section may be successful by rapidly removing the fetus, which is the dietary drain for the ewe. in goats, pregnancy toxemia is best treated by removal of the fetuses either by cesarean section or induction of parturition. parturition can be induced in does by either dexamethasone ( mg) or pgf a ( ~tg). in addition, goats may be treated with % dextrose ( to ml iv) or propylene glycol ( ml per os or times a day). adjunctive therapy includes normalizing acid base and hydration status, administration of vitamin b and transfaunation. heifers may be force-fed alfalfa gruels, given propylene glycol per os, placed on iv % glucose drips, and treated for concurrent disease. research complications. in research requiring pregnant ewes in late stages of gestation, for example, this disease should be considered if the animals are likely to bear twins and will be transported or stressed in other ways during that time. f hypocalcemia (parturient paresis, milk fever) etiology. hypocalcemia is an acute metabolic disease of ruminants that requires emergency treatment; the presentation is slightly different in ewes, does, and cows. clinical signs and diagnosis. in sheep, the disease is seen in ewes during the last weeks of pregnancy and is characterized by muscle tetany, incoordination, paralysis, and finally coma. as calcium levels drop, ewes begin to show early signs such as stiffness and incoordination of movements, especially in the hindlimbs. later, muscular tremors, muscular weakness, and recumbency will ensue. animals will frequently be found breathing rapidly despite a normal body temperature. morbidity may approach %, and mortality may reach as high as % in untreated animals. affected does become bloated, weak, unsteady, and eventually recumbent. cows are affected within - hr before or after parturition. cows initially are weak and show evidence of muscle tremors, then deteriorate to sternal recumbency, with the head usually tucked to the abdomen, and an inability to stand. tachycardia, dilated pupils, anorexia, hypothermia, depression, ruminal stasis, bloat, uterine inertia, and loss of anal tone are also seen at this stage. the terminal stage of disease is a rapid progression from coma to death. heart rates will be high, but pulse may not be detectable. hypocalcemia is diagnosed based on the pregnancy stage of the female and on clinical signs. it is later confirmed by laboratory findings of low serum calcium. with hypocalcemia in ewes, the plasma concentrations of calcium drop from normal values of - mg/dl to values of - mg/dl. in cattle, plasma levels below . mg/dl are hypocalcemic; at the terminal stages levels may be mg/dl. ewes during the last weeks of pregnancy or during the first few weeks of lactation. the disease is not as common in the dairy goat as in the dairy cow. high-producing, older, multiparous dairy cows are the most susceptible, and the jersey breed is considered susceptible. cows that have survived one episode are prone to recurrence. in addition, dry cows must be managed carefully regarding limiting dietary calcium. the disease is not common in beef cattle unless there is an overall poor nutrition program. ing at necropsy. there is no pathognomonic or typical find-pathogenesis. during the periparturient period, calcium requirements for fetal skeletal growth exceed calcium absorbed from the diet and from bone metabolism. additionally, dietary calcium intake is thought to be compromised because, in advanced pregnancy, animals may not be able to eat enough to sustain adequate nutrient levels, and intestinal absorption capabilities do not respond as quickly as needed. after parturition, calcium needs increase dramatically because of calcium levels in colostrum and milk. recent information suggests that legume and grass forages, high in potassium and low in magnesium, create a slight physiological alkalosis (at least in cattle), which antagonizes normal calcium regulation (rings et al., ) . thus, bone resorption, renal resorption, and gastrointestinal absorption of calcium are less than maximal. prevention and control. maintaining appropriate nutrition during the last trimester is helpful in preventing the disease. in cows and does, for example, limiting calcium intake by removing alfalfa from the diet is helpful. treatment. hypocalcemia must be treated quickly based on clinical signs; pretreatment blood samples can be saved for later confirmation. twenty percent calcium borogluconate solution should be administered by slow intravenous infusion. response will often be rapid, with the resolution of the animal's dull mentation. less severely affected animals will often try to stand in a short time. relapses are common, however, in sheep and cattle. hypermagnesemia and hypophosphatemia often coincide with hypocalcemia. these imbalances should be considered when animals appear to be unresponsive to treatment. hypocalcemia in the goat can be treated with - ml of calcium borogluconate. heart rate should be monitored closely throughout calcium administration. if an irregular or rapid heart rate is detected, then calcium treatment should be slowed or discontinued. calcium gels and boluses are also available for treatment (rings et al, ) . prognosis is generally good if the animal is treated early in the disease, but the prognosis will often be poor when treatment is initiated in later stages of the disease. etiology. urolithiasis is a metabolic disease of intact and castrated male sheep, goats, and cattle that is characterized by the formation of bladder and urethral crystals, urethral blockage, and anuria (murray, ) . the disease occurs rarely in female ruminants. clinical signs and diagnosis. affected animals will vocalize and begin to show signs of uneasiness, such as treading, straining postures, arched backs, raised tails, and squatting while attempting to urinate. these postures may be mistaken for tenesmus. male cattle may develop swelling along the ventral perineal area. affected animals will not stay with the herd or flock. small amounts of urine may be discharged, and crystal deposits may be visible attached to the preputial hairs. additionally, in smaller ruminants, the filiform urethral appendage (pizzle) often becomes dark purple to black in color. the pulsing pelvic urethra may be detected by manual or digital rectal palpation, and bladder distention may be noticeable in cattle by the same means. as the disease progresses to complete urethral blockage, the animal will become anorexic and show signs of abdominal pain, such as kicking at the belly. the abdomen will swell as the bladder enlarges, and rupture can occur within hr after development of clinical signs. bladder or urethral rupture may cause a short-lived period of apparent pain relief; subsequent development of uremia will eventually lead to death. the disease may progress over a period of - weeks, and the mortality is high unless the blockages are reversed. diagnosis is made by the typical clinical signs. abdominal taps may yield urine. calculi are usually composed of calcium phosphate or ammonium phosphate matrices. clinical disease is usually seen in growing intact or castrated males. the disease may be sporadic or there may be clusters of cases in the flock or herd. necropsy findings. necropsy findings include urine in the abdomen with or without bladder or urethral rupture. renal hydronephrosis may be evident. calculi or struvite crystal sediment will be observed in the bladder and urethra. histologically, trauma to the urethra and ureters will be present. etary, anatomical, hormonal, and environmental factors. male sheep and goats have a urethral process that predisposes them to entrapment of calculi. in cattle, the urethra narrows at the sigmoid flexure, and calculi lodge there most frequently. additionally, the removal of testosterone by early castration is thought to result in hypoplasia of the urethra and penis. this physical reduction in the size of the excretory tube may predispose to the precipitation of and blockage by the struvite minerals. grains fed to growing animals tend to be high in phosphorus and magnesium content. these calculogenic diets lead to the formation of struvite (magnesium ammonium phosphate) crystals. other minerals associated with urolithiasis include silica (range grasses), carbonates (some grasses and clover pastures), calcium (exclusively alfalfa hay), and oxalates (fescue grasses). differential diagnosis. grain engorgement colic, gastrointestinal blockage, and causes of tenemus, such as enteritis or trauma, are differentials. trauma to the urethral process should be considered. urinary tract infections are uncommon in ruminants. prevention and control. one case often is indicative of a potential problem in the group. urolithiasis can be minimized by monitoring the calcium:phosphorus ratio in the diet. the normal ratio should be : . additionally, increasing the amount of dietary roughage will help balance the mineral intake. increasing the amount of salt (sodium chloride, - %) in the diet to increase water consumption, or adding ammonium chloride to the diet, at gm/head/day or % of the ration, to acidify the urine, will aid in the prevention of this disease. palatability of and accessibility to water should be assessed as well as functioning of automatic watering equipment. treatment. treatment is primarily surgical (van metre et al. ) . initially, amputation of the filiform urethral appendage may alleviate the disease since urethral blockage often begins here. as the disease progresses, urethral blockage in the sigmoid flexure as well as throughout the urethra may occur. in more advanced stages, perineal urethrostomy may yield good results. the prognosis is poor when the condition becomes chronic, reoccurs, or surgery is required. research complications. young castrated and intact male ruminants used in the laboratory setting will be the susceptible age group for this disorder. rickets is a disease of young, growing animals but rarely occurs in goats. it is a metabolic disease characterized by a failure of bone matrix mineralization at the epiphysis of long bones due to lack of phosphorus. the condition can occur as an absolute deficiency in vitamin d , an inadequate dietary supply of phosphorus, or a long-term dietary imbalance of calcium and phosphorus. the syndrome must be differentiated from epiphisitis (unequal growth of the epiphyses of long bones in young, rapidly growing kids fed diets with excess calcium). clinical signs include poor growth, enlarged costochondral junctions, narrow chests, painful joints, and reluctance to move. spontaneous fractures of long bones may occur. animals will recover when dietary phosphorus is provided and if joint damage is not severe. a. copper deficiency (enzootic ataxia, swayback) etiology. chronic copper deficiency in pregnant ewes and does may produce a metabolic disorder in their lambs and kids called enzootic ataxia. in goats, this deficiency also causes swayback in the fetuses. clinical signs and diagnosis. this disease results in a progressive hindlimb ataxia and apparent blindness in lambs up to about months of age. additionally, because copper is essential for osteogenesis, hematopoiesis, myelination, and pigmentation of wool and hair, ewes may appear unthrifty, may be anemic, and may have poor, depigmented wool with a decrease in wool crimp. affected kids are born weak, tremble, and have a characteristic concavity to the spinal cord, leading to the name swayback. when the deficiency occurs later during gestation, demyelination is limited to the spinal cord and brain stem. kids are born normally but develop a progressive ataxia, leading to paralysis, muscle atrophy, and depressed spinal reflexes with lower motor neuron signs. diagnosis is based on low copper levels found in feedstuffs and tissues at necropsy. diagnosis is based on clinical signs, feed analysis, and pathological findings. epizootiology and transmission. enzootic ataxia is rarely seen in western states; most north american diets have sufficient copper levels to prevent this disease. copper antagonists in the feed or forage at sufficient levels, such as molybdenum, sulfate, and cadmium, however, may predispose to copper deficiencies. pathogenesis. the maternal copper deficiency leads to a disturbance early in the embryonic development of myelination in the central nervous system and the spinal cord. copper is part of the cytochrome oxidase system and other enzyme complexes and is important in myelination, osteogenesis, hematopoiesis (iron absorption and hemoglobin formation), immune system development, and maintenance and normal growth (smith and sherman, ) . differential diagnosis. the differential diagnosis for newborns includes [ -mannosidosis, hypoglycemia, and hypothermia. for older animals the differential should include caprine arthritis encephalitis (goats), enzootic muscular dystrophy, listeriosis, spinal trauma or abscessation, and cerebrospinal nematodiasis. prevention and control. copper deficiency can be prevented by providing balanced nutrition for pregnant animals. necropsy findings. gross encephalomalacia has been noted. histopathologically, white matter of the brain and spinal cord displays gelatinization and cavitation. extensive nerve demyelination and necrosis are evident. postmortem lesions include extensive demyelination and neuronal degeneration. treatment. because the condition is developmental, supplemental copper may improve clinical signs but not eliminate them. necropsy findings. common findings at necropsy include icterus; a soft, dark, friable, enlarged spleen; an enlarged, yellow-brown friable liver; and "gun-barrel" black kidneys. hemoglobin-stained urine will be visible in the bladder. copper accumulations in the liver reaching - ppm are toxic. pathogenesis. hemolysis occurs when sufficient amounts of copper are ingested or released suddenly from the liver and is believed to be due direct interaction of the copper with red-cell surface molecules. stresses such as transportation, lactation, and poor nutrition or exercise may precipitate the hemolysis. etiology: acute or chronic copper ingestion or liver injury often causes a severe, acute hemolytic anemia in weanling to adult sheep and in calves and adult dairy cattle. growing lambs may be the most susceptible. copper toxicosis is rare in goats. differential diagnosis. other causes of hemolytic disease include babesiosis, trypanosomiasis, and plant poisonings such as kale. arsenic ingestion, organophosphate toxicity, and cyanide or nitrate poisoning should also be considered as the source of poisoning. urethral obstruction and gastrointestinal emergencies should be considered for the abdominal pain. clinical signs and diagnosis. the clinical course in sheep can be as short as - days, and mortality may reach %. hemolysis, anemia, hemoglobinuria, and icterus characterize the acute hemolytic crisis, associated with copper released from the overloaded liver. some clinical signs are related to direct irritation to the gastrointestinal tract mucosa. weakness, vomiting, abdominal pain, bruxism, diarrhea, respiratory difficulty, and circulatory collapse are followed by recumbency and death. hepatic biopsy is currently considered the best diagnostic approach; serum or plasma levels of copper and hepatic enzymes such as aspartate aminotransferase (ast) and y-glutamyltransferase (ggt) may provide some information, but it is generally believed that these will not accurately reflect total copper load or hepatic damage. and goats is the range of - mg/kg, and for cattle it is - mg/kg. chronic poisoning in sheep may occur when . mg/kg is ingested. copper-containing pesticides, soil additives, therapeutics, and improperly formulated feeds may potentially lead to copper toxicity. phytogenous sources include certain pastures such as subterranean clover. feed low in molybdenum, zinc, or calcium may lead to increased uptake of copper from properly balanced rations. a common cause of the disease in sheep is feeding concentrates balanced for cattle; cattle feeds and mineral blocks contain much higher quantities of copper than are required for sheep. chronic ingestion of these feedstuffs leads to copper accumulation and toxicity. copper toxicosis has been reported in calves given regular oral or parenteral copper supplements, and in adult dairy cattle given copper supplements to compensate for copper-deficient pasture. pregnant dairy cattle may be more susceptible to copper toxicity. rare sources of copper ingestion may include copper sulfate footbaths. control and prevention. the disease is prevented by carefully monitoring copper access in sheep and copper supplementation in cattle. sheep and goats should not be fed feedstuffs formulated for cattle, and dairy calf milk replacer should not be used for lambs and kids. molybdenum may be administered to animals considered at high risk. molybdenum-deficient pastures may be treated with molybdenum superphosphate. herd copper supplementation should be undertaken with the knowledge of existing hepatic copper levels, and existing copper and molybdenum levels, in the feedstuffs. treatment. oral treatment for sheep consists of ammonium or sodium molybdenate ( - mg/day), and sodium thiosulfate ( . - . mg/day) for weeks aids in excretion of copper. oral d-penicillamine daily for days ( mg/kg) has also been shown to increase copper excretion in sheep. ammonium molybdenate has been administered intravenously to goats at . mg/kg for treatments on alternate days. cattle have been treated orally with sodium molybdenate ( gm/day) or sodium thiosulfate ( gm/day). treatment for anemia and nephrosis may be necessary in severe cases. merino crosses and the british breeds, may be more susceptible to copper toxicosis caused by phytogenous sources. (nutritional muscular dystrophy, nutritional myodegeneration, white muscle disease, stiff lamb disease) etiology. white muscle disease, also known as stiff lamb disease, is a nutritional muscular dystrophy caused by a deficiency of selenium or vitamin e. clinical signs and diagnosis. clinically two forms of the disease have been identified: cardiac and skeletal. the cardiac form occurs most commonly in neonates. in these, respiratory difficulty will be a manifestation of damage to cardiac, diaphragmatic, and intercostal muscles. young will be able to nurse when assisted. in slightly older animals, the disease is characterized by locomotor disturbances and/or circulatory failure. clinically, animals may display paresis, stiffness or inability to stand, rapid but weak pulse, and acute death. mortality may reach % (jensen and swift, ) . paresis and sudden death in neonates with associated pathological signs are frequently diagnostic. with the skeletal form, affected animals are stiff and reluctant to move, and muscles of affected animals are painful. young will be reluctant to get up but will readily nurse when assisted. peracute to acute myocardial degeneration may occur in the cardiac form, and animals may simply be found dead. serum selenium levels are usually below ppb (normal is - ppb) (nelson, ) . diagnosis may also include determination of antemortem whole blood levels of selenium and plasma levels of vitamin e. glutathione peroxidase levels in red blood cells can be measured as an indirect test. clinical biochemistry findings of significant elevations of aspartate aminotransferase (ast) in creatinine kinase (ck) are also supportive of the diagnosis. epizootiology and transmission. selenium deficiency has been associated with formulated diets deficient in selenium, forages grown on selenium-deficient soils in certain geographic regions, and forages such as alfalfa and clover that have an inability to efficiently extract available selenium from the soils. rumen bacterial reduction of selenium compounds to unavailable elemental selenium may also contribute to the disease. necropsy findings. necropsy lesions include petechial hemorrhages and muscle edema. hallmarks are pale white streaking of affected skeletal and cardiac muscle. these are due to coagulation necrosis. pale striated muscles of the limb, diaphragm, and tongue are also seen. antioxidants that protect lipid membranes from oxidative destruction. selenium is a cofactor for glutathione peroxidase, which converts hydrogen peroxide to water and other nontoxic compounds. lack of one or both results in loss of membrane integrity. differential diagnosis. in neonatal ruminants presenting with respiratory and cardiac dysfunction, differentials include congenital cardiac anomalies. differentials generally for weak neonates or sudden or peracute neonatal deaths should include septicemia, pneumonia, toxicity, diarrhea, and dehydration. prevention and control. awareness of regional selenium deficiencies is important. control involves providing good-quality roughage, vitamin e and selenium supplementation, and parenteral injections prior to parturition and weaning. treatment. affected animals may be treated by administering vitamin e or selenium injections. administering vitamin e or selenium to ewes in late pregnancy can prevent white muscle disease (kott et al., ) . the label dose for selenium is . - mg/ kg of body weight. combination products are available and can be used in goats at the sheep dose (smith and sherman, ) . proper mineral balance in the diet is critical. selenium toxicity occurs most frequently as the result of excessive dosing to prevent or correct selenium deficiency or as the result of ingestion of selenium-converting plants. the main preventive measure for the former is the use of the appropriate product for the species. secondarily, the concentration of the available product should be double-checked. in the united states, ruminants in the midwest and western areas may be subject to selenium toxicity when pastured in areas containing selenium-converting plants. signs of overdosing include weakness, dyspnea, bloating, and diarrhea. shock, paresis, and death may occur. initial clinical signs of excessive selenium intake from plants are observed in the distal limb, with cracked hoof walls and subsequent infection and irregular hoof growth. etiology. polioencephalomalacia (pem) is a noninfectious, noncontagious disease characterized by neurological signs. growing and adult ruminants on high-concentrate diets are typically affected. animals exposed to toxic plants or moldy feed containing thiaminases, feed high in sulfates, or unusually high doses of some medications are also at risk. clinical signs and diagnosis. an early sign may be mild diarrhea. acute clinical signs include bruxism, hyperesthesia, involuntary muscle contractions, depression, partial or complete opisthotonus, nystagmus, dorsomedial strabismus, seizures, and death. in subacute cases of the disease, animals may appear to walk aimlessly as if blind or may display head-pressing postures. hypersalivation may be present, but body temperatures and ocular reflexes are normal. morbidity and mortality may be high, especially in younger animals. diagnosis is suggestive from clinical signs and from response to intensive parental thiamine hydrochloride. epizootiology and transmission. pem is caused by a thiamin deficiency. the disease tends to be seen more frequently in cattle and sheep feedlots where the concentrates fed are high in fermentable carbohydrates. pastured animals are also vulnerable if grain is feed. thiaminase-containing plants, such as bracken fern, are often unpalatable so will less likely be a contributing factor. recent studies have also indicated that high levels of sulfate in the diet, such as in the fermentable, low-fiber concentrates, may play an important role. medications such as as amprolium, levamisole, and thiabendazole have thiaminantagonizing activity when given in excessive doses. sherman, ) . vitamin a deficiencies associated with hyperkeratosis have been reported, as well as vitamin e-responsive and selenium-responsive dermatitis. necropsy signs. cerebral lesions characterized by softening and discoloration are grossly observed in the gray matter. microscopically, neurons will exhibit edema, chromatolysis, and shrinkage. gliosis and cerebral capillary proliferation may be observed. a lack of thiamin results in inappropriate carbohydrate metabolism and accumulation of pyruvate and other intermediaries that lead to cerebral edema and neuronal degeneration. differential diagnosis. several important differentials include acute lead poisoning, nitrofuran toxicity, hypomagnesemia, vitamin a deficiency, listeriosis, pregnancy toxemia, infectious thromboembolic meningoencephalitis, and type d clostridial enterotoxemia. prevention and control. the disease can be prevented by monitoring the diet and by providing adequate roughage necessary to prevent overgrowth of thiaminase-producing ruminal flora and to maximize ruminal production of b vitamins. if excess sulfur is the primary factor, immediate removal of the source is critical. neonatal ruminants are born without immunoglobulins and must receive colostrum by hr after birth. the morbidity and mortality associated with failure of or inadequate passive transfer, such as enteric and respiratory illnesses, can be severe. measures to assure passive immunity for neonatal ruminants are covered in section ii,b, , and clinical signs of illness associated with lack of immunity are addressed in the discussions of bacterial diseases (e.g., escherichia coli infections) and, of viral diseases (e.g., diarrheas) in section iii,a, and iii,a, . generally, transfer of less than mg/dl of immunoglobulins in the serum is classified as failure of transfer, - mg/dl is partial, and above mg/dl is complete transfer. methods to determine success of transfer should be performed within a week of birth and include single radial immunodiffusion (quantitates immunogloblin classes); zinc sulfate turbidity (semiquantitative); sodium sulfite precipitation (semiquantitative); glutaraldehyde coagulation (coagulates above specific level); and, y-glutamyltransferase (assays enzyme in high concentration in colostrum and absorbed simultaneously with colostrum). treatment. early aggressive treatment is essential to save animals. the disease is treated by frequent parenteral administration of thiamine hydrochloride, the first dose being administered intravenously. dexamethasone, b vitamins, and diazepam may also be required. treatment is less successful when sulfur plays a prominent role in the etiology. research complications. this disease is preventable. although the disease is less likely to occur in smaller groups of confined ruminants, the risks of feeding concentrates or moldy feed, for example, with minimal good-quality roughage, should be kept in mind. vitamin d toxicity can result either from iatrogenic overadministration or ingestion of the plant trisetum flavescens. serum calcium levels may be high enough that blood in edta tubes will clot. laminitis is common in ruminants and can be caused by sudden changes in diet, excess dietary energy, and grain overload (or overeating). laminitis is also associated with mastitis and metritis. facility conditions, such as concrete flooring, poor manure management, and inadequate resting areas may also contribute to the pathogenesis of the disease. the complete pathogenesis of laminitis is poorly understood; however, it is thought that changes in the diet cause changes in rumen microbial populations, resulting in acidosis and endotoxemia. dramatic changes in the vascular endothelium result in chronic inflammation of the sensitive laminae of the hoof, separation of corium and hoof wall, and rotation of the third phalanx. affected animals may be reluctant to get up or walk, will shift their weight frequently, and will grind teeth or walk on carpi. chronically, the hoof wall takes on a "slipper" appearance. treatment consists of identifying the underlying cause, administering antiinflammatories (phenylbutazone, flunixin meglumin), feeding good-quality forages only, and regular foot trimming. in goats, nutritional deficiencies often manifest as a generalized poor coat that is dry, scaly, thin, and erectile. zincresponsive dermatitis has been reported in goats (smith and otherwise normal, well-managed lambs, kids, and calves can develop loose, pasty feces due to a nutritional imbalance caused by overfeeding and/or improper mixing of milk replacers. only milk replacer formulated for the particular species should be used. once nutritional imbalances are corrected, the feces readily return to normal. sudden changes in diet can also result in loose feces. photosensitization is an acute dermatitis associated with an interaction between photosensitive chemicals and sunlight. the photosensitive chemicals are usually ingested, but in some cases exposure may be by contact. animals with a lack of pigment are more susceptible to the disease. three types of photosensitization occur: primary; secondary, or hepatogenous; and aberrant. primary photosensitization is related to uncommon plant pigments or to drugs such as phenothiazine, sulfonamides, or tetracyclines. secondary photosensitization is more common in large animals and is specifically related to the plant pigment phylloerythrin. phylloerythrin, a porphyrin compound, is a degradation product of chlorophyll released by rumen microbial digestion. liver disease or injury, which prevents normal conjugation of phylloerythrin and excretion through the biliary system, predisposes to photosensitization. the only example of aberrant photosensitization is congenital porphyria of cattle (see section iii,b, ). pathologically, the photosensitive chemical is deposited in the skin and is activated by absorbed sunlight. the activated pigments transfer their energy to local proteins and amino acids, which, in the presence of oxygen, are converted to vasoactive substances. the vasoactive substances increase the permeability of capillaries, leading to fluid and plasma protein losses and eventually to local tissue necrosis. photosensitization can occur within hours to days after sun exposure and produces lesions of the face, vulva, and coronary bands; lesions are most likely to occur on white-haired areas. initially, edema of the lips, corneas, eyelids, nasal planum, face, vulva, or coronary bands occurs. the facial edema, nostril constriction, and swollen lips potentially lead to difficulty in breathing. with secondary photosensitization, icterus is also common. necrosis and gangrene may occur. diagnosis is based on clinical lesions and exposure to the photosensitive chemi-cals and sunlight. treatment is symptomatic. the prognosis for hepatogenous type may be guarded if hepatic disease is severe. from excessive straining associated with dysuria from the pressure of the fetuses and/or abdominal contents on the bladder. if the prolapse obstructs subsequent urination, rupture of the bladder may occur. the vaginal prolapse can be reduced and repaired if discovered early, and techniques in small and large ruminants are comparable. the animal should be restrained, and the prolapsed tissue should be cleansed with disinfectants. best done under epidural anesthesia, the vagina is replaced into the pelvic canal and the vulvar or vestibular opening is sutured closed (buhner suture). alternatively, a commercial device called a bearing retainer (or truss) can be placed into the reduced vagina and tied to the wool, thereby holding the vagina in proper orientation without interfering with subsequent lambing. vaginal prolapses may have a hereditary basis in ewes and cows and may prolapse the following year. these animals should be culled. vaginal prolapses may occur in nonpregnant animals that graze estrogenic plants or as a sequela to docking the tail too close to the body (ross, ) . uterine prolapses occur sporadically in postpartum ewes and cattle. the gravid horn invaginates after delivery and protrudes from the vulva. the cause is unknown, but excessive traction utilized to correct dystocia or retained placenta, uterine atony, hypocalcemia, and overconditioning or lack of exercise have been implicated. in cattle, the uterine prolapses usually develop within week of calving, are more common in dairy cows than in beef cows, and are often associated with dystocia or hypocalcemia. cows may also have concurrent parturient paresis. initially, the tissue will appear normal, but edema and environmental contamination or injuries of the tissue develop quickly. clinical signs will include increased pulse and respiratory rates, straining, restlessness, and anorexia. if identified early, the uterus can be replaced as for vaginal prolapses. electrolyte imbalances should be corrected if present. additional supportive therapy, including the use of antibiotics should always be considered. tetanus prophylaxis should be included. oxytocin should be administered to induce uterine reduction. vaginal closures are less successful at retaining uterine prolapses. preventive and control measures include regular exercise for breeding animals, and management of prepartum nutrition and body condition. vaginal and uterine prolapses occur in ewes, does, and cows. the conditions are not common in does. vaginal prolapses usually occur during late gestation and may be related to relaxation of the pelvic ligaments in response to hormone levels. in sheep, these are most common in overconditioned ewes that are also carrying twins or triplets. overconsumption of roughages, which distends the rumen, and lack of exercise leading to intraabdominal fat may predispose an animal to vaginal prolapse by increasing intra-abdominal pressure. the condition may result f rectal prolapse rectal prolapse is common in growing, weaned lambs and in cattle from months to years old. the physical eversion of the rectum through the anal sphincter is usually secondary to other diseases or management-related circumstances. rectal prolapses may occur secondary to gastrointestinal infection or inflammation, especially when the colon is involved. diseases that cause tenesmus, such as coccidiosis, salmonellosis, and intestinal worms, may result in prolapse. urolithiasis may result in prolapses as the animal strains to urinate. any form of cystitis or urethritis, vaginal irritation, or vaginal prolapse and some forms of hepatic disease may lead to rectal prolapse. abdominal enlargement related to advanced stages of pregnancy, excessive rumen filling or bloat, and overconditioning may cause prolapse. finally, excessive coughing during respiratory tract infections, improper tail docking (too short), growth implants, prolonged recumbency, or overcrowded housing with animal piling may lead to prolapses. diagnosis is based on clinical signs. early prolapses may be corrected by holding the animal with the head down, while a colleague places a pursestring suture around the anus. the mucosa and underlying tissue of prolapses that have been present for longer periods of time will often become necrotic, dry, friable, and devitalized and will require surgical amputation or the placement of prolapse rings to remove the tissue. rectal prolapse may also be accompanied by intestinal intussusceptions that will further complicate the treatment and increase mortality. occasionally, acute rectal prolapse with evisceration will result in shock and prompt death of the animal. prognosis depends on the cause and extent of the prolapse as well as the timeliness of intervention. in all cases of treatment, determination and elimination of the underlying cause are essential. gastrointestinal accumulations or obstructions of hair (and/ or sometimes very coarse roughage, forming bezoars) occur in cattle and sheep. cattle that are maintained on a low-roughage diet, that lick their coats frequently, that have long hair coats from outdoor housing, or that have heavy lice or mite infestations and associated pruritus will often develop bezoars. in addition, younger calves with abomasal ulcers have been found to be more likely to have abomasal tric. hobezoars as well. clinical signs may be mild or severe according to size, number, and location. ruminal trichobezoars rarely result in clinical signs. obstruction will be accompanied by signs of pain, development of bloat, and decreased fecal production. serum profiles will show hypochloridemia; other imbalances depend on the duration of the problem. diagnosis is also based on abdominal auscultation, rectal palpation, and ultrasound (useful in calves and smaller ruminants). treatment is surgical, such as paracostal laparotomy (for abomasal), paralumbar celiotomy with manual breakdown, or enterotomy. supportive care should be administered as necessary to correct electrolyte imbalances and to prevent inflammation and sepsis. prognosis is generally good if the condition is diagnosed and treated before dehydration and imbalances become severe and peritonitis develops. prevention includes providing good-quality roughage and treating lice and mange infestations. wounds may be sustained from poorly constructed pens or fences, or from skirmishes among animals. predators will usu-ally be sources of bite wounds. standard veterinary wound assessment and care are essential for wounds or bites. tetanus antitoxin may be indicated. use of approved antibiotics may be appropriate. the lesion should be cleaned with disinfectants and repaired with primary closure if it is clean and uncontaminated. thorough cleaning, regular monitoring, and healing by second intention are recommended for older wounds. abscesses may also occur in the soft tissues of the hooves (sole abscesses; see section iii,c, ) because of entrapped foreign bodies or hoof cracks that fill with dirt. preventive measures include improvement of housing facilities, pens, and pastures; monitoring hierarchies among animals penned together; and implementing predator control measures, such as sound fencing, flock guard dogs, or donkeys, in pasture situations. acute anaphylatic reactions in sheep, goats, and cattle are often clinically referable to the respiratory system. anaphylactic vaccine reactions cause acute lung edema; lungs are the primary site of lesions if collapse and death are sequelae. the animals will also be anxious and shivering and will become hyperthermic. salivation, diarrhea, and bloat also occur. immediate therapy must include epinephrine by intravenous infusion at ( ml of : per kg of body weight for goats and : , ( . mg/ml) or . mg/kg (about ml) for adult cows.) furosemide ( mg/kg) may be beneficial to reduce edema. prognosis is usually guarded. recovery can occur within hr. in a research environment, catheter sites or experimental surgeries may be sources of iatrogenic infection. traumatic injuries to peripheral nerves can cause acute lameness. improper administration of therapeutics can easily cause this type of lameness. injections given in gluteals or between the semimembranosus and semitendinosus can cause irritation to the sciatic nerve and subsequent lameness. contraction of the quadriceps results in the limb being pulled forward. injections in the caudal thigh can damage the peroneal nerve and cause knuckling at the fetlock. traumatic injury to the radial nerve can result in a "dropped elbow" (nelson, ) . husbandry procedures such as tail docking, castration, dehorning, dosing with a bailing gun, and shearing may result in superficial lesions, dermal infections, or cases of tetanus. bailing-gun injuries to the pharynx may lead to cellulitis with coughing, decreased appetite, and sensitivity to palpation. standard veterinary assessment and care are essential for these cases. local and systemic antibiotics with supportive care may be indicated. swelling around peripheral nerves caused by inoculations may be reduced by diuretics and anti-inflammato-ries. mild cases of peripheral nerve damage may recover in - days. personnel training, including review of relevant anatomy, preprocedure preparation, appropriate technique, careful surgical site preparation, rigorous instrument sanitation, and sterile technique will minimize the incidence of potential complications from surgical procedures. albumin values and foaming urine. the proteinuria also distinguishes amyloidosis (and glomerulonephritis) from other causes of weight loss and diarrhea in cattle such as johne's disease, parasitism, copper deficiency, salmonellosis, and bovine viral diarrhea virus infection. prognosis is poor, and no treatment is reported. neoplasia and tumors are relatively rare in ruminants. lymphosarcoma/leukemia in sheep has been shown to result from infection by a virus related (or identical) to the bovine leukemia virus. pulmonary carcinoma (pulmonary adenomatosis) and hepatic tumors are found in sheep. virus-induced papillomatosis (warts), discussed in section iii,a, ,s, and squamous cell carcinomas have also been reported in sheep. in goats, thymoma is one of the two most common neoplasias reported, although no distinct clinical syndrome has been described. cutaneous papillomas are the most common skin and udder tumor of goats, and although outbreaks involve multiple animals, no wart virus has been identified. persistent udder papillomas may progress to squamous cell carcinoma. lymphosarcoma is reported rarely in goats. although adrenocortical adenomas have been reported frequently and almost exclusively in older wethers, no clinical condition has been described. lymphosarcoma of various organ systems and "cancer eye" (bovine ocular squamous cell carcinoma, or oscc) are the most commonly reported cancers in cattle. lymphosarcoma is described in section iii,a, ,c. lack of periocular pigmentation and the amount and intensity of exposure to solar ultraviolet light are considered important factors in oscc. genetic factors may also play a role. many cases occur in herefords. this is a disease of older cattle; no case has been reported in animals less than years of age. the cancer metastasizes through the lymph system to major organs. treatment in either lymphosarcoma or oscc is recommended only as a palliative measure. the extent of ocular neoplastic involvement is a significant criterion for carcass condemnation. papillomatosis (warts) are common in cattle (see section iii,a, ,s). dental wear is seen most commonly in sheep. as sheep age, excessive dental wear may lead to an inability to properly masticate feed, manifesting as weight loss and unthriftiness. several factors predisposing to dental wear should be considered. the diet should be properly balanced for minerals, especially calcium and phosphorus, because primary or secondary calcium deficiency during teeth development results in softening of the enamel and dentin. dietary contamination with silica (i.e., hays and grains harvested in sandy regions) will lead to mechanical wear on the teeth. likewise, animals grazing or being fed in sandy environments will have excessive tooth wear. sheep older than about years of age are especially prone to tooth wear and should be checked frequently, especially if signs of weight loss or malnutrition are evident. managing the content and consistency of the diets can best prevent the disease. of the ruminants, cows are the most frequently affected by subsolar absesses. dirt becomes packed into cracks in the horny layer of the sole of the hoof, and contamination eventually extends into the sensitive areas of the hoof, with lameness and infection resulting. animals maintained in very soiled or muddy conditions, combined with poor hoof care, are more likely affected. fusobacterium necrophorum is often the pathogen involved. separation of the animal, supportive care, surgical drainage, and antibiotic treatment are indicated. amyloidosis amyloidosis in adult cattle is due to accumulations of amyloid protein in the kidney, liver, adrenal glands, and gastrointestinal tract. the disease has been classified as aa type, or associated with chronic inflammatory disease, although other unknown factors are believed to be involved in some cases. clinical signs include chronic diarrhea, weight loss, decreased production, nonpainful renomegaly, and generalized edema. the loss of protein in the urine contributes to abnormal plasma advances in sheep and goat medicine animals and animal products, subchapter a, animal welfare formulary for laboratory animals domestic animal behavior for veterinarians and animal scientists schlam's veterinary hematology diseases of sheep animal feeding and nutrition guide for the care and use of laboratory animals veterinary drug handbook veterinary medicine: a textbook of the diseases of cattle, sheep, pigs, goats, and horses sheep production and management animal and plant health inspection service (aphis), policy # , farm animals used for nonagricultural purposes goats the clinical syndromes caused by salmonella infection armed forces institute of pathology (afip) ( ) the effect of stress on the carrier state of salmonella typhimurium in goats bibliography of naturally occurring models of human disease clinical signs, treatment, and postmortem lesions in dairy goats with enterotoxemia: cases control of the estrous cycle the goat industry: feeding for optimal production neurologic disease in sheep and goats modern breeds of livestock the sheep gene map pasteurella haemolytica complicated respiratory infections in sheep and goats ungulates as laboratory animals diagnosis of lameness in sheep an overview of the influence of ace inhibitors on fetalplacental circulation and perinatal development protozoan infections (toxoplasma gondii, neospora caninum, and sarcocystis spp.) in sheep and goats: recent advances cloned transgenic calves produced from nonquiescent fibroblasts transgenic bovine chimeric offspring produced from somatic cell-derived stem-like cells use of an animal model of trichomoniasis as a basis for understanding this disease in women council report: vaccination guidelines for small ruminants (sheep, goats, llamas, domestic deer, and wapiti) ( ) maedi-visna and ovine progressive pneumonia pathophysiology of oestrus ovis infection in sheep and goats: a review experimental surgery in farm animals evaluation of an agar gel immunodiffusion test kit for detection of antibodies to mycobacterium paratuberculosis in sheep veterinary laboratory medicine induction of human tissue plasminogen activator in the mammary gland of transgenic goats pasteurella haemolytica infections in sheep coccidiosis and cryptosporidiosis in sheep and goats the major histocompatibility complex region of domestic animal species brucella melitensis infection in sheep: present and future hemoglobin switching epididymitis in rams current veterinary therapy: food animal practice livestock handling guide: management practices that reduce livestock bruises and injuries, and improve handling efficiency. livestock conservation institute synchronization of oestrus in the boer goat doe: dose effect of prostaglandin in the double injection scheme. south afr guide to the dissection of domestic ruminants reproduction in farm animals review of polyclonal antibody production procedures in mammal and poultry considerations in the design and construction of facilities for farm species clinical update: leptospirosis the sheep as an experimental animal bibliography oflnduced animal models of human disease bibliography of naturally occurring models of human disease postpartum assessment and care of the newborn ruminant animal genetics guide for the care and use of laboratory animals blackleg: a new perspective on an old disease protecting calves from viral diarrhea bovine leukemia virus. part : descriptive epidemiology, clinical manifestations, and diagnostic tests bovine leukemia virus. part : risk factors of transmission bovine leukemia virus. part : zoonotic potential, molecular epidemiology, and an animal model. in "infectious disease in food animal practice bovine leukemia virus. part : economic impact and control measures brucella abortus strain rb vaccine: its advantages and risks current veterinary therapy: food animal practice neural control of maternal behavior and olfactory recognition of offspring comparison of the burdizzo and rubber ring methods for castrating and tail docking lambs postpartum care of the cow and calf advances in the control of foot rot in sheep mastitis in ewes giardiasis in large animals laboratory diagnostic tests for retrovirus infections of small ruminants effects of dietary vitamin e supplementation during late pregnancy on lamb mortality and ewe productivity myotonia congenita (thomsen) and recessive myotonia genetic and environmental causes of variation in mastitis in sheep the gnrh system of seasonal breeders: anatomy and plasticity genetic organization, polymorphism, and function of the bovine major histocampaticulity complex scrapie in sheep biochemical and morphological expression of early prenatal caprine beta-mannosidosis respiratory infections of sheep dogs are the definitive hosts of neospora caninum a century of classical contagious caprine pleuropneumonia from original description to aetiology sheep and goat practice gene manipulation in goats through biotechnology minimizing morbidity and mortality from cryptosporidiosis managing dairy cows during the transition period: focus on ketosis environmental enrichment for dairy calves and pigs the trypanocidal cape buffalo serum protein is xanthine oxidase oral rehydration therapy for diarrheic calves neonatal ruminant diarrhea techniques for artificial insemination of cattle with frozenthawed semen noninfectious causes of lameness neosporosis: its prevalence and economic impact a review of yersinosis (yersinia pseudotuberculosis infection) dairy goat reproduction maedivisna virus in sheep: a review evaluation of a commercially available vaccine against corynebacterium pseudotuberculosis for use in sheep ruminant production management: control programs for gastrointestinal nematodes in sheep and goats fetal brain injury following prolonged hypoxemia and placental insufficiency: a review managing cryptosporidium and giardia infections in domestic ruminants milk fever: seeking new solutions to an old problem recent advances on ovine chlamydial abortion risk factors for abomasal displacement in dairy cows the spider syndrome: a report on one purebred flock development of ingestive behavior current state of in vivo preclinical heart valve evaluation dermatophilus congolensis infections in cattle and sheep ovine listeric encephalitis coccidiosis in ruminants principles of dairy science human factor ix transgenic sheep produced by transfer of nuclei from transfected fetal fibroblasts the major histocompatibility complex region of domestic animal species clinical reproductive anatomy and physiology of the doe immunobiology of the mammary gland coccidiosis brucella abortus strain rb : a new brucellosis vaccine for cattle use of age and serum gamma-glutamyltransferase activity to assess passive transfer status in lambs effect of congenitally acquired neospora caninum infection on risk of abortion and subsequent abortions in dairy cattle artificial control of breeding in ewes toxoplasmosis infection in sheep bovine reproductive biotechnology transgenic dairy cattle. genetic engineering on a large scale the effect of intra-mammary inoculation of lactating ewes with pasteurella haemolytica isolates from different sources bovine surgery and lameness reduction of myocardial myoglobin in bovine dilated cardiomyopathy intraosseous infusion of prostaglandin e prevents disuse-induced bone loss in the tibia estrous cycle synchronization the bronchopneumonias (respiratory disease complex of cattle, sheep, and goats) the cattle gene map treatment and control of gastrointestinal nematodes in sheep diagnosis, treatment, and management of enteric colibacillosis key: cord- -a ie fs authors: nan title: digestive system, liver, and abdominal cavity date: - - journal: the cat doi: . /b - - - - . - sha: doc_id: cord_uid: a ie fs nan these complex pathways highlight the need to consider the whole cat and not just the cat's gastrointestinal vomiting can be defined as the ejection of part or all of the contents of the stomach and/or upper intestine through the mouth, usually in a series of involuntary spasmodic movements. the disturbances in gastrointestinal (gi) motility are coordinated with respiratory and abdominal muscle contractions and mediated by the central nervous system (cns). vomiting begins with retching, a series of brief negative intrathoracic pressure pulses that coincide with positive abdominal contractions. these pressure changes occur as a result of repeated herniations of the abdominal esophagus and cardiac portion of the stomach into the esophagus. during retching, food freely moves back and forth in the esophagus, which is now dilated because of the ingesta. ultimately, the diaphragm rapidly moves cranially, resulting in positive intrathoracic pressure that leads to expulsion of these contents. vomiting is such an active process that it seems to involve the whole cat, and so it is little wonder that it concerns owners so much. since vomiting is mediated by the cns with input and influence from just about anywhere in the body, it is important to summarize this physiology so it can be appreciated when managing clinical cases. vomiting results from stimulation of the "vomiting center," which is located in the brainstem; there are four main pathways that stimulate the vomiting center, and these are summarized below and in figure (though not all older cats have grown out of this habit). some extragastrointestinal problems, such as hyperthyroidism and renal disease are more likely to occur in older cats. most texts and references instruct clinicians to distinguish between vomiting and regurgitation, with the latter noted as being quite passive. , , in practice, it can be hard to make this distinction, because it is the author's experience that cats with esophageal disease can have quite forceful, spasmodic movements when ejecting ingesta by regurgitation-although it is also possible for regurgitation to be a passive process. given that the physiology of vomiting, as described above, results in ingesta being forced to and then evacuated from the esophagus, it is hardly surprising that it can resemble regurgitation. fortunately, regurgitation and esophageal disease do vary from vomiting in other ways! vomiting . blood and urine testing . imaging (radiography, ultrasonography) . biopsy samples . treat and manage underlying problem the decision to proceed to steps and is based on the assumption that the prior steps have narrowed down the underlying cause as gastrointestinal, pancreatic, or hepatic in origin. the important aspects of the clinical history are given in chronic kidney disease. the author has found that some cats with dental disease can gorge their food, resulting in vomiting; so, paying attention to the state of the teeth and gums is important. of course, some cats have multiple problems, and correction of dental disease may not resolve vomiting if there is another process. in the examination, it is also important to note consequences of both the underlying process and the vomiting itself; these include the demeanor of the cat, hydration status, and abdominal pain. the physical examination findings, together with the clinical history, help determine the next appropriate steps. well cats that are not continually vomiting and are appropriately hydrated, with no other specific signs, may be treated as outpatients by fasting them for hours, then returning to food with a bland diet, such as plain cooked chicken or commercial, low-residue prescription diets designed for this purpose. follow-up is important to ensure signs do not progress. cats with nonspecific signs may require supportive care with subcutaneous or intravenous fluids and perhaps analgesia (with opioids). if clinical signs do not resolve, the pursuit of a specific diagnosis should be attempted. the practitioner must ask the following important questions: • are ancillary tests appropriate? • is supportive care necessary? • are any medications required? routine serum/plasma biochemistries, hematology, urinalysis, and total thyroxine (t ) (for older cats) testing is not only important to distinguish primary from secondary gastrointestinal disease but to look for consequences of vomiting that may need to be addressed, such as hydration status and electrolyte abnormalities. careful interpretations should be made. severe azotemia, even with hyperphosphatemia, can occur as a result of primary gastrointestinal disease, and the distinction from renal disease usually requires an assessment of urine specific gravity. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity (fpli) tests are useful markers of intestinal and pancreatic disease, , , , but it is important to note that they mostly do not give a precise diagnosis. more detail about the utility of these tests is noted below in the section approach to the cat with diarrhea. is usually preceded by the cat licking its lips, salivating, or making attempts to swallow. regurgitated ingesta is often in a tubelike structure and if undigested can be covered with frothy saliva. partially digested food suggests vomitus, and the presence of bile or digested blood confirms this. it is important to determine if the cat vomits regularly. many owners have seen their cats vomit on a regular basis with no evidence of the cat being unwell, and this is noted frequently in the veterinary literature. , hairballs can cause gastric irritation, and it may be that eating quickly also stimulates the peripheral sensory receptors that contribute to vomiting. if a cat does vomit regularly, it is important to assess if the cat is presenting for a change in the vomiting pattern (e.g., frequency or timing in relation to eating) and if the cat is unwell in any way, such as anorexia or weight loss. the pattern of vomiting is important in all cases, because cats presenting with acute gastritis usually have a sudden onset of frequent vomiting compared with those with chronic disease processes that may vomit every few days. the timing in relation to eating can be helpful, because the stomach should empty by to hours after a meal; so, vomiting longer than hours after a meal can suggest motility or retention disorders. the description of the vomitus can be helpful. if bile is present, the pylorus is not obstructed; the presence of blood (digested or fresh) indicates ulceration. hair in the vomitus can indicate hairball gastritis, and the possibility of trichobezoar obstruction should be considered. access to foreign bodies or toxins is an important aspect of the clinical history. has the cat been seen playing with an insect, mouse, or other prey? are there any medications unaccounted for (e.g., a dropped aspirin tablet)? are lilies present in the house? vomiting is the major sign of gastric disease, but given the number of potential organ systems that can be involved, a thorough physical examination should be undertaken. because linear foreign bodies are a common cause of vomiting, all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of string caught there. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see . a thorough examination may reveal specific signs, such as a palpable thyroid nodule and tachycardia in the case of hyperthyroidism or palpably small kidneys with and analgesia, many cats recover uneventfully. one survey assessed that % of cats undergoing exploratory laparotomy survived the hospitalization, and although complications occurred in % of cats, these were more likely to be associated with the underlying disease process and not surgery or anesthesia. laparoscopy is not readily available in all veterinary clinics. this alternative is less invasive and allows exploration of the abdomen but not as thoroughly as with laparotomy. organs are usually exteriorized for biopsy. there is the possibility of anesthetic complications associated with insufflating the abdomen. endoscopy is the least invasive procedure and is the only alternative that allows examination of the intestinal lumen. this option limits the parts of the gastrointestinal tract that can be biopsied; it does not allow examination or sampling of any other part of the gastrointestinal tract and does not enable full-thickness biopsy samples. one study found that, of cats investigated for gastrointestinal disease, of cats ( %) had no pathology recognized proximal to the jejunum (i.e., the effective length of diagnostic endoscopes would have precluded diagnosis), and other organs were affected in of cats with inflammatory bowel diseases and of cats with intestinal small cell lymphoma. careful case selection for endoscopy from survey ultrasonography can reduce the number of missed diagnoses from endoscopy, but the possibility still remains. the quality of endoscopically obtained biopsy samples varies greatly with the skill of the endoscopist. it has been stated that "it is exceedingly easy to take inadequate tissue samples with a flexible endoscope." in an assessment of endoscopically obtained biopsy samples, two laboratories were compared, one that received samples from any practitioner and the other that received samples only from practitioners trained to take, mount, and submit endoscopy samples. all slides were reviewed by three pathologists who found that, of samples from the first laboratory, % of the slides were considered inadequate for diagnosis, % were considered questionable, and only % were adequate. by comparison, in the second laboratory (with samples from experienced practitioners) % of slides were inadequate, % were questionable, and % were considered adequate for diagnosis. in the case of distinguishing between lymphocytic intestinal infiltrates (commonly known as inflammatory bowel disease) and lymphocytic neoplasia (small cell lymphoma), endoscopically obtained samples can give an incorrect diagnosis. many of these problems can be minimized with experienced operators and careful case selection from prior ultrasonography. radiography is most useful for identifying foreign bodies or signs of intestinal obstruction from other causes. the major findings are noted below in the section intestinal obstruction. contrast radiography can aid the diagnosis for both discrete and linear foreign bodies but should be used with caution, because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium irritates the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. ultrasonography is a useful diagnostic adjunct and helps to detect and characterize localized thickening of the stomach or intestinal wall, lymphadenopathy, radiolucent foreign bodies, and changes in the size and echogenicity of the pancreas, liver, kidneys, or spleen. abdominal effusions can be assessed and sampled. ultrasound-guided fine-needle aspiration can be used to sample masses, bile, or peritoneal fluid. it should be recognized that in most cases of gastrointestinal disease, imaging will not give a definitive diagnosis and biopsy will be required, usually using either endoscopy or laparotomy. ultrasonography can be a considered as a means to "survey the field," assessing • the nature of the underlying disease, such as • thickened intestines with or without discrete layers • lymph node involvement • other organ involvement • the location of disease, for example, • diffuse or focal • proximal duodenum (reachable by endoscope) versus distal ileum these factors may be used to assess the appropriateness of endoscopy versus laparotomy to obtain diagnostic samples. most commonly used antiemetics all control vomiting by different mechanisms and include mirtazapine, metoclopramide, dolasetron/ondansetron, maropitant, and the phenothiazines (tables - and - ) . metoclopramide functions both as an antiemetic and prokinetic in cats, while cisapride functions solely as a prokinetic. mirtazapine, a piperazinoazepine, antagonizes the presynaptic alpha -adrenergic receptor, increasing noradrenergic and serotonergic neurotransmission; the primary mechanism targeted for its use is as an antidepressant in humans. mirtazapine is also a potent antagonist of the postsynaptic serotonergic receptors ( -ht and -ht ) and histamine h receptors. because of its antiserotonergic and antihistaminic effects, mirtazapine is used as an entiemetic and appetite stimulant in cats. anorexia is a common clinical problem in ill cats, and in some anorexic or partially anorexic cats the use of an appetite stimulant as adjunctive therapy to nutritional support (i.e. feeding tubes) may be of clinical benefit. prior to the development of mirtazapine, cyproheptadine was used as an appetite stimulant in cats, with variable clinical results. recently, the pharmacokinetics and pharmacodynamics of mirtazapine have been reported in cats. in a group of healthy cats, mirtazapine was found to be an effective appetite stimulant, with a shorter half-life than that reported in humans. the recommended oral dose is . mg/cat every hours. a in humans, age and kidney and liver dysfunction affect mirtazapine metabolism (hepatic cyp enzymes) and clearance (excreted in urine and feces), suggesting that dose adjustment may be necessary. a side effects reported in cats treated with mirtazapine include behavior changes (vocalization and interaction), tremors, muscle twitching, and hyperactivity. a, a metoclopramide is both an antiemetic and prokinetic drug that acts peripherally on the gastrointestinal tract and centrally within the central nervous system (cns). at low doses metoclopramide inhibits dopaminergic (d ) transmission, and at higher doses it inhibits serotonergic -ht receptors in the chemoreceptor trigger zone (crtz). , metoclopramide also acts peripherally as a prokinetic at the level of the gastrointestinal smooth muscle of the stomach and duodenum, triggering gastric emptying and duodenal contractions. multiple mechanisms mediate metoclopramide's prokinetic activity, including augmentation of acetylcholine release and increased smooth muscle sensitivity to cholinergic neurotransmission, which may in part be because of antagonism of dopamine, but more recently, serotonergic ht receptor activation has been suggested. , metoclopramide has been reported to increase the lower esophageal sphincter tone in humans, although in cats metoclopramide's affect on the lower esophageal sphincter is reported to be weak. adverse central nervous system, extrapyramidal signs occur secondary to dopamine (d ) antagonism, including excitement and behavior changes. extrapyramidal signs are most often seen at the higher doses needed to block -ht receptors. because of metoclopramide's prokinetic properties, an intestinal obstruction should be ruled out prior to its use. dopamine is a less important neurotransmitter in the chemoreceptor trigger zone of cats than alpha adre nergic and -ht -serotonergic receptors, suggesting that d -dopaminergic antagonist may be a less effective antiemetic in cats. clinically metoclopramide commonly controls vomiting in cats, although this clinical response may be secondary to -ht antagonism and/ or its prokinetic effects. , extrapolated from the short elimination half-life of metoclopramide in dogs ( minutes), frequent be clinically significant side effects. phenothiazines have the potential to lower the seizure threshold; their use is not recommended in patients with a known seizure history. other cns-associated side effects linked to d antagonism occur at higher doses and produce extrapyramidal signs, including rigidity, tremors, weakness, and restlessness. antagonism of the histaminergic receptors carries the risk of sedation. because of the need for frequent dosing ( . to . mg/ kg subcutaneously every hours) and the risk of hypotension and sedation, the clinical use of phenothiazine antiemetics is limited to hospitalized patients with refractory vomiting and should be avoided in patients who are dehydrated or hypotensive. cisapride is a serotonergic -ht agonist that increases propulsive gastrointestinal motility from the lower esophageal sphincter to the colon. cisapride binds serotonergic -ht receptors in the myenteric plexus, increasing the release of acetylcholine in gastrointestinal smooth muscle. in dogs cisapride has greater prokinetic activity in the stomach relative to metoclopramide. cisapride has no direct antiemetic effect, although it is indicated in a vomiting cat with colonic dysmotility secondary to megacolon. colonic distention can trigger the vomiting reflex in cats. cisapride induces colonic smooth muscle contractions in cats with megacolon that is dependent on the influx of extracellular calcium and is only partially cholinergic dependent. other potential indications include refractory generalized ileus or gastroesophageal reflux. dosage recommendations based on the pharmacokinetics in healthy cats is . mg/kg orally every hours. prior to the use of cisapride, an intestinal obstruction should be ruled out because of its strong prokinetic effects. side effects reported in humans are cramping and diarrhea. potentially life-threatening side effects include qt prolongation and ventricular arrhythmias, the primary concern in humans that led to cisapride's removal from the market in the united states. in cats qt prolongation associated with cisapride administration requires times the therapeutic dose. because of the risk of prolongation of the qt interval and ventricular arrhythmias, the concurrent use of cisapride and dolasetron is not recommended. other potential drug interactions associated with cisapride include concurrent therapy with azole antifungals (ketoconazole and itraconazole), because of their inhibition of hepatic cyp a isoenzyme system and the inhibition of cisapride metabolism. diet trials are commonly used in cats with idiopathic gastrointestinal signs or in cats with suspected or known intermittent dosing or delivery by a constant rate infusion (cri) is necessary. empirical dosing in cats is . to . mg/kg subcutaneously or orally every hours or to mg/kg/day as a cri. approximately % of metoclopramide is excreted in the urine, thus dose reduction is recommended in cats with underlying renal azotemia. dolasetron and ondansetron are selective serotonin antagonists that inhibit central and peripheral -ht receptors. their main antiemetic effect is through antagonism of the peripheral -ht receptors in the gastrointestinal tract. in cats -ht antagonism of the crzt is also likely important in the antiemetic effect of dolasetron and ondansetron. dolasetron and ondansetron were originally used for vomiting secondary to chemotherapy because of their superior clinical efficacy. the clinical use of dolasetron and ondansetron in cats has not been associated with reported side effects, and experimental studies report minimal toxicity in animals at doses times the antiemetic dose. side effects reported in humans include headaches, elevated liver enzymes, rare hypersensitivity reactions, prolongation of the qt interval, and arrhythmias. , dolasetron is commonly used for parenteral administration and ondansetron for oral administration, dictated primarily based on the tablet sizes available and cost. recommended dosing of dolasetron is . to mg/kg intravenously every hours and ondansetron . mg/ kg orally every hours. maropitant is a neurokinin- (nk- ) receptor antagonist, blocking the binding of substance p to the nk- receptors located in the emetic center, crtz, and the enteric plexus. in cats maropitant has been reported to be efficacious in treating xylazine-induced vomiting and motion sickness. recommended dosing in cats is mg/ kg intravenously, subcutaneously or orally every hours for up to days. maropitant is reported to be well tolerated in cats. prochlorperazine and chlorpromazine are considered broad-spectrum antiemetics by antagonism of d dopaminergic, histaminergic (h and h ), and cholinergic (muscarinic) receptors within the crtz and, at high doses, the alpha-adrenergic receptors (alpha and alpha ) within the vomiting center. in cats alpha -receptors play a key role in emesis (recall xylazine is the emetic of choice in cats), suggesting cats may be more sensitive to the antiemetic effects of the phenothiazines. prochlorperazine and chlorpromazine produce an antiemetic effect at relatively low doses, thus avoiding profound sedation; although, because of antagonism of the alpha-receptors, vasodilation and hypotension can hyperacidity alone is not considered a common cause for vomiting in cats, but famotidine is effective in treating vomiting in cats associated with gastric ulcers or gastritis. recommended dosage in cats is . mg/kg every to hours. ranitidine is also a competitive inhibitor of the h receptor associated with gastric parietal cells. in addition, ranitidine increases lower esophageal sphincter tone and functions as a prokinetic agent (increasing gastric emptying and stimulating intestinal motility, including colonic motility), because of its anticholinesterase food hypersensitivities. dietary strategies used to control vomiting in cats focus on either a highly digestible diet or an elimination (novel protein/carbohydrate or hydrolyzed protein) diet. the empirical use of elimination diets in cats is reported to be relatively successful, with approximately % of cats with idiopathic gastrointestinal signs responsive to a novel protein/carbohydrate diets within to days. interestingly, traditional diet trials are recommended for a minimum of to weeks, but in this group of diet-responsive cats with chronic gastrointestinal disease, clinical improvement was reported within days. thus if a cat is going to be diet responsive, clinical improvement to a diet trial should be noted relatively early. highly digestible diets enable more effective absorption and assimilation of nutrients in the face of a compromised digestive tract. these diets contain highly digestible proteins and carbohydrates, moderate to low fat, soluble fiber but low concentrations of insoluble fiber, and are supplemented with omega- fatty acids. these diets are recommended when food allergy or intolerance is suspected. these diets contain a single highly digestible novel carbohydrate source and novel protein source. alternatively, diets formulated with hydrolyzed proteins can be used as an alternative to novel protein/carbohydrate diets. see tables - and - for information on gastrointestinal ulcers. famotidine has no direct antiemetic effect but is a competitive inhibitor of the histamine (h ) receptors associated with the gastric parietal cells. the h -receptor is the dominant receptor involved in gastric acid secretion. h receptor antagonism is reported to result in a % to % reduction in acid production. famotidine is more effective at suppressing gastric acid secretion relative to ranitidine. famotidine is well tolerated, although, with chronic therapy, there is the potential for hypoacidity and gastric bacterial overgrowth. in humans dose reduction is recommended in association with renal dysfunction. famotidine is not an inhibitor of the hepatic microsomal cytochrome p- enzyme system, therefore significant drug interactions are not anticipated. activity. , significant drug interactions associated with hepatic microsomal cytochrome p- enzyme system inhibition are not a clinical concern with ranitidine. an adverse effect to be aware of in cats treated with ranitidine is transient hypotension associated with ranitidine administered as an iv bolus. in humans dose reduction is recommended in patients with renal azotemia. ranitidine is effective in decreasing gastric acid in cats. ranitidine would be a logical choice in a cat with gastrointestinal ulceration and/or atony. the reported dosage recommendation for ranitidine in cats is . mg/ kg orally every hours or . mg/kg intravenous every hours. omeprazole omeprazole is a proton pump inhibitor that targets the h + /k + atpase pump on the luminal surface of partial cells. omeprazole is effective at suppressing parietal cell acid secretion, and its effects persist for ≈ hours after drug withdrawal because of drug accumulation in the parietal cell (by ion trapping). indications for omeprazole therapy are for the treatment and prevention of nonsteroidal antiinflammatory drug (nsaid)-induced ulcers. omeprazole is enteric coated to prevent its degradation by gastric acid; therefore oral formulations should not be crushed. based on human studies, omeprazole is a hepatic microsomal cytochrome p- enzyme inhibitor with known drug interactions with diazepam. the extent of clinically significant drug interactions in cats has yet to be studied. omeprazole is reported to be effective in reducing gastric acid secretion in cats. the recommended empirical dosage in cats is . to mg/kg orally once daily. long-term use in humans and dogs is associated with gastric polyps and parietal cell hyperplasia, respectively, but the effect of long-term use in cats is currently unknown. sucralfate is a disaccharide complexed with aluminum that dissociates to sucrose octasulfate and aluminum hydroxide upon exposure to gastric acid. the sucrose octasulfate spontaneously polymerizes, producing a viscous material capable of binding ulcerative lesions in the gastric mucosa. once bound to the exposed mucosa, it prevents back diffusion of h + , inactivates pepsin, absorbs bile acids, and increases mucosal prostaglandin synthesis, collectively supporting ulcer healing. sucralfate is not systemically absorbed but does prevent the absorption of drugs capable of chelating with aluminum, including fluoroquinolones, tetracyclines, and digoxin. if sucralfate is indicated in a cat being treated concurrently with fluoroquinolones, tetracyclines, or digoxin, the recommendation is to administer the other drug hours prior to the administration of sucralfate to optimize drug absorption. clinical indications for the use of sucralfate in cats are for the treatment of gastric ulcers and esophagitis. dosage recommendation in cats is mg orally every hours. sucralfate can be crushed, suspended in water, and administered as slurry. diet trials are used in some cats with diarrhea if the underlying cause is from known or suspected food hypersensitivities. dietary management includes either a highly digestible diet, an elimination (novel protein/ carbohydrate or hydrolyzed protein) diet (see above for both), or a diet high in fiber. high-fiber diets contain a mixture of both soluble and insoluble fiber that can be beneficial in patients with signs of large bowel diarrhea. insoluble fiber, such as cellulose, functions to increase the bulk of the stool, bind fluid, and regulate intestinal motility. soluble fiber, including fruit and vegetable pectins and beet pulp, functions as a source of butyric acid that can be used by the colonic mucosa and decreases proinflammatory cytokines. , cobalamin cobalamin (vitamin b ) is an essential vitamin needed by a number of different enzymes, including key enzymes involved in methionine metabolism and the conversion of methylfolate to tetrahydrofolate needed for dna synthesis. cobalamin and folate are intimately linked, and hypocobalaminemia can lead to a functional deficiency of folate. ingested cobalamin requires intrinsic factor binding for enterocyte absorption at the level of the ileum. hypocobalaminemia is commonly associated with distal small intestine diseases in cats, including inflammatory bowel disease. in addition, low cobalamin has a negative impact on enterocyte function; therefore in many cats with intestinal disease and hypocobalaminemia, cobalamin supplementation is necessary for resolution of clinical signs. , quantification of serum cobalamin levels is recommended in cats with clinical signs of small bowel diarrhea, ones suspected to have an infiltrative disease of the small intestine (inflammatory bowel disease or gastrointestinal lymphoma), or ones with pancreatic dysfunction. when hypocobalaminemia is identified, supplementation is recommended mannanoligosaccharides, inulin, chicory, and lactosucrose. reports on the use of prebiotics in cats are limited to their use in healthy cats; healthy cats fed fructooligosaccharides were reported to have a trend toward an increase in fecal concentrations of lactobacilli and a decrease in concentration of c. perfringens and e. coli relative to the controls. to date no reports are available on the use of prebiotics in cats with gastrointestinal disease. probiotics and prebiotics potentially have a supportive role in the treatment of gastrointestinal disease in cats. the important clinical consideration in the use of probiotics as an adjunctive therapy is to ensure the use of live nonpathogenic microorganisms that have been documented to colonize the intestinal tract of cats. gastrointestinal flora co-evolve with their host. gastrointestinal microorganism colonization varies among species and within each individual animal. the distribution of fecal microflora for a given individual is considered unique but stable over time. antimicrobial and antiparasitic therapies for the treatment of feline diarrhea are indicated based on the specific diagnosis of infectious diarrhea, bacterial enteritis, or as adjunctive therapy for inflammatory bowel disease. infectious pathogens more commonly associated with feline diarrhea include bacterial enteropathies (clostridium, campylobacter), protozoal enteropathies (tritrichomonas foetus, giardia spp.), and helminthic enteropathies associated with ascarids, hookworms, whipworms, and tapeworms. only the more common anthelminthic, antimicrobial, and antiprotozoal therapies are discussed below (tables - and - ) . more information about antimicrobials and antiparasitics is found under specific infections in the discussions of infectious enteritis and gastrointestinal parasites. fenbendazole is an anthelmintic used to treat common helminth infections, including ascarids, hookworms, whipworms, and a single species of tapeworm, taenia pisiformis. giardia spp. are also considered susceptible to fenbendazole. fenbendazole binds beta-tubulin subunits of microtubules, interfering with their polymerization. side effects include vomiting and diarrhea, although both are considered rare. fenbendazole is not approved for use in cats in north america but is commonly used clinically, and an empirical dosage of mg/kg ( µg/cat every days) while the underlying cause of cat's malabsorption is being investigated and at initiation of targeted therapy. probiotics probiotics are ingested live microorganisms intended to benefit the host, specifically to support the microflora environment of the gastrointestinal tract as well as to provide an overall benefit to the body's immune function by immunomodulation. , , probiotics chemically modify ingesta and intestinal mucus, as well as affect immune cells, enterocytes, and goblet cells within the intestinal mucosa through direct receptor interactions and indirectly through the action of cytokines. the microorganisms commonly used are nonpathogenic bacteria and yeast that have a vital role in gastrointestinal health, including lactobacillus spp., enterococcus faecium, bifidobacterium spp., and saccharomyces spp. for example, lactobacilli synthesize b vitamins, digestive enzymes, and folate coenzymes. clinical indications for the use of probiotics are diverse, including primary gastrointestinal disease, chronic renal disease, and pancreatitis. the rational use of probiotics in the treatment of gastrointestinal diseases include their ability to modulate gastrointestinal flora, minimize colonization by pathogenic bacteria, and decrease the likelihood of bacterial translocation. in healthy cats, lactobacillus acidophilus is reported to reduce fecal clostridium counts. when lactobacillus acidophilus was used adjunctively with antimicrobial therapy, fecal shedding of campylobacter was reduced in cats with campylobacter-induced diarrhea relative to cats treated with antimicrobials alone. specifically, in cats with gastrointestinal disease, available research supports the probiotic enterococcus faecium as clinically beneficial in resolving diarrhea in kittens. relative to the control group, the kittens treated with probiotics had increased fecal bifidobacteria and blood iga concentrations and decreased fecal counts of clostridium perfringens. prebiotics are dietary supplements used to select for the more beneficial enteric flora, support gastrointestinal function, and prevent the overgrowth of pathogenic bacteria, including salmonella, escherichia coli, clostridium, or campylobacter. for a food additive to be considered a prebiotic, it must be nondigestible by the gastrointestinal tract (resistant to gastric acidity, gastrointestinal hydrolysis and absorption), yet fermentable by gastrointestinal microflora to short-chain fatty acids to stimulate the growth of "good" intestinal bacterial. prebiotics include nondigestible oligosaccharidescommonly, oligofructose, fructo-oligosaccharides, pyrantel pamoate is a nicotinic anthelmintic used primarily for the treatment of ascarids, but its spectrum of activity also includes hookworms and the stomach worm, physaloptera spp. pyrantel is toxic to susceptible parasites through its selective action on their nicotinic acetylcholine receptors, resulting in depolarization and spastic paralysis. pyrantel is not approved for use in cats but is considered safe in cats and is commonly used clinically. the dosage recommendation in cats is mg/ kg orally once, repeat in weeks, and finally repeated in months. metronidazole is a nitroimidazole antibiotic with an anaerobic antibacterial spectrum with antiprotozoal activity against giardia spp. in an anaerobic environment, metronidazole is converted to unstable intermediates (nitroso free radicals) that disrupt bacterial dna synthesis. immunomodulatory properties capable of inhibiting cell-mediated immunity have been described for metronidazole, although its immunomodulatory properties are reported at dosages well beyond what is recommended for clinical use, raising questions about the clinical use of metronidazole as an adjunctive therapy for treating inflammatory bowel disease. , resistance to metronidazole is considered rare. the most common adverse reaction is gastrointestinal upset, including inappetence, anorexia, nausea, and vomiting. profuse salivation can occur in cats after oral administration of metronidazole base (formulation used in standard tablets), which has lead to the use of metronidazole benzoate (a compounded formulation not approved by the food and drug administration) in some cats because of its better oral palatability. at high doses (> mg/ kg/day) benzoic acid is reported to be neurotoxic in cats, but with appropriate clinical dosing of metronidazole benzoate benzoic acid toxicity is unlikely. dose-related metronidazole toxicity in cats results in cerebellovestibular ataxia secondary to gamma-aminobutyric acid (gaba) inhibition at dosages greater than or equal to mg/kg/day , ; clinical signs include nystagmus, head tilt, ataxia, seizures, and obtundation. in cats with inflammatory bowel disease, the dosage recommendation for the metronidazole base is to mg/kg/day. metronidazole benzoate contains approximately % metronidazole base by weight, translating to an empirical dosage of mg/kg/day of metronidazole benzoate (equivalent to . mg/kg/day of metronidazole base). little is known about the safety of chronic metronidazole use in cats, but oral metronidazole has been reported to disrupt dna within feline peripheral mononuclear cells following days of therapy. this metronidazole-induced genotoxicity is reversible and is no longer detected days after antibiotic therapy is discontinued. ronidazole is a nitroimidazole antibiotic (similar to metronidazole) and available as a powder-on-feed antibiotic. ronidazole is not approved for use in cats but has immunosuppressive therapies used in cats with inflammatory bowel disease include glucocorticoids, cyclosporine, and chlorambucil (tables - and - ). more information on the treatment of inflammatory bowel disease is found elsewhere in this chapter. glucocorticoids are considered first-line therapy in the treatment of cats with inflammatory bowel disease. glucocorticoids bind their intracellular glucocorticoid receptors, modifying the expression of genes with glucocorticoid response elements. immunomodulation is achieved through inhibition of cytokine release and response, including decreasing leukocyte phagocytosis, chemotaxis, and antigen expression. the more common side effects in cats include gastrointestinal ulceration, opportunistic infections (e.g., urinary tract infections), pancreatitis, and diabetes mellitus. cats are less susceptible to iatrogenic hyperadrenocorticism than dogs. initial therapy is usually with oral prednisone or prednisolone. prednisone is a prodrug that is metabolized to its active form prednisolone. cats are reported to be less efficient in the conversion of prednisone to prednisolone ; therefore prednisolone may be preferred in cats, especially in cats refractory to prednisone therapy. been used off-label to effectively treat tritrichomoniasis in naturally and experimentally infected cats ( mg/kg orally every hours for days). t. foetus reduces nitroimidazoles to their nitroso free radicals. ronidazole has been reported to have better in vitro and -fold higher in vivo activity against t. foetus relative to metronidazole. , , ronidazole resistance is beginning to be reported in t. foetus isolates from cats with diarrhea. side effects include hepatoxicity and neurotoxicity. neurotoxicity is associated with high doses and has been reported in cats. the use of ronidazole is recommended only for confirmed cases of t. foetus, and dosing should not exceed mg/kg once daily in cats, especially in cats at risk for neurotoxicity. ronidazole is not registered for human or veterinary use in the united states; therefore its use in cats requires owner informed consent and client education of the potential human hazards. immunosuppressive therapies are considered the standard of care for cats with gastrointestinal biopsies consistent with inflammatory bowel disease (lymphoplasmacytic or eosinophilic inflammation). the common alternative forms of glucocorticoids can be considered in specific patient populations. in patients with severe malabsorption, injectable dexamethasone may provide improved bioavailability and clinical response. also dexamethasone maybe preferred in patients with a history of heart failure, fluid retention, or hypertension because of its lack of mineralocorticoid activity relative to prednisone/prednisolone. dexamethasone's potency is to times that of prednisolone; therefore a dose reduction is necessary when prescribing dexamethasone (the dexamethasone dose is one seventh that of prednisolone). , budesonide is an oral, locally active, highpotency glucocorticoid that is formulated to be released in the distal gastrointestinal tract (based on the ph differential between the proximal and distal small intestine), where it is absorbed and is locally immunomodulating at the level of the enterocyte. the amount of systemically absorbed budesonide is minimized, because % to % of the budesonide absorbed from the gastrointestinal tract undergoes first-pass metabolism in the liver. some systemic absorption does occur, as evidenced by a blunted adrenocorticotropic hormone (acth) stimulation test in dogs treated with budesonide at mg/m for days. , the use of budesonide in cats remains anecdotal, with a suggestive empirical dose of . to mg/cat/day. initial glucocorticoid therapy for cats with inflammatory bowel disease consists of antiinflammatory ( . to mg/kg/day) to immunosuppressive ( to mg/kg/ day) dosages, with dosages based on the potency of prednisone/prednisolone. the goal of therapy is to achieve clinical remission and slowly taper the dose of glucocorticoids to the lowest dose that will control the cat's clinical signs. some cats may be completely weaned off therapy, while others require long-term lowdose therapy. the tapering of therapy should be slow, with a % to % dose reduction every to weeks. cyclosporine is considered a second-tier immunosuppressive drug used to treat inflammatory bowel disease in cats. use of cyclosporine in the treatment of diarrhea associated with inflammatory bowel disease in cats is extrapolated from its use in dogs to treat glucocorticoid refractory inflammatory bowel diarrhea. cyclosporine suppresses t-lymphocyte-mediated inflammation in the gastrointestinal tract secondary to suppression of inflammatory cytokines. specifically, cyclosporine attenuates t-lymphocyte activation and proliferation through the inhibition of interleukin- (il- ) production. side effects of cyclosporine in cats include dose-dependent inappetence and vomiting, which may occur at the onset of therapy and are generally responsive to dose reduction. other less common side effects reported in cats are opportunistic infections, including toxoplasmosis and hepatoxicity. the microemulsion formulation of cyclosporine has higher oral bioavailability and less variable pharmacokinetics. a suggested initial dosage of cyclosporine is mg/kg every or hours. serum cyclosporine levels can be used to monitor for excessive trough plasma concentration (> ng/ml) as determined using a highperformance liquid chromotography (hplc) analytical method. chlorambucil is a slow-acting nitrogen mustard that alkylates and effectively cross links dna, leading to altered protein production. the immunosuppressive effects of chlorambucil are the result of its cytotoxic effect on lymphocytes, similar to other nitrogen mustards. bone marrow suppression is considered mild to moderate and is rapidly reversible. neurotoxicity and myoclonus has been reported in a cat accidently overdosed with chlorambucil. chlorambucil is used as a second-tier drug in cats to treat immune-mediated disorders, in part because of ease of administration and its low risk of myelosuppression. for the treatment of inflammatory bowel disease, the recommended dosing in cats is mg/cat every hours in cats greater than kg and mg/cat every hours in cats less than kg. chlorambucil is commonly used in combination with glucocorticoids in the treatment of immune-mediated diseases, including inflammatory bowel disease, overlooked. awareness about feline esophageal diseases is low, the clinical signs are often not specific, and imaging beyond survey radiographs may be required for diagnosis. the esophagus is composed of four layers (from inner to outer): mucosa, submucosa, muscularis, and adventitia (there is no serosal layer). in the dog, the muscle layer is entirely composed of skeletal muscle, but in cats, the distal third of the esophagus is composed of smooth muscle. the upper esophageal sphincter prevents reflux of esophageal contents into the pharynx and minimizes aerophagia. the lower esophageal sphincter prevents gastroesophageal reflux and relaxes during swallowing to allow food and fluid to enter the stomach. clinical signs of esophageal disease include drooling, dysphagia, pain on swallowing (odynophagia), and, most classically, regurgitation. weight loss may occur secondary to inadequate food intake when disease is severe or chronic. other clinical signs, such as anorexia, cough, dyspnea, and fever, may occur if complications such as aspiration pneumonia or esophageal perforation occur. regurgitation is passive expulsion of food or fluid from the esophagus. the food is undigested and often accompanied by mucus and saliva. mucosal erosions may produce frank blood in the regurgitated material. regurgitation must be differentiated from vomiting (table - esophageal disease is uncommon in the cat when compared with dogs, but it is also likely that problems such as esophagitis and esophageal strictures are often salivation, retching, and abdominal contractions. the vomitus consists of partially digested food from the stomach and/or intestines and may be mixed with bilestained fluid. some cats will have both vomiting and regurgitation. expectoration may also be confused with vomiting or regurgitation. expectoration is associated with coughing, but cats that cough excessively may also stimulate vomition so that a careful history is needed to characterize the clinical signs correctly. coughing may also occur in cats that have aspirated as a result of regurgitation. drooling, dysphagia, and odynophagia are most commonly seen with conditions of the oropharynx and/or proximal esophagus. odynophagia is most commonly associated with esophagitis and foreign bodies. dysphagia and regurgitation together most commonly indicate oral or pharyngeal dysfunction; if regurgitation is not accompanied by dysphagia, esophageal dysfunction is likely. regurgitation in cats with esophageal disease is caused by obstruction or muscular dysfunction. causes of obstruction include vascular ring anomaly, foreign object, stricture, and neoplasia. causes of muscular dysfunction include congenital disease, esophagitis, myopathies, neuropathies, and dysautonomia. regurgitation may occur immediately after eating if the lesion is in the proximal esophagus. however, a dilated esophagus provides a reservoir for food and fluid so that regurgitation may not be associated in time with eating. young cats with signs of esophageal disease should be suspected of congenital defects, such as vascular ring anomaly, or a foreign body. adult cats with esophageal disease may have a recent history of general anesthesia, administration of certain oral medications, or ingestion of irritant chemicals. acute onset of clinical signs may suggest a foreign body, while chronic, slowly worsening signs may indicate a stricture or tumor. all cats suspected of esophageal disease should have a minimum database as part of the diagnostic plan (complete blood cell count, serum chemistries, urinalysis, and other tests as indicated by age or concurrent diseases, such as serum total t and blood pressure measurement). an important part of diagnosis is observation of the cat while eating food, to localize the location of the dysfunction. if the cat is unwilling to eat while in the veterinary clinic, the owner can make a video of the cat eating at home for the clinician to view. the general diagnostic approach to regurgitation in cats is found in figure - . plain and contrast radiography and endoscopy are important diagnostic tools for esophageal disease. fluoroscopy is valuable for the diagnosis of motility disorders, but availability is limited to universities and referral centers because of the cost of equipment. ultrasonography is limited to evaluation of * references , , , , , . the cervical esophagus and a small segment of abdominal esophagus between the cardia of the stomach and the diaphragm. the entire esophagus should be evaluated with cervical and thoracic radiographs. thoracic radiographs may also show evidence of complications such as aspiration pneumonia or esophageal perforation. the normal esophagus is not visualized on plain radiographs, but may be seen if food or fluid are retained or a foreign body or mass is present. radiographic contrast agents useful for esophagrams in cats include liquid or paste barium. a water-soluble iodinated contrast agent (e.g., iohexol, gastrografin) is preferred if there is any risk the esophagus is perforated, because these agents are less irritating and more rapidly reabsorbed. esophagrams are most useful for diagnosis of luminal obstructions, extraluminal compression, mucosal irregularities, and possibly alterations in motility. dilute liquid barium can be administered with a syringe or it may be mixed with canned food, especially if a motility disorder or stricture is suspected. multiple lateral radiographs are taken rapidly, starting within seconds of swallowing the contrast agent. contrast is rapidly cleared from the normal esophagus by peristalsis. if the contrast in the esophagus terminates abruptly, an obstruction is likely. if the contrast is retained throughout the esophagus, muscular dysfunction is suspected. some conditions, such as esophagitis, are difficult to diagnose radiographically, because contrast agents may or may not adhere to ulcerated mucosa. flexible endoscopy is a noninvasive diagnostic tool for esophageal disorders and is often used if plain and contrast radiographs have failed to establish a diagnosis. it is most sensitive for diagnosis of masses, ulcers, perforations, and obstructions. in addition, it is often possible to retrieve foreign bodies using endoscopy as well as to assist with dilatation of strictures or placement of gastrostomy feeding tubes if required. biopsy of the esophageal mucosa is more difficult than biopsy of gastric or intestinal mucosa and is not commonly performed with the exception of mass lesions. esophagitis may result from various causes of inflammation, such as contact irritation from foreign bodies (including trichobezoars lodged in the esophagus), chemical irritants or caustic medications, gastroesophageal reflux, persistent vomiting, hiatal hernia, or general anesthesia. inflammation disrupts the esophageal mucosa and exposes the submucosa. an important part of the treatment plan is identification and treatment of the underlying cause. clinical signs include dysphagia, regurgitation, salivation, and repeated swallowing, although signs may be absent in cats with mild esophagitis. cats with odynophagia may repeatedly extend the head and neck while swallowing. if the esophagitis or underlying disease is severe, weight loss and dehydration may occur secondary to anorexia. if the submucosa and muscularis are damaged, strictures may form as a result of the production of fibrous connective tissue and compromise the esophageal lumen. neoplasia is an important cause of esophageal stricture in humans, but not in cats. most cases have single strictures, but multiple strictures are possible. in two studies, the mean stricture diameter was reported as mm. , most strictures are less than cm in length. clinical signs associated with strictures appear to days after the esophageal injury and may be present for weeks before definitive treatment is pursued. regurgitation typically occurs immediately after eating, although if the stricture is long standing, a pouch may form cranial to the lesion where food accumulates. survey radiographs may be normal in cats with esophagitis and strictures, but are useful to rule out other causes for the clinical signs, such as a foreign body, or to detect related problems, such as aspiration pneumonia. in some patients, dilation of the esophagus with fluid or air may be seen. a contrast esophagram may disclose irregularities of the mucosa in cats with severe esophagitis. segmental dilation may occur with severe inflammation. strictures may be diagnosed with an esophagram (figure - ) ; however, in some cases, it may be difficult to differentiate a stricture from intramural thickening (e.g., because of neoplasia). endoscopy is useful for diagnosis of esophagitis; findings include mucosal erythema, hemorrhage, and erosions or ulcerations. if gastroesophageal reflux is present, the lesions will be most severe in the distal esophagus, and the lower esophageal sphincter may be dilated. endoscopy is often used for definitive diagnosis of esophageal stricture as well as to visualize the lesion during treatment by bougienage or balloon catheter dilation. strictures appear as a ring of white fibrous tissue that narrows the esophageal lumen. if endoscopy is performed after a barium esophagram, hours should be allowed to elapse between the procedures or the barium will obscure visualization with the endoscope. general anesthesia is an important cause of esophagitis (sometimes leading to stricture formation) in cats, probably because gastroesophageal reflux appears to occur commonly in anesthetized cats.* for example, in a series of seven cats with benign esophageal stricture, recent anesthesia for ovariohysterectomy was the suspected cause in five cases. clinical signs appeared up to days after anesthesia. abnormal esophageal tissue was performed in two cases. the authors noted that the esophageal mucosa may appear grossly normal, but submucosal inflammation may be found on histopathologic examination of biopsies. a consequence of chronic severe gerd in humans is the development of metaplastic columnar epithelium (barrett esophagus) that replaces the normal squamous epithelium. one case series reported on barrett-like esophagus in three cats. two cases were associated with hiatal hernia and one with cardial incompetence. drug-induced esophageal damage and stricture formation is well known in humans and cats (see . in humans over drugs have been implicated, and most are antibacterials or nsaids. implicated drugs in the cat include tetracycline, doxycycline, and clindamycin in tablet or capsule form administered without a food or water bolus. , , , , clinical signs (dysphagia, regurgitation, salivation, anorexia) appear to days after drug treatment is started. strictures commonly form in the midcervical esophagus or over the heart base in the thoracic esophagus. doxycycline hyclate is most commonly associated with esophageal strictures in cats, and the principle reason for its irritating properties is an acidic ph. the monohydrate salt of doxycycline is less irritating and is marketed as tablets and a palatable paste licensed for use in dogs and cats in some countries. in humans esophageal ulceration after doxycycline therapy is more common than stricture formation. although the development of strictures in cats would appear to be uncommon, it seems possible the incidence of esophagitis is underestimated, because the clinical signs (e.g., odynophagia, chest pain) may go unrecognized. esophageal transit studies of normal cats have shown that the passage time of dry-swallowed tablets and capsules is often prolonged (longer than seconds). , complete entrapment (retention for more than minutes) in the midcervical region occurs commonly. however, a small bolus of food or water is sufficient to ensure immediate passage of the medication into the stomach. , the risk of esophageal retention can also be lessened by coating a tablet or capsule with butter or a gel dietary supplement (nutri-cal; vétoquinol, fort worth, tex.). one study determined that tablets or capsules administered using a one-step pill gun with flavored liquid (flavorx pill glide; flavorx, columbia, md.) or a pill delivery treat (greenies pill pockets; nutro products, franklin, tenn.) ensured an average transit time of seconds or less. delayed esophageal transit of medications allows tablets and capsules to disintegrate within the esophagus, exposing the mucosa to irritating chemicals. cats may be at risk of delayed esophageal transit, because they do not typically drink water with medication, and they do not have an upright posture. in addition, medications are often given to sick or dehydrated patients many preanesthetic drugs and induction agents reduce lower esophageal sphincter pressure. , other predisposing factors may be intraabdominal surgery and a head-down position on the surgery table. reflux fluid with a ph less than is likely to cause esophageal mucosal damage, as is prolonged contact time. esophageal defense mechanisms include clearance of the reflux fluid by peristalsis and neutralization of the acidic ph by the bicarbonate present in saliva. in a study of kittens less than weeks of age, risk of gastroesophageal reflux during anesthesia was evaluated with use of a laryngeal airway mask versus endotracheal intubation. gastroesophageal reflux was observed in % of kittens with use of the laryngeal airway mask but more importantly in % of kittens with endotracheal intubation. the reflux episodes occurred shortly after anesthesia induction. in a study of cats anesthetized with thiopentone or propofol, gastroesophageal reflux occurred in %. reflux also occurred shortly after anesthesia was induced and lasted for a mean of minutes. it is unknown why esophageal strictures form only in a small number of cats that experience gastroesophageal reflux during anesthesia. gastroesophageal reflux disease (gerd) is a commonly reported cause of esophagitis in humans, but it is rarely reported in cats when not associated with general anesthesia. , the true incidence is unknown, and diagnosis may be hampered by scant knowledge about the clinical presentation and diagnosis. clinical signs and diagnostic procedures are as for other causes of esophagitis. in one case series of three cats, diagnosis of gerd was based on clinical signs, contrast radiography, and endoscopic findings. biopsy and histopathology of obtained from a compounding pharmacy in most countries and can only be given orally. h -receptor antagonists are competitive inhibitors that block parietal h receptors and decrease the amount of gastric acid produced. proton pump inhibitors are noncompetitive inhibitors that act on the h + /k + atpase enzyme system at the secretory surface of gastric parietal cells. they are considered superior for decreasing gastric acid secretion and are therefore the first choice, despite their greater cost. a drawback of proton pump inhibitors is that they must be administered orally. sucralfate may be beneficial for reflux esophagitis, because it binds to mucosal erosions in an acid environment and provides a protective barrier. it is given as oral slurry, ideally separate from meals or other medications. antibiotics are not commonly recommended unless aspiration pneumonia is present or the eroded mucosa is at risk of bacterial infection in a patient with severe disease or a compromised immune system. corticosteroids are often recommended for cats with esophagitis to reduce esophageal inflammation and impair the formation of fibrous connective tissue. however, the benefit of corticosteroids in cats with esophagitis has not been investigated and administration must be weighed against potential adverse effects, especially in patients with aspiration pneumonia. treatment of esophageal stricture typically requires dilation with either bougienage or a balloon catheter; both are used with endoscopic visualization under general anesthesia. appropriate analgesia should be provided, because dilating the stricture is painful. it does not appear that placement of a gastrostomy feeding tube is specifically required to recover from dilation procedures, although a tube may be placed in some anorexic cats to ensure nutritional intake and administer oral medications. a bougie is a long, narrow, oblong, mechanical dilator available in various sizes (typically -to -mm sizes are used in cats) that is gently passed through the stricture, usually over a guide wire. established criteria for selection of bougie diameter and dilation end points are not available. in one study, the initial bougie chosen was approximately the same size as the estimated diameter of the stricture, or no more than mm larger. once the first bougie is passed, subsequent bougies of increasing diameter are employed. two to four bougies of increasing size may be passed in a single session, with the goal of dilating the stricture without causing esophageal tear or perforation. determining when dilation should be stopped is a matter of clinical judgment. the procedure may be repeated as needed to maintain improvement; the total number of procedures required is variable. in one retrospective case series of eight cats treated with bougienage, the median number of procedures was . , and a good outcome was achieved in % of the cases. in some cases, the endoscope tip itself has been used for that may be at greater risk of esophageal retention of medication. all oral medication given to cats in tablet or capsule form should be followed with food or a liquid. mild esophagitis will resolve on its own, especially if an underlying cause can be removed or treated. frequent meals of canned food should be provided. cats with moderate to severe esophagitis will require medical therapy, and those with difficulty eating or weight loss may also require gastrostomy tube feeding. esophagostomy or pharyngostomy feeding tubes should be avoided in these patients. treatment is provided to control inflammation and promote healing while reducing gastric acid secretion and increasing lower esophageal sphincter tone. the length of medical treatment will vary from about one week to several weeks, depending on the underlying cause and severity of disease. medications indicated for esophagitis include prokinetics, h -receptor antagonists, proton pump inhibitors, and sucralfate (table - ) . prokinetic drugs enhance gastric emptying and increase lower esophageal sphincter tone. metoclopramide also has antiemetic effects, which may be beneficial in patients with chronic vomiting. it can be administered by the subcutaneous (sc) route, an advantage in a vomiting or regurgitating patient. cisapride may be more effective at enhancing both gastric emptying and lower esophageal sphincter tone, but it must be stent placement has recently been described in cats with esophageal strictures with variable results. a -year-old cat presented with a -week history of dysphagia and regurgitation caused by a single cervical esophageal stricture after treatment with oral clindamycin. guided balloon dilation was performed times over a period of weeks, but stricture formation always recurred. a self-expanding metal stent was placed using endoscopy and fluoroscopy after another dilation procedure. the cat did well eating a canned diet from an elevated position for months, but by months, the cat was no longer able to eat even liquid food and was euthanized. on necropsy, the stent had migrated and was obstructed by swallowed hair. in another case, a biodegradable self-expanding stent was used to successfully treat an -year-old cat that presented with a stricture in the cervical esophagus after anesthesia for dentistry. balloon dilation was performed twice, but regurgitation recurred days after the last procedure. the stricture was dilated a third time with a balloon catheter, and a tubular selfexpanding polydioxanone stent was placed with fluoroscopic guidance. the life span of the stent was estimated to be to weeks, sufficient time to allow healing of the esophagus. foreign bodies are less commonly found in the esophagus of the cat than in other gastrointestinal locations. reported foreign bodies include string, needles, fish hooks, and bones. trichobezoars may cause obstruction when they become lodged in the esophagus during vomiting ( figure - ). recurrent esophageal trichobezoars have been infrequently reported in the literature. , it is not known if an esophageal motility disorder is the underlying cause for recurrent obstructions. in one case, an esophageal diverticulum developed in association with recurrent trichobezoars. treatment for recurrent trichobezoars includes prokinetic drug therapy (e.g., cisapride), moderate to high-fiber diets, and shaving of long-haired cats. common areas for foreign bodies to lodge include the thoracic inlet, the heart base, and the esophageal hiatus in the diaphragm. obstruction of the esophageal lumen may be complete or partial. clinical signs include acute onset of gagging, salivation, repeated swallowing, dysphagia, and regurgitation. however, chronic esophageal foreign bodies have been reported in cats with dysphagia, intermittent regurgitation, and weight loss over a period of weeks or months. bougienage when bougies or balloon catheters were not available. balloon catheter dilation has become a popular method in recent years. , , although some clinicians feel this is a safer procedure than bougienage, there is no data in the literature to support this assumption. the catheter can be placed through the endoscope biopsy channel, alongside the endoscope, or with the aid of a preplaced guide wire. as for bougienage, established criteria for selection of balloon diameter and dilation end points are not available, and the clinician's best judgment must be used. various balloon sizes are available; in one study, the size was selected so that the inflated diameter was mm larger than the stricture diameter. the balloon is passed into the stricture with endoscopic guidance. it is then inflated to a predetermined pressure for to minutes to stretch the stricture, usually with saline, but contrast agents may also be used if fluoroscopy is used. as for bougienage, some cases may require more than one dilation procedure (typically two to four). cuffed endotracheal tubes are not appropriate substitutes for balloon catheters. regardless of the method used, after the dilation procedure, the endoscope should be used to look for other strictures and should be passed into the stomach to look for potential causes, such as causes of chronic vomiting. after treatment, medical management to decrease ongoing gastroesophageal reflux, resolve inflammation, and prevent further stricture formation should be instituted (as described previously). most cats are able to eat the day following the dilation procedure. corticosteroid treatment after dilation is controversial, and no controlled studies in animals are available. antibiotics are not routinely recommended. the prognosis for cats undergoing esophageal dilation is generally good based on the ability to eat canned food with minimal episodes of regurgitation. however, published studies show % to % of cats died or were euthanized despite multiple episodes of dilation, and up to % could only be fed liquid diets. , , , even among cats with good outcomes, a return to a dry kibble diet may not be possible. the dilation technique employed may be dictated by the clinician's experience, the equipment available, and the cost. potential complications of both methods include esophageal tear or perforation, hemorrhage, infection, and aspiration. esophageal tears or perforations may lead to pneumothorax or pneumomediastinum. repeated stricture formation is also possible, leaving only less desirable treatment options, such as long-term percutaneous gastrostomy tube feeding or surgery. esophageal surgery is generally avoided whenever possible, because it is difficult and invasive (requiring a thoracotomy), with risk of serious complications, such as failure of anastomosis, necrosis, and stricture formation. closure of incisions in the esophagus is following uncomplicated foreign body removal, the esophagus should be carefully inspected for lesions and bleeding before the endoscope is withdrawn. food and water should be withheld for to hours. supportive care includes fluid therapy and analgesia; a gastrostomy feeding tube may be required in selected cases for nutritional support. broad-spectrum antibiotics are administered to control bacterial infection and therapy for esophagitis should be instituted as described previously. careful follow-up should include evaluation for stricture formation. if an esophageal perforation has occurred, conservative management may be sufficient if the defect is small. a broad-spectrum antibiotic should be administered along with other supportive care, such as fluid therapy and analgesia. feeding through a gastrostomy tube for several days is recommended as well as close monitoring for complications such as pleuritis. large perforations require thoracotomy for surgical repair. megaesophagus is a diffuse hypomotility disorder that may be classified as congenital versus acquired or idiopathic versus secondary to other diseases. it is uncommon in cats compared with dogs. at least two dog breeds have been identified with heritable congenital megaesophagus. a heritable form of megaesophagus has been suggested for cats, particularly for siamese cats, although no detailed studies have been performed. , it is often frustrating to determine the underlying cause of acquired megaesophagus. megaesophagus may be a manifestation of neuromuscular diseases, such as dysautonomia or myasthenia gravis (see chapter ) . megaesophagus may also develop secondary to esophagitis from chronic vomiting or gerd. , other uncommon causes of megaesophagus are found in the literature. one case report describes a young cat with megaesophagus secondary to a large nasopharyngeal polyp that extended into the cervical esophagus. megaesophagus resolved once the polyp was removed. in another report, a young cat with diaphragmatic hernia was diagnosed with megaesophagus and gastric dilation. megaesophagus resolved with medical treatment and surgical correction of the diaphragmatic defect. clinical signs are typically those of esophageal dysfunction; regurgitation is the most consistently found sign. regurgitation may not be closely related in time to eating if the esophagus is markedly distended and holds food. cats with long-standing disease may suffer from weight loss or secondary rhinitis. the appetite is typically normal or increased. additional signs may occur if systemic neuromuscular disease is present. aspiration pneumonia may cause fever, dyspnea, and cough. two case reports describe cats with idiopathic cough, mucopurulent nasal discharge, and fever may be found if aspiration has occurred. trauma to the esophagus may cause esophagitis and even esophageal stricture. perforation of the esophagus by the foreign body may lead to pneumothorax, pneumomediastinum, or pyothorax with signs of depression, anorexia, fever, and dyspnea. if the perforation occurs in the cervical esophagus, swelling, cellulitis, and drainage of serous or purulent material may be noted. many foreign bodies are readily diagnosed with survey radiographs, especially if they are radiopaque. other radiographic findings include an esophagus dilated with fluid or air. radiolucent objects may be detected with an esophagram. care must be taken when performing esophagrams on cats that may have an obstruction, because aspiration is a concern. if abnormalities that could be consistent with an esophageal perforation (e.g., periesophageal gas or fluid, pleural effusion) are detected on survey radiographs, an aqueous iodine contrast solution should be used. removal of esophageal foreign bodies should be performed as soon as possible to minimize esophageal trauma and pressure necrosis. endoscopy can be used to confirm the diagnosis and often to remove the object. both rigid and flexible endoscopes may be used along with accessories such as various forceps and foley catheters. care should be taken to remove the object as atraumatically as possible, especially if the object is sharp or pointed. if the object is in the caudal esophagus and it cannot be grasped and removed, an attempt should be made to gently push it into the stomach, where it can be retrieved using laparotomy and gastrotomy. if esophageal perforation has occurred, esophagotomy is recommended and is described elsewhere. , removal of fish hooks may require a combination of surgery and endoscopy. , a surgical approach to the esophagus is made, but the esophagus is not incised; rather, the portion of the hook protruding through the esophagus is cut and removed, and the endoscope is used to retrieve the remainder. clinical sign is regurgitation, and most patients are underweight. a distended cervical esophagus may be palpated, and secondary aspiration pneumonia may occur. a history of regurgitation since weaning is very suggestive of a vascular ring anomaly, but other causes of regurgitation must be ruled out. survey radiographs show a dilated esophagus cranial to the heart, while the caudal esophagus is usually normal. the bulge of the aortic arch normally seen on a ventrodorsal radiographic view is absent. an esophagram is used to confirm the location of the obstruction and the severity of disease. definitive treatment is surgical repair of the vascular defect (i.e., ligation and transection of the ligamentosum arteriosum). some patients will require nutritional support through gastrostomy tube feeding and treatment for aspiration pneumonia before surgery. early diagnosis and surgical intervention brings the best prognosis for return of normal esophageal function. some affected cats are left with residual esophageal hypomotility, which is managed as for idiopathic megaesophagus. esophageal neoplasia is rare in the cat as in the dog. although parasitic granulomas caused by spirocerca lupi are associated with esophageal neoplasia in dogs, this parasite does not infect cats. both primary and metastatic esophageal tumors can occur in the cat. squamous cell carcinoma is the most common primary esophageal tumor in cats and is often found in the caudal two thirds of the esophagus. , , , affected cats are middle aged or older. clinical signs are typically those associated with esophageal obstruction, such as regurgitation, dysphagia, odynophagia, and salivation. patients with advanced disease may suffer anorexia, depression, and weight loss. on physical examination, an esophageal mass may or may not be palpable. survey and contrast radiographs reveal esophageal dilation, a soft tissue mass, or periesophageal lesions that displace the esophagus. computed tomography is useful to identify periesophageal or intraluminal masses. definitive diagnosis is made with endoscopy and biopsy. mucosal biopsies are difficult to obtain, because the esophageal mucosa is tough; exfoliative cytology may also be helpful. treatment is rarely undertaken, because disease is often advanced at the time of diagnosis, and many patients have complications such as aspiration pneumonia. palliation may be attempted with chemotherapy or radiation, although data on efficacy is unavailable. in general, squamous cell carcinomas in other anatomic locations respond poorly to treatment. surgical resection may be attempted if anastomosis can be accomplished without excessive tension. megaesophagus and chronic vomiting associated with intermittent gastroesophageal intussusception. , survey and contrast radiographs may identify a dilated esophagus (figure - ), but contrast fluoroscopy is the diagnostic tool of choice when available, because it allows for assessment of peristalsis. care must be taken with contrast studies because of the risk of aspiration. treatment of megaesophagus is largely symptomatic and supportive unless an underlying disorder can be identified and treated. frequent small meals are offered with the cat feeding in an upright position. the upright position should be maintained for at least minutes after eating to allow for gravity-assisted passage of food into the stomach. this is best accomplished by having the owner hold the cat over their shoulder so that the esophagus is in a vertical position. different types of diets should be offered to determine which is best for the individual patient; calorically dense diets may be beneficial for patients with weight loss. prokinetic drugs, such as cisapride, stimulate smooth muscle, but since most of the esophagus is skeletal muscle, the efficacy of such drugs is questionable for treatment of megaesophagus. prokinetic drugs also increase lower esophageal sphincter tone and may increase esophageal transit time, neither of which is desirable in patients with megaesophagus. vascular ring anomalies are congenital malformations of the great vessels that entrap the thoracic esophagus and cause obstruction. the most commonly reported anomaly is persistent right aortic arch. the esophagus is entrapped by the aorta on the right, the ligamentum arteriosum and the pulmonary trunk on the left, and the heart base ventrally. other vascular anomalies are rarely described in cats, such as a double aortic arch described in a siamese cat. onset of clinical signs occurs around the time of weaning to solid food so that most affected cats are presented at less than months of age. the most common surgery. surgery is the treatment of choice for large defects, especially in young cats with congenital disease or cats that have failed medical management. various reconstructive surgical techniques have been described. disorders of the hiatus are rare in cats. hiatal hernia is protrusion of the distal esophagus and stomach through the esophageal hiatus of the diaphragm into the thoracic cavity; the protrusion may be intermittent ("sliding") or persistent. other organs are occasionally involved, such as the omentum. this is distinct from a gastroesophageal intussusception where the stomach is prolapsed into the lumen of the distal esophagus. , both congenital and traumatic hiatal hernias have been described in cats. , , , , congenital hernias appear to be more common than acquired hernias, and affected cats typically present with clinical signs before year of age. it is suspected that increased inspiratory effort associated with upper airway obstruction, such as a nasopharyngeal polyp, may also lead to development of hiatal hernia. hiatal herniation reduces lower esophageal sphincter pressure. clinical signs associated with hiatal hernia, such as intermittent vomiting and regurgitation, may be because of reflux esophagitis, hypomotility, or obstruction. large hernias and secondary aspiration pneumonia may be associated with respiratory distress. survey radiographs may reveal a gas-filled soft tissue density in the caudal dorsal mediastinum. an esophagram will show the gastroesophageal junction and gastric rugae cranial to the diaphragm (figure - ) . both fluoroscopy and endoscopy may be useful for diagnosis but are not typically necessary. the prognosis for cats with hiatal hernia is considered to be good. a trial of medical management (as for reflux esophagitis) for month has been recommended before the stomach is a frequent site for gastrointestinal problems in cats, and the most common gastric problems are described in this chapter. some conditions such as gastric dilatation-volvulus are often reported in dogs but rarely reported in cats. in one report of three feline cases, all were associated with diaphragmatic hernia. gastric parasites, the diagnostic approach to the vomiting cat, the gastric emptying time of normal cats is shorter than that of other mammals. in one study, the gastric emptying half-time for solid food in normal cats was . to . hours. this implies prolonged fasting (longer than hours) in preparation for anesthesia and surgery is unnecessary. the main clinical sign of gastric disease is vomiting, but it is important to note that vomiting is also associated with many nongastric problems, including concurrent intestinal disease, such as enteritis or colitis. vomiting patients therefore require a thorough physical examination and diagnostic plan to determine the cause. vomiting must be distinguished from regurgitation, which is primarily associated with esophageal disease (see table - ). vomitus often contains food, hair, refluxed bile, and therapeutics for vomiting are covered elsewhere in this chapter. the anatomy of the feline stomach is similar to that of other mammals having a simple glandular stomach. most of the stomach is situated on the left side of the abdominal cavity. it has five regions, starting from the lower esophageal sphincter: cardia, fundus, body, antrum, and pylorus ( figure - ) . the pylorus of the cat is unique compared with other species in that it is narrow and has high resistance in order to maintain a tight seal ( figure involve a wide variety of objects, including linear objects (e.g., dental floss, thread with or without a needle, tinsel, string). the owner may or may not be aware of the ingestion. ingestion of multiple foreign bodies may be seen in cats with pica ( figure - ). in one case report, a young domestic shorthair cat required gastrotomy for removal of copper pennies. some patients require multiple surgeries, because of repeated foreign body ingestion. in such cases, a behavioral diagnosis should be sought and treatment instituted (see chapter ) . trichobezoars (large masses of hair) also represent a type of foreign object. both long-and shorthaired cats may be affected. hair is normally ingested during grooming and is eliminated in vomitus and feces. cats lack the strong peristaltic contractions ("housekeeper" contractions) that clear the stomach of undigested contents normally found in other species. this may explain why cats seem to be susceptible to gastric trichobezoars. gastric motility dysfunction is suspected to cause repeated gastric trichobezoars in some cats. intestinal , and esophageal , obstruction with trichobezoars has also been documented. traditional treatments for cats with recurrent trichobezoars include regular grooming, shaving the hair coat of long-haired cats, flea control, or blood. fresh blood may appear as large or small clots. older blood clots have a brown "coffee ground" appearance. gastric bleeding may also cause melena. other clinical signs may be associated with gastric disease, such as anorexia, weight loss, pain, lethargy, bloating, and nausea. gastritis may be acute or chronic in nature, and this distinction may be useful in assessing the potential cause. for example, cats with acute gastritis may be suspected of foreign body or plant ingestion, drug or toxin exposure (see chapter ), or dietary indiscretion. cats with chronic gastritis may be suspected of parasitism, helicobacter spp. infection, or dietary intolerance or hypersensitivity (see chapter ) . chronic lymphocytic plasmacytic gastritis of unknown etiology is also a common cause of chronic vomiting. whenever possible, a specific underlying cause should be sought and treated. patients with sudden onset of vomiting may have an obvious cause in the history (e.g., dietary indiscretion), but in many cases, the cause is not apparent. abdominal radiographs should be taken if foreign body ingestion is possible, especially in a young cat. if the patient is systemically well, further diagnostic testing may be postponed pending response to therapy. treatment for uncomplicated acute gastritis is symptomatic and supportive. clinical signs are expected to resolve in to hours; if signs persist, re-evaluation and further investigation is warranted. subcutaneous fluid therapy using an isotonic balanced electrolyte solution may be used to correct mild fluid deficits (< %). oral intake of fluids and food should be discontinued for up to hours. a highly digestible diet, either commercial or homemade, is introduced with a gradual transition back to the normal diet over the next several days. antiemetic therapy may be indicated for acute uncomplicated gastritis if the vomiting is frequent or the cat has signs of nausea (see table - ). protectants, such as kaolin and pectin, are difficult to administer to cats and are without proven efficacy. bismuth subsalicylate is controversial; it is considered contraindicated by some experts, because of the cat's sensitivity to salicylates, yet is commonly used in clinical practice. cats ingest foreign bodies less commonly than dogs. in one study of cases of gastrointestinal foreign body ingestion, only % were in cats. foreign body ingestion is most likely to be seen in young cats and may a b taken just before surgery to ensure the object has not moved further down the gastrointestinal tract. postoperative management after gastrotomy includes maintenance of hydration and electrolyte balance. hypokalemia is common with anorexia and vomiting and should be treated by supplementation of iv fluids with to meq/l potassium chloride (not to exceed . meq/kg/hour). refractory vomiting should be treated with an antiemetic. a highly digestible diet can be introduced the day after surgery. in general, the prognosis for recovery is good. in one study, % of cats with gastrointestinal foreign bodies survived to discharge. those cats that did not survive had linear foreign bodies of long-standing duration with subsequent peritonitis. helicobacter are spiral or curved gram-negative bacteria that inhabit the glands, parietal cells, and mucus of the gastric antrum and fundus. helicobacter contain large amounts of urease, which alters the ph in the vicinity of the bacteria and allows for colonization of the acidic environment of the stomach. in the early s, the discovery of the association of helicobacter pylori with gastric disease (gastritis, peptic ulcers, and neoplasia) in humans revolutionized treatment of those diseases. since then, helicobacter spp. have been associated with gastric disease in various veterinary species, including cats and dogs. several helicobacter spp. (e.g., h. heilmannii, h. bizzozeronii, h. felis) have been identified in cats, some of which have the potential to infect humans, although transmission is thought to be rare. , the prevalence of helicobacter infection in cats varies geographically and may be very high (> %) in some locations. , , , , the importance of helicobacter as a cause of gastric disease is cats is unclear; the bacteria may be found in the stomach of both clinically normal cats and cats with gastritis. the prevalence of helicobacter infection is not higher in cats with gastritis compared with normal cats. determination of the role of helicobacter is also hampered by the paucity of controlled clinical trials that evaluate eradication of gastritis and clinical signs in infected cats. an immune response to infection characterized by gastric lymphoid hyperplasia is common, although the local immune response in cats is generally less severe than the response in humans infected with h. pylori. to date gastrointestinal ulcers have not been associated with helicobacter infection in cats. recent studies have suggested a possible association between helicobacter infection and gastric lymphoma in cats, although more research is needed to confirm the association and understand the pathogenesis. , helicobacter spp. may be commensal in most cats, and perhaps loss of tolerance explains the development of gastritis in some individuals. another possibility is that the inflammatory response is normally well managed and disease may treatment of underlying dermatologic disorders, and administration of semisolid petroleum laxatives. more recently, commercial diets have been formulated for control of trichobezoars. cats with recurrent trichobezoars causing illness and suspected motility disorders may benefit from treatment with prokinetic drugs such as cisapride. clinical signs of gastric foreign bodies are variable but typically involve intermittent or persistent vomiting because of gastric outflow obstruction, distention, and mucosal irritation. gastric obstruction may be partial or total. patients with complete obstruction will present with more dramatic signs, including anorexia and depression. the base of the tongue should always be examined, because linear foreign bodies are sometimes anchored either in this location, or they may be lodged in the pylorus, causing intestinal plication. gastric foreign bodies may also be asymptomatic and found incidentally. physical examination may be unremarkable or may reveal dehydration or abdominal pain. if the stomach is markedly distended, the foreign body may be palpable in some patients. survey radiographs are always indicated when foreign body ingestion is suspected. radiopaque foreign bodies may be readily diagnosed, although some, along with radiolucent objects, will require a contrast study for diagnosis ( figure - ) . barium is commonly used as a contrast agent, although if gastric perforation is suspected, an aqueous iodinated agent is preferred. ultrasonography is also useful for detection of gastrointestinal foreign bodies. removal of some foreign bodies can be attempted endoscopically, particularly if the object does not have sharp edges and is not too large. successful removal of fish hooks, particularly single-barb hooks, using endoscopy has been described. otherwise, foreign objects are best removed using gastrotomy through a ventral midline laparotomy. a radiograph should always be to know when treatment should be attempted. one expert recommends treating only patients with clinical signs of gastritis that have biopsy-confirmed helicobacter infection with a treatment regimen of amoxicillin ( mg/kg, every hours, po), clarithromycin ( . mg/ kg, every hours, po) and metronidazole ( mg/kg, every hours, po) for days. a common dilemma would be determining the treatment of choice for patients with lymphoplasmacytic inflammation of the stomach and small intestine and confirmed helicobacter infection. are such patients best treated for inflammatory bowel disease, helicobacter infection, or both? currently, guidelines for determining the best treatment approach are lacking. also, few studies on the efficacy of combination therapy have been conducted in cats. long-term eradication of infection may be difficult, and histopathologic resolution of gastritis may not be possible, which raises the question of whether helicobacter is the true underlying cause. , in one study, two cats with clinical gastritis and helicobacter infection were treated with oral metronidazole, amoxicillin, and bismuth subsalicylate for weeks and were also fed a commercial elimination diet. posttreatment gastric biopsies were obtained a mean of weeks after the cessation of treatment. resolution of clinical signs occurred rapidly, and clearance of helicobacter spp. was achieved at that time point, but gastric inflammation persisted in post-treatment biopsies. in another study, cats with asymptomatic helicobacter infection were treated with oral omeprazole, amoxicillin, metronidazole, and clarithromycin for days. treatment failed to eradicate infection in of the cats based on molecular analysis of post-treatment gastric biopsies. it is unclear if treatment failure is because of recrudescence or reinfection. the reader is referred to excellent reviews of helicobacter in cats for more information. , , chronic gastritis chronic gastritis is common in cats with chronic intermittent vomiting. ollulanus tricuspis is a worm that infects the stomach of cats, causing chronic gastritis, and it is difficult to diagnose (see below, gastrointestinal parasites). the worm is occasionally found on histologic examination of gastric biopsy samples. it is reasonable to treat empirically (fenbendazole mg/kg, once daily, po × days) for this parasite when the cause of gastritis is not apparent. the frequency of vomiting in cats with chronic gastritis is highly variable, ranging from once or twice per week (and not necessarily every week) to more than once daily. most patients are otherwise well, although other clinical signs (inappetence, anorexia, depression, or weight loss) are possible depending on disease severity. results of routine laboratory testing are typically normal but may show neutrophilic leukocytosis, result when there is an abnormality of the immunoregulatory system. the most commonly used methods for diagnosis of helicobacter infection in cats are based on gastric specimens obtained during endoscopy (or laparotomy): exfoliative cytology, histopathologic examination of biopsy specimens, and rapid urease testing of biopsy specimens. however, it is important to note that even when helicobacter organisms are identified, the infection may not be the cause of the patient's clinical signs, and other causes of vomiting should always be evaluated. exfoliative cytology is the least expensive and most easily performed diagnostic test. in one study, it was also the most sensitive diagnostic method when compared with urease testing and histologic examination. brush cytology samples gathered during endoscopy are airdried on microscope slides and stained with wright's stain. the slide is examined at × magnification under oil immersion. spiral bacteria are readily seen if present. at least oil-immersion fields on two slides should be examined before determining a specimen is negative for helicobacter-like organisms. since helicobacter produce abundant urease, a rapid urease test (e.g., clotest, ballard medical products, draper, utah) may be used for diagnosis. the kit consists of an agar gel impregnated with urea and a ph indicator. a gastric biopsy sample is applied to the gel, and if urease is present, ammonia will form and change the ph (and thus the color) of the gel. the gel may change color rapidly (within minutes), but hours must elapse before the test can be considered negative. the more rapidly the color changes, the higher the bacterial load. both false-positive and false-negative results are possible with rapid urease testing for various reasons, giving the test a sensitivity of % to %. , histopathologic examination of gastric biopsy samples using hemotoxylin and eosin (h&e) or silver stains is highly sensitive and specific in human studies for detection of helicobacter-like organisms. the organisms are not equally distributed; so, examination of biopsy specimens from multiple sites will increase sensitivity. the bacteria may be seen in mucus on the surface epithelium as well as in the gastric pits, glandular lumen, and parietal cells. organisms may also be seen submucosally within gastric lymphoid follicles. histopathologic examination of biopsy samples also allows for assessment of other abnormalities. mild to severe lymphocytic-plasmacytic or lymphocytic gastritis may be present. in humans combination therapy with antibiotics and antisecretory drugs is recommended to reduce the risk of gastric ulcers and cancer from h. pylori infection. treatment is highly successful at eradicating both clinical signs and histologic changes in the gastric mucosa. since helicobacter infection is common in cats, yet no clear pathogenic role has been established, it is difficult cases. depending on the underlying cause and severity of disease, abdominal pain, anorexia, lethargy, pale mucous membranes, and drooling may also be seen. cats with neoplastic disease may have prolonged clinical signs and are more likely to present with anorexia and weight loss. cats with perforated ulcers may or may not present with signs of shock. diagnosis may be problematic because the clinical signs and physical examination findings are often not specific, even in cats with perforated ulcerations. the causes of gastric ulceration in cats are not well characterized. in dogs the most common cause is the administration of ulcerogenic drugs, particularly nsaids, either alone or in combination with corticosteroids. several cases of nsaid-induced gastroduodenal ulceration or perforation have been reported in cats. , , additional cases may be reported in the future, because long-term administration of these drugs is gaining in popularity for treatment of chronic diseases such as osteoarthritis. nsaids cause direct mucosal damage and interfere with prostaglandin synthesis. although inhibition of the cox- enzyme is thought to be the cause of adverse effects, such as gastric ulceration, even cox- -selective drugs have been associated with adverse effects, and safety in sick cats is not well evaluated. recently, guidelines for the long-term use of nsaids in cats were published by the international society of feline medicine and the american association of feline practitioners. the recommendations include administering nsaids either with or shortly after food, withholding therapy if inappetence or anorexia develops, determining dose based on lean body weight, and titrating to the lowest effective dose. neoplastic causes of gastric ulceration include systemic mastocytosis, mast cell tumor, lymphosarcoma, adenocarcinoma, and gastrinoma (zollinger-ellison syndrome). cats with chronic renal disease may suffer mucosal damage from uremic toxins and increased gastric acid production secondary to hypergastrinemia (because of decreased renal metabolism of gastrin). hepatic disease is a cause of gastric ulceration in dogs but is uncommonly reported in cats. recent anesthesia and surgery have been implicated as a cause of gastric ulceration and perforation, perhaps through hypovolemia, hypoperfusion, or stress. , other non-neoplastic causes reported for gastric or gastroduodenal ulceration in cats include parasites (e.g., ollulanus tricuspis, toxocara cati, aonchotheca putorii, gnathostoma spp.), bacterial infections, toxins, inflammatory bowel disease, and foreign bodies. one case report describes a cat with severe gastric ulceration caused by intoxication with dieffenbachia leaves. in some case reports, the cause for the gastric ulcerations could not be determined. a minimum database should be collected for cats suspected of gastric ulceration, to identify underlying diseases. anemia, usually regenerative, may be present. eosinophilia, or hypoproteinemia. survey and contrast radiographs are often normal. the most common finding on histopathologic examination of biopsy samples is lymphocytic plasmacytic (lp) gastritis ( figure - ) . some patients will also have concurrent evidence of lp inflammation in the small intestine, pancreas, and/or liver. such patients will be treated for their concurrent problem; treatment of inflammatory bowel disease, pancreatitis, and cholangiohepatitis is covered elsewhere in this chapter. some cats with chronic lp gastritis respond to treatment for dietary intolerance or hypersensitivity with a limited antigen diet (see chapter ) . patients with moderate to severe lp gastritis may be best treated with a limited antigen diet and immunosuppressive therapy (prednisolone to mg/kg/day, po tapering to every other day at the lowest dose that controls clinical signs). patients that fail this initial treatment approach may require additional immunosuppressive therapy, such as chlorambucil (see table - ) . occasionally, cats with chronic gastritis are diagnosed with eosinophilic inflammation on histopathologic examination of biopsy specimens. treatment is similar to that for lp gastritis, although such patients should be evaluated for evidence of hypereosinophilic syndrome and eosinophilic enteritis. eosinophilic fibrosing gastritis was suspected to be caused by toxoplasmosis in one case report. gastric or gastroduodenal ulcerations are uncommon in the cat compared with the dog and may be caused by a variety of disorders, both gastric and nongastric. classical clinical signs include vomiting, hematemesis, and melena. however, in one review of eight cats, hematemesis and melena were present in less than one third of suturing of the ulcer site as well as collection of biopsy samples for histopathologic examination. the prognosis for recovery was excellent in two studies, particularly for cats with non-neoplastic causes of gastric or gastroduodenal ulceration. , in one study of seven cats with perforated gastric or duodenal ulcers, the survival rate was low ( %). disorders of gastric motility are better characterized in dogs than in cats. the most common clinical sign is vomiting of undigested food hours or more after a meal. if outflow obstruction is present, vomiting may be projectile. there may also be a history of recurrent trichobezoars. various disorders are associated with impaired gastric motility, such as chronic gastritis, drug therapy (e.g., anticholinergic and narcotic drugs), dysautonomia, gastric neoplasia, metabolic disorders (e.g., hypokalemia), and temporary postsurgical gastroparesis. in some cases of chronic motility dysfunction, no cause can be identified. outflow obstruction may be caused by neoplasia, foreign bodies, and extragastric masses. pyloric stenosis is infrequently documented in young cats, often siamese cats. , , since the range of underlying disorders is diverse, the diagnostic approach should allow for detection of both gastric and nongastric disorders. a minimum database (cbc, serum chemistries, urinalysis, feline leukemia virus [felv] and feline immunodeficiency virus [fiv] serology) is used to establish overall health status. radiographs are used to confirm presence of food in the stomach for longer than hours. ultrasonography may detect gastric lesions, such as masses. endoscopy is used to identify outflow obstruction as well as other lesions, such as ulcers, and evidence of gastritis. assessment of gastric emptying using nuclear scintigraphy is the most accurate method but is limited to referral centers. gastric emptying times for liquids, canned food, and dry diets have been established using nuclear scintigraphy. , , however, emptying times are variable, depending on the amount and type of diet fed as well as the amount of water ingested. even the shape of kibble affects emptying time. radiographic contrast series are widely used, but gastric emptying times are variable for barium in either liquid form or mixed with canned food. contrast radiography using liquid barium ( to ml/kg) is performed in a fasted patient. radiographs are taken immediately after administration of the barium and again at and minutes, in some cases, also at and hours. liquid barium is expected to enter the duodenum no more than minutes after administration, and the stomach should be completely empty of barium within hours. the clinician should be aware that some cats with gastric motility disorders will have other findings will be dependent on the presence of underlying diseases; for example, azotemia and isosthenuria may indicate renal disease. electrolyte and acidbase abnormalities may be because of chronic vomiting and anorexia. survey and contrast radiographs and ultrasonography are primarily useful to rule out other causes for the clinical signs, such as foreign bodies. cats with perforated ulcers may have evidence of pneumoperitoneum (sometimes severe) on plain radiographs or ultrasonographs, and this is an indication for surgical exploration. , , , , evidence of peritonitis on imaging studies should be followed with peritoneal fluid analysis. a definitive diagnosis may be made using endoscopy, which allows direct visualization of lesions and collection of biopsy samples. however, some cats with gastric ulceration present in poor condition, which may preclude the use of endoscopy because of anesthetic risk and risk of ulcer perforation. the location of ulcers is typically pyloroantral or fundic in cats with nonneoplastic disease. , areas of erosion may appear pale or hemorrhagic; the mucosa is often friable and bleeds easily. fresh or clotted blood may be seen in the stomach lumen. in some cases, mucosal ulceration must be distinguished from ulcerated tumors. nsaid-induced ulcers are typically found in the antrum and do not have marked mucosal thickening; ulcerated tumors frequently have thickened edges and surrounding mucosa. biopsy samples should be taken at the periphery of the ulcer to avoid perforation. treatment should be directed at any underlying disorder. treatment for nsaid toxicity is described in chapter . general supportive measures include fluid therapy and electrolyte replacement; blood transfusion may also be required (see chapter ) . gastric acid production can be decreased with the use of h -receptor blockers or proton pump inhibitors, and sucralfate is used as a mucosal protectant (see table - ). sucralfate may inhibit absorption of other oral medications and should be given hours apart from other drugs. if vomiting is severe or persistent, antiemetic therapy is warranted (see table - ). analgesia should be provided for painful patients; a good choice is the opioid buprenorphine (see table - ). broad-spectrum antibiotic therapy is indicated for patients with significant mucosal barrier dysfunction, perforation, leukopenia and/or neutrophilia, fever, and melena. surgical intervention is warranted for patients with life-threatening hemorrhage, failure to respond to medical management, or evidence of perforation. the entire abdominal cavity and gastrointestinal tract should be thoroughly explored to locate extragastrointestinal lesions, non-perforated ulcers, and multiple ulcers. in one case series, nonperforated ulcers were detected at laparotomy by association with adhesions or a gastric mass. surgical management includes débridement and months. physical examination findings are nonspecific, although occasionally a gastric mass or gastric thickening may be palpated if the stomach is markedly enlarged. results of routine diagnostic testing are generally nonspecific; anemia may be associated with ulceration. survey or contrast radiography may reveal a mass ( figure - , a); other findings include delayed gastric emptying, impaired motility, and mucosal ulceration. ultrasonography is also useful for diagnosis and can be used to guide needle aspirates of masses ( figure - , b). endoscopy allows for visualization of lesions as well as the ability to obtain partial thickness biopsy samples. problems with interpretation of endoscopic biopsy samples include detection of necrosis, inflammation, and ulceration rather than the primary lesion. in dogs some neoplastic lesions are submucosal, making it very difficult to obtain diagnostic samples by endoscopy. therefore several biopsies should be taken and masses should be biopsied multiple times in the same place to sample deeper tissues. the center of ulcerated lesions should not be biopsied. surgical biopsies are more reliable for diagnosis. a normal gastric emptying time with liquid barium. barium can also be mixed with canned food and fed as a meal; retention of barium-containing food in the stomach for more than to hours is abnormal. gastric emptying time may also be established with the use of barium impregnated polyspheres (bips; med i.d. systems, grand rapids, mich.) and radiography. gastric emptying times for bips have been established in healthy fasted and fed cats as well as in sedated cats, , but the values do not correlate well with scintigraphic studies. a mixture of small ( . mm) and large ( mm) spheres are administered with food, and two to four radiographs are taken over the next hours. the small spheres are intended to mimic liquid transit time and the large spheres solid transit time. however, studies assessing the clinical relevance of this method are lacking. one review concluded that bips are probably sufficiently sensitive to detect grossly delayed gastric emptying. treatment of gastric emptying disorders is directed at identifiable causes. treatment for gastric ulcers, chronic gastritis, and foreign bodies is described elsewhere in this chapter. pyloric stenosis is managed surgically. if no outflow obstruction exists, treatment with prokinetic agents, such as metoclopramide or cisapride, may be beneficial (see table - ). gastric tumors account for less than % of malignancies in dogs and cats. benign gastric tumors are even less common than gastric malignancies. gastric smooth muscle hamartoma has been reported in one -year-old cat. although adenocarcinoma is the most common gastric cancer of the dog, lymphoma is the most common gastric cancer in the cat. feline gastrointestinal lymphoma occurs as two major types: small cell (lymphocytic) and the more aggressive large cell (lymphoblastic) form. small cell lymphomas are more frequently enteric. in one study of cats with gastric lymphoma, diffuse large b-lymphocyte tumors of immunoblastic nuclear type predominated. gastric lymphoma is not associated with felv, and the role of helicobacter in the development of gastric lymphoma in cats requires investigation. adenocarcinoma, , , plasmacytoma, and gastric carcinoid have also been described. the siamese cat may be predisposed to adenocarcinoma. , as would be expected, most cats with gastric neoplasia are older cats. as for most gastric diseases, vomiting is the most common clinical sign of neoplasia. the vomitus may contain blood and melena may be present. other clinical signs include anorexia, weight loss, bloating, and depression. perforation of the tumor may occur, leading to pneumoperitoneum or septic peritonitis. clinical signs present gradually and are often present for weeks to surgical resection is the most common treatment for gastric neoplasia other than lymphoma ( figure - ). the prognosis for most patients is poor, typically because of debilitation, concurrent diseases, and recurrent or metastatic disease. the success of chemotherapy for lymphoma depends on cell type, with small cell tumors carrying a better prognosis than large cell tumors. in diarrhea can be defined as increased volume and/or increased frequency of defecation of stools with increased water content. approaches to diarrhea, as for any clinical sign, need to take into account the individual animal. for example, neoplasia is much less likely to occur in a kitten than in a geriatric cat. in many cases, the precise diagnosis of gastrointestinal disease cannot be reached without biopsy samples. the decision to obtain biopsy samples should follow a logical pathway that is appropriate to the cat's condition. these are summarized in figure - . for example, many cases of acute diarrhea in a well cat can resolve with limited or no intervention, and so do not require a precise diagnosis. the diagnostic steps are . signalment and clinical history . physical examination . fecal assessment . blood and urine testing . imaging (radiography, ultrasonography) . biopsy samples these steps do not include treatment/diet trials or other empiric therapies that are appropriate in many cases. steps and are often undertaken at the same time, and there is no definite order for these steps. they are divided here for reasons of clarity. in a younger cat, where infectious causes are more likely, thorough fecal testing is more important; in an older cat, extragastrointestinal diseases, such as hyperthyroidism, are more likely; so, blood and urine testing is more important, but fecal assessment should not be neglected. the decision to proceed to step (and each subsequent step) should take into account several considerations. the main considerations in assessing and managing a cat with diarrhea are • is there an acute onset or a chronic time course? • are there any dietary changes or indiscretion? consider treatment trials: antibiotics (e.g., amoxicillin/ clavulanate ؉ metronidazole). food trials with novel proteins (e.g., rabbit, kangaroo, venison). • is the cat well or unwell? • is there primary or secondary gastrointestinal disease? • is there small or large bowel diarrhea? the components of the clinical history for cats with diarrhea are detailed in table - . after establishing the cat's age, breed, vaccination, and deworming history, it is important to establish the duration and nature of the diarrhea. chronic diarrhea is usually defined as greater than weeks in duration and mostly warrants at least some degree of a diagnostic workup, whereas acute diarrhea is often self-limiting in a well cat. a description of the feces helps determine whether the diarrhea is small or large bowel in origin (table - ); this will affect how any investigations might proceed. important questions to ask concern frequency of defecation (and how this compares with the normal state), tenesmus (straining usually indicates large bowel diarrhea, since an irritated colon leads to urgency), volume of feces (smaller volumes are typical of large bowel diarrhea; larger volumes are more typical of small onset and duration of diarrhea acute versus chronic? acute diarrheas are abrupt in onset and of short duration, and generally they are self-limiting. chronic diarrheas persist usually longer than weeks and fail to respond to symptomatic therapy. appearance of diarrhea quantity and quality of the stool (color, consistency, character, presence of blood or mucus)? loose to watery feces that contain fat droplets, undigested food, melena, and variable colors suggests small intestinal disease. the volume is always increased with small intestinal disease. loose to semisolid feces containing excess mucus and fresh blood (hematochezia) indicates large intestinal disease. the volume may be normal to slightly decreased with large intestinal disease. description of defecation process tenesmus (straining) and dyschezia (painful defecation)? these are hallmarks of large intestinal disease (e.g., inflammatory or obstructive lesions of the colon, rectum, or anus). frequency is normal to slightly increased with small bowel disease, but greatly increased with large bowel disease. associated physical signs vomiting, anorexia, weight loss, and dyschezia may help localize the disorder to a specific part of the gastrointestinal tract. clinical signs relating to problems in other organs or body systems should be noted and may suggest a more generalized disease. vomiting may occur as a consequence of small intestinal inflammation in some cats with diarrhea. weight loss may result from decreased caloric intake (anorexia), decreased nutrient assimilation (maldigestion/malabsorption), or excessive caloric loss (protein-losing enteropathy or nephropathy). weight loss is observed uncommonly with large bowel disease. in many cases, the answers to these questions are obvious. for example, a cat may seem well but has had access to lilies (the author has seen diarrhea as a primary presenting sign for this!) or has a palpable abdominal mass. substantial weight loss is an indicator that further investigations are warranted sooner rather than later. if the decision is made for empiric management and outpatient care, it is vital to follow up either by scheduling a recheck visit or calling the client, because simple acute problems can turn into complicated chronic problems. if the diarrhea has been present for less than a week and the cat has no weight loss, dehydration, fever, or palpable abdominal abnormalities, it is appropriate to manage the cat as an outpatient. even in the absence of fecal testing, it is appropriate to deworm the cat (see the section gastrointestinal parasites). the cat should be fasted for hours ( hours, if less than months old) and then fed a bland diet (such as plain, cooked, skinless chicken, or low-residue prescription diets designed for cats with gastrointestinal problems). it is appropriate to maintain the cat on the low-residue diet for at least to days and then slowly reintroduce the regular diet. fecal assessment is mostly used to assess infectious agents, such as parasite-associated diarrhea, but the importance of assessing feces, even when parasitic or bacterial infections are not suspected, should not be underestimated. gross examination of feces can determine if melena or fresh blood or mucus are present to help distinguish large from small bowel disease when the owner's observations may be misleading. occult fecal blood can be an indicator of gastrointestinal inflammation in cases of subtle disease, and undigested starches and fats can indicate maldigestion or malabsorption. for assessment of feces for parasites, the fecal sample should ideally be fresh (< hour old). refrigeration (for no longer than one week) can preserve ova, oocysts, and cysts but not protozoal trophozoites. feces should be assessed by a. to assess for trophozoites bowel), how formed the stool is (from soft stool to cow-pat consistency to liquid tea; usually more watery stool relates to small intestinal disease), color (darker indicates digested blood), and presence of any mucus or blood (presence relates to large bowel). most household toxins, such as plants, cause signs additional to diarrhea such vomiting or neurological signs, but it is important to ascertain if the cat has had access to anything unusual. likewise, it is important to find out if the cat has had any possible exposure to dietary indiscretions; this can include if the cat has been seen with or is known to hunt prey including insects. cockroaches carry pathogenic bacteria , and other prey such as birds and rats can carry salmonella; salmonellosis in cats has been dubbed songbird fever. simple causes of self-limiting diarrhea include dietary change (either a new flavor or a new style of food, such as dry food for the first time); so, the owner must also be quizzed if anything new has been offered, either new cat food or treats (such as greasy fish or chicken). although the physical examination will usually determine how unwell a cat is, the owner's impressions are also important, because cats can hide signs from strangers, particularly in a practice setting. lethargy and inappetence are important signs, as ill cats typically do not eat well. the cat's general demeanor can be an indicator of how unwell a cat is and therefore dictate the extent of diagnostic testing required. this can be noted by assessing how interested the cat is in its surroundings or any behavior changes from previous visits, such as if a normally difficult-to-handle cat is placid. body weight should be assessed and, if possible, compared with that of previous visits (even those noted on a clinical record from another veterinarian). the body condition score (bcs) should also be assessed and can be very important when there is no prior weight information. dehydration is usually a sign that a cat needs more involved management. abdominal palpation should be performed to assess pain (where?), any masses (foreign bodies, lymph nodes, or even focally thickened intestines, such as with neoplasia), or turgid intestines. fever often indicates infection but can also reflect neoplasia or other inflammatory changes. a thorough examination of all body systems should always be performed, no matter what a cat presents for. in the case of diarrhea, extragastrointestinal signs can be of vital importance, such as a palpable thyroid and tachycardia suggesting hyperthyroidism. after the clinical history has been taken and the physical examination performed, the veterinarian must make the important decisions of whether any interventions are required and whether the patient should be factors affecting interpretation include whether the growth is a heavy and pure growth of a known pathogen, such as salmonella, campylobacter, yersinia, or clostridium difficile. further information about the relevance of culture and pcr results is contained below in the section infectious enteritis. investigations begin by assessing if the diarrhea is the result of primary gastrointestinal disease or secondary to another process, by performing routine serum/plasma biochemistries, hematology, urinalysis, and total t (for older cats). in most cases of secondary gastrointestinal disease, diarrhea is not usually the primary presenting complaint, but since the approach to investigations and management diverge so much, this is an important step to take. biochemistry and urine tests may also show the consequences of diarrhea, such as dehydration and electrolyte abnormalities. a. can aid in the visualization of internal structures of some protozoa . fecal flotation (preferably with centrifugation) a. to find cysts, oocysts, and ova fecal culture should be undertaken with the understanding that bacteria will be cultured; so, interpretation is based on the relevance of the positive culture result. used to evaluate the smear for the presence of trophozoites, such as giardia spp. and tritrichomonas foetus. . place peppercorn size amount of feces on a warm slide and mix with a drop of . % saline (smear must not be too thick, because trophozoites will be easily missed). . apply coverslip. . evaluate systematically for motile organisms using the × magnification. . confirmation at × magnification. adding iodine to a wet mount through the edge of the coverslip can aid in the visualization of internal structures of some protozoa. the direct wet preparation must be examined without any stain for motility first, because staining the preparation kills the organism. methylene blue is useful for identifying trophozoites, particularly those of entamoeba histolytica. this method has little to no diagnostic value for the diagnosis of bacterial-associated diarrhea. used to find cysts, oocysts, and ova in feces. standing (gravitational) flotation methods are easier and quicker but have much poorer sensitivity than centrifugation methods. solutions used in centrifugation flotation methods include zinc sulfate and sheather sugar. . weigh out to g of feces. . mix feces with approximately ml of flotation solution. . pour mixture through a tea strainer into a beaker or fecal cup. . pour strained solution into a -ml centrifuge tube. . fill tube with flotation solution so that a slight positive meniscus forms, being sure not to overfill the tube. . place a coverslip on the tube, and put the tube in the centrifuge. . make sure the centrifuge is balanced. . centrifuge at rpm ( × g) for minutes. . remove the tube and let stand minutes. . remove the coverslip, and place it on a glass slide. systematically examine the entire area under the coverslip at × magnification (i.e., × objective). you may wish to use the × objective lens to confirm your diagnosis and make measurements; however, with practice, most parasites can be identified using the × objective ( × magnification). (fpli) are useful markers of intestinal and pancreatic disease, [ ] [ ] [ ] [ ] but it is important to note that they typically do not give a precise diagnosis. cobalamin and folate are water-soluble vitamins and are readily found in commercial cat foods so that dietary insufficiency is rare, and decreased levels are almost always because of gi disease. these vitamins are taken up by specific receptors in different areas of the small intestine. chronic inflammatory gastrointestinal disease may damage the receptors and lead to decreased serum concentrations of one or both vitamins, provided the disease process is severe and long standing enough to deplete body stores. serum cobalamin and folate concentrations may also be decreased in cats with exocrine pancreatic insufficiency (epi). trypsin-like immunoreactivity is a pancreasspecific marker, and assessment of serum tli is used for diagnosis of epi and pancreatitis in the cat, although the sensitivity of the assay for pancreatitis is low. pli is a marker for pancreatic inflammation and is more sensitive than tli for the diagnosis of pancreatitis. since inflammation of the small intestine may be seen concurrently with pancreatitis, serum tli and pli are useful adjunctive tests in the diagnosis of diarrhea. tli, pli, and cobalamin are stable in serum at room temperature for several days, but folate is unstable so that samples for cobalamin/folate analysis should be frozen (table - ) . samples submitted for folate concentration should not be hemolyzed, because red blood cells contain high levels of folate. in addition, folate is light-sensitive, and samples should be wrapped to exclude light. severe lipemia may interfere with common assays for tli and pli. the main utility of these tests are to indicate that further investigation of gastrointestinal disease is warranted. when a cat presents for weight loss with no overt signs of gi disease, decreased cobalamin or folate can indicate that further investigations with imaging and, ultimately, biopsy sampling are warranted. many clients are more willing to proceed with hematology can be normal in some cats, with changes expected, and so should not be used to rule out any condition. it can be useful, for example, if there is a left shift neutrophilia, indicating acute infection, or eosinophilia, reflecting parasitism. monocytosis can suggest chronic disease that was not suggested by the clinical history. in the case of acute onset diarrhea, the cat may be unwell as a consequence of the diarrhea (e.g., from dehydration) and not because of the cause of the diarrhea. if rehydration is required (with intravenous or subcutaneous fluids, depending on severity of illness), then it is important that biochemistry tests are performed before fluid administration so that any diagnostic clues are not lost by alteration of the profile from the fluid therapy. fever and neutrophilia may indicate the need for antibiotic therapy. if infection is suspected, fecal sampling (see step ) should occur before starting antibiotics. if a cat is unwell from dehydration, then further testing may not be warranted. the clinician should be alert that linear foreign bodies can result in diarrhea (see the section intestinal obstruction). diarrhea of chronic duration (greater than weeks) does require a more thorough investigation at the outset. however, if clinically well, the cat can be managed as an outpatient in the first instance, at least while waiting for results of diagnostic testing. a diet trial with a novel protein is appropriate for a well cat with stable weight. as with any patient managed as an outpatient, follow-up is vital and, in this scenario, includes scheduling revisits. cobalamin, folate, feline trypsin-like immunoreactivity (ftli), and feline pancreatic lipase immunoreactivity invasive diagnostics when a specific marker of the disease in the organ involved has been recognized. caution should be exercised, because either cobalamin or folate may not be reduced with gi disease. in one study of small cell lymphoma, only % of cats were hypocobalaminemic, meaning that if this was the only instigating factor to investigate, nearly one fourth of cats would not have been investigated further. also, cobalamin may be reduced in nonalimentary illness. . to detect hypocobalaminemia that may indicate the need for supplementation for clinical improvement. to recognize pancreatic pathology when fpli is increased. it is important to note that an elevated value gives no indication of the nature of the pancreatic pathology. . to make a diagnosis of epi when the ftli is low. it should be noted that epi can result from other pathology that may require further investigations. veterinarians if the patient is new to the practice) are usually helpful. body condition scoring (using a -point or -point scale) for every cat seen is helpful in recognizing those that are underweight. weight loss often occurs with loss of muscle mass in cats, and muscle mass can be assessed over the ribs and pelvis as well as scapulae and nuchal crest. thickened intestines are also a subjective finding; it is the author's opinion that thickened intestines are actually intestines with increased turgidity, since differences between normal intestines and those with inflammatory infiltrates can be as little as . mm. perhaps more important during the history taking and physical examination are those signs that can point to extragastrointestinal disease. when confronted with a cat showing weight loss or vomiting or diarrhea (or a combination of signs), the clinician should start with trying to distinguish the signs as being either primary gastrointestinal or secondary signs. examples of clues pointing to extragastrointestinal diseases include tachycardia and palpable thyroid nodule, indicating hyperthyroidism, or polydipsia/polyuria, which has a variety of causes but is not typical of primary intestinal disease. inflammatory bowel disease has traditionally been considered an immune-mediated disease. the local immune system of the intestinal mucosa no doubt plays an important role, but recent work has also shown the importance of the normal bacterial population in perpetuating and, perhaps, even initiating pathology. it is known for certain that ibds are an expression of an overanxious immune response, with a recent study indicating increases in inflammatory (il- ), type- immunity (il- p ), and immunomodulatory (transforming growth factor [tgf]-beta, il- ) cytokines. other researchers have found an association with bacterial counts (enterobacteriaceae, e. coli, and clostridium spp.) and abnormalities in mucosal architecture, indicating that mucosal bacteria are involved in the etiopathogenesis. we can summarize these theories by saying that ibds are likely to be a consequence of hypersensitivity reactions to antigens from the intestinal lumen (e.g., bacterial, parasitic, or dietary antigens). this hypersensitivity may occur because of failed immunoregulation (suppressive function) of the gut-associated lymphoid tissue (galt). it is known that granulomatous colitis in boxer dogs is associated with infection, and pathogens may well be found in at least some cases of ibd in cats that cause the immune response and subsequent inflammatory infiltrate of the lamina propria typically seen. although not described specifically in cats, chronic intestinal inflammatory change can impair motility. inflammatory bowel disease (ibd) refers to intestinal inflammatory infiltrates of the small or large intestine (or both) of unknown etiology. the term ibd should strictly be applied to mean idiopathic ibd, thus excluding inflammatory enteritis because of food sensitivities, although common usage has led to ibd referring to intestinal inflammatory infiltrates of both known and unknown causes. ibd is not a diagnostic end point but a description of a series of intestinal diseases that have similar histopathology. recent efforts by the world small animal veterinary association (wsava) gastrointestinal standardization group have led to both diagnostic and classification guidelines , , that encompass chronicity, nonresponse to symptomatic treatment, no specific cause found, as well as histologic confirmation of non-neoplastic intestinal inflammatory changes. there are no obvious breed or gender predispositions, and although cats of any age can be affected, inflammatory intestinal diseases are more likely to occur in middle-aged to older cats ( to years of age or older) than in younger cats. presenting clinical signs include vomiting, diarrhea, and weight loss with increased or decreased appetite. these signs can occur in isolation or together. weight loss without vomiting or diarrhea deserves special mention because not only have several studies , shown this to be the most common presenting sign for ibd, but many veterinarians do not consider primary intestinal disease without the presence of vomiting or diarrhea. weight loss despite normal to increased caloric intake can represent poor absorption of food because of small intestinal disease, although it can also represent maldigestion associated with exocrine pancreatic insufficiency or increased metabolism associated with hyperthyroidism, or even lack of energy utilization associated with diabetes mellitus. conversely, appetite may be reduced, most likely because of nausea. if the large bowel is affected, signs are typically discomfort when defecating, resulting in frequent small volumes of diarrhea, often with mucus and blood; if the large bowel alone is affected, there may be no weight loss. physical examination findings are often nonspecific, but the most consistent findings for small intestinal disease are weight loss (or being underweight in a cat not seen previously) and palpably "thickened" intestines. noting a cat as underweight can be subjective, and prior recorded weights (even from previous paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma than those with ibd, but this change was also seen in % of cats with normal small intestine. inflammatory bowel diseases require histologic findings obtained from biopsy samples for diagnosis, but diagnosis should not be made solely on these findings. the wsava international gastrointestinal standardization group has proposed "an all encompassing definition of inflammatory bowel disease" that comprises clinical criteria, imaging criteria, as well as pathophysiologic criteria. the clinical criteria for the diagnosis of ibd include there are no typical laboratory findings in ibd, and many cats may have entirely normal results from routine biochemical and hematologic investigations. moderate liver enzyme elevations may be seen , , , even in the absence of recognizable hepatic pathology, and this may reflect subclinical secondary hepatic disease, secondary cholestasis, or showering of the liver with inflammatory cells from the small intestine through the portal circulation. other changes can reflect consequences of the intestinal disease, such as azotemia or hemoconcentration reflecting dehydration, or hypokalemia reflecting inappetance. the chronic inflammation may be reflected by neutrophilia, monocytosis, , , , or hyperglobulinaemia. hypocobalaminemia can reflect ileal inflammation, and low serum folate can reflect proximal small intestinal inflammation. typical ultrasonographic findings consistent with ibd are focal or diffuse intestinal wall thickening ( figure - ) ; normal wall thickness is less than or equal to . mm for the duodenum and less than or equal to . mm for the ileum, and large mesenteric lymph nodes with hypoechoic changes may be seen. one study found that ultrasonographic findings correlated with histologic grade of ibd. there is no clear distinction between ultrasonographic changes from ibd and those from small cell lymphoma. one recent therapeutic trial, follow-up visits are vitally important. many cats with small intestinal disease may show initial improvement simply because of the diet having lower residue, since there is decreased substrate for intestinal bacteria to digest and lower osmotic potential. the corollary of this is that failure of one novel protein diet does not mean that all novel protein diets will fail. when food sensitivities are responsible for gastrointestinal clinical signs in cats, the responsible food ingredient is usually a dietary staple. commonly incriminated ingredients are beef, fish, wheat, and corn gluten. a careful dietary history is therefore important. large bowel inflammation typically improves with higherfiber diets, , and attempting a trial with such a diet is certainly appropriate. immune suppressive therapy is the mainstay of ibd treatment, and glucocorticoids, such as prednisolone, are most commonly used. sulfasalazine use for large bowel signs has not been critically evaluated but seems safe and effective. in cats with substantial weight loss or severe clinical signs, such as chronic diarrhea, the author prefers to start with corticosteroid therapy, even if dietary causes have not yet been ruled out. the diet should also be changed to one containing a novel protein, and, if and when clinical signs resolve, an attempt is made to wean the cat from corticosteroid therapy, hopefully to the point of being discontinued. a diet challenge can then be used to confirm the diagnosis of food sensitivity. there are no universal guidelines for doses of corticosteroids. the author prefers the use of orally administered prednisolone to reduce the chance of side effects and will choose the starting dose based on the severity of disease. the starting dose is usually mg/kg, once daily, po ( mg/cat/day for most cats) starting days after biopsies have been obtained to allow time for the mucosa to heal. if there is an improvement noted after a recheck at weeks, the higher dose is maintained for a further to weeks, at which point, many cats are back to their normal weight and are not exhibiting clinical signs. if this is the case, the corticosteroid dose can be weaned down to mg/kg, po (often mg/cat/day) for several months, with continued rechecks scheduled to assess weight, clinical signs, and diet. the goal is to wean down to the lowest effective dose. if hypocobalaminemia is present, cobalamin supplementation may be required. cobalamin is administered parenterally at µg/cat subcutaneously weekly for weeks, then every second week for weeks, then monthly. owners can be shown how to inject their cats (as practitioners routinely do with diabetics). clinicians often consider the assessment of histologic samples to be out of their hands; however, it is important to work with the pathologist by providing good quality samples and a good clinical history, as well as having an open dialogue if the findings are not within expectations. for example, with lymphocytic/plasmacytic infiltrations, the pathologist has the difficult task of distinguishing diseased from normal tissue in a site that is laden with lymphocytes in the healthy state. once deciding the tissue has pathology, the pathologist's next task is distinguishing inflammatory infiltrate from neoplastic infiltrate with normal, mature lymphocytes (as seen in small cell lymphoma). inflammatory change also results in changes to normal tissue architecture, with thickened villi, edema, or erosion of the epithelium being typical changes. clinicians should expect morphologic descriptions as well as assessments of degree and type of inflammation. these difficulties are further compounded with the recognition that histologic grading of mild, moderate, or severe does not necessarily correlate with severity of clinical signs. this means that a cat with severe clinical signs of weight loss and vomiting or diarrhea may have only mild histologic changes (and vice versa). concurrent inflammation of the pancreas and liver with intestinal inflammation was first described in the mid- s, and despite constant reference to this phenomenon at conferences and veterinary websites, there has been little description since then, though one study found % of ibd cases had liver inflammation and % had pancreatic inflammation. the term "triaditis" has frequently been used, but the author prefers to spell this "tri-iditis" to distinguish it from inflammation of the hepatic portal triads. there has been no assessment of prognosis when the pancreas and/or liver are involved, but the author has found no difference in prognosis. many cases diagnosed with intestinal inflammatory infiltrates have these changes because of dietary sensitivity. in one study, % of cats with histologic gastrointestinal changes improved with dietary elimination therapy alone. interestingly, improvement was noted within days compared with the longer duration of weeks often recommended for improvement of dermatologic manifestations of food sensitivities. this careful study made note of the cat's prior diets and likely dietary causes of sensitivities. another study found dietary therapy to be unsuccessful in of cats but no specifics of diets tried are noted. as with any national cancer institute working formulation (nci wf) system. for most veterinarians in practice, the most important distinction is the histologic grade, because low-grade (lymphocytic or small cell) lymphoma has a much better prognosis (and requires different treatment) compared with high-grade (often lymphoblastic) or intermediategrade lymphoma. for the purposes of simplicity and practicality, only small cell lymphoma and high-grade lymphoma will be addressed here. the prognosis and treatment for intermediate-grade intestinal lymphoma should be considered as for high-grade lymphoma. small cell lymphoma was first described in human pathology in . earlier, small lymphocytes were considered end-stage cells without the ability to divide. in cats small cell lymphoma is most commonly associated with the gastrointestinal tract or skin. small cell neoplasia can be a confusing concept, since our traditional ideas of malignant neoplasia focus on rapidly dividing cells. the confusion is compounded by various terms used in the literature, such as lymphocytic lymphoma, low-grade lymphoma, well-differentiated lymphoma, or diffuse lymphoma; another term, epitheliotropic malignant lymphoma predominantly applies to small cell lymphoma, and other papers fail to distinguish these lymphomas from lymphoblastic lymphosarcoma (the traditional, aggressive form). "small cell lymphoma" seems to be most widely used term, though the author prefers "lymphocytic lymphosarcoma," since it is more descriptive. intestinal small cell lymphoma can be considered as a severe lymphocytic intestinal infiltrate, the most common form of which is commonly called ibd. not only is lymphocytic ibd hard to distinguish histologically from lymphocytic lymphosarcoma, but the approaches and treatments are similar. several reports have suggested a relationship between the two conditions in that inflammatory infiltrates may become neoplastic over time. , , prevalence the true prevalence of intestinal small cell lymphoma is unknown, but several recent studies have indicated similar rates to inflammatory bowel diseases, with kleinschmidt et al noting small cell lymphoma cats compared with with intestinal lymphocytic infiltrates, evans et al reporting cases compared with with ibds , and baral et al diagnosing cases compared with with ibds. traditionally, % of feline lymphosarcoma is regarded as intermediate or high grade, but this may not be the case within the gastrointestinal tract. fondacaro et al found % of gastrointestinal lymphoma to be lymphocytic ; a more recent paper found some cats seem resistant to conventional therapy. if this is the case, the diagnostic findings should be re-assessed to ensure no steps were missed or findings disregarded; the cat should be reexamined to look for emergence of other signs; and the pathologist who reads the histology should be contacted to recheck the findings. some cases of apparently resistant ibd are actually food sensitive, but it can be difficult to find the incriminating diet source, and commercial diets are not always effective. if underlying infectious causes have been entirely ruled out and the practitioner is certain of the diagnosis of idiopathic disease, immune suppressive therapy can be increased by either increasing the dose of prednisolone or using other agents, such as chlorambucil, typically at mg/cat, po, every second day. it has been suggested that cats with eosinophilic inflammation may be more likely to be refractory to standard therapy. side effects of immunosuppressive therapy are rare but include inducing diabetes mellitus, immune suppression, delayed healing, and gastrointestinal ulceration. reported doses of sulfasalazine to manage large bowel ibd are to mg/kg, po, once daily for to days. because this drug is usually only available as mg tablets, one eighth of a tablet, providing a dose of . mg, is usually appropriate for most cats. in some countries, it is possible to have a compounding pharmacist formulate the drug into more convenient tablet sizes or as an oral suspension. cats are generally regarded as susceptible to salicylates, and possible side effects include vomiting or diarrhea, or anemia. the exact pharmacodynamics of this drug are not known; so, caution for extended use should be exercised and the drug withdrawn if any possible adverse signs are noted, but there are anecdotal reports of extended use of this drug without adverse consequences. a survey of the online veterinary cancer registry (http://www.vetcancerregistry.com) identified % of all submitted feline tumors to be intestinal tumors. approximately % of reported feline small intestinal tumors were lymphomas. adenocarcinomas accounted for %, and other tumor types reported included mast cell tumors and leiomyosarcomas. "lymphoma in veterinary medicine: no longer a oneword diagnosis" was the title of an editorial in a recent issue of the veterinary clinical pathology journal, and this is nowhere truer than in the feline gastrointestinal tract! a recent study classified cases of feline gastrointestinal lymphoma both histologically and immunophenotypically, and it found eight different categories according to the revised european and american lymphoma/world health organization (real/who) classification system and six categories according to the approximately equal numbers of high-grade and lowgrade gastrointestinal lymphoma. older cats are more at risk of small cell lymphoma, with mean or median ages reported from to years. younger cats with the disease have, however, been recognized. , , , no breed or gender predispositions have been definitively recognized. two larger studies have suggested a skew to males with males compared with females in one report, and males compared with females in the other ; most other studies looking at gender and breed did not clearly distinguish between lymphoblastic and lymphocytic neoplasia. clinically, it is impossible to distinguish cats with ibds from cats with small cell lymphoma. this is hardly surprising when even histologic distinction can be difficult! therefore cats will present with weight loss or vomiting or diarrhea at a similar frequency to those with ibd. weight loss has been recognized as a presenting sign in % to % of cases, diarrhea in % to % of cases, and vomiting in % to % of cases, with various combinations of these signs also possible. other variable signs are lethargy and inappetence or, conversely, polyphagia. , , , these findings can be summarized by stating that cats with gastrointestinal small cell lymphoma can present with any combination of signs relating to the gastrointestinal tract. intestinal small cell lymphoma is typically a diffuse disease, and therefore multiple areas of the alimentary tract are usually affected. in studies where different locations of the small intestine were assessed, the jejunum was most commonly affected ( %), with the ileum frequently affected ( % to %), and duodenal pathology slightly less prevalent ( % to %). , although the numbers of cats assessed in these studies are small, the important fact that the duodenum is not always affected needs to be recognized, which has important implications for how biopsy samples are obtained, because lesions beyond the duodenum are likely to be beyond the reach of an endoscope. further difficulties in precise diagnosis may arise, since non-neoplastic lymphocytic infiltrates (e.g., ibd) are often found in other locations along the intestinal tract. , , the stomach is also affected in % to % of small cell lymphoma cases. , , although not fully assessed, involvement of the colon appears rare. local lymph node involvement is common, being noted in up to % of cases. this percentage may be even higher, because many studies assessed lymph node cytology from ultrasound-guided fine-needle aspirates, which may miss spread to the lymph node, because the population of neoplastic lymphocytic cells is indistinguishable from the normal population of lymph node cells. histology is required to assess changes in lymph node architecture. liver involvement is not uncommon but not thoroughly assessed. one study noted liver lymphocytic neoplasia in of cats with small intestinal lymphocytic neoplasia, another found of affected cats in which the liver was biopsied, another noted of cats had liver involvement, and a further study detected neoplasia "in the lymph nodes, liver, or both" in all cats with intestinal small cell lymphoma. the pancreas may also be involved. , this may be akin to the noted association of lymphocytic inflammation of intestine, pancreas, and liver that has been dubbed tri-iditis. ultrasound findings may not suggest extragastrointestinal involvement. in the case of liver pathology, ultrasonography may show no changes in as many as % to % of cases. , focal nodular changes and he patomegaly have been recognized as ultrasonographic signs of hepatic small cell lymphoma. both lymphocytic ibd and lymphocytic neoplasia are often recognized simultaneously in the same cat, , and numerous authors have suggested that lymphocytic ibd may be a precursor to intestinal lymphoid neoplasia. , if this is the case, then antigenic factors, such as bacterial population changes or food sensitivities, could be considered primary initiating factors for small cell lymphoma since they are potential underlying etiologies of ibds. however, neoplasia also requires genetic mutations to occur (often affecting regulation of cell death and cell survival), and these may be initiated by the inciting antigenic factors or the ongoing inflammatory changes. as opposed to other feline lymphoid neoplasia, no association has been made with felv infection. , , , intestinal lymphocytic lymphosarcoma begins in the superficial mucosa and progresses to involve the entire mucosa and submucosa; then advancing in a perivascular pattern into the tunica muscularis, eventually infiltrating all four intestinal tunics. lymph node and other organ (such as liver or pancreas) involvement likely represent metastasis through lymphatics and perhaps hematogenously. more distant metastasis is not reported. serum or plasma biochemistry and hematologic findings are typically nonspecific. however, this testing is important as part of the diagnostic workup to rule out extra-gi disease, such as hyperthyroidism or diabetes mellitus. common biochemistry findings are mild to moderate increase of liver enzymes, such as alanine aminotransferase (alt), aspartate aminotransferase (ast), and/or alkaline phosphatase (alp). , , , as with ibds, these liver enzyme changes may or may not represent overt hepatic disease. albumin may be reduced but is normal in most cases , , ; azotemia may be present and may be of prerenal origin or represent concurrent renal disease. in one study, of cats were hypocobalaminemic; of cats had low folate, but of had elevated folate; and of cats had increased ftli. hematologically, a mature neutrophilia with or without monocytosis is sometimes present, representing the inflammatory response; lymphopenia may be present as a stress response. anemia may be present and may occur as a result of chronic slow gi blood loss, and in some cases, ulceration, or it may be because of chronic disease; hemoconcentration is also possible, reflecting dehydration. , , , palpable or ultrasonographically visible thickened intestines ( % to % of cases) , , , or mesenteric lymph nodes ( % to % of cases) , , , are no more or less likely to be present in comparison with ibds. there are no defined ultrasound guidelines for cats with intestinal small cell lymphoma, because most prior papers do not distinguish between small cell and lymphoblastic neoplasia. , a more recent paper found of cats undergoing ultrasound examination had diffuse small intestinal wall thickening, with a mean of . mm (range, . to . mm; median, . mm), and focal mural thickening of mm was noted in one cat. in many cases, against expectations, intestinal wall layering was preserved. these findings also mean that of cats had ultrasonographically normal intestinal wall thickness (≤ . mm for the duodenum and ≤ . mm for the ileum). if affected, jejunal lymph nodes may appear as hypoechoic and enlarged; in the same study, of cats had lymph node changes with a mean diameter of . mm (range, . to mm; median, mm) compared with the normal diameter of less than or equal to . mm. none of these findings can definitively distinguish small cell lymphoma from ibds; although one recent paper has suggested that ultrasonographic thickening of the muscularis layer is more likely in cats with intestinal small cell lymphoma (figure - ) than those with ibd, this change was also seen in % of cats with a normal small intestine. however, thickening of the muscularis layer together with lymphadenopathy was recognized in % of those cats with small cell lymphoma compared with % of those with ibd and % of cats with no small intestinal pathology. biopsy samples and histopathology are required for definitive diagnosis. an example of jejunal and mesenteric lymph node appearance at laparotomy is shown in figure it is difficult to distinguish between lymphocytic inflammation and small cell lymphocytic neoplasia in any location; some histopathologic features that might help in differentiating the ends of the spectrum may include therefore become known as the fondacaro protocol. this consists of a combination of prednisolone and chlorambucil given orally by the client at home (table - ). the rationale is that a slow alkylating agent, such as chlorambucil, is more appropriate to use for the slowly dividing, well-differentiated lymphocytes that cause disease. this can be contrasted to the aggressive chemotherapeutic agents required for the rapidly proliferating cells in lymphoblastic neoplasia that is typically associated with lymphosarcoma. reported response rates to this protocol are excellent, with % to % of cats achieving complete clinical remission, reported median survival times ranging from to months for those cats responding to therapy, and reports of individual cats surviving as long as months. , , the original reported protocol comprised prednisolone ( mg/cat, po or mg/kg, po) given daily with chlorambucil pulsed by administration of mg/m for days every weeks. a more recent study dosed prednisolone similarly, but chlorambucil was given as continuous therapy of mg/cat, po every second or third day. no mucosal congestion, edema, or fibrosis in lymphocytic neoplasia, compared with ibd . epitheliotropism, or homing of neoplastic t lymphocytes to the mucosal epithelium in lymphocytic neoplasia these features can be seen in figure - . each of these criteria may be useful but are unlikely to be definitive. further studies that may not be routinely available but which may be helpful are immunophenotyping; most reports have found purely t lymphocytes in most cases of intestinal small cell lymphoma , , , (figure - ). . clonality; the detection of a clonal population of cells, as recently described for intestinal lymphocytic lymphosarcoma, would be closest to providing the basis for definitive diagnosis. effective treatment of feline intestinal small cell lymphoma was brought to light by fondacaro et al and has cat to be weaned off corticosteroids, with chlorambucil continued as monotherapy (as is often the case with humans). iatrogenic diabetes mellitus usually needs to be managed with insulin therapy, at least initially (see chapter ) . high-grade lymphoma or lymphosarcoma is the traditional style of aggressive, rapidly dividing lymphoid neoplasia that carries a much poorer prognosis than small cell lymphoma. most early studies do not distinguish grade of neoplasia; so, the prevalence of low-grade and high-grade alimentary lymphoma are difficult to assess. several recent studies found a similar prevalence of each, , but the seminal paper describing small cell lymphoma found only cases of lymphoblastic lymphoma compared with cases of small cell lymphoma. this ratio of approximately one high-grade gi lymphoma case for every three low-grade cases more closely approximates the rate found in the author's practice. the reported median ages of affected cats range from to years, but cats as young as year old have been diagnosed. most papers note that males are overrepresented, and siamese cats may also be overrepresented although most affected cats are domestic shorthairs. , , , , precise signalment is difficult to determine from the literature, because many papers assess all anatomic locations of lymphoma without necessarily breaking down epidemiologic data for each anatomic site. also, there are few comparisons to a reference population. the association of lymphoma with felv infection is well established and documented and is covered in chapter ; fiv has also been shown to be lymphomagenic. , since the control of felv through vaccination began in the s, nonretroviral-associated lymphoid neoplasia has become more common, and the rates of intestinal lymphoma have, in fact, increased since felv infection rates have decreased. the underlying causes for this increase are not known. the association with inflammation from ibds was noted for small cell lymphomas, and perhaps there is a spectrum from lymphocytic ibd to small cell lymphoma to high-grade lymphoma. that some cats are more likely to have inflammatory changes become neoplastic is suggested by a paper noting higher lymphoma rates in cats with vaccine-associated sarcomas (a neoplastic condition where the role of chronic inflammation is well noted). similar protocols are used in humans with both lowgrade (i.e., lymphocytic) lymphosarcoma and chronic lymphocytic leukemia. , some studies with humans have indicated that continuous therapy with chlorambucil results in prolonged survival, although metaanalyses have not been able to determine optimum dosing and scheduling of administration of chlorambucil or other alkylating agents in these conditions in humans. , although we do not have enough data to critically compare pulsed therapy to continuous dosing, the study assessing continuous dosing appeared to have a lower number of cats completely responding, although those cats that did respond had a longer median survival than those in the studies assessing pulsed chlorambucil dosing. , the differences may also relate to the definitions used for complete response. the chlorambucil dose of mg/cat, po every second day (or third day) is often chosen because of the ready availability of mg coated tablets, the breaking of which can expose the owner to these cytotoxic medications. chlorambucil can be compounded into smaller doses, thus allowing daily dosing of mg capsules. the author has used this dose to apparent good effect, but there has been no critical assessment. it is unknown whether involvement of lymph nodes or other organs, such as the liver, affects prognosis. the only study of substantial size to include extra-gi locations found anatomic location was not prognostic for response or survival time. in another study, of the five cats with liver involvement, two cats did not survive more than months, yet the other three lived longer than years, with two surviving longer than years. a study of hepatic small cell lymphoma suggests the density of neoplastic lymphocytes may influence survival, and density may relate to the stage of the disease when diagnosis occurs. adverse effects of chlorambucil are rare, but gastrointestinal signs, myelosuppression, and myoclonus have all been reported. gastrointestinal signs, such as vomiting, diarrhea, or inappetence, can be difficult to distinguish from continuation of the gastrointestinal disease diagnosed. these signs are usually self limiting. myelosuppression is also possible with thrombocytopenia reported. , monoclonus has been reported on one occasion. it is ideal to check hematologic parameters every months for cats receiving chlorambucil. continuous therapy using lower doses of chlorambucil may be less likely to lead to these adverse effects. high doses of corticosteroids can induce diabetes mellitus, and thus blood glucose should be checked regularly. if diabetes occurs, the author has found that budesonide ( mg budesonide is generally considered to be equivalent of mg prednisolone) can be substituted for prednisolone, since it has reputed lower systemic effects (though no assessments of this drug's effectiveness in cats have been made). an alternative is for the of distinction of intestinal layering as shown in figure - . the area of lymphomatous infiltration is hypoechoic, because it contains a uniform cell population without much reactionary fibrous tissue. mesenteric lymphadenomegaly is common (figure - ) , as are changes in other organs, such as kidney, liver, or pancreas. ascites may also be seen. , although ultrasonographic distinctions predominate, there is considerable overlap between ultrasonographic findings with small cell lymphoma and high-grade lymphoma. the clinician must not lose sight of the fact that microscopic distinctions are required to diagnose either condition. cytologic diagnosis of high-grade lymphoma from fine-needle aspirates (fna) is much more likely than with small cell lymphoma. this is because there is usually a focal lesion, and the neoplastic cells are a monomorphic population of large, immature cells (i.e., that are not normally seen in tissue). sometimes, mixed lymphoid whether the underlying cause is retroviral or chronic inflammation or anything else, the pathogenesis of highgrade intestinal lymphoma, as with small cell lymphoma and other neoplasia, depends on chromosomal changes that affect regulation of cell growth and death, resulting in malignant transformation and clonal expansion of immature lymphocytes. metastasis can occur in one third to two thirds of cases, , with involvement of mesenteric lymph nodes most commonly noted, but spread to liver, spleen, kidneys, and thorax is also possible. a recent survey of gastrointestinal lymphoma found that most cases ( of ) involved the small intestine (including that also involved the stomach and that also involved the large intestine), and of cases involved the large intestine only. cats with high-grade alimentary lymphoma often present similarly to those with other gastrointestinal diseases. typical clinical signs are weight loss, anorexia, lethargy, vomiting, diarrhea, or a combination of these signs. repeated studies have found cats with no vomiting or diarrhea; in one study, of cats had only anorexia or weight loss on presentation. cats with large bowel pathology usually present, as with other causes of colitis, with increased urgency, and small, frequent amounts of diarrhea, often with blood or mucus. cats with large bowel neoplasia of any form can present for constipation caused by intestinal obstruction. palpation of an abdominal mass has been recognized in % to % of cases, , but the corollary of this is that % to % of cases did not have a palpable mass. it is also important to note that up to % of cats with intestinal small cell lymphoma, and a number with ibds, have palpable mesenteric lymph nodes; so, a palpable abdominal mass is not a specific indication of high-grade neoplasia. many cats have palpably thickened bowel loops. hematology and plasma or serum biochemistry findings are also nonspecific. increased liver enzymes may or may not indicate liver involvement. anemia may be recognized and can be non-regenerative, reflecting chronic disease or slow blood loss, or regenerative if there is more substantial blood loss associated with mucosal ulceration. hypoalbuminemia can be because of blood loss or intestinal protein loss. hypercalcemia of malignancy is a possibility but not commonly reported. despite nonspecific signs, laboratory testing is important to rule out extra-gi diseases and help manage consequences of enteric disease, as with small cell lymphoma and ibds. ultrasonography commonly shows a focal intestinal thickening (of to mm) with partial or complete loss although noted as the next most common intestinal neoplasia, after the various forms of lymphoma, adenocarcinoma is seen relatively infrequently in practice. most cats are more than years old, , , males may be overrepresented, and several studies have recognized a distinct overrepresentation of siamese cats. , three distinct forms have been described : cats typically present with nonspecific signs of gastrointestinal disease but can present with obstructive signs. cats with large bowel neoplasia can present for tenesmus or hematochezia and even constipation, if the lesion is obstructive (or partially obstructive). on physical examination, an abdominal mass is palpable in approximately % of cases, but other findings are usually nonspecific. anemia can be found if mucosal ulceration has occurred, but there are no distinctive laboratory findings. lesions can occur anywhere along the intestinal tract. one study of cases found % of feline intestinal adenocarcinoma lesions were present at the ileum or ileocolic junction. twenty-five to percent of cases have metastasis at the time of the diagnosis, and this is a poor prognostic indicator. , , radiology may show a mass lesion or intestinal obstruction, and ultrasonography can localize lesions to an intestinal origin. the ultrasonographic appearance of the proliferative, circumferential, outwardly expansile populations (of immature lymphoblasts and mature lymphocytes) are seen if a germinal lymphoid follicle is aspirated, and precise diagnosis may be difficult if there are a large number of lymphoblasts. fna samples are best obtained with ultrasound guidance. the cytologic sample quality is greatly improved by not aspirating when the needle is visualized in the mass but merely "pecked" into the mass so that the needle is merely acting to finely "core" the mass. on removing the syringe and needle, the hub of the needle is removed before drawing air into the syringe, the hub is replaced, and the sample within the needle is expressed onto a slide. usually, the decision to diagnose by cytology from fna is based on the ultrasonographic appearance of a mass. since there is substantial crossover of ultrasonographic appearance of intestinal masses, laparotomy for excision is often performed with the affected bowel submitted for histology. except when intestinal obstruction has resulted, there is no therapeutic benefit of excising a gastrointestinal lymphoma (which requires excision and anastomosis), but there is minimal room for doubt when a histologic diagnosis is achieved. the response to therapy for high-grade intestinal lymphoma is significantly worse than that for small cell lymphoma. , further, response to therapy for highgrade intestinal lymphoma appears to be worse than for lymphoma in other anatomic locations. precise remission rates and survival times are difficult to quantify, because many studies assess lymphoma from multiple locations and do not necessarily differentiate response of gastrointestinal lymphoma or report the grade of lymphoma. with remission rates reported from % to % , , and a median survival time of up to weeks (range, to weeks), it can be said with some certainty that some cats respond to therapy for reasonable durations. multiple authors have noted that the best prognostic indicator is response to an initial treatment cycle, , , which should prompt clinicians to encourage owners to start therapy and decide whether to continue based on the cat's response. there are several published chemotherapeutic protocols, , , , , but all follow the same principles of using medications to target specific phases of the cell division cycle (such as l-asparaginase and vincristine) with other medications that interrupt multiple phases of the cell cycle (cyclophosphamide and doxorubicin). targeting the cancer cell in different ways enables more cells to be killed, reduces the toxicity of the individual drug used, and reduces the likelihood of resistance to a specific drug. several authors have noted increased success with the addition of l-asparaginase and doxorubicin to protocols. , , chemotherapy for lymphoma is covered in more detail in chapter , oncology. look promising, but only two cats assessed had gastrointestinal mast cell neoplasia. lomustine was used unsuccessfully in one cat with sclerosing mct; another cat with sclerosing mct received eight treatments of vinblastine and had a survival time of greater than years. adenomatous polyps have been reported in the duodenum and ileum and can result in intussusception. cats of asian ancestry, predominantly siamese, are greatly overrepresented, and most reported cases have been males. cats usually present for vomiting or hematemesis that, surprisingly, can be very acute in onset; complete intestinal obstruction may result. , , resection is curative, with survival times of more than years reported. eosinophilic sclerosing fibroplasia has recently been described in a series of cases and is not strictly neoplasia. the ulcerating mass lesions that can occur anywhere from the stomach to the colon are often grossly and histologically mistaken for neoplasia. there appears to be no breed predisposition or age predisposition (with ages ranging from weeks to years), but of cases ( %) were castrated males cats compared with of ( %) female spayed cats. eighty-four percent of cats presented for vomiting, % presented for weight loss, and of ( %) cats had peripheral eosinophilia. all cases had a palpable abdominal mass. the pyloric sphincter was the most common site, and lesions in this location were mostly considered unresectable. fourteen of cats ( %) had bacterial colonies within microabscesses and necrotic foci within the lesion. the bacteria recognized were predominantly gram-negative rods, but antibiotics did not seem to be clinically effective. the bacteria are suspected to initiate the lesions, having been embedded after foreign body penetration. there are no specific treatment recommendations, but excision, where possible, would be prudent; corticosteroids appear to be helpful adjunctive therapy. survival times are difficult to estimate since many cats were euthanized because neoplasia was suspected and follow-up times were short (up to months) for the remaining cats. there are very few reports of intestinal leiomyosarcomas in cats, , which have been reclassified as gastrointestinal stromal tumors. these tumors may be more likely to arise from the ileocecocolic junction. resection, if possible, is usually recommended, with survival times of to months reported before recurrence. the author owns a cat with this tumor where resection was not form is better described than the annular, constrictingband form with minimal outward enlargement. in these cases, sonographically, a solitary segmental intestinal mural mass is present and characterized by circumferential bowel wall thickening with transmural loss of normal sonographic wall layers. the thickening can vary in echogenicity but may be hypoechoic and may be symmetric or asymmetric. there is no definitive distinction, however, from lymphosarcoma, mast cell tumor, smooth muscle origin tumors, or even segmental benign inflammatory bowel disease. surgical resection is the treatment of choice. there seem to be two distinct groups in terms of survival time postresection: . short-duration survival (euthanasia or death within weeks of surgery) . long-duration survival (mean survival time of months, with a number of cats surviving greater than years) , because clean margins improve prognosis, for large bowel adenocarcinoma, subtotal colectomy may be required for complete excision. because of the potential for success after resection, it is recommended to excise unidentified masses at the time of surgery. other forms of intestinal neoplasia are recognized infrequently, and include intestinal mast cell tumors, adenomatous polyps, eosinophilic sclerosing fibroplasia, gastrointestinal stromal tumors (leiomyosarcoma), and hemangiosarcoma. mast cell tumors (mct) are often cited as the third most common form of feline gastrointestinal tumor, but the intestines are a far less common site than cutaneous, splenic, or hepatic mast cell neoplasia. , masses are usually segmental nodular thickenings that occur in older cats. the masses are indistinguishable ultrasonographically from other tumors, such as lymphoblastic lymphosarcoma. a recent series of cases described a variant of feline intestinal mast cell tumor, dubbed sclerosing mast cell tumor, for which neoplastic cells form a trabecular pattern with dense stromal collagen. additionally, eosinophilic infiltrates were moderate to marked in most cases. these cases can be confused histologically with eosinophilic enteritis, gastrointestinal stromal tumor, or fibrosarcoma. surgical resection is recommended, but lesions are commonly infiltrative or metastasize widely, and there are few reports of successful treatment. lomustine (dosed to mg/m , po every to weeks) has recently been assessed as adjunctive chemotherapy for mast cell neoplasia in various locations, and results recognized in that many intestinal bacteria can be found in healthy animals. further antibiotic administration can result in increase of other bacteria. fungal causes of diarrhea are usually recognized from histology of biopsy samples. it remains to be seen whether the recent ready availability of pcr panels looking at a number of infectious causes of diarrhea will be beneficial for recognizing pathogens that had previously been misdiagnosed or a hindrance for readily recognizing commensal organisms not necessarily causative of the clinical signs being investigated. the most common viral, bacterial, and mycotic causes of diarrhea in cats are described below. parasitic gastrointestinal diseases are covered later in this chapter. viral causes of diarrhea are not usually specifically diagnosed, since, with the exception of the canine fecal elisa possible, and the cat appears healthy months past diagnosis (figure - ). cats with intestinal hemangiosarcoma often present with anemia, and the disease appears to be highly metastatic. the intestines appear grossly thickened by dark red tissue. the small and large intestines seem to be affected with similar frequency. removal of macroscopic disease is recommended, but often the full extent of the severity is only recognized at surgery. the prognosis is poor. suspicions of infectious causes of diarrhea should be aroused in younger cats, cats from shelters, or cats with immune suppression. when considering infectious causes of diarrhea, clinicians should assess whether the diarrhea is large bowel or small bowel in origin and correlate this with specific pathogens that are likely to cause clinical signs as shown in table - . to increase the diagnostic yield of fecal examination for parasitic causes of diarrhea, wet smears and appropriate fecal flotations should be performed on fresh fecal samples (< hour old). it is appropriate to administer broad-spectrum anthelminthics, even if fecal tests are negative. bacterial and viral causes of diarrhea should be considered when the cat is systemically unwell with fever. fecal culture should be performed in these circumstances, but the limitations of this testing need to be yersinia enterocolitica current theory of fip pathogenesis involves initial infection with fecv and then mutation to fipcv in small numbers of susceptible individuals. , routine serologic testing for fecv in cats with diarrhea would neither prove correlation with the clinical signs nor affect how the disease is managed and so is not recommended. cats with fecv diarrhea should be managed with symptomatic therapy of fasting, then reintroducing a bland diet and supportive care with fluid therapy if necessary. other viruses, such as astrovirus, reovirus, rotavirus, and torovirus-like agent, have been recognized to cause diarrhea in cats, but their roles as pathogens are unclear. they are not routinely recognized in practice, since electron microscopy of fecal samples is necessary for diagnosis and is not routinely performed. management is supportive care with appropriate fasting, then reintroduction of bland diets and fluid and electrolyte replacement if necessary. successful identification of a known bacterial pathogen from a fecal sample does not necessarily mean that the agent found is the cause of disease in the cat. although a number of bacterial pathogens have been demonstrated to cause diseases when specific pathogen-free (spf) cats are experimentally infected, these same organisms can be found in healthy cats. the differences between healthy and diarrheic cats that have bacteria found in their feces may relate to virulence factors of the organism, or host factors (local or systemic immunity) of the cat. there is no definitive answer for this quandary. the author's opinion is that • if a diarrheic cat is systemically unwell and has a fever, then feces should be cultured. • if an organism is isolated that is known to cause signs consistent with those the cat is showing, the cat should be treated appropriately. campylobacter diarrhea is usually caused by c. jejuni. clinical signs of infection are poorly documented, but most cats are asymptomatic. younger cats are more likely to have clinical signs and hemorrhagic, mucoid diarrhea has been reported. diagnosis can be from culture of feces or swabs, and the organism is quite hardy; so, it usually survives transport to the laboratory. in individual cases, the organism has not been cultured after antibiotic treatment, , but it is not definitively proven that antibiotic therapy affects the natural course of the disease. antibiotics that can be used are amoxicillin-clavulanate ( mg/kg, every hours, po) for parvovirus, routine definitive tests are not available. clinical signs of panleukopenia (feline parvovirus infection) are more likely to occur in kittens, with the highest morbidity and mortality occurring between and months of age. subclinical cases in older (susceptible) cats probably go unrecognized. the organism is very stable in most environments, and infections mostly occur from environmental contact. peracute cases can result in death within hours, with little or no warning signs. acute cases often have fever, depression, and anorexia, with signs beginning approximately to days before presentation. vomiting is usually bile tinged and unrelated to eating. diarrhea does not always occur, and when it does, it is usually later in the course of the illness. leukopenia is not pathognomonic, because this can also occur with acute bacterial infection (e.g., salmonellosis can present identically). commercially available elisa tests for canine parvovirus antigen in feces can detect feline parvovirus; however, shedding may have ceased by the time clinical signs occur, and vaccination can result in positive test results for up to weeks. aggressive fluid therapy, usually at twice maintenance rates, is usually required. broad-spectrum antibiotic coverage is used to prevent or treat secondary bacterial infection from viral injury of intestinal mucosa. parenteral antibiotics are preferred to prevent the possibility of further gastrointestinal irritation. the author recommends calculating iv doses and introducing appropriate amounts of antibiotics to the fluids bag to create a constant rate infusion (cri); cefazolin can be used in this way at mg/kg/ hours, and betalactam cris are commonly used in human medicine. aminoglycosides or fluoroquinolones can be used concurrently at routine doses if fever persists after hours or the cat is moribund on presentation, but care must be used with these agents. aminoglycosides are potentially nephrotoxic, and fluoroquinolones have been reported to result in cartilage damage in growing animals, although this has not been demonstrated clinically in cats. fluoroquinolone retinal toxicity has been seen in all animals. cats that survive the first week usually recover, and prior infection imparts lifelong immunity. vaccinations are highly effective for disease prevention. feline enteric coronavirus (fecv) mostly causes mild, self-limiting diarrhea and must be distinguished from feline infectious peritonitis coronavirus (fipcv), which is essentially always fatal and for which diarrhea is not a typical sign (but is possible). the most widely accepted or fluoroquinolones, such as enrofloxacin ( mg/kg, once daily, po) for durations of to days. macrolides, such as erythromycin ( to mg/kg, every hours, po), are regarded as the drug of choice for humans but can cause gastrointestinal side effects. clostridium difficile has been recognized in up to % of diarrheic cats. clinical signs are typically acute onset watery diarrhea and anorexia. diagnosis has been made with detection of toxin a or toxin b in fecal samples using elisa. although these tests have not yet been validated for cats, they may prove to be a useful aid to diagnosis and are available for testing of equine feces at some commercial laboratories. nontoxigenic strains exist; so, positive culture alone does not ensure diagnosis. metronidazole ( mg/kg, every hours, po) for approximately days is the treatment of choice. clostridium perfringens typically results in large bowel diarrhea with tenesmus, mucus, and hematochezia, but small bowel signs can also be seen. pcr testing for enterotoxin a is commercially available and may prove to be a useful adjunct in diagnosis. antibiotics that can be used include metronidazole ( mg/kg, every hours, po), tylosin ( to mg/kg, twice daily, po), or amoxicillin-clavulanate ( mg/kg, every hours, po) for days. escherichia coli is a ubiquitous organism within the feline intestinal tract, and it would be unusual not to successfully culture e. coli from the feces of both healthy and unwell cats. when e. coli is associated with clinical signs of gastrointestinal disease, it is mostly as an opportunistic pathogen, with overgrowth resulting from changed environmental conditions, such as inflammation from other pathology or another pathogen. there are also specific strains of e. coli that are true pathogens because of virulence factors not present in commensal e. coli; these include enteropathogenic e. coli and enterotoxigenic e. coli, which both induce a watery diarrhea, and enterohemorrhagic e. coli, which produces a diarrheal syndrome with copious bloody discharge and no fever. pcr testing is commercially available to identify pathogenic strains of e. coli , ; although not offered at routine veterinary laboratories, this testing is available to veterinarians, and laboratories offering these services can readily be found online. diagnosis should also document histologic lesions corresponding to the strain of e. coli identified. there is emerging resistance to e. coli worldwide in all species of animals, including humans. this includes the typical therapies for gram-negative bacteria of beta-lactamenhanced penicillins and fluoroquinolones. a major risk factor is prior antibiotic usage, because commensal organisms are exposed to antibiotics. pcr testing does not enable antibiotic sensitivity testing, and fecal culture may not be able to distinguish pathogenic from nonpathogenic strains; so, sensitivities may not be an accurate reflection of the pathogenic organism. pcr testing for genes that impart resistance to e. coli have recently been described but are not yet commercially available. in some circumstances, supportive care with fluid and electrolyte replacement may be all that is required while the cat's immune system combats the infection. empiric therapy could include beta-lactam-enhanced penicillins (such as amoxicillin-clavulanate at mg/kg, every hours, po), fluoroquinolones (such as enrofloxacin at mg/kg, once daily, po), or cefovecin ( mg/kg, every weeks, sc), but the clinician must be aware of possible drug resistance. salmonella typhimurium infection is possible from ingestion of infected prey, infected food sources, or from a contaminated environment, including the veterinary hospital. the resulting clinical signs depend on the number of infecting organisms, the immune status of the cat, and the presence of concurrent diseases. infection rates in cats (and humans) have been correlated with seasonal bird migrations, and the illness has been dubbed songbird fever, but there is no distinction between this and other salmonella infections. clinical signs usually begin to days after exposure, starting with fever (often > ° c [ ° f]), malaise and anorexia, and progressing to diarrhea, vomiting, and abdominal pain. hematology can show leukopenia with a left shift and nonregenerative anemia, and biochemistry results are usually nonspecific. diagnosis is based on isolation of the organism by culture or identification with pcr, but care should be taken to correlate pathogen identification with clinical signs since, as with most gi pathogens, the organism can be isolated from healthy animals. as with e. coli, antibiotic resistance is widespread, with one united kingdom survey finding the multiple drugresistant strain dt to be the most frequent bacteriophage type identified. treatment should be reserved only for those cats showing systemic signs, because routine antibiotic use in treating salmonellosis induces drug-resistant strains and prolongs the convalescent excretion period. antibiotic choice should be based solely on sensitivity findings, since resistances are so widespread and unpredictable. this means that if the organism has been identified by pcr, then culture of feces must also be undertaken. the duration of treatment must be long enough to eliminate fecal excretion of the organism, prevent the chance of relapse, and reduce the chance of resistance developing; up to days has been advocated. , these cautions are particularly important because of the zoonotic potential of salmonellosis. linear foreign bodies have traditionally been considered more common than discrete foreign bodies in cats, , , but a study from a primary care facility indicated only % of foreign body cases were because of linear foreign bodies. the larger case load of linear foreign bodies at referral institutions noted in earlier studies may indicate the abilities of primary care practitioners to recognize and effectively deal with discrete foreign body obstructions. most studies have found that cats with intestinal foreign bodies are generally younger (mean, . to . years), with a notable exception being obstruction from trichobezoars where three of five cats in one study were years or older; the greatest risk factor appears to be length of hair coat. no specific breed predispositions have been described but siamese and siamese-related cats have been noted to have oral fixations and so may be expected to be overrepresented with intestinal foreign bodies. clinical signs will vary depending on the type of foreign body (linear or discrete), the position of obstruction, and the time since obstruction. most cats present for anorexia or vomiting. partial obstruction can result in diarrhea (which can be bloody). foreign body obstruction is typically considered an acute condition, with duration of obstruction because of a linear foreign body, measured from the onset of clinical signs to diagnosis, reported to range from to days. , , however, one paper demonstrated chronic, intermittent, gastrointestinal disease from a linear foreign body of a -month duration demonstrating that partial obstruction can result in a chronic course. physical examination may or may not reveal abdominal pain, palpable abdominal mass (or plication), dehydration, or fever. all cats presenting for anorexia or vomiting should have the underside of the tongue evaluated for the presence of a linear foreign body. applying gentle pressure with a thumb in the intermandibular space to elevate the tongue is an effective way to visualize lesions or foreign bodies in the sublingual area (see figure - ) . life-threatening consequences can result from the interactions of local and systemic factors that arise from intestinal obstruction. locally, damage to the mucosa from traction and pressure of the foreign object can cause hemorrhage, ischemia, and necrosis. systemically, hypovolemia, toxemia, and acid-base and electrolyte imbalances can ensue. complete intestinal obstruction by discrete masses results in gas and fluid distention of the lumen proximal other bacterial causes of diarrhea have been reported in cats, such as yersinia enterocolitica, yersinia pseudotuberculosis, clostridium piliforme (tyzzer's disease), and anaerobiospirillum sp. specific diagnosis of these (and other bacterial infections) may be found in the course of investigation. management follows the principles of supportive care and appropriate antibiosis based on sensitivity testing. small intestinal bacterial overgrowth (sibo) has not been specifically described in cats. the criteria defined for dogs is a fasting bacterial count in duodenal juice of greater than organisms/ml and is often recognized with other chronic gastrointestinal diseases. healthy cats appear to have at least this number of upper intestinal bacterial with a range of to /ml recognized. bacterial overgrowth could potentially occur with ileus or intestinal inflammation of any underlying cause. foul-smelling small bowel diarrhea with no specific pathogen recognized may be an indicator of this condition, as could an increase in bacterial metabolites, such as folate. if suspected, it is appropriate to manage with broad-spectrum antibiotics, such as metronidazole ( to mg/kg, every hours, po) or amoxicillin ( mg/ kg, every hours, po) for an extended duration such as to days. alterations in intestinal flora have been recognized after such treatment ; however, any advice for this "condition" is entirely empirical. all efforts should be directed at identifying a precise underlying cause. mycotic and other infectious agents are only rarely recognized as intestinal pathogens in cats. diagnosis is made by histologic and microbial analysis of samples obtained at biopsy. possible agents include histoplasma capsulatum, aspergillus spp., candida albicans, and pythium insidiosum. intestinal obstructions arise most commonly as a result of neoplasia in older cats and foreign body ingestion predominantly in younger cats. , , less common causes include intussusception and granulomatous inflammation (e.g., from fip) ; tapeworm infection, with greater than worms acting as a linear foreign body, has also been reported. other listed causes are volvulus, intestinal torsion, incarceration of bowel in a hernia, adhesions or stricture, intramural abscess or hematoma, and congenital malformations. may not occur if the obstruction is partial or intermittent, or if vomiting results in less fluid present. since most foreign body obstructions in cats are proximal, identifiable dilatation may not be recognized for this reason. linear foreign bodies present further challenges for radiographic recognition; the following typical radiographic signs , may or may not be present: to the obstruction. most gas accumulation is a result of swallowed air, which is predominantly nitrogen that cannot be absorbed by the intestinal mucosa. gas also arises from bacterial fermentation. fluid accumulates as a result of increased secretions (saliva, bile, and secretions of gastric, pancreatic, and small intestinal origin) and retention of fluid already ingested, and it can be augmented by local hemorrhage. since most intestinal obstructions in cats do not reach the midjejunum, reabsorption of fluids that normally occurs at the jejunum and ileum is impaired. linear foreign bodies, such as string, dental floss, or elastic toys, require proximal anchoring, usually under the tongue or in the pylorus (for example, by part of a toy attached to elastic). peristalsis moves the free end of the "string" through the intestinal tract, resulting in pleats of intestines around the foreign body. as the foreign body is forced against the intestinal mucosa, the mucosa becomes edematous, and even partial penetration affects mucosal integrity, allowing systemic entry of bacteria. intraluminal bacterial populations increase for both discrete and linear foreign bodies as a result of stasis. mucosal permeability can be affected by prolonged luminal distention, allowing entry of bacteria and toxins systemically or into the peritoneal cavity. direct entry of bacteria to the peritoneal cavity, causing septic peritonitis, can result from perforation of the intestinal wall from linear foreign bodies or sharp discrete foreign bodies, such as toothpicks or plastic toys. definitive diagnosis requires identification of the foreign body retrieved at surgery or in some cases, by endoscopy. this may be aided greatly prior to surgery by diagnostic imaging. however, imaging findings, particularly in the case of partial obstructions, may be subtle enough that obstruction of no identifiable cause is recognized or no overt signs are apparent. laboratory findings are not helpful in the precise diagnosis but are important to assess fluid and electrolyte balances that must be corrected. cats rarely help practitioners by ingesting radiopaque objects, but on the rare occasions that they do, these can be observed easily on plain radiographs. nonopaque foreign bodies depend on dilatation of the intestine from gas and fluid accumulation proximal to the obstruction for radiographic recognition (figure - ) . one study has suggested that if the jejunal diameter is greater than . times the length of the cranial end plate of the second lumbar vertebra, then intestinal obstruction is the most likely abnormality. care must be taken that the jejunal and not duodenal diameter is measured and that the radiographs must be positioned strictly lateral, because an oblique view can alter the measurement of the lumbar vertebra. however, dilatation of an obstructed intestine successful treatment of foreign body obstruction requires evacuation or removal of the foreign body as well as correction of any bacteremia or endotoxemia, acid-base or fluid imbalances. discrete foreign body obstruction requires surgery or endoscopy to remove the object. in some specific circumstances, linear foreign body obstruction may be managed conservatively by cutting the anchor point below the tongue and allowing the cat to pass the foreign body by peristalsis. however, the decision to manage a cat conservatively must be done with the cat hospitalized, with fluid therapy and antibiotic coverage and a clear recognition on behalf of the practitioner and the owner that surgery may subsequently be required. cutting a sublingual linear foreign body may be achieved in a conscious cat by applying pressure with the thumb of one hand in the intermandibular space to elevate the tongue and gently grasping it using gauze with the other hand while a second person cuts the line with a suture cutter. there is a chance of a small nick on the sublingual surface. if the cat will not tolerate the procedure, sedation is appropriate. when cutting the line, the nature of the linear foreign body should be assessed (i.e., is it more or less likely to cut mucosa). in one study, cats with linear foreign bodies were managed conservatively with cats subsequently requiring surgery. the authors of that paper created guidelines that will be adapted here. conservative management should be attempted if the cat • is presented acutely (within days) after known ingestion of a linear foreign body • has a sublingually fixed linear foreign body that can be cut • has no overt signs of peritonitis • altered gas pattern with luminal gas collecting in small bubbles instead of normal curved tubular columns. this can be subtle when there is only minimal involvement of the intestine but overt when involving the entire small intestine. commashaped gas patterns are more likely to occur with linear foreign bodies. contrast radiography can aid diagnosis for both discrete and linear foreign bodies but should be used with caution because intestinal perforation may be present. nonionic iodinated agents that are typically used for myelography (such as iopamidol or iohexol) should be used, since barium is irritating to the peritoneum and oral iodine compounds are hypertonic. hypertonic compounds may draw fluid into the stomach and intestines after oral administration, with the potential of creating further fluid and electrolyte imbalances in an already compromised patient. ultrasonography is a very useful diagnostic tool, particularly for discrete foreign bodies, where, in most cases, there is overt distention of the small intestines with intraluminal fluid apparent (figure - ) . this modality has not been extensively assessed as an adjunct to diagnosis of foreign body intestinal obstruction in cats specifically, although there are several papers assessing dogs and small numbers of cats that agree with its utility. , , linear foreign bodies are more difficult to assess ultrasonographically, but plicated bowel can be recognized, sometimes with the foreign body seen as a hyperechoic line centrally. si a technique has been described for removal of linear foreign bodies by making a single enterotomy incision proximally and passing a red rubber catheter over the linear foreign body aborally, milking the foreign body within the catheter through the colon for retrieval from the cat's anus by an assistant. this technique is not always effective, because it can be hampered if the foreign body is knotted or does not run smoothly through the red rubber catheter. if the affected bowel segment demonstrates evidence of necrosis or perforation on the mesenteric border of the intestine, resection and anastomosis should be performed. necrosis is indicated by dark discoloration, thin intestinal wall, poor arterial pulsation, poor capillary bleeding, or lack of peristalsis. end-to-end anastomosis can be accomplished using a simple interrupted appositional pattern or a modified simple continuous appositional pattern with the same type of suture material used for enterotomy closure. , intraabdominal masses causing intestinal obstruction are often presumed to be neoplastic but can also be of infectious origin. resection, where possible, is always recommended, because resection of neoplasia (if no metastasis) can offer a good prognosis, , , , and infectious causes may be managed with adjunctive therapy after definitive diagnosis. intestinal obstruction in older cats is more likely to be secondary to neoplasia. any neoplasia can cause obstruction, but adenocarcinoma , and adenomatous polyps , are reported to cause obstruction more often surgical intervention is mandatory if • clinical signs (e.g., vomiting or anorexia) persist or deterioration occurs with conservative management • the cat has overt signs of peritonitis • the linear foreign body is fixed at the pylorus some authors disagree with attempting conservative management, since a perforated intestine from a linear foreign body reportedly carries a % mortality rate, , and early surgical intervention is never an incorrect decision. this should be balanced with the observation that cats can carry a linear intestinal foreign body, such as an elastic cord for a -month duration without intestinal perforation. however, fishing line, for example, would not be so forgiving! surgery to remove an intestinal foreign body (figures - and - ) should be considered an exploratory laparotomy. that is, the aim of the surgery is not only to remove the foreign body but to assess the entire intestinal tract and abdomen for other foreign bodies or pathology. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction, thus delaying healing and creating the potential for surgical dehiscence. linear foreign bodies require multiple enterotomy incisions, since pulling the object out through a single incision could create iatrogenic intestinal perforation. the anchor point (either sublingual or pylorus by gastrotomy) must be released in the first instance. enterotomy incisions are closed with / synthetic, monofilament, absorbable suture material, such as polydioxyanone (pds) or equivalent, in either a simple interrupted or simple continuous pattern. , removal of a discrete foreign body (a piece of leather) at laparotomy. enterotomy to remove discrete foreign bodies should always be distal to the obstruction, because the intestine is likely to be compromised proximal to as well as overlying the obstruction. this is the same cat as in the radiology image in figure - . affected bowel is required, with anastomosis of the healthy tissue. there appears to be no benefit to enteroplication, which can result in significant ileus. there is no benefit to performing resection-anastomosis if the intussusception does reduce manually. , the prognosis depends on the underlying disease process and the chronicity of the intussusception, and therefore how debilitated the cat is at presentation, however, prognosis is mostly good, with survival reported in up to % of cases, though recurrence can occur in some cats with idiopathic disease, often at different locations of the intestinal tract. constipation is defined as infrequent or difficult defecation associated with retention of feces within the colon and rectum. prolonged constipation results in harder than other types of neoplasia. please refer to the sections on intestinal neoplasia earlier in this chapter for more details. granulomatous inflammation causing a single focal intestinal lesion can lead to obstruction in the same way that neoplastic change can. feline infectious peritonitis (fip) can present as focal lesions, often in the colon or ileocecocolic junction. in the case of fip, the focal lesion is usually an indicator of multisystemic disease; so, resection does not help prognosis. the fungus-like organism, pythium insidiosum has also been reported to cause granulomatous lesions, resulting in intestinal obstruction from large extraluminal masses that are approximately fist sized. resection with adjunctive itraconazole ( mg/kg) for months after surgery was a successful treatment. intussusception refers to invagination or prolapse of one portion of the intestine into the part of the tract that either precedes or follows it. there is a bimodal age distribution with intussusceptions in older cats, most likely associated with neoplasia (or ibd in some cases) ; underlying causes for younger cats are ill defined and may be idiopathic in many cases, , but associations with parasitism and, in one case, a linear foreign body, have been made. siamese and burmese cats seem to be overrepresented. the most common locations are the ileocolic region and the jejunum. , , , affected cats present with nonspecific signs of gastrointestinal disease, such as anorexia and lethargy. vomiting is not necessarily a presenting sign; diarrhea may occur. abdominal palpation reveals a mass in most cases. plain and contrast radiography only show evidence of obstruction and usually do not help define that the bowel has intussuscepted. , , , ultrasonography is very useful for diagnosis, because a distinctive pattern of alternating hypoechoic and hyperechoic concentric rings (figure - ) is present in transverse sections. , sometimes, the target lesion seen can be hard to distinguish from the pathology of other intraabdominal masses, such as lymph nodes, and in these cases, the size of the lesions can help, because the width will always be greater than mm with an intussusception (because the sum of at least four intestinal wall widths cannot be less) and is often greater than mm. surgical correction is always required, and manual reduction is typically not possible because there is usually significant venous infarction, edema, and congestion (figure - ) as well as adhesions from fibrin and effusions from the affected bowel. , if the intussusception does not reduce manually, resection of the and drier feces that become impacted, and this is known as obstipation. chronic, recurrent constipation and obstipation can result in megacolon, which refers to persistent increased bowel diameter that is not responsive to therapy. megacolon is not a specific disease entity; it may be considered the most advanced stage in the spectrum of chronic constipation. in most cases, constipation can be managed quite simply if the underlying cause is determined and dealt with. a comprehensive list of causes of constipation is noted in table of course, multiple factors can interact. for example, an older cat may have renal disease and so will be dehydrated to some degree and have arthritic hips and so be reticent to squat. the presenting signs of constipation are usually evident to owners and include straining in the litter box and producing hard dry feces, if at all. sometimes, however, owners can misinterpret signs. cats can strain because of lower urinary tract problems, and, if no urine is produced, some owners assume the problem is because of constipation. some constipated cats can intermittently have diarrhea because of direct colonic irritation from hard dry feces and so may present for diarrhea and not constipation. cats can also present for less specific signs, such as anorexia, lethargy, weight loss, and even vomiting. , vomiting can occur because of colonic receptors stimulating vagal afferent endings, which, in turn, can stimulate the chemoreceptor trigger zone. sometimes owners are concerned that their cat is defecating less, but the cat has just changed its diet to a much lower-residue diet and so is producing less feces. a full dietary history is an important aspect of the initial assessment. physical examination should confirm presence of feces in the colon and assess the degree of impaction. the presence of feces can usually be confirmed by abdominal palpation. in constipated cats, the colon is often palpated as a long firm tube extending cranially; sometimes, feces can be palpated to and around the colic flexure. alternatively, the feces may be palpated as large, discrete fecal concretions (that can sometimes be hard to distinguish from intraabdominal masses such as lymph nodes). if there is any doubt of the presence or degree of fecal impaction, survey abdominal radiographs should be taken. a lateral view taken in a conscious cat should be adequate to confirm the diagnosis. the physical examination should also assess for contributing causes, including musculoskeletal conditions. any recent trauma should be taken into account. the hips and lumbosacral region should be assessed for pain. the degree of flexion and extension of the hips should be gently assessed. the lumbosacral spine can be assessed by running two fingers on either side of the spinous processes. the cat will flinch in painful areas. any arthritic change is magnified in an underweight cat, since there may be less muscle mass and the joints may bear a heavier load. any suspicions of underlying musculoskeletal abnormalities can be confirmed with radiographs. neurologic assessment should also be performed. subtle changes just affecting colonic innervation will not be apparent on physical examination alone. however, an assessment of proprioception, placing reflexes, and gait should at least be performed to assess for lumbosacral spinal cord disease. anorectal abnormalities or lesions should be evaluated. impacted or infected anal sacs can lead to reticence to defecate; and therefore anal sacs should be assessed and expressed. because this is painful for most cats, the cat should be held by an experienced assistant. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb positioned externally. a rectal exam can be performed with a well-lubricated (gloved!) middle finger, feeling over the pelvic rim for masses as well as assessing if the colon closes over (squeezes) the finger. if the colon feels open around the finger, this can be an indicator of impaired colonic innervation but does not imply that this is a permanent change. if there are impacted feces continuing to the anus, rectal examination is not possible until this has been cleared. if a cat finds anal gland expression or rectal examination too painful to tolerate (based on the clinician's judgment), these procedures should be done under sedation. hydration and electrolyte status are also important factors in the constipated cat. chronic renal disease is defined by azotemia (in conjunction with inadequately concentrated urine), which means the cat must be dehydrated to some degree. plasma or serum biochemistry and urinalysis can be used to diagnose renal disease, assess degree of renal disease, or recognize prerenal dehydration. electrolyte changes including hypokalemia and hypocalcemia may also contribute to reduced colonic smooth muscle function. in young to middleaged cats of apparent good health and hydration, blood difficulty defecating and pass hard, dry stools but do not have fecal impaction at the time of examination. after the obstructing feces have been removed, steps must be taken to ensure colonic motility and smooth passing of feces. medical management of constipation traditionally involves laxatives and prokinetic agents. these may not be required in straightforward cases. as long as there is no obstructive lesion, cisapride at . mg/ cat, every hours, po is very safe and can be instituted with a view to reducing the dose to once daily after to days and discontinuing if signs remain abated. doses of up to . mg/cat, every hours, po have been reported. cisapride is only available from compounding agencies in most countries. an osmotic or lubricant laxative (table - ) may be used concurrently at reduced doses as necessary. reducing the fecal bulk produced is an important part of long-term management. traditional dietary recommendations are to increase the amount of fiber. , , , increased dietary fiber results in production of shortchain fatty acids, which have been demonstrated to stimulate feline colonic smooth muscle contractions. however, dietary fiber is also classified as a bulk laxative and so, by definition, will increase fecal bulk. in humans dietary fiber has been considered a mainstay of therapy for constipation, but a recent review concluded that many patients with more severe constipation have worsening symptoms when increasing dietary fiber intake. because megacolon is believed to be the end result of chronic dilatation, , it is the author's firm belief that initial dietary efforts should be directed to reducing fecal bulk and thus introducing a low-residue diet. reduced dry matter intake reduces stool volume, and the author has found that recurrence rates of constipation reduce greatly when cats are transitioned to entirely wet food diets. wet food diets also help ensure adequate water and urine assessments are usually not required at an initial presentation for constipation. in all cases, the same principles of management apply: . ensure removal of obstructing feces . ensure colonic motility and smooth passage of feces . reduce fecal bulk . ensure adequate hydration . manage underlying problems the first step is to ensure obstructing feces are removed. in simple cases, the cat will evacuate feces after use of a glycerin or sorbitol pediatric rectal suppository. another option is administration of a microenema, such as microlax (mcneil consumer healthcare, fort washington, pa.), which contains ml of sodium lauryl sulfoacetate. these products act to lubricate the colon wall and therefore facilitate the passage of feces. the author prefers to use one or two of these within the consult room to observe the cat defecating (the cat must be provided with a litter tray!). the outside tube should be lubricated with the suppository contents before carefully inserting and then expressing the rest of the contents. there are also stimulant laxatives (containing bisacodyl) and emollient laxatives (containing sodium docusate) that have reportedly been used. if a rectal suppository vial cannot easily be inserted because hardened fecal content obstructs its entry, a more substantive enema will be required (sometimes requiring sedation or anesthesia), and this is covered in the next section on management. some cats present for the abdominal wall with the other hand, but great care must be taken with this maneuver, because the devitalized colon can be perforated more easily. , enemas as described are painful for the cat, and opioid analgesia is recommended at the time of anesthesia. opioids can reduce peristalsis in humans, but having evacuated the bowel, the pain relief is more important than this transient effect. an alternative to enemas is administration of an oral polyethylene glycol (peg ) solution (e.g., colyte, golytely). a nasoesophageal tube is placed and the solution is given as a slow trickle ( to ml/kg/hour) over to hours. defecation usually results in to hours. in a retrospective study of cats, median time to defecation was hours and the median total dose of peg was ml/kg. a no adverse effects were noted. a cat that has been obstipated needs supportive therapy when discharged. there are no controlled comparisons of the various therapies noted in table - ; the author prefers cisapride . mg, every hours to every hours, po (first thing in the morning, when the owner returns from work, when the owner goes to bed), and lactulose syrup ml/cat, every hours, po. a cat that has been so severely obstipated that an enema under anesthesia is required can be expected to continue these medications lifelong. to reduce fecal bulk and decrease the opportunity for recurrence, low-residue canned foods (or sachets) are preferred for cats that have become obstipated. some cats may benefit from high-fiber diets. as with simple initial episodes, canned food helps maintain adequate hydration, and at home subcutaneous fluids may be used additionally in cats with chronic kidney disease. with repeat episodes or severe obstipation, investigations for an underlying cause should be thorough and include evaluation for colonic mass obstructions. a review of published cases indicated that % of cases of megacolon are accounted for by idiopathic megacolon ( %), pelvic canal stenosis ( %), nerve injury ( %), or manx sacral spinal cord deformity ( %). although most cases are idiopathic, an attempt should be made to identify and treat any specific underlying causes. megacolon is not specifically defined in cats. it has been described as "generalized colonic dysfunction manifesting as severe colonic dilation and fecal impaction," or a "severely and irreversibly dilated and hypomotile" colon and "a subjective evaluation of the diameter of the colon, usually based on radiographic assessment." there are specific radiographic guidelines for humans with megacolon, in that a colonic diameter of more than . cm at the level of the pelvic brim is considered diagnostic. intake and therefore help maintain hydration. however, increased dietary fiber is beneficial for some cats, and trial and error may be required to determine whether a high-fiber or low-residue diet will be of benefit to each individual cat. in one report, cats with recurrent constipation refractory to traditional medical and dietary management were successfully treated with a psylliumenriched dry extruded diet. a after month on the diet, cats had no clinical signs of constipation. the remaining cat was clinically normal after months on the diet. improvement was noted in of cats after only days of dietary therapy. measures should be taken to ensure adequate hydration. maintaining adequate hydration is particularly relevant for cats with chronic kidney disease that have impaired ability to conserve water. changing to wet food diets helps increase water intake. some cats with chronic kidney disease may need additional fluid support, such as subcutaneous fluids administered by the owner at home on a regular basis. underlying problems may be minor and simple to manage, such as an anal gland abscess, or more involved, such as reduced pelvic outflow, as a result of prior trauma. arthritis is a common underlying factor in many older cats and may be managed with prudent use of nonsteroidal agents (see chapter ) . in cases of obstipation, the cat is more likely to be debilitated to some degree; so, laboratory investigations to assess plasma or serum biochemistry parameters as well as hematology and urinalysis are ideal. any hydration deficit or electrolyte abnormalities should be corrected before the anesthesia that is often required to remove the obstructing feces. rectal suppositories and microenemas are usually ineffective in obstipated cats. enemas are often required to remove impacted feces in such circumstances. the enema solution must be warmed and introduced slowly to avoid vomiting. the typical volume required is to ml/kg (so, up to approximately ml/cat). the enema solution can be an isotonic electrolyte solution or tap water, and mild soap can be added (but any soap used must not contain hexachlorophene, which is neurotoxic if absorbed); mineral oil can be used ( to ml/cat) as a lubricant or docusate as an emollient ( to ml/cat), but the two agents must not be used together since docusate promotes mucosal absorption. sodium phosphate-containing enemas must not be used, because they can induce severe hypernatremia, hyperphosphatemia, and hypocalcemia in cats. often, the enema solution alone is insufficient to reduce the fecal mass, and manual manipulation of the feces by abdominal palpation is required. sometimes the feces must be broken down by a gloved finger perrectum while the colon is massaged manually through radiographically, in the lateral view, the normal colon should be approximately the same diameter as the length of the body of the second lumbar vertebra. in cats, however, "there are no published guidelines for determining megacolon, so, diagnosis of abnormal colonic dilatation is subjective." however, one author has suggested that "as a rule of thumb, the diameter of the colon should be less than the length of the body of the seventh lumbar vertebra (l )." this author continues, "enlargement of the diameter of the colon beyond times the length of the body of l is indicative of chronic large bowel dysfunction and an explanation must be sought." a recent paper found that of cats with no gastrointestinal disease had a colon diameter greater than the length of l ; however, no assessment of constipated cats was made. in practice, many cats with megacolon have a colonic diameter far exceeding this guideline ( figure - ). one study of cats with megacolon found the mean diameter of the colon was . times greater than the length of the seventh lumbar vertebra (median, . ; range, . to . ), but in general, objective descriptions of this condition are lacking in the veterinary literature. the definition of megacolon in cats should include functional as well as radiographic guidelines. in the absence of broadly recognized radiographic recommendations, the author proposes that the o'brien rule-ofthumb guidelines (as noted above) be introduced until a more comprehensive study can establish other radiographic diagnostic criteria (or confirm these). the author therefore proposes to define megacolon as dilatation of the colon, to more than . times the length of the seventh lumbar vertebra, which is refractory to medical and dietary management. practitioners can expect the radiographic assessment of colonic dilatation to exceed this guideline in cats with megacolon and, conversely, there are likely to be cats having colonic distention greater than this amount that will respond to medical and dietary management and can therefore not be defined as having megacolon. by the definition used above, megacolon is refractory to medical and dietary therapy; so, to be defined as having megacolon, a cat may have had several episodes of obstipation managed by enema as well as dietary trials (with both low-residue and high-fiber diets) and medical therapy with cisapride and an osmotic or emollient laxative; yet the cat will still obstruct with feces. in these circumstances, the only possible therapy is subtotal colectomy. subtotal colectomy refers to surgical excision of % to % of the colon, whether it is grossly diseased or not with preservation of the ileocolic junction (icj). this approach has resulted in a more favorable clinical response than when the icj is also excised. , when preserving the icj, it has been noted that, in some rare cases, it can be difficult to join the proximal segment of colon to the distal piece of descending colon because of the tethering effect of the ileocecocolic blood vessels. in these cases, sacrificing these vessels and removing the icj (i.e., total colectomy) is recommended to facilitate approximation of the ileum to the distal colonic segment. a recently described technique using a biofragmentable anastomosis ring, compared with sutured anastomoses, showed no discernible effect on prognosis. prognosis following subtotal colectomy is generally good. a review of multiple papers, totaling over cats that had undergone subtotal colectomy, found the most commonly reported perioperative complication was diarrhea or loose stools immediately after surgery. in the majority of individuals, stool consistency improves without further treatment so that within to weeks of the surgery soft, formed stools are developed. diarrhea can persist in a small number of cases. in the longer term, in some cats, constipation can eventually return, but this can usually be managed by dietary and medical therapies. pathology of the rectum or anus is relatively rare in cats and therefore poorly described in the veterinary literature. consequently, published information is often not referenced, suggesting it expresses the authors' opinions. readers are directed to surgical texts for details and approaches about surgical corrections. the anal sacs are paired cutaneous evaginations situated between the internal and external sphincter muscles. these sacs store secretions from alveolar and sebaceous glands that reside within the sacs. each anal gland has an associated duct that opens to the skin surface just lateral to the anus. , normal anal gland secretions have only very recently been described and vary markedly; the color can be white, brown, orange, yellow, tan or gray, and consistency can range from watery to thick and creamy, with two thirds of cats having solid portions within the secretion. on microscopic examination, epithelial cells are commonly seen, with most cats having some neutrophils present. bacteria are commonly recognized as are, on some occasions, yeasts. bacteria seen in this study were mainly gram-positive cocci ( %) or gram-negative cocci ( %). gram-negative or grampositive rods were also seen but were rarely the dominant bacterial population. with such a wide range of normal secretions, it is difficult to diagnose any pathology from the nature of the secretion alone. however, blood is infrequently recognized, and neutrophils are typically present in only small numbers in normal secretions. anal sac diseases described in cats include impaction, inflammation (sacculitis), infection, abscessation, and neoplasia (essentially the same as in dogs). , it has been contended in dogs that sacculitis and abscessation are an extension of impaction. it is not known in dogs or cats what the predisposing causes are, but suggested underlying reasons are loose stools (that are less effective at expressing the sac during defecation), local swelling or edema occluding the duct, and obesity. the author's observations have also indicated that constipation can result in anal sac impaction because of less frequent expulsion of the sac contents; the resultant pain of the anal sac impaction can lead to further constipation, thus establishing a cycle. the retention of secretions may predispose to sacculitis, but impacted anal sacs do not always result in inflammation. abscessation is a likely sequel to sacculitis. cats usually present for licking, scratching, or biting at the perineal area and can present for scooting (or dragging their anus) as dogs do. other presenting signs can be inability to sit or settle, a lump seen by the owner, or a generally unwell state. expression is the only management required for impacted (and not infected) anal sacs. the author prefers expressing one anal sac at a time with well-lubricated gloves and the index finger within the rectum and the thumb externally. this is painful for most cats; so, the cat should be held by an experienced assistant, and it is sometimes not possible without some degree of sedation. with frequent episodes, underlying causes should be investigated. sometimes, trial-and-error diet change to manipulate the nature of the feces to either more (highfiber diets) or less (low-residue diets) bulk help reduce the frequency of episodes. obesity should be managed by reduced caloric intake, but dietary management for this should also take into account the nature of the feces. overt infection may be recognized by pus secretion from the anal sacs, which will have a high numbers of neutrophils. this can be managed by broad-spectrum antibiotics, such as amoxicillin/clavulanate or cephalosporins. a single treatment with a nonsteroidal antiinflammatory drug, such as meloxicam, can be given in animals with appropriate hydration and without other illness. anal sac abscesses often present already open and draining. many heal well by secondary intention with antibiotic treatment until they are closed over; so rechecks are required before the completion of an antibiotic course. large abscesses may require surgical drainage with the insertion of a penrose drain and management as for a cat fight abscess. it must be remembered that wounds in this area are easily re-infected by fecal contamination. recurrent impaction, sacculitis, or infection may require anal sacculectomy (as in dogs). this procedure should be delayed until infection is cleared. the procedure is similar to that performed in dogs. reports of anal sac/gland neoplasia were confined to sporadic case reports, , until a large case series was recently published. in this study, cases of anal gland carcinoma were recognized at a private diagnostic laboratory during a -year period, with submissions from practices. this indicates that, for most practices, this condition will be seen, at most, once every years. affected cats ranged in age from to years (median and mean, years); female (mostly spayed) cats were overrepresented ( % of cases), and siamese cats may have been over-represented ( . % of cases). the number of siamese cats with anal sac neoplasia was times greater than the number of siamese cats in the laboratory reference population. affected cats presented for dyschezia, recurrent constipation, change in the nature or volume of feces, b). a low-residue wet diet is recommended to reduce fecal bulk during the healing period. rectal prolapse occurs as a result of a disease process that causes chronic straining, such as intestinal conditions that result in diarrhea and tenesmus . conditions that result in constipation or other intestinal obstruction . lower urinary tract diseases . dystocia and/or perineal swelling or ulceration, sometimes with purulent or hemorrhagic discharge. most tumors were originally interpreted as and initially managed as anal sac abscess. presumptive metastasis in liver, lung, or abdominal lymph nodes was recognized by physical examination or radiography in six cats; one cat was hypercalcemic. excision appeared to be curative (with a -to -year follow-up period) in of cats undergoing surgery for resection or debulking (others had only incisional biopsy performed). for the remaining cats with known postsurgical outcome, median survival was only months, with a % -year survival rate (with none of these cats surviving to years). atresia ani is a developmental defect of the anal opening or terminal rectum (see figure - ). kittens usually present within days or weeks of birth with abdominal distention, discomfort, tenesmus, restlessness, vomiting, and/or loss of appetite. there are several anatomic variations : • type i: a membrane over the anal opening remains, with the rectum ending as a blind pouch just cranial to the closed anus. • type ii: the anus is closed as in type i, but the rectal pouch is located somewhat cranial to the membrane overlying the anus. • type iii: the rectum ends as a blind pouch cranially within the pelvic canal (rectal atresia), whereas the terminal rectum and anus are normal. • type iv: occurs in females and atresia ani exists with a persistent communication between the rectum and the vagina (rectovaginal fistula). this fistula can occur with a normal anal opening as well. most reported cases have been type iv, , , , , and this has also been recognized with concurrent sacrococcygeal agenesis. surgical correction has been described for type ii and type iv , atresia ani in cats. the reader should consult these references for surgical advice; possible complications include megacolon after prolonged obstruction, postsurgical anal stricture, and fecal incontinence because of sphincter dysfunction. foreign bodies in cats rarely obstruct the gastrointestinal tract distal to the jejunum ; however, large fecal balls resulting from constipation can, additional to constipation or obstipation, cause distention of the anus. this distention can result in inflammation of the anal sphincter with loss of tone ( figure - , a) , which, in the author's experience, is temporary with correction of the underlying cause of constipation. it can take some weeks for the dilated anus to return to normal (figure - , with antibiotics, such as cephalosporins, and regular cleansing. prolapses are usually classified in three ways. first degree: prolapse of only mucous membrane . second degree: prolapse of full rectal wall thickness . third degree: prolapse is sufficient to bring mesorectum outside the anus the prolapsed rectum is obvious but must be differentiated from ileocolic intussusceptions, which have been described with neoplasia. this distinction can be made by inserting a thermometer through the anus alongside the prolapsed mass. insertion will not be possible for an intussusception but will be for an anorectal prolapse. the prolapsed tissue must be assessed for viability, and management must include determining and managing the underlying cause as well as management of the prolapse. in simple cases where the mucosa is viable, the prolapse can be reduced with lubrication and gentle pressure. a temporary purse-string suture may be required to prevent recurrence. perineal dermatitis is often confused with gastrointestinal or urogenital disease, because there are often copious sebaceous secretions that can mimic fecal or urinary secretions. perineal dermatitis can result from flea or other allergies but also fecal or urine scalding associated with diarrhea or urinary incontinence, respectively. skin fold dermatitis can also occur in obese cats ( figure - ). episioplasty has been described to correct this, but the author has found that stringent dieting can result in improvement while managing the skin fold dermatitis these populations. the reported prevalence for each parasite varies greatly with the population studied, the geographic location of the population, and the sensitivity of the diagnostic test used to study that population. the presence or absence of diarrhea is not a reliable predictor of whether a particular cat is infected with or shedding a parasite. in fact, most cats with diarrhea do not harbor enteric protozoa. on the other hand, most cats with diarrhea because of enteric pathogens will shed those organisms, often intermittently. it is important to remember that infection with most gastrointestinal parasites may not cause clinical signs. therefore detection of a pathogenic parasite in a cat with diarrhea does not necessarily prove causation. a search should always be undertaken to identify other causes of diarrhea prior to convicting a cat of having diarrhea because of a particular parasite. in addition, co-infections or the presence of other noninfectious causes of diarrhea can result in more severe diarrhea that is often refractory to treatment for the parasite. treatment will be more rewarding if all potential causes of diarrhea are identified in the patient. enteric parasites with zoonotic potential occur commonly enough that cats, particularly those with diarrhea and who are owned by immunocompromised persons, should be evaluated for those pathogens. , the following is a discussion of the most common enteric parasites found in cats. for more on parasite prevention and control, see chapter , and for more on zoonotic enteric parasites, see chapter . ollulanus tricuspis is an almost microscopic nematode worm infecting the stomach of domestic and wild cats. the worm measures less than mm long. the larvae of o. tricuspis develop and hatch within the uterus of the female worm. they develop to maturity in the stomach of the cat where it is capable of re-infecting the host. the worm is transmitted to other cats that ingest the vomitus of an infected cat. clinical signs shown by infected cats include vomiting, anorexia, and weight loss. , histologic findings in infected cats include lymphocytic-plasmacytic gastritis, lymphoid hyperplasia, and mucosal fibrosis. gross lesions may be absent, or the cat may develop nodular gastritis. one report suggested the parasite may have been a contributing factor in the carcinogenesis of a gastric adenocarcinoma in an infected cat. . a common theme when discussing the prevalence of most gastrointestinal parasites in cats is that they occur more commonly in younger cats and in cats housed in crowded conditions, such as catteries and shelters. it is likely an increased chance for transmission exists in lungs. after further development in the lungs, the parasite migrates up the trachea and is swallowed. adult s. felis and s. planiceps burrow into the wall of the small intestine, while adult s. tumefaciens lives in the colonic mucosa. ova may be shed in the feces or hatch in the intestinal tract. autoinfection occurs if larvae become infective and penetrate the intestinal wall before being shed. ova and larvae that are shed develop into freeliving adult worms. the prepatent period is between and days. , clinical signs and diagnosis signs of a strongyloides spp. infection are usually absent. , lung migration may cause cough or respiratory distress. the presence of the parasite in the intestinal tract may result in diarrhea and weight loss. strongyloides tumefaciens is associated with the formation of small, worm-filled nodules in the colon. identification of strongyloides spp. larvae using the baermann fecal concentration technique is required to diagnose most infections. unless the infection is heavy, examination of a fresh fecal smear is insensitive for identification of these larvae. the nodules formed by s. tumefaciens infection can be visualized during colonoscopy. histopathology of the biopsied nodules should reveal many adult worms. infection with strongyloides spp. can be treated with fenbendazole, pyrantel pamoate, thiabendazole, , or ivermectin. to evaluate efficacy, repeat a fecal examination to days after the treatment ends. because of the presence of free-living adult worms in the environment and the ability of larvae to cause infection by penetrating intact skin, prevention is difficult. keeping cats indoors in warm, humid climates may be an owner's only means of preventing infection with strongyloides spp. parasites. infections with trichuris vulpis rarely occur in cats and are considered to be clinically unimportant. , the two species of roundworms commonly infecting cats are toxocara cati (figure - ) and toxascaris leonina (figure - ) . the latter also has the ability to infect dogs. cats are infected with t. cati in several ways. most commonly, infection is by ingestion of contaminated food, water, or infected paratenic hosts such as rodents. transuterine transmission has not been reported. the diagnosis of infection with o. tricuspis is difficult, because ova are not shed in the feces; rather, the vomitus must be examined for worms or larvae. the worms may also appear in gastric mucosal biopsy samples. a report of cats undergoing endoscopic examinations found the parasite in gastric biopsy samples from cats. fenbendazole may be effective in treating infections with o. tricuspis. preparations with febantel may also be expected to successfully treat these infections. transmission can be prevented by appropriately treating infected cats. other cats should not be allowed to ingest infected vomit. this parasite is of no zoonotic concern. physaloptera another parasite rarely inhabiting the stomach in cats is in the genus physaloptera. larger than ollulanus tricuspis, this blood-sucking worm infects cats that have ingested intermediate hosts, such as cockroaches, crickets, or flour beetles. preying on transport hosts, such as mice that have eaten an intermediate host, is another way cats become infected with this parasite. clinical signs of infection with physaloptera spp. include vomiting, anorexia, and melena. a diagnosis of physaloptera infection can be made after identifying the ova in the patient's feces or adult worms in the vomitus. occasionally, the worms may be seen during gastroscopy. the adult worms must be differentiated from ascarids. infection can be treated with ivermectin, pyrantel pamoate, or fenbendazole. because there is no migratory phase of the life cycle, the treatment does not need to be repeated. three species of strongyloides infect cats. strongyloides felis infects cats in india and tropical australia, , s. tumefaciens is a rare parasite of cats in the southeastern united states, and s. planiceps is found in cats in malaya and japan. strongyloides stercoralis, found in dogs and humans, produces experimental infections in cats, but natural infection with this species has not been observed. feline infection with strongyloides spp. is considered by most to be rare. however, one report from australia identified s. felis in of necropsied cats. infection with strongyloides spp. occurs after ingestion of infective larvae. infection can also take place after the larvae penetrate the skin of the cat. ingested larvae penetrate the intestinal wall and migrate through the diaphragm into the lungs. after cutaneous penetration, the larvae enter the venous circulation and enter the clinical illness because of roundworm infection is uncommon. illness, when it does happen, most often occurs in kittens signs may be mild and can include vomiting, diarrhea, weight loss, poor growth, and a "pot belly." a heavy infection with t. cati can result in catarrhal enteritis. severe infections can lead to intestinal obstruction and, possibly, perforation. much less dramatic changes arise after infection with t. leonina, although enteritis may occur. roundworms are frequently diagnosed with a fecal floatation. the centrifugal floatation technique is more sensitive than the simple fecal floatation technique many hospitals use. occasionally, adult worms will be passed with the feces. the goals of treating roundworms include disease prevention in an individual cat or kitten, prevention of environmental contamination by cats defecating outside, and the prevention of zoonotic infections. many effective and safe anthelmintics are available (table - ) . benzimidazoles, such as fenbendazole, act on the parasite's microtubular structure, leading to disintegration of the worm's intestines, muscular layer, and hypodermis. pyrantel in the pamoate formulation is poorly absorbed and causes paralytic parasite death. macrocyclic lactones, such as milbemycin, also lead to paralytic parasite death. these compounds act on the parasite's gamma-aminobutyric acid (gaba)-and glutamate-controlled ion channels. these channels are lacking in tapeworms, accounting for the lack of efficacy against these parasites. lastly, emodepside (a cyclic octadepsipeptide) has been combined with praziquantel in the product profender (bayer animal health). this topical parasiticide has been shown to be both safe and effective. these drugs appear to be so safe that overdosing is almost impossible. kittens can be dewormed starting at two weeks of age and again at , , , , and weeks. older kittens and adults can be dewormed every month to months. because of the safety of these drugs, the possibility of false-negative tests and, more importantly, the zoonotic potential of these infections, perhaps all kittens should be dewormed, not just those testing positive. roundworm ova are very hardy and can remain infective for years. they survive sewage treatment and composting, and there is no practical means of decreasing the ova population once the environment is contaminated. thus it is best to attempt to prevent contamination in the first place. when practical, keeping cats indoors allows appropriate control of potentially transmammary infection occurs, but only if the queen is acutely infected late in pregnancy. chronically infected queens do not pass t. cati ova in their milk. after ingestion, t. cati larvae migrate through the small intestinal wall, into the liver, and then to the lungs where they are coughed up and swallowed. these larvae then infect the small intestine. some of the migrating larvae become encysted in the cat's muscle tissue. larvae from ova ingested through the milk tend not to undergo migration and mature directly in the small intestine. the prepatent period is approximately weeks. infection with t. leonina occurs after ingestion of infective ova or an infected paratenic host. unlike t. cati, very few t. leonina larvae migrate through the cat's tissues. most develop in the wall of the small intestine. the prepatent period is to weeks. toxascaris leonina ova can become infective within days of being passed in the feces when the ambient temperature is ° c but normally require to weeks. lungs, and kidneys. ocular larval migrans results in granulomatous retinitis that is often misdiagnosed as retinoblastoma in older children. this can lead to unnecessary enucleation. toxocara cati appears, however, to be less important than t. canis as an infection in humans. the species of hookworms that infect cats are ancylostoma tubaeforme and ancylostoma braziliense (see figure - ). they are reported to be an uncommon infection in cats. , ancylostoma braziliense can also infect dogs. hookworm infections occur after ingesting food or water contaminated with hookworm larvae or eating contaminated fecal material. if the pet cat is allowed outdoors, attempts at preventing hunting may reduce the possibility of infection. keep children's play areas, such as sand boxes, inaccessible to cats when children are not at play. feeding only well-cooked food can prevent infection by contaminated food. finally, empirical, preventative deworming for cats that go outdoors should be performed to times yearly. any less frequently does not lead to an appreciable decrease in the prevalence of the parasite. roundworms easily infect humans who ingest the ova, particularly children. visceral larval migrans occurs after infection with toxocara canis in humans. infection can lead to the formation of nodules in the brain, liver, tapeworm infections are well tolerated by the cat. usually there are no signs of infection other than finding segments on the feces or attached to perianal hair. because both d. latum and spirometra tapeworms absorb vitamin b across the cuticle, megaloblastic anemia is possible, but unlikely. tapeworm infections are diagnosed by identifying the typical appearance of the segments or the egg packets within the segments. the segments of t. taeniaeformis are flat, while those of d. caninum have been described as appearing like a grain of rice. the segments should be handled carefully, because they are friable and rupture may result in exposure of the handler. the operculated ova of d. latum and spirometra spp. must be differentiated from trematode ova. even though tapeworm infections are well tolerated, cats should be treated for reasons of owner discomfort and public health concerns (see table - ) . these infections are easily treated, because drug treatment is highly effective. re-infection must be controlled using preventative measures, especially flea control to prevent re-infection with d. caninum. praziquantel and infected paratenic hosts. the larvae can survive for months in the tissues of paratenic hosts. infection also occurs after larval migration through the skin. in either case, the worm matures in the small intestine. unlike dogs, transmammary infection has not been reported in cats. , the prepatent period is between and days, depending on the route of infection. the time to patency after transcutaneous infection is longer than for direct colonization. infective l larva develop to days after the ova are passed. developing larvae attach to the mucosa of the small intestine where they ingest copious amounts of blood. because the worms can remove a significant volume of blood from kittens, weakness from iron-deficiency anemia or blood-loss anemia may be noted. melena and diarrhea may also be recognized. signs are uncommon in adult cats. identification and treatment of hookworm infections are similar to that for roundworm infections (see table - ) . hookworm larvae are not as hardy as roundworm eggs. soil contamination may be a temporary problem in areas that experience a hard frost. hookworm larvae will not develop in temperatures less then ° c or greater than ° c. frequent, appropriate disposal of feces, cleaning surfaces with a % bleach solution, and deterring hunting may prevent infections. migration through the skin of persons coming into contact with the larvae of a. braziliense is the most common cause of cutaneous larval migrans, particularly in the southeastern united states. this is an erythematous, pruritic skin eruption often found on the soles of the feet of infected children. the tapeworms most commonly found in cats are dipylidium caninum and taenia taeniaeformis. diphyllobothrium latum, spirometra spp., and echinococcus multilocularis occasionally infect cats. the latter is important, because it can lead to alveolar echinococcosis in humans. spirometra tapeworms are found in north america (s. mansonoides) and far-east asia (s. mansoni and erinacei), while d. latum prefers temperate climates. unnoticed, but the cat may cough or experience hemoptysis. diagnosis involves demonstration of fluke ova in the feces. although therapy may be unnecessary, praziquantel or epsiprantel are effective in eliminating the intestinal population of the fluke. platynosomum spp. are flukes living in the gall bladder, bile ducts, and pancreatic ducts. these flukes are most prevalent in the southeast united states and caribbean islands and require two intermediate hosts. the first host is a snail, while the second intermediate host is a lizard, toad, gecko, or skink. cats become infected with this fluke after ingesting an infected second intermediate host. the prepatent period for the fluke is weeks. most infections are subclinical. if clinical signs do occur, they may include weight loss, vomiting, diarrhea, icterus, hepatomegaly, or abdominal distention. diagnosis involves identification of ova shed in the feces using a fecal sedimentation method or by finding adult flukes in the gall bladder or bile ducts during abdominal surgery. treatment involves administering praziquantel ( mg/kg, q h, po for to days) and/or surgical removal of the flukes. two species of coccidians are the most common to infect cats, isospora felis and isospora rivolta (figure - ) . the genus isospora may be renamed cystoisospora. these are epsiprantel are safe and effective. fenbendazole is effective against t. taeniaeformis, but not d. caninum. without controlling exposure to intermediate hosts, tapeworm infections are difficult to eliminate. flea control is imperative in eradicating infections with d. caninum. controlling predation helps prevent ingestion of t. taeniaeformis-infected rodents. infection with d. caninum occurs in young children who are most likely to eat fleas. infection results in only minimal signs of illness. the larval stage of t. taeniaeformis is of little zoonotic importance. although cats are uncommonly infected with echinococcus multilocularis, potentially life-threatening alveolar damage occurs in north american humans infected with this tapeworm. plerocercoids of spirometra spp. can penetrate the mucous membranes or open skin wounds of humans and migrate around the subcutaneous connective tissue, forming nodules, a condition called sparganosis. megaloblastic anemia, as a result of vitamin b deficiency, may occur in humans infected with d. latum or spirometra spp. tapeworms. alaria marcianae flukes reside in the intestinal tract of cats and the mammary glands of lactating queens. miracidia hatch underwater from ova shed in the feces and penetrate the skin of a snail. after further development, cercariae penetrate the skin of leopard frog tadpoles and are able to survive the metamorphosis to the adult frog. if the tadpole is eaten by a snake, bird, or mammal, the parasite enters the host's tissues but does not undergo further development. after a male or nonlactating female cat ingests the infected intermediate host, the parasite penetrates the wall of the small intestine, passes through the diaphragm, and enters the lungs for further development. finally, the parasite is coughed up and swallowed to complete maturation and reproduce in the small intestine. if, however, an infected host is ingested by a lactating queen, the parasite migrates through the tissues to the mammary glands, rather than the lungs. once shed in the milk, the parasites develop into mature adults in the kittens. some of the mesocercariae remain in the mammary glands to infect future litters. clinical signs associated with worms in the small intestine are uncommon. migration through the lungs often goes species-specific obligate intracellular parasites. , they are able to survive in the environment for months. a detailed description of the coccidial life cycle can be found elsewhere. , simply put, direct transmission is by ingesting oocyst-contaminated food or water or by grooming contaminated body parts. indirect transmission occurs after ingesting a mechanical vector or the infected tissues of paratenic hosts. after ingestion by a cat, the oocyst excysts in the small intestine and enters the enterocyte where further development occurs. the parasite may also migrate through the intestinal wall to form cysts in mesenteric lymph nodes. these cysts may serve as a source for reinfection. , the prepatent period is to days and the shed oocyst becomes infective after several days of exposure to warmth and moisture. infection with isospora spp. is usually subclinical. signs, if they occur, range from mild, transient watery diarrhea to severe mucohemorrhagic diarrhea with vomiting and resultant dehydration and weight loss. , signs are most commonly recognized in severely infected neonatal kittens, particularly those with concurrent illness, and arise because of small intestinal congestion, mucosal erosion, or villus atrophy. signs may also be noted in immunosuppressed adult cats. isospora species are readily found in fecal floatation or wet-mount examinations. shedding can be intermittent, but most cats with diarrhea caused by coccidial infection shed large numbers of oocsyts. fortunately, in most cats, the diarrhea from isospora spp. infection is self-limiting. in fact, if a kitten is persistently shedding oocysts despite appropriate treatment or the parasite is identified in an adult cat with chronic diarrhea, attempts should be made to identify co-infections or other diseases that may cause diarrhea. anticoccidial drugs are either coccidiostatic or coccidiocidal (table - ) . coccidiostatic drugs are the most commonly used drugs for individual pet cats. trimethoprim-augmented sulfadiazine (tribrissen; intervet/schering-plough animal health, summit, nj) or another sulfa-containing antibiotic, sulfadimethoxine (albon; pfizer animal health, madison, nj), can be used. supportive care for severely affected kittens, such as parenteral rehydration, should be used as needed. coccidiocidal drugs are often reserved for use in densely populated situations such as catteries or shelters. however, many veterinarians are now using them as a first-line defense against isospora spp. infection. ponazuril (marquis oral paste; bayer animal health, shawnee mission, kan.), formulated for horses, is effective and can be safely administered to cats. for more on the use of ponazuril in cats, see chapter . a related drug, diclazuril, is also available and may be administered once at mg/kg po. while not available in north america, toltrazuril (baycox, bayer animal health) may be administered once at mg/kg po or mg/kg po once daily for days. a a second course of therapy days later may be required to completely eliminate the oocysts. sanitation is very important, because the oocyst requires several days to become infective. frequent removal of feces, preferably daily, is recommended to prevent re-infection and transmission to other cats. controlling a cat's ability to hunt reduces the chance of ingesting an isospora-infected rodent. control of mechanical vectors, such as cockroaches and flies, is also useful. since a cat can become infected after grooming an infected cat's perineum, consideration should be given to treating all cats in contact with the patient. in addition, catteries and shelters should ensure all food is well cooked, litter boxes are cleaned daily, and surfaces are well cleaned with steam or % ammonia. where recurrent isospora spp. infections are a problem, prophylactic treatment of all -to -week-old kittens with ponazuril should be considered. despite all wellintentioned efforts at hygiene and treatment, isospora spp. infection can still be transmitted to other cats. because these are species-specific parasites, transmission of i. felis and i. rivolta from cats to humans does not occur. the flagellated protozoal parasite, giardia duodenalis, has seven microscopically indistinguishable genotypes or assemblages. assemblages a and b infect humans, while assemblage f is harbored by cats. cats will occasionally harbor assemblages a and b. infection with g. duodenalis occurs after ingesting cystcontaminated feces, by grooming an infected cat or from contaminated fomites. re-infection may occur by selfgrooming. only a small number of cysts need be ingested to establish an infection. in humans as few as cysts are required to cause infection. after ingestion of infective cysts, trophozoites begin to excyst in the stomach. this process is completed in the proximal duodenum. the trophozoites adhere to enterocytes along the length of the small intestine using the ventral suction disk. intermittent shedding of immediately infective cysts begins to days after infection. proteins released during encystment of the trophozoites are detected by the fecal antigen tests. cysts may adhere to the perianal region, facilitating re-infection by self-grooming. occasionally, trophozoites are found in examinations of fresh, watery feces. these do not survive for long and are not infective. the mechanisms of disease induced by g. duodenalis are still unclear. after the trophozoite attaches to the brush border of the enterocyte, the tight junction between cells is disrupted, increasing intestinal permeability. the brush border becomes attenuated, further exacerbating malabsorption of water, electrolytes, and other nutrients. the alteration in intercellular adhesion results in t-lymphocyte activation and mucosal cell injury. infection also promotes mucosal cell apoptosis (preprogrammed cell death). in addition, small intestinal bacterial overgrowth may accompany g. duodenalis infections, resulting in more severe clinical signs. fortunately, most cats infected with g. duodenalis show no clinical signs. , the most common sign is acute, transient, small bowel diarrhea without systemic illness, such as fever or vomiting. less commonly, a cat might have profuse, watery malodorous diarrhea with mucus. also possible, but uncommon, is weight loss , or abdominal pain. the severity of clinical signs exhibited in an individual cat depends on the age and general health of the cat. cats co-infected with cryptosporidium felis or tritrichomonas foetus may have more severe diarrhea that is more difficult to control, as will the presence of bacterial overgrowth. the diagnosis of g. duodenalis requires demonstration of trophozoites or cysts in a fecal examination, or detection of encystment proteins or giardial dna in a fecal sample. a reliable diagnosis may be difficult to obtain for several reasons. cysts are small, easily missed, and must be differentiated from plant debris or yeast. trophozoites are short lived outside the body and can only be found in very fresh, watery feces or, better yet, in diarrheic feces collected directly from the cat's rectum. shedding of cysts is usually intermittent, and the intensity of shedding varies greatly. , because of these difficulties, the absence of the organism in a fecal sample does not eliminate it as the cause of diarrhea. it is often necessary to test multiple fecal samples, using at least two different techniques in order to find the organism. , the easiest test to perform is a fecal smear or wet mount examination to identify trophozoites or cysts (figures - and - ). the sample examined should be very fresh, warm, diarrheic feces. one drop of feces is placed on a slide along with a drop of . % saline or lugol iodine. trophozoites are identified by their characteristic structure (table - ) . the motile trophozoites have a motion described as appearing like the back and forth rolling motion of a falling leaf. since lugol iodine stain kills the trophozoite, there will be no motion to detect. this test is not very sensitive; however, with trained examiners, the test has a high specificity. increased sensitivity can be gained by performing a centrifugal flotation using zinc sulfate. the sample should be warm, fresh feces or feces refrigerated for no more than days. the processed sample is examined for the same structures as the wet mount. the sensitivity of examining one sample is % and increases as more samples are examined. the sensitivity of looking at three samples is % , ; therefore the test is not considered negative until three specimens have been found free of the organism. a fecal antigen test that identifies the encystment protein is available. the snap giardia antigen test (idexx laboratories) uses fresh or frozen feces, or feces refrigerated for less than days. since the antigen is continuously shed, this test avoids the problem of intermittent shedding of the whole organism. the sensitivity of the test is %, with a specificity of %. by combining the antigen test with a zinc sulfate fecal centrifugal flotation, the sensitivity improves to . %. it is unknown how long the antigen remains in the feces after treatment. thus a zinc sulfate centrifugal flotation examination should be used to evaluate therapeutic efficacy. , the use of this test in cats without diarrhea is controversial, because these cats are unlikely to shed cysts. the zoonotic significance of a positive antigen test in a cat not shedding cysts is unknown and may cause confusion. polymerase chain reaction detection of giardia dna is available, but the test has not been standardized across all diagnostic laboratories. one needs to ensure the laboratory performing the test has validated it for assemblage f. the test may also be used to identify cats harboring the zoonotic assemblages a and b. the sensitivity of this test is unknown. two commonly available drugs are used most frequently to treat infections with g. duodenalis (see table - ) . fenbendazole may be effective and can be used in pregnant queens and in cats co-infected with roundworms, hookworms, and taenia spp. tapeworms. however, in one small study, only four of eight cats infected with both g. duodenalis and cryptosporidium felis stopped shedding giardia permanently after receiving fenbendazole. febantel, in the combination product drontal plus (bayer animal health), is converted to fenbendazole. when six experimentally infected cats received . mg/ kg of febantel q h po for days, four of them stopped shedding g. duodenalis cysts. metronidazole has been the traditional drug used to treat g. duodenalis in pets. the drug is also useful for treating concurrent small intestinal bacterial overgrowth and clostridial infections. the administration of metronidazole may eliminate shedding in % of cats. neurologic side effects may occur at the dose recommended for treatment of giardia (see above, therapeutics for vomiting and diarrhea). the use of a giardia vaccine was ineffective in clearing infection by itself. the combination of fenbendazole and metronidazole has been suggested as the initial treatment of choice for g. duodenalis infections. although controlled studies are lacking, they may work synergistically by acting on two different targets within the parasite. febantel would be expected to have the same synergism with metronidazole. drug therapy may not be necessary in cats without diarrhea that are infected with g. duodenalis, because it is uncommon for a cat to carry the assemblages required to infect humans. the veterinarian may be obligated to treat a healthy cat if the owner wants to treat, the owner is immunocompromised, or the goal is eradication of an infection from a multicat home or prevention of parasite transmission to giardia-naïve cats is attempted. what may appear to be treatment failure is more likely to be re-infection. in addition to drug therapy, steps should be taken to prevent re-infection. all cats with diarrhea positive for g. duodenalis should be treated along with their housemates. sanitation is imperative in the fight against re-infection and transmission of g. duodenalis. dispose of old litter pans and scoops and use disposable litter boxes during treatment. when the infection is eliminated, not just controlled, new litter boxes and scoops may be purchased. bathe all cats during treatment to remove cysts from the hair coat. since giardia spp. cysts are susceptible to desiccation, blowdry all cats using a warm air blower, paying particular attention to the perineal area. disinfect bowls, housing, and other utensils with bleach. in addition to antiprotozoal drugs and sanitation, supportive care may become necessary. probiotics and a highly digestible, bland diet may be offered to cats with small bowel diarrhea, while a high-fiber diet may be useful for those few cats with large bowel diarrhea. where required, hydration and electrolyte imbalances must be corrected and antiemetics used to control vomiting. therapy can be evaluated by retesting feces with a zinc sulfate centrifugal flotation examination to days after the end of treatment and again weeks later. a positive test immediately posttreatment is most likely because of therapeutic failure. if the cat is negative immediately after treatment ends, but is positive weeks later, re-infection is likely. since the fecal antigen test may remain positive long after the infection is eradicated, this test is inappropriate for evaluating therapy. , re-treatment of fecal flotation-positive, recovered cats may be handled in a manner similar to the positive healthy cat mentioned above. cats with diarrhea that continue to shed cysts may be re-treated for g. duodenalis infection along with dietary modification and empirical treatment for other common intestinal parasites. however, serious consideration should be given to investigation into other potential causes of diarrhea. the giardia vaccine has been found to be ineffective in preventing infection and production has been discontinued. this means prevention of giardia infection involves avoiding exposure, stress and re-infections. providing a clean environment, feeding only processed foods, and controlling potential transport hosts will help reduce the chances of exposure. isolation of cats with diarrhea may be important, too. municipal sanitation control is difficult as the cyst survives for weeks in cool, moist environments. cysts are also able to survive water treatment and can pass through attempts at water filtration. giardiasis is associated with debilitating diarrhea in some humans, particularly those who are immunocompromised. however, cats do not commonly carry the assemblages needed to infect humans. transmission of g. duodenalis from cats to humans is rare and unproven. still, it seems prudent to consider the owner's health when contemplating management of giardial infections in cats. to avoid human health risks, cats with diarrhea that test positive for g. duodenalis should be treated with the goal of controlling the diarrhea. since no treatment for g. duodenalis is completely effective or % safe, treatment of positive cats without diarrhea should only begin after a discussion of the benefits and risks of the treatment with the owner. tritrichomonas foetus is best known for causing bovine reproductive infections. it is an obligate anaerobic parasite that also colonizes the lower intestinal tract of cats. there are enough differences between the two isolates that the feline isolate does not cause disease in heifers and vice versa. the parasite depends on the host's normal intestinal flora and secretions for obtaining nutrition. a report from the united states of purebred cats tested at an international cat show found t. foetus in of the cats tested, a prevalence of %. this parasite seems to have a higher prevalence in purebred cats than nonpurebred cats. a study of pet cats visiting veterinary hospitals across the united states reported of purebred cats were positive for t. foetus, while only of nonpurebred cats were positive. in this same study, of the positive tests were from purebred cats. a study from the united kingdom of diarrheic fecal samples sent to a veterinary diagnostic laboratory reported similar results. purebred cats represented of the cats testing positive for t. foetus. the u.k. study also found the siamese and bengal breeds each represented of positive cats; only two other breeds tested positive. transmission like most other protozoal parasites, t. foetus is transmitted by ingestion of the parasite, in this case, the trophozoite. unlike most of the other parasites, t. foetus does not form cysts and only survives up to days outside the body in moist feces. a cat becomes infected through the use of a shared litter box with an infected cat. after walking into the box, the parasite is transferred from the infected feces of one cat to the paws of the other. infection then occurs through ingestion of the trophozoites during grooming. after infection, t. foetus colonizes the distal ileum and colon, followed by shedding of infective trophozoites to days later. there are several mechanisms by which t. foetus causes diarrhea. these include alteration of the cat's normal bacterial flora population, increases in local inflammatory cytokine concentrations, production of enzymes, and direct mucosal injury. the resulting injury leads to plasmacytic-lymphocytic and neutrophilic colitis. although most infections involve only the mucosa of the colon, one study reported two of seven cats with diarrhea and t. foetus infections as having trophozoites in deeper layers of the colonic wall. co-infection with cryptosporidium felis or giardia duodenalis can be associated with increased numbers of t. foetus trophozoites and increased severity of diarrhea. signs of infection are most frequent in kittens and young cats, although infections without clinical signs can occur. adult cats, however, may also show signs of t. foetus infection. the most common sign is a foulsmelling large bowel diarrhea with increased frequency of defecation, mucus, blood, and flatulence. the consistency of the diarrhea may wax and wane, but the presence of diarrhea does not. cats with diarrhea are otherwise in good health and maintain their body condition. , severe diarrhea can result in anal swelling and fecal incontinence. diarrhea may respond to the use of antibiotics because of changes in the cat's intestinal microbial flora. however, it always returns at the cessation of therapy. , many cats experience a spontaneous resolution of the diarrhea within years of diagnosis. , since t. foetus causes reproductive infections in heifers and bulls, there is speculation the parasite also infects the reproductive tract in cats. tritrichomonas foetus was found in the uterus of a queen with pyometra. however, in a study of breeding male and female cats from catteries, no cytologic or molecular evidence of t. foetus was found in the reproductive tract. the authors reported colonic infection with t. foetus in of the cats representing of the catteries. detection of the trophozoites in a sample of feces is the most expedient means of diagnosing an infection with t. foetus (figure - ). an index of suspicion is required, because the clinical presentation of t. foetus infection is often mistaken for infection with giardia duodenalis. if a cat is not responding to treatment for that parasite, consider t. foetus as a cause of the diarrhea. the sample required for the diagnosis of t. foetus is a fresh, nonrefrigerated sample of watery feces. refrigeration kills the trophozoites, and they are not found in normal feces. the sample may be freshly passed diarrhea, feces collected using a wire loop passed into the colon, or collected by a colonic flush using a red rubber catheter and ml of saline. a wet mount or smear examination of the feces should be performed on all cats with diarrhea. examination of multiple samples may be required to find the t. foetus trophozoites with this technique because it is insensitive. the trophozoites must be differentiated from giardia duodenalis based on structural differences and motility patterns (see table - ). the trophozoites of t. foetus can be cultured using the inpouch tf system (biomed diagnostics). this test is more sensitive than the fecal wet mount examination and detects trophozoites per sample. the number of parasites shed by a cat with diarrhea is high enough to be routinely detected with this method. the test should be performed in-house, because the parasite is unlikely to survive the trip to the laboratory. the test pouch is inoculated with µg of freshly collected feces, about the size of a peppercorn. any more than this increases the chances of bacterial overgrowth. the pouch is incubated at ° c and examined under the microscope for motile trophozoites every other day for days. the pouch should be tapped gently to dislodge the parasites, which tend to collect along the seams. the test is considered negative if parasites are not found after days. one benefit of this system is that it does not support growth of giardia duodenalis or pentatrichomonas hominis. if a fecal wet mount examination and culture are both negative and infection with t. foetus is still under consideration, a pcr test can be performed. this test detects dna from live or dead trophozoites, but is more expensive than other diagnostic methods. this test is more sensitive than the other two methods and can detect parasites per sample. the sample size is mg of feces not contaminated by litter preserved in to ml of rubbing alcohol shipped at room temperature. trophozoites of t. foetus are sometimes found in colonic biopsy samples adhered to the surface or in the lumen of crypts. the most effective drug for the treatment of t. foetus in cats is ronidazole. the drug has a bitter taste and should be compounded into capsules. veterinary staff and owners should use gloves when handling ronidazole. if a confirmed relapse occurs, another course of treatment may eliminate the parasite. diarrhea may take several weeks to resolve after elimination of the parasite, because significant colitis is often present. effectiveness of treatment can be evaluated by performing fecal pcr tests and weeks after the end of treatment. apparent treatment failures may occur because of re-infection, co-infection with giardia duodenalis or cryptosporidium felis, or the presence of another concurrent diarrhea-causing disorder. a more worrisome cause for treatment failure is a recent report of parasite resistance to ronidazole in two cats. fortunately, diarrhea ultimately resolved in both cats despite the continued presence of the parasite. if the cat retests negative and the diarrhea is not improving after weeks, consider the possibility that another disease may exist. nonspecific treatment for diarrhea is unhelpful and may prolong the duration of diarrhea. diarrhea may respond to antibiotics as they alter the intestinal flora population; however, once treatment is stopped, the diarrhea will return. an important and potentially serious adverse effect of ronidazole administration in cats is a reversible neurotoxicity. onset of signs often begins within week of the onset of therapy and may last between and weeks after cessation of therapy. these signs can include depression, ataxia, seizures, behavioral changes, weakness, hyperesthesia, and trembling. neurotoxicosis usually requires only supportive care along with discontinuation of the drug. the neurologically affected cat should be retested for the parasite, because it may have been eliminated. because of the potential for neurotoxicity, the use of ronidazole should be restricted to cats with confirmed infections with t. foetus. crowded conditions should be avoided, because transmission of t. foetus trophozoites is more efficient in these settings. cats testing positive should be isolated from other cats during treatment. providing a clean environment will help prevent transmission of trophozoites. although there is a report of an infection in one immunocompromised person, transmission of t. foetus trophozoites from cats to healthy humans has not been reported. still, prudence dictates handling feces infected with t. foetus trophozoites carefully. recent genetic evaluations have shown that most feline infections with cryptosporidium spp. are with c. felis; not, as previously thought, with c. parvum. cryptosporidium parvum seems to be limited to farm animals. cryptosporidium felis is an obligate intracellular parasite infecting the small intestine. infective oocysts are ingested from contaminated feces during self-grooming of contaminated body parts and from contaminated food and water. , after infection, the parasite attaches to the brush border of the enterocyte. the prepatent period is to days, and the oocysts are infective as soon as they are shed, making this a very contagious disease. like most intestinal parasites, shedding is often intermittent. the pathogenic effects of c. felis infections are not well understood. direct cytotoxicity and inflammation causes villus atrophy and decreased surface area for absorption of water, electrolytes, and other nutrients. , apoptosis (preprogrammed cell death) of the mucosal cells may be accelerated, adding to the malabsorption. most infections with c. felis are subclinical. signs, if present, range from a mild, self-limiting small bowel diarrhea to chronic intermittent small bowel diarrhea. severe diarrhea with weight loss and anorexia may also occur. , clinically apparent infections are most common in kittens, adult cats with concurrent gastrointestinal diseases, and cats co-infected with giardia duodenalis or tritrichomonas foetus. cats with co-infections may experience more severe clinical signs. a fecal flotation, which should be performed on all cats with diarrhea, may reveal c. felis if there are large numbers of oocysts (figure - ) . the fecal floatation test, however, is often negative because of intermittent shedding. the parasite is small and floats in a higher plane than helminth ova; the high-power lens and appropriate adjustment of the microscope stage is required to find the parasite. the small size of the oocyst makes identification difficult, particularly if the examiner is not specifically looking for them. a modified ziehl-neelsen stain of a thin fecal smear may help in the identification of the oocysts. this technique works well in humans with large numbers of oocysts. once signs resolve or the oocyst numbers decline, a single examination of a stained smear becomes insensitive. when only one sample is available, testing for c. felis antigen is a good choice. the prospect microplate assay (alexon biomedical, sunnyvale, calif.) is more sensitive and specific for the diagnosis of c. felis than is the examination of a stained smear. immunofluorescent antibody testing is available from some laboratories. fecal c. felis dna can be detected using pcr testing. this test is available at many veterinary diagnostic laboratories; however, at present, there is no test standardization among laboratories. the clinical and zoonotic significance of a positive pcr test combined with an oocyst negative test is unknown. therefore a positive pcr test in a cat without diarrhea presents a confusing situation for the attending veterinarian with regard to recommendations for the owner. unfortunately, there are no completely effective and safe treatment protocols available for c. felis. , a concerted attempt to find other causes of diarrhea should take place prior to convicting a cat of having diarrhea solely from c. felis infection. most reports on therapy for c. felis are uncontrolled and anecdotal. a number of drugs have been discussed. azithromycin for at least days appears safe but produces variable results. paromomycin, an oral aminoglycoside, may be effective. however, one study reported acute renal failure in of cats receiving the drug. deafness also occurred in three of those four cats. nitazoxanide is a drug approved for treating humans with diarrhea caused by cryptosporidium spp. infections. the administration of nitazoxanide to cats at mg/kg q h po for at least days up to days may be effective. however, nitazoxanide is a gastrointestinal irritant and commonly results in vomiting and foul-smelling diarrhea. co-infections with giardia duodenalis and/or tritrichomonas foetus are more difficult to control. if diarrhea from c. felis infection improves but does not resolve at the end of therapy, the duration of treatment may be prolonged. additional diagnostic testing should also be performed to ensure the only cause of the diarrhea is infection with c. felis. environmental control of c. felis is difficult, because it is extremely hardy. it is resistant to chlorination and most disinfectants. oocysts remain viable at temperatures above freezing up to ° c. the parasite is difficult to filter and survives treatment at municipal water treatment facilities. steam-cleaned housing and utensils may be beneficial in controlling parasite numbers, and they are susceptible to % ammonia solutions; however, the required contact time is hours. cryptosporidium spp. are relatively species specific, and there are no reports of waterborne outbreaks of human cryptosporidiosis associated with c. felis. cryptosporidiosis can cause life-threatening diarrhea in hivpositive persons. fortunately, humans are rarely infected with c. felis. in fact, the zoonotic species most commonly found in humans (often veterinary students), is c. parvum found in young heifers. regardless of a person's health, feces from a cat with diarrhea should be handled carefully. if a cat infected with cryptosporidium spp. is owned by an immunocompromised person, a pcr test may be useful in determining the species of the parasite and its zoonotic risk. like other coccidians, toxoplasma gondii is an obligate intracellular parasite. domestic cats and other felids are the only animals that shed oocysts. any warmblooded animal, including humans, can be infected with this parasite. toxoplasma gondii can be transmitted by ingestion of infective oocysts in fecally contaminated food or water after ingestion of tissue cysts through carnivorism, or by transplacental or trans-mammary transmission of the parasite. the parasite enters into one of two cycles, depending on the host species. the enteroepithelial cycle only occurs in cats and results in shedding of oocysts after sexual reproduction of the parasite. after a cat ingests an infective oocyst or a tissue cyst, the parasite enters the mucosal cells of the small intestine, where it may undergo development and sexual reproduction, after which oocysts are shed. the prepatent period after ingesting an infective oocyst is to days, while shedding after ingesting tissue cysts starts in to days. fecal shedding, which occurs only after initial infection, lasts for to weeks , and the oocysts become infective to days after they are shed. the extraintestinal cycle occurs in any animal, including cats. after ingestion, the parasite penetrates the cells of the small intestine and rapidly replicates in the enterocytes and associated lymph nodes into tachyzoites. after hematogenous and lymphatic spread, tachyzoites infect cells in all tissues of the body. tissues most commonly infected include the brain, liver, pancreas, and lungs. if a pregnant queen becomes infected, tachyzoites cause placentitis, after which they infect the fetus. in weeks, the host's immune response slows parasite replication, and the resultant bradyzoites form tissue cysts in the brain, striated muscle, and liver, and they remain viable for the life of the animal. immunosuppressive drugs or disease may dull the suppression of parasite division by the host immune system and allow the slowly dividing bradyzoites in tissue cysts to begin rapid division, thereby reactivating the infection with tachyzoites. none of the forms of t. gondii produces a toxin. rapid replication of tachyzoites within a cell leads to rupture of the cell and necrosis of the tissue in which they are located. the most commonly injured tissues are the brain, lungs, liver, and pancreas. prenatal infection leads to more severe illness, because the immature immune system is unable to slow down replication by tachyzoites, allowing continued damage to tissues. prenatal infection is more likely to result in ocular infections, and neonatal death is usually caused by pulmonary or hepatic infection. type ii and iv hypersensitivities may be involved in the pathogenesis of chronic disease from bradyzoites in tissue cysts. kittens infected perinatally can be stillborn or die shortly after birth. they may also suffer from hepatomegaly and ascites, central nervous system signs resulting from encephalitis, respiratory distress, or uveitis. , clinical signs of infection in healthy adult cats are uncommon (box - ). diarrhea from enteroepithelial development of the parasite is rare. cats that develop clinical disease often have an episodic course with vague signs that depend on the body system affected. onset of illness may be acute or chronic, and the most commonly affected organs include the brain, lungs, liver, heart, pancreas, and the eyes. signs are the result of spread of tachyzoites after initial infection or after reactivation of tissue cysts. cats suffering from uveitis may develop lens luxation and glaucoma. the best way to identify a cat shedding t. gondii oocysts is to demonstrate them with a centrifugal fecal flotation technique using sheather sugar solution. the oocysts are about a quarter of the size of isospora felis oocysts ( figure - ). oocysts of t. gondii are morphologically indistinguishable from hammondia or besnoitia spp. oocysts. detection of fecal t. gondii dna using a pcr test can be used to definitively differentiate t. gondii oocysts from similar coccidians. it is probably best, however, to assume suspicious oocysts are those of t. gondii until proven otherwise. proving infection with t. gondii is responsible for a cat's systemic illness is also difficult. finding tachyzoites in cytology samples is uncommon. they are most likely to be identified from body cavity effusions. the most common method of identifying an infected cat is by detecting t. gondii-associated immunoglobulins using immunofluorescent antibody or elisa techniques. since cats are infected for life, a seropositive cat has been infected at some point in its life. however, use of serology alone is insufficient to diagnose an active t. gondii infection. serum immunoglobulin m (igm) is produced within to weeks after infection, but increased igm titers may persist for months to years. serum immunoglobulin g (igg) begins to rise later; in some cats, igg may not be detectable for to weeks. by the time igg is detectable, shedding will have ceased. maternally acquired igg persists in kittens for to weeks. a rising igg titer is associated with an active infection, but the degree of increase is not associated with the severity of the clinical signs. if a cat becomes seronegative, it is more likely the titer has fallen below the sensitivity of the test rather than the parasite has been eliminated from the body. because of the vague nature of the clinical signs, many cats are presented later in the course of the disease. by this time, they may have switched from igm to igg production or passed the time of maximal igg production. thus a negative igm titer or a lack of rising igg titer does not rule out t. gondii infection. also, reactivation of tissue cysts is rarely associated with rising igg titers. ultimately, the diagnosis of an active systemic t. gondii infection requires demonstration of an igm titer greater than : or a fourfold increase in igg titers over a -to -week period along with signs consistent with toxoplasmosis, the exclusion of other disorders that may cause the clinical signs, and response to appropriate anti-t. gondii treatment. although serum igm titers may be increased in otherwise healthy cats, increased igm titers in cerebrospinal fluid or aqueous humor only occurs in cats with active cns or ocular infections. the goals of treating a cat infected with t. gondii are to reduce shedding of oocysts and to control the clinical signs in sick cats. shedding can be reduced by using ponazuril, toltrazuril, or high doses of clindamycin. the drug options for treating a sick cat include clindamycin, trimethoprim-augmented sulfadiazine, or azithromycin for at least weeks (see table - ) . recurrences are more common if the cat is treated for less than weeks. , the antifolate drug pyrimethamine may be more effective than trimethoprim, but megaloblastic anemia develops in many cats. supplementation with folinic acid ( mg/cat, once daily, po) or brewer's yeast ( mg/kg, once daily, po) may prevent or reverse the anemia. no drug clears all of the tissue cysts; so, cats remain infected for life. if uveitis is also present, use appropriate topical, oral, or parenteral corticosteroids. for a cat with proven t. gondii-associated uveitis alone, a topical ocular glucocorticosteroid is the only required treatment; no antibiotics are necessary unless the uveitis is persistent or recurrent. • wash hands after handling cats, especially if you are pregnant or immunocompromised. • remove fecal material from the home environment daily, since shed oocysts require a minimum of hours to become infective. • do not have immunocompromised persons clean the litter box. if they must clean the litter box, they should wear gloves and wash hands thoroughly when finished. • use litter box liners, and periodically wash the litter box with scalding water and detergent. • wear gloves when gardening, and wash hands thoroughly when finished. • cover children's sandboxes when not in use to avoid fecal contamination by outdoor cats. • only feed cats cooked or commercially processed food. • control potential transport hosts, such as flies and cockroaches, that may bring the organism into the home. • filter or boil water from sources in the environment. • cook meat for human consumption to ° c for minutes minimum (because of uneven heating, microwave cooking does not kill all t. gondii ). • freeze meat at − ° c for hours. • wear gloves when handling meat, and wash hands thoroughly with soap and water when finished. clinical signs such as malaise, fever, and muscle pain should begin to resolve in to days. if there is no response within days, switch to or add another drug. if there is still no response, search for another condition that may cause the observed clinical signs. however, ocular and cns signs resolve more slowly and thoracic radiographic changes may take weeks to resolve. some cns changes may never completely resolve. cats co-infected with feline immunodeficiency virus (fiv) do not respond to anti-t. gondii treatment as well as fivnegative cats respond. feeding cats commercially processed cat food and avoiding undercooked or raw meat can prevent exposure to t. gondii. controlling hunting reduces access to paratenic hosts with infective tissue cysts. access to mechanical carriers of t. gondii, such as earthworms or cockroaches, should be minimized. human infection with t. gondii is common, more so in warm, humid climates where the prevalence of t. gondii seropositive persons approaches %. the number of persons seropositive for t. gondii is estimated to be around , , worldwide. infective oocysts are hardy and may remain viable in the environment for up to months. human infection most often occurs after eating raw or undercooked meat infected with tissue cysts or by transplacental infection. seropositive cats are finished shedding and are unlikely to resume shedding even if the infection becomes reactivated. cats found to be shedding oocysts should be quarantined at a veterinary hospital until shedding ends. oocysts of t. gondii have not been found on the hair coat ; so, transmission of toxoplasmosis does not occur after touching a cat. pregnant women infected with t. gondii for the first time, or chronically infected women who are also hiv positive, can transmit the parasite to their unborn child. transplacental infection can result in stillbirths, cns, or ocular disease. more severe fetal disease may occur if the infection happens in the first half of the woman's pregnancy. toxoplasma gondii infection of immunocompetent humans usually results in a self-limiting fever and malaise. steps useful in preventing transmission of t. gondii to humans can be found in box - . pancreatitis refers to inflammation of the pancreas only, with no implication of the underlying cause or pathology. for example, acute necrotizing pancreatitis (anp) with pancreatic auto-digestion, requiring predominantly supportive care by maintaining fluid and electrolyte balances and pain relief, must not be confused with chronic pancreatitis (cp) caused by lymphocytic infiltration, and commonly associated with lymphocytic inflammatory bowel disease (ibd), and often requires corticosteroids to manage. these two conditions (and others) can only be definitively distinguished histologically. in many cases, the clinical signs of cats with acute pancreatitis will resolve with supportive care before a precise diagnosis is reached and will thus remain undiagnosed. there are no formal classifications for feline pancreatitis, but most authors , , use the terms • acute pancreatitis • acute necrotizing pancreatitis, characterized by severe peri-pancreatic fat necrosis • acute suppurative pancreatitis, characterized by neutrophilic infiltration • chronic pancreatitis, characterized by lymphocytic infiltration the exact prevalence of feline pancreatitis is unknown. necropsy studies from the s to s reported prevalence of feline pancreatitis ranging from . % to . %. , a more recent study found % of cats had evidence of pancreatitis. however, this included pancreatic pathology in % of apparently healthy cats, which suggests that mild pathology is unlikely to cause clinical signs. these studies all show lymphocytic pancreatitis to be significantly more prevalent than acute pancreatitis. this may underestimate the true prevalence of acute pancreatitis, since it is understood that no permanent histopathologic changes are present after resolution of acute pancreatitis. it is also possible that studies assessing pathology in necropsy cases do not reflect clinical practice. there are no specific age, breed, or sex predispositions. although one study reported siamese cats to be at increased risk of acute pancreatitis, subsequent studies have recognized the majority of cases are domestic shorthair cats, suggesting no specific breed predispositions. , , , most studies have indicated older cats ( to years of age) are more likely to be affected, , , , but these studies most likely underrepresent cats with less severe clinical disease for which definitive diagnosis may not be reached and which may be younger. no association has been made with a high-fat diet or obesity. in most cases of both acute and chronic pancreatitis, no specific cause is found, and the disease is primarily considered to be idiopathic. , there are, however, some specific underlying causes that are sporadically recognized. these include infections with herpesvirus, calicivirus, , feline infectious peritonitis (fip), liver fluke and pancreatic fluke, , and toxoplasmosis. however, a recent paper found no association between serum feline pancreatic lipase immunoreactivity (fpli) concentrations and toxoplasma gondii serology. pancreatitis has also been recognized subsequent to trauma and organophosphate poisoning. the association of pancreatitis with inflammatory bowel disease and cholangitis is frequently mentioned (triaditis) but poorly described in the literature. one study found % of ibd cases to have histologic evidence of pancreatic involvement, and another found fpli concentrations were elevated in % of cases with histologically confirmed ibd. it is the author's experience that many cases of pancreatitis recognized with ibd have no specific clinical signs attributable to pancreatitis and should therefore be diagnosed and treated as intestinal disease. diabetes mellitus is a recognized co-morbidity of pancreatitis in cats. a recent study found fpli concentrations were significantly higher in diabetic cats compared with non-diabetics. no association could be made between fpli concentrations and the degree of diabetic control. one study found of cats ( %) histologically diagnosed with hepatic lipidosis were also histologically diagnosed with acute pancreatitis. it is not known if pancreatitis is a cause, consequence, or coincident disease of hepatic lipidosis. for example, anorexia associated with acute pancreatitis could predispose to fatty infiltration of the liver. however, the high rate of concurrent disease has important implications for ensuring cats with pancreatitis receive adequate caloric intake. ongoing or recurrent pancreatitis may lead to pancreatic cysts or exocrine pancreatic insufficiency, which are both covered later in this chapter. although pancreatitis has been experimentally induced in cats, , , the pathophysiology of spontaneous pancreatitis remains unknown. acute pancreatitis is initiated by an increase in secretion of pancreatic enzymes that leads to inappropriate cellular activation of trypsin and subsequently other digestive zymogens. these activated digestive enzymes lead to local effects including inflammation, hemorrhage, acinar cell necrosis, and peripancreatic fat necrosis. , , chronic pancreatitis may result from any of several underlying processes: ongoing, low-grade acute pancreatitis episodes may instigate chronicity; chronic pancreatitis, with a predominance of lymphocytic inflammation has been induced experimentally within weeks by narrowing the main pancreatic duct to approximately % of its normal diameter ; and the association with ibd may suggest an immune-mediated cause. the clinical signs of pancreatitis in cats are nonspecific. a review of eight prior series totaling cases of acute pancreatitis in cats found anorexia ( % of cases) and lethargy ( %) to be the most common historical findings. vomiting was recognized in % of cases, diarrhea in %, and weight loss in %. physical examination findings were similarly nonspecific with dehydration ( %) being the major finding; fever was recognized in only % of cases and abdominal pain in %. it is important to note that vomiting and abdominal pain, key features of pancreatitis in dogs, are not consistently recognized in cats. similar, nonspecific findings indistinguishable from ibd are recognized in cats with chronic pancreatitis. , diagnosis because the presenting signs and physical examination findings are nonspecific, the diagnosis of pancreatitis can be challenging, requiring not only clinical suspicion but a combination of diagnostic modalities. for the most part, hematology and plasma biochemistry findings are unremarkable, although a combination of findings may increase clinical suspicion. for example, moderate elevations in liver enzymes, bilirubin, and glucose are present in approximately % of cases and hypocalcemia in approximately two of three of cases; hypocalcemia infers a poorer prognosis. hypoalbuminemia is seen in approximately one of three of cases and has important implications for fluid therapy. amylase and lipase elevations are not reflective of pancreatitis in cats. feline trypsinlike immunoreactivity (ftli) is the diagnostic test of choice of exocrine pancreatic insufficiency, but elevations in pancreatitis are not seen consistently enough to warrant use of this test for this purpose. , , the biggest recent advance in feline pancreatic diagnostics has been the characterization of feline pancreatic lipase, leading to the development of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity (fpli). it must be remembered, however, that an increase in fpli only tells the clinician that pancreatic pathology is present, but not the cause of pathology, which may be, for example, neutrophilic or lymphocytic pancreatitis or neoplasia, and it may or may not involve the intestines or liver. fpli should therefore be used as a screening test, with elevated results not suggesting a diagnostic end point. further, the high interassay variability of this test would suggest that mild cases may be missed as shown in one study and that the test may not be appropriate for serial monitoring. fpli is currently available as "spec fpl" from commercial laboratories and has a sensitivity of % and a specificity of % when . µg/l is used as the diagnostic cut off compared with . µg/l, which is the listed reference range high point. in an acutely unwell cat (less than days) with only mild to moderate signs of disease, further diagnostics may not be warranted, and many cats will improve with supportive therapy of balancing fluid and electrolytes, pain relief, and antinausea/vomiting therapy. cats with chronic duration of signs and acutely unwell cats that do not improve with supportive therapy warrant further diagnostics. the underlying disease process cannot be assumed from an elevated fpli; in one study of cases, acute necrotizing pancreatitis could not be distinguished from chronic nonsuppurative pancreatitis by signalment, duration of signs, or clinical findings. the major utility of diagnostic imaging is to rule out other differential diagnoses, such as an intestinal foreign body, and perhaps confirm that the pancreas is affected. radiography is non-specific for diagnosis of pancreatitis, but findings may include decreased abdominal detail (sometimes associated with ascites), soft tissue density in the right cranial quadrant of the abdomen, hepatomegaly, or gas-filled intestines , , (see . additionally, thoracic radiographs may show pleural effusion. one study found of cats with pancreatic necrosis had such a change ; the mechanisms resulting in pleural effusion are not precisely defined. ultrasonography has high specificity (> %) but low sensitivity (< %) for recognizing pancreatitis in cats, , , , with findings dependent on operator skills, quality of equipment, and severity of lesions. typical findings are hypoechogenicity of the pancreas, which may be enlarged or irregular; hyperechogenicity of the peripancreatic fat; the possible presence of abdominal effusion; and abnormal findings with other organs, such as liver or intestine, may add to the clinical picture , , , . one study indicated that contrast-enhanced doppler ultrasonography can provide further diagnostic insights. a recent study suggested that endosonography may be useful in cases where transabdominal ultrasonography is difficult, for example, because of obesity, hyperechoic mesentery, or excessive intestinal gas. for more than years, computed tomography (ct) has been a commonly used modality to confirm pancreatitis in humans, but this reliability has not been demonstrated in cats, where sensitivity may be as low as %. , definitive diagnosis of pancreatitis, including differentiation of the inflammatory process, can only be made by cytologic assessment of pancreatic tissue. in most cases, ultrasound-guided fine-needle aspiration (fna) of the pancreas is technically difficult because of the small dimension of the feline pancreas; there appears to be no assessment of feline pancreatic fna findings in the literature. gross inspection of the pancreas and samples for histologic assessment can be obtained during laparotomy , (see or laparoscopy. , because pancreatitis often occurs concurrently with pathology of other organs, thorough evaluation of the abdomen by ultrasonography or gross inspection is recommended, as are multiple biopsies of, for example, intestines, liver, and mesenteric lymph nodes, where appropriate. clinicians may be reluctant to biopsy the pancreas because of perceived risks of deleterious effects. studies of pancreatic biopsy in healthy cats dispel the concern that the pancreas is unforgiving to mild manipulation and biopsy , a and the author's clinical experience is consistent with these findings. supportive care comprising correction of fluid/ electrolyte imbalances, pain management, and nutritional support are the mainstay of therapy for cats with figure - gross appearance of pancreas at laparotomy; this was histologically diagnosed as chronic pancreatitis (i.e., lymphocytic infiltration was recognized). gross appearance of pancreas at laparotomy; this pancreas was found to be histologically normal. it does look smaller than is typically seen; pancreatic atrophy can look similar to this, grossly. pancreatitis. , , specific underlying causes, when diagnosed, should be managed, as should concurrent diseases. follow-up evaluation is determined on a case-by-case basis; reduction or resolution of clinical signs is the main criterion for success of therapy. serial fpli values may be monitored when initial results are extremely high but are of limited value for mild increases because of assay variability. dehydration, acid-base and electrolyte abnormalities should be corrected during the first to hours. hypocalcemia, if present, should be treated with a calcium gluconate infusion of to mg/kg during to hours, with continued assessment of plasma calcium concentrations. plasma transfusions can be considered in cats with hypoalbuminemia. , , although abdominal pain is not commonly described in cats with pancreatitis, it is likely to be present in most cases and may contribute to anorexia. historical concern about exacerbation of pancreatitis with opioids is no longer accepted, and this class of drugs is considered appropriate. meperidine ( to mg/kg sc or im) every to hours, butorphanol ( . to . mg/kg sc) every hours, or sustained-release buprenorphine ( µg/kg sc) every hours are alternatives. , , the author uses one dose of methadone ( . to . mg/kg sc, im, or iv) initially and places a fentanyl patch for longer-term pain management. the traditional recommendation for management of pancreatitis across all species has been nil per os for several days. this recommendation is appropriate for cats with severe vomiting, but there is no evidence to support this approach in cats that are not vomiting and that are eating normally. further, nutritional support is vital for those cats with concurrent hepatic lipidosis. if the cat is not eating voluntarily, nutritional support by tube feeding is often warranted. , , a recent paper found nasogastric tube feeding of cats with pancreatitis was tolerated well and resulted in few clinically significant complications. other reported nutritional strategies for cats with pancreatitis incorporate partial parenteral nutrition (ppn; . % amino acids, % lipids), or total parenteral nutrition (tpn; % amino acids, % lipids, % dextrose), or both instead of enteral feeding. , , cats do not seem to benefit from feeding of specially formulated low-fat diets; commercially available, veterinary liquefied diets appear to be well tolerated despite their high-fat contents. other therapy may be appropriate in individual cases. all cats with pancreatitis that are vomiting should be treated with antiemetics. examples of drugs that can be used are -ht antagonists, such as dolasetron ( . to . mg/kg iv or po, once to twice daily); ondansetron ( . to . mg/kg iv every to hours); and maropitant, an nk -inhibitor ( . to . mg/kg sc once daily). these drugs are covered in detail earlier in this chapter under therapeutics for vomiting and diarrhea. dopaminergic antagonists, such as metoclopramide, are less effective antiemetic agents in cats than the other choices mentioned. , in most cases, pancreatitis begins as a sterile process, and antibiotic therapy is controversial. pancreatic necrosis and inflammation may predispose to bacterial colonization of the pancreas as demonstrated in experimental models. , this has not been demonstrated in spontaneous disease, and no comparison of outcomes has been made of cats with pancreatitis treated with or without antibiotics. cefotaxime ( to mg/kg iv, im) has been used to prevent bacterial colonization in experimental models. other broad-spectrum cephalosporins or ampicillin may act similarly. antibiotic considerations are possibly more important for acute pancreatitis than for treatment of chronic disease. cats with demonstrated lymphocytic pancreatitis, with or without concurrent ibd or lymphocytic cholangitis, should be treated with corticosteroids (e.g., prednisolone, to mg/kg once to twice daily) with tapering to the lowest effective dose. there is no justification for use of corticosteroids in cats with acute necrotizing or acute suppurative pancreatitis, or cats for which the cause of pancreatitis has not been diagnosed histologically. use of corticosteroids in cats with pancreatic disease creates a risk of iatrogenic diabetes mellitus. surgical intervention is warranted to relieve any bile duct obstruction that may result or for the débridement of pancreatic abscesses or necrotic tissue; in many cases, cats will survive multiple years after such corrective surgery. pancreatic cysts, pseudocysts, and bladders have been described sporadically in cats.* pancreatic cysts are lined by a single layer of cuboidal epithelium and do not communicate with the pancreatic duct; pseudocysts are enclosed by a wall of fibrous tissue, lacking the epithelial lining characteristic of true cysts and can form secondary to pancreatic inflammation; cystic dilations of the pancreatic duct are referred to as pancreatic bladder. true pancreatic cysts have been described in three cats , , ; a congenital pancreatic cyst with associated inflammation was described as an incidental finding in an adult cat ; multiple pancreatic cysts were described in a cat with concurrent polycystic disease in the kidney and liver ; and a another cat had multiple recurrent pancreatic cysts with concurrent mild pancreatic inflammation and atrophy associated a with rapid clinical course resulting in diabetes mellitus. cysts, pseudocysts, and bladders may be identified ultrasonographically or by ct. they may be benign, but the associated pancreatic inflammation and other sequelae, such as diabetes mellitus, may need to be managed. pancreatic bladders may result in biliary obstruction, and surgical correction may be required. pancreatic nodular hyperplasia is recognized quite frequently as an incidental finding in older cats or at necropsy. neoplasia of the exocrine pancreas is rare in cats. its frequency was assessed in the s when one study estimated . cases per , patients per year at risk, and another found pancreatic tumors in of feline necropsies. a more recent study recognized, from , feline admissions over a -year study period, only two cats with pancreatic adenomas ( . % of admissions) and eight with pancreatic adenocarcinomas ( . % of admissions). adenomas appear as small, solitary or multifocal nodules and are not typically associated with adjacent pancreatic inflammation. they do not cause clinical signs, unless large, when any clinical signs result from the physical size and are usually an incidental finding. , few generalities can be made about the presentation for pancreatic adenocarcinoma. the age range is large ( to years), there is no sex predisposition, and no clear breed predispositions are present. , only cytology or histopathology can distinguish pancreatitis from pancreatic carcinoma in cats antemortem, yet it is important to differentiate the two conditions, because, in contrast to adenomas, pancreatic adenocarcinoma is associated with a grave prognosis. the presence of lesions consistent with metastases on radiography or ultrasonography may suggest malignancy, but one study could not distinguish neoplasia from pancreatic nodular hyperplasia ultrasonographically based on the appearance of the pancreas alone (figures - and - ) . pancreatic adenocarcinomas in cats can result in a paraneoplastic dermatologic condition consisting of nonpruritic, symmetric alopecia affecting the face, ventral body, and medial aspect of the limbs of cats. the skin is usually glistening but not fragile, and there can be crusty lesions on the footpads.* the pathogenesis of this dermatologic disease is unknown. in one case, surgical excision of the pancreatic carcinoma resulted in resolution of dermatologic disease, indicating that the process is reversible (although signs recurred as the tumor re-emerged). diabetes mellitus is a recognized complication of pancreatic adenocarcinoma. the mechanism is unknown and may simply be secondary to compression or invasion of islet cells by the tumor. in some cats, diabetes is recognized ahead of pancreatic neoplasia. , , obstructive jaundice has also been described with pancreatic adenocarcinoma. most cases of pancreatic adenocarcinoma in cats have metastasized by the time of diagnosis, and most reported cases die or are euthanized within days of diagnosis. surgical excision is a potential option if neoplasia is confined to one limb of the pancreas, but recurrence is possible even if there is no evidence of metastasis and excision seems complete at the time of surgery. exocrine pancreatic insufficiency (epi) is a condition caused by insufficient synthesis and secretion of pancreatic digestive enzymes from the exocrine portion of the pancreas. in humans it has been reported that % of pancreatic acinar cells must be lost before clinical signs of epi are seen. epi is considered rare in cats but is perhaps being recognized more frequently because of increased awareness. there are less than fifty cases described in the veterinary literature* with one of these papers describing only cases from five institutions, with prevalence described as . % to . % of cats seen over a -year period. in contrast to this, the gastrointestinal laboratory at texas a&m university recognized samples with serum ftli concentrations at or less than . µg/l, which is diagnostic for epi, out of , submissions, which equates to . % of cats with known or suspected gastrointestinal disease. all studies indicate a wide age range of cats can be affected, from kittens less than months of age to cats more than years old, with a median age of approximately years. there is no apparent breed predisposition. , , one paper recognized of ( . %) cats to be male, and another recognized of ( %) male cats, suggesting a possible sex predisposition. chronic pancreatitis is believed to be the most common cause of epi in cats, acinar atrophy (paa) is recognized as the most common cause of epi in dogs, and has been definitively described in two feline cases and mentioned as a cause for three other cases. other potential causes of epi include disruption of pancreatic enzyme flow at the duodenal papilla following duodenal resection and pancreatic fluke infection (eurytrema procyonis), , and amyloid deposition and neoplasia are other possible causes of pancreatic cell damage that have not definitively been described in cats. congenital pancreatic hypoplasia or aplasia has not definitively been reported in cats, but reports of epi in cats as young as months of age , suggest this possibility. since chronic pancreatitis is a common cause of epi and chronic pancreatitis has a strong association with ibd, many cats may have concurrent lymphocytic pancreatitis and enteritis. , , therefore cats failing to respond to therapy for epi may require further diagnostics and management of an underlying condition. further, destruction of functional exocrine pancreatic tissue can also affect pancreatic endocrine tissue, resulting in concurrent diabetes mellitus. several studies have indicated that all cats with epi will have weight loss when diagnosed, unless a kitten, in which case ill-thrift is recognized. , diarrhea is not necessarily present, being described in % to % of cats; the nature of feces can vary from voluminous, malodorous stools that can be discolored (yellow or pale), sometimes with steatorrhea, to normal feces in other cats. increased frequency of defecation and the presence of mucus in the feces of some cats can lead to the diarrhea being characterized as large bowel. only about % to % of cats are polyphagic, some described as having a ravenous appetite; conversely, some cats present with anorexia. vomiting has also been described. since cats with epi often have concurrent disorders, such as ibd, the clinical signs recognized may reflect the concurrent disease and not necessarily epi alone. physical examination findings are similarly nonspecific, with thin/ emaciated body condition being the most common finding. hematologic findings are non-specific, but a mild nonregenerative, normocytic, normochromic anemia may be recognized as well as lymphopenia or neutrophilia. plasma biochemistry results may show a mild to moderate increase in alanine aminotransferase (alt) and a mild increase in alkaline phosphatase in some cats. mild to moderate hyperglycemia may be seen, as may mild hypoglycemia or normoglycemia. , , hypocobalaminemia is recognized in nearly all cats with epi. , , , , this may be because of insufficient production of intrinsic factor, a cobalamin-binding protein only produced by the pancreas in cats and necessary for ileal absorption of cobalamin ; it may also be because of failure of pancreatic enzymes to liberate cobalamin from binding by r protein in the duodenum or small intestinal bacterial overgrowth (sibo), not yet specifically described in cats. folate concentrations may be reduced (because of concurrent intestinal malab sorption), normal, , or increased, which may relate to reduced pancreatic bicarbonate secretion, secondary to severe hypocobalaminemia, or associated with sibo. none of these presenting complaints, physical examination findings, or routine testing results are specific to epi. therefore epi requires a degree of clinical suspicion and/or thorough diagnostics to ensure the diagnosis is not missed. a low level of serum ftli is diagnostic for epi. , , samples can be sent to the gastrointestinal laboratory at texas a&m university from anywhere worldwide (with instructions about sample handling requirements on their website: http://vetmed.tamu.edu/gilab/). the reference range for serum ftli is to µg/l, with concentrations at or less than . µg/l diagnostic for epi. since the clinical signs and routine laboratory findings are nonspecific for epi, it is ideal to test serum for ftli in any cat with weight loss or ill-thrift. the texas a & m gastrointestinal panel also includes testing for levels of cobalamin, folate, and fpli, ensuring concurrent hypocobalaminemia will not be missed and potentially providing indications of other gastrointestinal disease. conversely, although a low level of serum ftli confirms a diagnosis of epi, it is not necessarily a diagnostic end point, since epi is so often recognized concurrently with other gastrointestinal disease. failure to respond to therapy should prompt the clinician to consider and investigate further for concurrent processes. most cats with epi can be successfully managed with dietary supplementation of pancreatic enzymes. commercial products (e.g., viokase [axcan pharma, birmingham, ala.], pancrezyme [virbac, fort worth, tex.], and creon [abbott laboratories, abbott park, ill.]) are available, and powder is considered more effective than tablets or capsules (some capsules can be opened and the contents sprinkled onto food, like powder). the required dose can vary quite substantially from cat to cat. it is appropriate to start with one teaspoon of powder with food twice daily, and adjustments can be made depending on the response; most cats accept the powder readily if it is mixed thoroughly through canned food, but other flavors (e.g., fish oil or brine from canned tuna) can be used to disguise the taste if necessary. raw pancreas (e.g., from beef or pork) may also be used, with to g twice daily an appropriate starting dose. since most cats with epi are hypocobalaminemic, supplementation by subcutaneous injection is required (oral supplementation is not effective since cobalamin deficiency leads to cobalamin malabsorption). an appropriate dose for most cats is µg, and it is usually given weekly for weeks, then every second week for a further six doses; it is appropriate to continue dosing every month beyond that. owners can be taught to inject their cats at home (as owners of diabetic animals are taught to do with insulin). because some cats may have sibo, antibiotics such as metronidazole ( to mg/kg po every hours for days) may be warranted. an elevation of folate may arouse suspicion of sibo, but it is appropriate to try antibiotics in a cat failing to respond to enzyme and cobalamin supplementation. concurrent diseases, such as lymphocytic, chronic pancreatitis, or ibd may need to be managed with corticosteroids, or diabetes mellitus with insulin. no studies have assessed specific dietary requirements in cats with epi. most cats respond to appropriate treatment, with a return to normal weight and normal feces. with ongoing therapy, cats can lead normal lives for a full life span. the feline liver is a large, complex organ involved in a variety of essential metabolic, functional, and detoxification processes that can be affected, individually or collectively, by disease or dysfunction. cats have a unique set of liver diseases that occur more commonly in this species compared with the typical diseases that occur in dogs, and these include hepatic lipidosis, feline cholangitis syndrome, and infectious hepatopathies (e.g., fip, flukes, histoplasmosis, toxoplasmosis). , , , , nevertheless, these conditions often present with characteristic clinical, laboratory, and histopathologic changes that are necessary for proper diagnosis and management. the goal of this section is to review the interpretation of clinical and laboratory changes that occur in these feline liver diseases, provide an approach for separating the more common diseases by their clinical footprint, and then discuss therapy of each liver disease based on our current level of understanding of hepatoprotectants, antioxidants, and drugs used for specific therapeutic purposes. the clinical signs of liver disease in cats are often vague and nonspecific; however, recognition of certain clinical and laboratory abnormalities and their association with liver disease can greatly aid the diagnostic process. the most common early clinical signs observed in cats with liver disease are anorexia, lethargy, and weight loss, which are signs present in many (if not most!) feline diseases. , because these early indicators of disease do not point specifically toward liver disease, a delay in diagnosis will occur unless the clinician carefully considers all possibilities and performs other tests to further evaluate the situation. for example, feline hepatic lipidosis is the most common form of liver disease in cats in the united states, united kingdom, japan, and western europe, occurring with a prevalence of nearly % in one study. however, the most common, and often only, clinical sign associated with onset of this condition is anorexia; the signs of serious hepatic disease (especially jaundice and vomiting) do not occur until later (days or weeks) in the course of the disease. , recognition that anorexia in a cat, even for a few days, is a risk factor for development of hepatic lipidosis is essential, and this risk is increased in obese cats. , further, the clinical signs of liver failure develop much more slowly; many cats with hepatic lipidosis present alert and responsive until much later in the course of the disease, thus delaying onset of appropriate therapy. a similar clinical situation exists for the second most common form of liver disease in cats, feline cholangitis syndrome. , , this complex of diseases in the cat can be associated with signs ranging from anorexia and lethargy to vomiting and jaundice, and these signs can vary in severity and prevalence. the key point is that except for development of jaundice, there is no constellation of clinical signs that are classic clinical indicators of liver disease in cats. , as with many feline diseases, the subtle clinical signs of anorexia, lethargy, or inactivity are often the only signs of illness and should be further investigated. there are few changes that occur in the complete blood count that are specific indicators of primary liver disease in cats. the most common finding is the presence of poikilocytes, which are red blood cells with an irregular shape, speculated to be caused by changes in membrane lipids as a result of liver dysfunction. other abnormalities may occur, such as anemia of chronic disease or neutrophilia, but these findings are nonspecific and occur with variable frequency. perhaps the most important reason for obtaining a hemogram is in icteric cats, because this test is essential to help rule out hemolysis as the cause of the hyperbilirubinemia. the serum chemistry profile can be very helpful, but there are several critical points in interpretation of these values that are important to review. the hepatic transaminases (alanine aminotransferase [alt] and aspartate aminotransferase [ast]) are leakage enzymes but do not discriminate among hepatobiliary disorders, nor do they provide an indicator of severity or disease origin. thus although increases in alt may be noted in cats with liver disease, they are also present in a variety of other systemic infectious, inflammatory, neoplastic, and and protein-losing nephropathies can also cause loss of albumin and affect cholesterol, it is essential to evaluate the cat for these problems when interpreting these results. finally, bilirubin metabolism is a critical function of the liver, but interpretation of hyperbilirubinemia requires a careful consideration of bilirubin disposition. hyperbilirubinemia develps because of one of three possible causes: ( ) excessive hemolysis of red blood cells (rbc) (also known as prehepatic icterus)-high bilirubin in the blood stream occurs because of an overload of the mononuclear/phagocyte system with heme pigments from rbc destruction, ( ) hepatic parenchymal disease or insufficiency (also known as hepatic icterus)-resulting in lack of normal bilirubin metabolism in hepatocytes and regurgitation of the pigments into the blood stream when they are not taken up into cells and excreted in bile, and ( ) disease of gall bladder, biliary tract, or pancreatic duct (also known as posthepatic icterus)resulting in obstruction of the bile ducts or loss of bile into the abdomen (duct or gall bladder rupture and bile peritonitis). the bottom line is that in any cat with hyperbilirubinemia, an assessment of the packed cell volume and rbc morphology should be completed to determine whether icterus is caused by hemolysis. once hemolysis is ruled out, then assessment of primary endocrine diseases, including hyperthyroidism, feline heartworm disease, fip, and neoplasia.* alternatively, the cholestatic membrane-associated enzymes alkaline phosphatase (alp) and gamma glutamyltransferase (ggt) are especially useful for recognizing disorders involving biliary or pancreatic ductal components. unlike the dog, these enzymes will only increase modestly in cats, even in severe disease, and there is no glucocorticosteroid or drug induction of the enzymes to influence interpretation. , thus increases in alp in the adult cat represent a release of enzyme from the hepatobiliary tree and should be considered clinically important. both alp and ggt are produced in other tissues than the liver, with the highest ggt activity present in the kidney and pancreas; however, sources other than the liver do not contribute to the activity of these enzymes in health. recent studies of the effects on these enzymes in cats with pancreatitis, cholangitis, extrahepatic bile duct obstruction (ehbdo), and hepatic lipidosis reveal some important characteristics in interpreting increases in these enzymes. first, both alp and ggt are increased in cats with pancreatitis, cholangitis, or ehbdo, because inflammation in the biliary tree also affects the pancreatic ducts (and vice versa, figure - ) , and if the fold increases in these enzymes are similar, the diagnosis is likely one of the three. conversely, in cats with hepatic lipidosis (without concurrent inflammatory disease of the biliary or pancreatic duct system), large increases in alp are observed, but ggt will remain normal or only slightly increased. thus if the increase in alp is to times, while ggt is not increased or is only increased to times, then the likely diagnosis is hepatic lipidosis. [ ] [ ] [ ] other than enzymes on the biochemistry panel, which are of limited value for assessing liver function, there are several key tests that can be used to help assess liver function cats with elevated liver enzymes. these five tests found on most routine biochemistry panels are helpful functional indicators: cholesterol, bilirubin, glucose, albumin, and urea nitrogen (bun). however, none are immune to outside influences on their interpretation, including bilirubin and cholesterol, which are the most liver specific. in cats with severe liver disease or failure, bilirubin levels tend to be quite elevated, while bun, albumin, cholesterol, and glucose concentrations tend to be significantly decreased, reflecting inability to metabolize urea (lack of arginine), inability to produce albumin or cholesterol, and abnormal metabolization of glucose. however, these changes represent severe loss of liver function and thus are not sensitive indicators of liver function because the changes occur quite late in the course of the disease. nevertheless, in cats with elevated liver enzymes and clinical signs of liver disease, these values should be carefully assessed. because gi disease of hepatic failure. in nonicteric cats with severe liver disease or in young cats suspected of having a portosystemic shunt, serum bile acids are the more reliable indicator of hepatic insufficiency. the measurement of serum bile acid concentrations, preprandially and postprandially, is the most reliable, readily available, and sensitive test of hepatic function in nonicteric cats. , that being said, although increases in bile acids are accurate indicators of hepatic insufficiency, the levels cannot be used to assess severity of disease or the type of dysfunction. further, bile acid assays are most effective when paired samples (preprandial-and postprandial) are compared, because single, fasting, or random bile acid samples can result in a falsenegative (normal) result. however, cats will often not eat in the hospital or when they are sick, and this prevents collection of a postprandial sample. however, this does not invalidate the results, because if the result of the single bile acid sample is abnormal, it does reliably indicate liver dysfunction. an alternative to using serum for testing bile acids in cats is urine bile acid analysis. healthy cats excrete a small percentage of conjugated bile acids in the urine ; however, in cats with liver disorders that cause increased serum bile acids (and especially cholestatic liver diseases) a significant increase in urine bile acid excretion occurs. when urine bile acids (uba) were collected to hours after a meal and measured (normalizing the value with urine creatinine: uba/ucr) and compared with serum bile acids in a study of cats with hepatic disease, cats with nonhepatic disease, and normal cats, the results were highly correlated. the utility of the urine bile acid test is that it does not require a paired sample (postprandial test), and it is not as affected as the serum test is by hemolysis or lipemia of the blood sample. normal cats will have an uba/ucr of less than . µmol/mg, while values greater than . are considered evidence of significant hepatic dysfunction. it is well known that the liver plays a central role in coagulation homeostasis and is the single site of synthesis of many coagulation proteins, anticoagulant proteins, and fibrinolytic factors. vitamin k is one of the most common factors found to be inactive or deficient in cats with liver dysfunction, and it is essential for normal functioning of factors ii, vii, ix, and x; protein c and s; and thrombin. insufficient or inactive vitamin k can occur for a variety of reasons, including dietary restriction (e.g., anorexia or diet deficiency), disruption of the enteric microflora that synthesize vitamin k (e.g., chronic antibiotic therapy), diseases causing fat malabsorption (e.g., ibd, exocrine pancreatic insufficiency), ingestion of vitamin k antagonists, or liver dysfunction. for example, in cats with hepatic lipidosis, approximately % will have an increased prothrombin time (pt), % will have an increased partial thromboplastin time (ptt), but % of cats will have increased pivka parenchymal disease versus disease of the biliary tree is completed by evaluating the clinical presentation, laboratory values, and imaging of the biliary tree and abdomen for possible evidence of biliary or pancreatic disease. a urinalysis is also an important part of the minimum database, and it is no different in a sick cat with suspected liver disease. in cats the presence of hyperbilirubinuria is abnormal at any urine concentration, because they do not conjugate bilirubin in their renal tubules. however, like bilirubinemia, presence of bilirubin in the urine can occur because of any of the three possible causes of hyperbilirubinemia: prehepatic, hepatic, and posthepatic; thus further evaluation is necessary once bilirubin is detected. ammonium biurate crystalluria suggests the presence of hyperammonemia, which in the cat is either because of a congenital portosystemic shunt (less common in cats than in dogs) or because of severe, end-stage liver disease resulting in portal hypertension, which is typically caused by cirrhosis or advanced polycystic liver disease. , the most common feline liver diseases are hepatic lipidosis and feline cholangitis syndrome, which are two diseases that often result in development of clinical or biochemical icterus. thus because hyperbilirubinemia is a more sensitive indicator of liver function than bile acids or other liver function tests, the need for further testing is moot. however, there will be circumstances when further assessment of liver function is indicated, and for this, serum bile acids, blood ammonia levels, and urine bile acids may be needed. there are several situations where liver function testing may be indicated, but the most common indications for additional testing would be a cat with persistently elevated liver enzymes of unknown origin, a cat that develops urethral obstruction because of urate stones (suggestive of portosystemic shunting) or a cat with possible polycystic liver disease. one of the oldest tests of liver function, because of its association with development of hepatoencephalopathy, is measurement of blood ammonia levels. however, although this test is the only practical way to diagnose hepatoencephalopathy in dogs, the test has a number of limitations, including differences in ammonia levels between arterial and venous (lower) samples and significant sample handling issues (ammonia is labile and results are affected by improper sample handling or lack of immediate measurement) that make its use difficult in practice. in cats hyperammonemia is even less common than in dogs likely because of their highfunctioning urea cycle pathways ; the assays have not been validated for feline blood in most laboratories, and as such, the test is not recommended as the sole indicator uncommon in cats, the most common causes are neoplasia (primarily of the pancreas, but cholangiocarcinomas can occur) or chronic pancreatitis, which can occur concurrently with cholangitis in cats, resulting in both intrahepatic and extrahepatic cholestasis in some cats. , the bile ducts are affected in cats with chronic pancreatitis, because the feline biliary system and pancreatic duct system merge at the level of the pancreas to form a single duct that empties into the duodenum. thus in cats with either pancreatitis or biliary disease, recent evidence has shown that the inflammation affects both organs. , further, in chronic pancreatitis, either persistent inflammation or development of fibrosis can result in dilation or obstruction of the common bile duct. in cats with chronic ehbdo, the common bile duct will become widely dilated and tortuous, a finding easily seen on abdominal ultrasonography but a problem not easily managed (figures - and - ) . interestingly, the gallbladder is often not enlarged, and may in fact be small in cats with this condition, because the remaining fluid in the gallbladder is white bile (highly concentrated mucinous bile from which the pigment has been resorbed). in addition, variable filling of the gall bladder is a normal phenomenon; thus gallbladder size is not an indicator of ehbdo. (proteins induced by vitamin k antagonists or absence). nevertheless, although pivka is a very sensitive test for abnormalities of vitamin k function, most cats with liver disease that have a normal pt/ptt, but abnormal pivka do not represent clinical evidence of bleeding. in any case, abnormalities in the clotting cascade related to vitamin k deficiency in cats with liver disease are common, whether or not they show evidence of active bleeding. and because the balance of the coagulation system in a cat with liver disease can be disrupted by a procedure that initiates small amounts of bleeding (e.g., a biopsy), all cats with liver disease should be given vitamin k as a precautionary measure before and after invasive procedures, even if the clotting times (pt and ptt) are normal. this may be especially important in cats with hepatic lipidosis, because their vitamin k clotting status is likely to be even more affected by the concurrent anorexia and disruption of enteric microflora. the dose of vitamin k (phytonadione, aquame-phyton [merck, west point, pa.]) used prophylactically is . mg sc, im, or po q h for to days, then weekly until recovered. see box - for a summary of the causes of icterus. cholestasis is the reduction of bile flow, which can occur at any point along the biliary tree; bile production occurs in hepatocytes, and flow is connected to the distal concentrating components (gallbladder and common bile duct) by the bile ductules. thus cholestasis can occur inside the liver's biliary tree (intrahepatic cholestasis) or outside the liver in the gallbladder and common bile duct (extrahepatic cholestasis). intrahepatic cholestasis most often occurs in diseases involving hepatocellular damage, leakage, or swelling, such as infections (e.g., bacterial cholangiohepatitis, toxoplasmosis, fip, or other diseases causing inflammation), infiltrative diseases (e.g., lymphoma), metabolic diseases (e.g., hepatic lipidosis), or diseases causing disruption of architecture (e.g., cirrhosis or severe polycystic disease). intrahepatic cholestasis occurs in zone of the liver lobules (periportal zone); at the level of hepatocytes, canaliculi or bile ductules; and is damaging to cells because of the emulsifying properties of lipid on membrane lipids. however, because the liver has a large reserve capacity, clinical icterus (e.g., jaundice) only occurs in the most severe cases when the liver is affected diffusely. thus severe or persistent intrahepatic cholestasis can serve to perpetuate the inflammation and cell damage if it is not corrected. extrahepatic cholestasis or extrahepatic bile duct obstruction (ehbdo) is less common than intrahepatic cholestasis and is most commonly associated with obstruction of the common bile duct. since gallstones are icterus is the result of cholestasis, and the underlying cause can be either hemolysis or hepatobiliary disease, for which further clinical examination will be needed to determine if rbc destruction or liver disease is occurring. in most hepatobiliary diseases of cats, cholestasis is occurring, but there may be no clinically apparent icterus because the degree of hyperbilirubinemia must be at least to times greater than the normal values to exceed the capacity of the liver to process the excess bilirubin. in cats with hyperbilirubinemia not caused by hemolysis, whether it is clinical or subclinical, there is no need for further evaluation of liver function (e.g., bile acid assays), because bilirubin is a more sensitive indicator of liver function than bile acids. the degree of hyperbilirubinemia does not suggest differentiation of intrahepatic versus extrahepatic cholestasis; however, the presence of acholic feces (white feces) is diagnostic for extrahepatic bile duct obstruction (ehbdo), because lack of stercobilinogen (the brown/black pigment in feces) is only found in cats with complete obstruction of the bile duct. finally, the presence of intrahepatic cholestasis and clinical icterus in a cat indicates a diffuse hepatobiliary disease, such as cholangitis or hepatic lipidosis, as focal liver disease, even if severe, will not cause clinical hyperbilirubinemia because of the tremendous reserve capacity of the liver for bilirubin uptake. portal hypertension is an abnormally high venous pressure in the portal system and is typically caused by increased resistance to portal blood flow. there are potentially three regional causes of portal hypertension: prehepatic (disease in the portal vein itself), hepatic (intrahepatic diseases causing compression or decreased flow), and posthepatic (diseases of the caudal vena cava, right heart or pulmonary vasculature). the most common cause of portal hypertension in the cat is cirrhosis or portal venous thrombosis, because portal vein hypoplasia (formerly known as microvascular dysplasia) is known to occur only in the dog, and the other causes of portal hypertension (budd-chiari syndrome, heartworm caval syndrome, pulmonary hypertension) are rare and more likely to occur in the dog. , in any case, the clinically recognizable effects of portal hypertension are development of ascites (unusual in the cat), acquired portosystemic shunting (reported in cats), and development of hepatic encephalopathy (less common in cats than in dogs, because of their profound ability to handle protein wastes). , , most cats and dogs that develop hepatic encephalopathy (he) secondarily to portal hypertension do so because of reduced liver function (because of portosystemic vascular shunting [pss] or cirrhosis and the acquired shunting that develops). cats can develop another form of chronic he because of hepatic lipidosis, but this is believed to be because of the combination of liver failure and prolonged fasting, resulting in arginine deficiency and impaired ammonia detoxification. portosystemic vascular anomalies, also called portosystemic shunts or portovenous shunts (pss), although less common than in dogs, also occur in cats. these vascular anomalies can be either congenital or acquired, single or multiple in number, and occur as extrahepatic vascular shunts or within the liver itself (intrahepatic shunts). the shunting of blood around the liver is the cause of hepatic atrophy and reduced hepatic function that results in an accumulation of toxins, particularly ammonia that leads to the development of hepatoencephalopathy. the two most common veins that serve as the connection point for the shunting portal venous blood are the caudal vena cava and the azygous. in cats a single, extrahepatic, portocaval shunt is the most commonly reported form, and occurs in % of cats with pss. as in dogs, specific breeds of cats may have pss more commonly, and these include domestic shorthair cats, burmese, siamese, persian, and himalayan breeds. in contrast to dogs, males may be more predisposed to pss than females, but the clinical signs relate to the three body systems most affected: the central nervous system, gi tract, and urinary tract. the most common presenting complaints in cats are weight loss or poor/stunted growth, and dull, bizarre or lethargic behavior, especially after eating. signs of gi disease common in dogs, such as vomiting, diarrhea, or inappetence, are less common in cats, but in one report, % of cats with pss drooled. finally, cats with pss often present with signs of lower urinary tract disease (e.g., hematuria, stranguria, or even obstruction) because of the development of urate uroliths (which are radiolucent, thus difficult to detect). because the most common signs of he are apathy, listlessness, and decreased mental alertness, they are often not recognized specifically as indicative of brain dysfunction but as part of the constellation of signs of the liver disease. however, with progression of the has not been reported. the clinical presentation is typically nonspecific (the most common signs are vomiting, lethargy, and anorexia), and there are no laboratory changes that are suggestive of hepatic neoplasia. thus the diagnosis must be made by identification of disease, other signs will develop, including ataxia, salivation, stupor, or coma. the best and only practical diagnostic test for he is plasma measurement of ammonia levels. however, as previously noted, the test has many technical issues that make its clinical utility in the practice setting difficult at best, and there are few laboratories that have validated ammonia measurement in the cat. cancer of the liver can occur as a primary disease (table - ) or as a result of metastasis of neoplastic disease occurring elsewhere and, most typically, the abdominal cavity. the most common neoplastic infiltration of the liver that is not a primary liver tumor is lymphoma (figures - and - ), followed by visceral mastocytosis. as with many other types of cancer, hepatobiliary neoplasia is most common in middle-aged to older cats, and it is relatively rare, with a reported incidence of . % to . %. benign tumors, such as biliary cystadenoma ( figure - ) , carry a good prognosis if they are amenable to surgical resection. the incidence of metastatic neoplasia (including lymphoma and mast cell tumors) the most common clinical signs are related to spontaneous rupture of the enlarged and friable liver. affected cats may present with lethargy, anorexia, pale mucous membranes, and a heart murmur secondary to anemia. clinical signs of liver disease are usually absent. hepatomegaly and hypotension may also be found. results of routine laboratory testing (mild to marked increases in alt and globulins while alp and ggt are typically normal) and ultrasonographic examination (hepatomegaly, generalized increase in hepatic parenchymal echogenicity) a of the liver may be supportive, but definitive diagnosis relies on histopathologic examination of a liver biopsy. fna of the liver is not helpful because amyloid is rarely detected with this method. hemostasis should be evaluated carefully before any biopsy procedure is planned. the most important differential diagnoses are fip, hepatic lipidosis, and hepatic lymphoma. scintigraphic imaging using i- serum amyloid p component has potential as a noninvasive test. a there is no specific treatment for amyloidosis in cats, so therapy is primarily supportive care (antioxidants, vitamin k, blood transfusion). attention should be paid to identification and control of any underlying chronic inflammatory disease. unfortunately, the long-term prognosis is poor as most affected cats die of intra-abdominal bleeding. survey abdominal radiography is the simplest and most readily available imaging modality to assess structures in the abdominal cavity. radiographs are most useful to assess liver size, will reveal large hepatic masses, and provide evidence of radiopaque masses or other abnormalities in the abdomen. however, the preferred imaging modality used to assess hepatic structures in cats with suspected liver disease is abdominal ultrasonography (aus). the reasons why ultrasonography is a more useful tool for assessment of the liver in cats are numerous, but because feline liver diseases are primarily diffuse, infiltrative, or metabolic diseases that also affect the biliary tree, ultrasonography is the only imaging tool that will give reliable diagnostic information. this widely available diagnostic tool can be helpful in determining liver size and parenchymal echogenicity, in identifying mass lesions, evaluating the biliary tree and gallbladder, quantifying flow (doppler techniques), and identifying vascular anomalies. as with all diagnostic modalities, the skill and experience of the operator is vital to accurate procurement and interpretation of the images. further, it is important to remember that although ultrasonographic images are extremely useful in the clinical evaluation of a cat with possible liver disease, the images themselves do not represent a histologic diagnosis. structural abnormalities by hepatobiliary imaging and subsequent examination of the tissue either by fna or biopsy techniques. historically, amyloidosis has been recognized as primarily a renal disease, especially in abyssinian cats. more recently, cases of hepatic amyloidosis without renal involvement have been diagnosed in siamese and related breeds, as well as in nonpedigreed cats. a, a, a the majority of cases have been described in australia, the united kingdom, and europe. amyloid a is deposited in the liver, probably in response to chronic inflammation in another organ. in the siamese breed, a genetic component may contribute. a the amyloid a protein occurring in the siamese breed differs from that known in the abyssinian breed. a contraindicated in cats, because they may cause a lethal shock reaction. a similar reaction may be seen with penetration of the larger bile ducts or gallbladder with a large-bore biopsy needle, because these tissues have a significant autonomic innervation in the cat that may result in bradycardia and shock following the procedure. , , it is particularly important to recognize this as a risk in cats with ehdbo or dilated bile ducts, and this risk factor reiterates the need for ultrasound examination of the liver prior to making biopsy decisions. nonetheless, owners should be informed of these potential risks, in addition to the risk of bleeding from biopsy sites in any cat undergoing liver sampling. , biopsy techniques liver biopsies, whether they are obtained by needle, laparoscopy, or surgical means, should be taken from a location that represents the primary liver pathology, handled appropriately to ensure accurate interpretation of the sample, and the histopathologic description should be interpreted according to the guidelines set by the wsava standards for clinical and histologic diagnosis of canine and feline liver disease. , guidelines for obtaining and handling surgical biopsies of the liver are reviewed elsewhere and will not be further discussed. because needle aspirates/biopsies, tru-cuttype biopsies, and laparoscopic biopsies are commonly used to obtain liver tissue in cats, the benefits and limitations of each of these techniques will be discussed. as a general rule, the more tissue that can be obtained, the better the pathologist's interpretation of the tissue abnormalities will be. for example, most pathologists believe that at least six portal areas are necessary to make a diagnosis of inflammation liver disease in cats. this will require either a -or -gauge needle size or larger piece of tissue than is obtained with smaller needles or an aspirate. the amount of tissues required to view at least six portal areas is approximately mg, and mg will be required for culture of the tissue. if other analyses of the tissues are considered (e.g., metal analysis), approximately to mg of liver is needed. a typical laparoscopic cup biopsy forceps will provide mg of liver tissue, a -g tru-cut-type biopsy needle provides to mg, and an -g needle biopsy provides only to mg of liver tissue. thus, depending on the clinical circumstances and considered differentials, the best approach for obtaining the needed tissue must be considered prior to planning the procedure. fine-needle aspiration to obtain liver tissue for cytologic examination is commonly performed in cats with liver disease for good reason. the procedure is inexpensive, easy to do, is relatively low risk, and often requires only sedation to complete. further, samples obtained by this method can be diagnostic for hepatic lipidosis, hepatic lymphoma or other round cell tumors, and in for the most common liver diseases of cats (hepatic lipidosis, feline cholangitis syndrome, and neoplasia/ lymphoma), aus examination provides a useful means of obtaining clinical clues and tissue to support or refute the differentials. for example, in cats with hepatic lipidosis, the liver is quite enlarged and typically diffusely hyperechoic, while in cholangitis or other inflammatory diseases, the liver is more often diffusely hypochoic. however, these sonographic findings are very nonspecific and can easily lead to errors in diagnosis if the tissue is not subsequently sampled for confirmation. , thus one of the most important utilities of the aus is the ability to obtain liver tissue (either by aspiration or guided-needle biopsy) and for aspiration of the gallbladder to obtain bile for culture. , these techniques alone have made the aus an extremely important diagnostic tool in the evaluation of liver disease in cats. the diagnosis of most liver diseases requires a histopathologic sample of liver tissue, and this is particularly true in the most common feline liver diseases, which tend to be diffuse diseases affecting the entire liver. cats with one of these diffuse diseases can be sampled randomly using any one of these commonly employed techniques: ultrasound-guided fine-needle aspirates (fna), ultrasound-guided needle biopsy, laparoscopic biopsies, or biopsies obtained surgically. some types of neoplasia (particularly round cell tumors) and vacuolar hepatopathies (hepatic lipidosis) can often be diagnosed by cytology using fna techniques. however, differentiation of liver cell tumors (adenomas and carcinomas) and inflammatory diseases of the liver cannot be diagnosed without a larger sample of tissue and histopathologic examination. , further, even in cats with classic hepatic lipidosis changes, concurrent diseases such as cholangitis or lymphoma can be missed if only fna techniques are employed. thus it is essential to consider that in many liver diseases the lesions, although typically diffuse, may also have focal components; for example, inflammation may be throughout the liver, but fibrosis will be present only in focal areas. thus the results of fna or tru-cut needle biopsies should always be considered in the light of the clinical, laboratory, and ultrasonographic evidence. prior to scheduling a cat for a biopsy, the risk-tobenefit ratio of performing a liver biopsy should always be considered. this is primarily because heavy sedation or anesthesia will be essential in most cats undergoing a liver fna, and for all cats undergoing a liver biopsy (needle or otherwise). in addition to anesthesia risks, the use of automatic spring-loaded biopsy guns to obtain ultrasound-guided biopsies of liver tissue is equipment, the interested reader is referred to several recent reviews on the subject. , to maximize the histopathologic accuracy, biopsies taken at laparoscopy or surgically should be taken from both normal-appearing and abnormal areas in the liver. further, if there is a need to obtain samples from the deeper tissues, the laparoscope can be used to direct a tru-cut needle biopsy to the best location for sampling. one of the major advantages of the laparoscopic technique is that it allows the operator to observe the biopsy sites for excessive bleeding, which is unusual, but if observed can be staunched by using pressure on the site, gelatin coagulation material placement, or electrocautery. with experienced operators, the complication rate for laparoscopy is very low (less than %), and most complications were because of anesthesia, bleeding, or air embolism. finally, although not necessary to have direct visualization to obtain an aspirate of gallbladder bile, laparoscopy allows easy sampling of bile for culture, which is important in all cats with suspected inflammatory liver disease or hepatobiliary disease. once a diagnosis of liver disease is made in the cat, specific therapy for the cause (if available) should be instituted; however, for many feline liver diseases, no specific therapy is available, and thus hepatoprotective therapy is used concurrently to aid in the recovery of the liver from the insult. in this section, therapy of two of the most common diseases of the feline liver will be considered, with a special emphasis on nutritional aspects of treatment, nutraceutical therapy, and the unique needs of cats. the most common liver disease of cats is idiopathic hepatic lipidosis (figures - and - ) , a disease that results in liver failure because of a combination of factors including hepatic lipid accumulation, insulin resistance, fasting, and protein (especially arginine) deficiency. , , , thus, unlike many diseases of the liver, the primary focus of therapy and the essential component for recovery is nutritional support. as in any patient with serious liver disease, initial therapy is always aimed at correction of any fluid or electrolyte abnormalities that may exist, because these may be profound if the cat has been vomiting. in addition, normalization of electrolytes is particularly important in cats that have been anorexic for an especially long time ( to weeks), because refeeding syndrome may be triggered with the initiation of feeding, resulting in sudden drops in potassium, phosphate, and magnesium. although this phenomenon is less common and usually less profound in cats fed enterally versus areas where appropriate, definitive diagnosis of certain infectious diseases (e.g., histoplasmosis). however, even with these relatively straightforward diseases, fna of liver tissue has significant limitations, the most important of which is the failure to accurately identify the primary disease. for example, although it is easy to make a diagnosis of hepatic lipidosis using this technique, a paper recently showed four cats that were incorrectly diagnosed with hepatic lipidosis instead of lymphoma because the fna samples were obtained from areas that did not have lymphoma infiltration. in another study, reviewing the agreement between fna cytologic samples of liver and the histopathologic diagnosis, only % of the cases had overall agreement. thus although cytology of fna samples of liver tissue in cats with diffuse hepatic disease remains a useful first step, it is important for the clinician to carefully interpret the results and discuss the potential limitations of this technique with owners. there are several needle biopsy techniques available for sampling liver tissue, but not all are suitable or safe for use in cats. the menghini technique is one such approach that is not suitable for use in cats, because it is a blind procedure using a large-bore needle that cannot be used with ultrasound guidance. the second option among the needle biopsy techniques that is not recommended for cats is the biopsy gun device. tru-cut biopsy guns are operated by a triggering device that can result in the induction of a lethal vagotonic shock reaction in the cat immediately following the procedure. for most ultrasound-guided liver biopsy procedures, either the manual or, preferably, the semiautomatic tru-cut device is recommended for use in obtaining needle biopsies from cats. as a general rule, the tru-cut device will advance into the liver to a depth of cm; so, it is essential to carefully note the amount of liver tissue available during the ultrasound assessment before advancing the needle for tissue collection. properly obtained tru-cut needle biopsies are a valuable technique for obtaining a representative sample of liver tissue ; however, because of the risk for bleeding or liver fracture with any movement, it is essential that cats be anesthetized for this procedure. laparoscopy is an intermediate step between needle biopsy and surgical laparotomy for obtaining liver tissue for histopathology in cats. , this technique is becoming more widely used as more specialists are trained for this procedure that allows visualization of tissues to be biopsied without opening the entire abdomen. although this technique does require general anesthesia, the limited degree of invasiveness, the large biopsy sample size, and rapid patient recovery make laparoscopy a valuable tool for obtaining liver tissue, and it can be used to obtain biopsies from the spleen, pancreas, kidneys, lymph nodes, or to aspirate the gallbladder. for a detailed discussion of laparoscopic techniques and lipidosis. the echogenicity of the parenchyma is uniformly increased, which is more apparent when compared with other ultrasonographic images presented in this chapter. additionally, the gall bladder is distended. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease. (courtesy dr. randolph baral.) liver gb figure - gross appearance of liver from a cat with hepatic lipidosis. note the pale tan and exaggerated reticular pattern. in most cases, the edges appear more rounded than is evident here. hepatic lipidosis in this cat was secondary to anorexia associated with primary intestinal disease (same cat as in figure - ) . (courtesy dr. randolph baral.) those started on intravenous nutrition, it can be a significant source of morbidity if electrolyte replacement and monitoring are not carefully attended. once the cat is hemodynamically stable, the next step in treatment planning in cats with hepatic lipidosis is re-introduction of nutrition, which must include placement of a feeding tube (box - ). however, because many of these cats are extremely ill and are not good candidates for anesthesia, placement of a nasoesophageal (ne) tube to allow initiation of enteral feeding is often the most appropriate step for the first few days. when administering food through a feeding tube, there are several important points: . the food should be room temperature (not too hot or cold). . the tube should be flushed with water following feeding, to remove any particles of food or medication that may cause the tube to clog. . if the cat is volume sensitive, it is important to carefully calculate how much water is used for flushing the tube, because a significant volume of fluid can be infused, creating a potential fluid overload. if the cat is fluid sensitive, the total amount of fluids (amount in the food, amount added to food if blenderized, and amount of flush) must be determined, and the amount of fluid used in flushes or food preparation may have to be reduced. force feeding is to be strongly discouraged in these sick cats for several reasons: • it is highly stressful and will further increase the stress response and insulin resistance phenomena that are perpetuating the hepatic lipidosis. • it can be dangerous to the cat (aspiration) or operator (scratches/biting). • it is rarely able to meet the necessary nutritional goals set for the patient. • it may induce food aversion, a phenomenon unique to cats, but creating a profound aversion to the chosen food that can be lifelong. although ne tubes are excellent choices for short-term feeding of cats unwilling to eat, there are several disadvantages to their long-term use, including the nasal irritation that occurs, the relative ease with which cats can (and will) remove them, and the need to use liquid enteral diets. thus once the cat is deemed stable enough for general anesthesia, a long-term feeding tube solution is needed, and this typically is either an esophageal (e) tube ( figure - ) or percutaneous endoscopic gastrostomy (peg) tube. , both feeding options are generally well tolerated methods for providing long-term feeding, but e tubes have the advantage of being placed without the need for any specialized equipment, and if complications occur, they are generally easily addressed, because the most common complications are infection at the tube site or premature removal of the tube by the cat. placement of a peg tube, although relatively easy to learn to place, requires having the appropriate endoscopic equipment, and if complications occur as a result of infection or tube removal, more significant morbidity can result. because there is no advantage to placement of peg tubes easiest to use, and are an acceptable choice in most situations. finally, because many cat stomachs are volume sensitive with initiation of feeding, it is very important to start conservatively with small-volume feeding on a more frequent schedule. with prolonged fasting, the stomach volume of a cat with hepatic lipidosis may be reduced dramatically, preventing normal expansion and limiting intake to as little as % of normal. thus to avoid vomiting when feeding, the starting volume may have to be as small as to ml every to hours. a good rule of thumb is to start with estimation of resting energy requirement (rer) ( to kcal/kg is a good estimate of rer), and then attempt to meet % of rer the first day. if no problems are encountered, increase the amount to % of rer the second day, and so on, but during this period, keep the frequency as high as possible (feed four to six meals per day) so that the volume remains relatively small at each meal. once full rer has been achieved with multiple meals per day, the frequency of feeding can be gradually reduced to three to four meals per day. most cats will eventually tolerate three meals per day well, and some can tolerate two meals per day, but this is quite variable and should not be attempted during the first weeks of feeding. in general, most cats with hepatic lipidosis will require tube feeding for a minimum of to weeks before they will show interest in food and begin eating again on their own. the tube should be retained until the cat has been eating on his or her own for at least week or longer and can be maintained for a longer duration if it is being used to administer medications, because cats can eat normally with the e tube in place. the other therapeutic considerations for cats diagnosed with hepatic lipidosis are directed toward dealing with the complications of the disease and reducing the oxidative stress on the liver with hepatoprotective therapy (table - ) . in cats that are vomiting, in cats versus e tubes, placement of e tubes is advocated as the best approach for most practice situations. interested readers are referred to several recent reviews on tube placement for specific details on each method and to chapter . , diet selection is the next step in treatment planning for cats with hepatic lipidosis. in contrast to the belief that animals in liver failure need lower quantities of protein to reduce the workload on the liver, cats with hepatic lipidosis actually need protein to recover. in fact, the work of biourge and coworkers showed that protein was the essential nutrient in reducing hepatic lipid accumulation, was essential to eliminate the negative nitrogen balance, and also appeared to minimize muscle catabolism. further, diets high in protein can improve insulin sensitivity and assist weight loss in recovery from obesity. , conversely, although carbohydrates are a readily available energy source, they are often associated with gastrointestinal distress (diarrhea, abdominal cramping) and hyperglycemia (secondary to the insulin resistance in place as a result of obesity and hepatic lipidosis). thus diets selected for cats with hepatic lipidosis should ideally be high in protein (> % metabolizable energy [me]) and have lower amounts of carbohydrates (< % me), with the remaining calories coming from fat. the diets that best fit this profile are the diets formulated for diabetic cats; however, kitten food, many adult cat foods, and some of the enteral recovery diets have this high protein/low carbohydrate profile. many of the intestinal diets are not higher protein and are higher in carbohydrates, and so would not be the ideal choice. the key to using any of the foods that are not designed for use in a feeding tube is to blenderize them (and if necessary, strain the food) so that the food will easily go through the -or -g feeding tube without clogging it. enteral diets designed for use in feeding tubes are the because the primary starting point of the inflammatory disease in cats is the bile ducts (cholangitis), with inflammation extending to the hepatic parenchyma (cholangiohepatitis) only with time and severity, the term cholangitis syndrome has become the preferred terminology. the disease syndrome has been further classified by the wsava liver diseases group into one of three primary types: neutrophilic or suppurative, chronic lymphoplasmacytic (figures - and - ) , and lymphocytic (non-suppurative). each of the forms appears to behave quite differently clinically as well as in their progression and outcome. in general, cats with the suppurative form of cch typically have an acute onset of illness, which often includes fever, anorexia, and vomiting, and they may become icteric quite quickly (figure - ) . , the nonsuppurative form of cch (lymphocytic form) tends to be a more chronic condition, with affected cats showing nonspecific signs of illness that may include partial anorexia and lethargy, but the signs may wax and wane or are non-progressive. , because of the feline pancreatic and bile duct anatomy, it is common for cats with cch to have pancreatitis and vice versa, and in some cases, cats will also have concurrent ibd; the constellation of the three conditions occurring together is called triaditis. this combination is increasingly recognized in cats, and recent reports suggest from % to % of cats with one syndrome have all three diseases. , , , at this time, the etiology of each of these syndromes and the pathogenesis is not well understood; however, the enteric microflora are presumed to play an important role in the suppurative form, and immune mechanisms are presumed to be the cause of the chronic inflammation found in the chronic nonsuppurative antiemetic therapy is beneficial, because it is imperative that the cat continues to receive some food, and vomiting will complicate this. metoclopramide is often used in cats because of its ready availability and low cost, but it is a very weak antiemetic in cats and thus may not be the best choice. in most cats, the novel nk- receptor antagonist maropitant has been a safe and effective choice. the most commonly used antiemetics in the author's feline practice are maropitant ( mg/kg iv, sc or through the e tube q h), ondansetron ( . mg/kg iv q - h), or dolasetron ( . mg/kg iv, sc q h). in addition to control of vomiting, all cats with hepatic lipidosis should be given vitamin k ( . mg/cat po, sc) daily for a week, then weekly until the cat has recovered, and vitamin b (cobalamin) ( µg/cat sc) weekly for weeks, then monthly until blood values are normal. other vitamins may become deficient, such as some of the b vitamins and vitamin e; however, feeding is likely to rapidly replenish these deficiencies if they exist. this is also likely true of amino acid deficiencies, but supplementation of l-carnitine ( mg/day po) may be beneficial by improving fatty acid oxidation. finally, hepatoprotectant and antioxidant therapy with s-adenosylmethionine (same) ( mg/kg po q h) has been advocated to increase glutathione and may be beneficial in cats with hepatic lipidosis. , , it is important to note that if same is given through the tube (and thus the tablets must be crushed), the dose must be increased by approximately % to allow for the loss of absorption from loss of the enteric coating. because drug metabolism is often impaired in cats with hepatic lipidosis, appetite stimulants, such as mirtazapine, cyproheptadine, and clonazepam, should not be used in cats because dosing and side effects can be unpredictable. benzodiazepine agonist drugs (e.g., diazepam) should be completely avoided in cats with possible lipidosis-induced hepatoencephalopathy, because they will exacerbate the signs and may cause fulminant liver failure. , fortunately, most cats with idiopathic hepatic lipidosis that receive immediate and aggressive therapy and feeding for their disease will recover completely. cats that develop hepatic lipidosis secondary to other serious diseases (e.g., lymphoma) have a much lower chance of complete recovery and often die of their disease or its complications. the most common inflammatory liver disease in the cat is a complex syndrome with multiple subgroups of disease previously termed cholangitis/cholan giohepatitis complex (cch) but currently recognized under the terminology feline cholangitis syndrome. this disease is quite variable in both its presentation and severity, and it may occur as a primary process or secondary to/ concurrent with other diseases (e.g., pancreatitis, ibd). ultrasonographic appearance of liver with lymphocytic/plasmacytic inflammation. note the varying echogenicity throughout the hepatic parenchyma; areas of hypoechogenicity likely reflect inflammatory cell infiltration. the gall bladder is distended; its shape is distorted by pressure from the transducer. (courtesy dr. randolph baral.) liver gb [ mg/kg po q h]), and if pancreatitis is concurrent, pain control with opioid pain relievers (e.g., buprenorphine . to . mg/kg po, sq q - h). if culture is not possible, combination therapy with enrofloxacin ( mg/kg po q h) and metronidazole ( mg/kg po q h) is reasonable. in cats with chronic lymphoplasmacytic forms of cholangitis, management must be tailored to the individual situation and often requires therapy with either immunosuppressive doses of prednisolone ( to mg/kg po q h) or chlorambucil ( mg/m po q d), along with the hepatoprotectants and cholerectics, and concurrent treatment of other diseases (pancreatitis or ibd) that may be occurring. the lymphocytic or lymphoplasmacytic forms of cholangitis may wax and wane in intensity over time, and may require long-term continuous or intermittent therapy to control the disease. there is no specific diet that is recommended for cats with inflammatory liver disease, but protein restriction should not be initiated unless the cat has clear evidence of severe hepatoencephalopathy. the diet should be selected based on other conditions (such as ibd), for which the diet may be more critical in the management. monitoring of serum chemistry values (especially glucose), clotting times, cobalamin levels, and pli/tli concentrations are recommended every few months, as well as careful monitoring of the cbc for all cats on chlorambucil. in all cats with chronic inflammatory liver disease, prior to initiation of immunosuppressive therapy, a careful assessment of the cat for other possible causes of inflammation should be completed (box - ). as in dogs, if a cat with pss can have surgical closure of the shunting vessel (ligation, placement of an ameroid constrictor, intravenous coiling), the long-term forms. however, whether or not these syndromes are related, a continuum of disease or completely different diseases remains undetermined. once a definitive diagnosis is obtained by histopathology of the liver tissue and culture of bile, treatment can be tailored to needs of the cat. cats with the more aggressive suppurative form of cholangitis often require intravenous fluid therapy, antibiotic therapy (based on results of culture whenever possible), and supportive therapy (antiemetics, vitamin k , hepatoprotectants such as same [ important antioxidant and stabilizes membrane functions • n-acetylcysteine-a precursor to glutathione and antioxidant, also improves tissues oxygen delivery • ursodeoxycholic acid (tertiary bile acid)-used to replace hepatotoxic, hydrophobic bile acids and increase bile flow • silymarin (milk thistle)-a free radical scavenger and anti-inflammatory/antifibrotic agent • vitamin e-an antioxidant and antiinflammatory vitamin* although few clinical trials of these nutraceuticals have been performed in feline liver disease, a few studies have recently appeared showing that same, ursodeoxycholic acid, silymarin, and n-acetylcysteine all are hepatoprotective, have few adverse side effects, and may be beneficial in many types of liver disease in cats. , , , , feline liver disease is a common problem that requires careful consideration of the presenting complaint, clinicopathologic findings, imaging results, and, if available, histopathologic interpretation to be able to provide an accurate diagnostic and therapeutic plan. a variety of insults can be responsible for liver dysfunction or failure, but hepatic lipidosis and feline cholangitis syndrome remain the most common reasons for cats to present prognosis for function and quality of life is generally very good. however, even if surgical correction is anticipated, and especially if surgical correction is impossible or not completely successful, medical management of he is indicated. see table - for the basic therapeutic approach to medical management of cats with he resulting from pss. because hepatocytes, by their position in the body between the gi tract and rest of the body, as well as their critical role in metabolism and detoxification, are uniquely susceptible to oxidative injury and reactive intermediates of metabolism, they must be able to protect themselves. the natural defenses of the liver include superoxide dismutase and glutathione, free-radical scavengers such as vitamin e and ascorbate, and other prosurvival signaling pathways that are controlled by hormones and growth factors. however, in injury or overwhelming infection or inflammation, the natural defenses of the liver can be overwhelmed, and then it is essential for medicines and nutraceutical therapy to be included in the treatment plan to help reduce inflammation and fibrosis, protect against oxidant injury, and enhance bile flow. the cytoprotective agents most commonly used in liver diseases to assist in these processes (table - ) are: • s-adenosylmethionine (same)-a precursor in the synthesis of glutathione and an important methyl donor to dna and proteins, is an with icterus or liver failure. therapy must be tailored to the individual, but nutritional support is critical in the management of hepatic lipidosis, and appropriate supportive therapy with hepatoprotectants may be crucial to treatment success. resorption from that space. effusion accumulation is therefore correlated to increased capillary hydrostatic pressure, widening of the oncotic pressure gradient, increased endothelial permeability, increased interstitial hydrostatic pressure, or loss of effective lymphatic drainage or a combination of these factors. , , peritonitis of any cause results in vascular dilation, increased capillary permeability, and the migration of inflammatory cells into the peritoneum in response to immunomodulatory mediators. the inflamed peritoneum becomes a freely diffusible membrane, allowing a massive outpouring of fluid and plasma proteins from the circulation. , ascites is not commonly seen in practice; one study recognized ascites in only three cats out of admissions to an american veterinary teaching hospital, but the prevalence may be greater in primary care practice. in that study, dilated cardiomyopathy (dcm) was the most common disease associated with peritoneal effusion; however, dcm was diagnosed in most of these cats before , when taurine deficiency was found to be a primary cause of this form of cardiomyopathy in cats. neoplasia was the most common cause after . feline infectious peritonitis (fip) was by far the most common cause of feline ascites diagnosed over a -year period at the feline centre at the university of bristol, comprising % of all cats with recognized ascites. cats with ascites usually present with nonspecific clinical signs, such as anorexia or lethargy. the owners may present the cat because they recognize abdominal enlargement ( figure - ), but in many cases, owners perceive this as weight gain. clinicians should be aware that sudden weight gain in a chronically underweight cat may be because of fluid accumulation (which can be intrathoracic fluid if ascites is not present), particularly if muscle mass seems reduced. ascitic cats presenting subsequent to trauma may have intraabdominal hemorrhage or urinary tract rupture. fever in a young ascitic cat will often suggest fip, and cats with fip may or may not be jaundiced. presence of jugular distention or even a jugular pulse can suggest right-sided heart failure. a palpable fluid thrill can help to distinguish ascites from other causes of abdominal enlargement, such as organomegaly, abdominal masses, bladder distention, abdominal wall weakness, obesity or, occasionally, accumulations of gas within the abdominal cavity (table - ) . recognizing a fluid thrill involves gently tapping one side of the abdominal wall with the fingers of one hand while feeling for a sensation of fluid movement the peritoneum is the serous membrane lining the abdominal cavity, as well as covering the organs of the abdomen. it comprises a single layer of squamous mesothelial cells resting on a deeper layer of loose connective tissue. the layer of peritoneum that lines the inner surface of the abdomen is called parietal peritoneum; the abdominal organs are lined by visceral peritoneum. the total surface area of the peritoneum is one to one-and-ahalf times that of the total cutaneous area of the body. , the peritoneal cavity contains a small amount of fluid (less than ml/kg body weight) that reduces friction between the abdominal organs as they slide over each other. the fluid is a pure transudate and contains solutes in the same concentration as serum (box - ). this fluid is absorbed from the abdominal cavity predominantly through lymphatic vessels lying beneath the mesothelial basement membrane on the surface of the diaphragm. lymphatic drainage occurs predominantly to the sternal lymph nodes. , ascites is the abnormal effusion and accumulation of fluid in the abdominal cavity. fluid exchange across the capillary bed is determined by starling forces, that is, the balance between hydrostatic pressure, which causes transudation of fluid out of blood vessels, and the colloid osmotic pressure, which acts to retain fluid within blood vessels. the amount of peritoneal fluid is therefore determined by the balance of these, as well as vascular permeability, with excess fluid drained by the lymphatic system. accumulation of fluid within a body cavity results when the rate of filtration of fluid into a space is greater than the rate of fluid routine laboratory findings are usually nonspecific but may provide clues to the underlying cause of ascites. for example, neutrophilia may point towards septic peritonitis but can also occur with fip; most cats with hemoperitoneum are anemic at presentation ; uroperitoneum often results in azotemia and electrolyte abnormalities; hypoglycemia may reflect sepsis with septic peritonitis, and a recent study recognized % of cases of septic peritonitis had ionized hypocalcemia ; elevated liver enzymes may be associated with inflammatory, infectious or neoplastic hepatopathies including fip; elevation of serum globulins occurs in many cats with fip but can also be associated with neoplasia or septic peritonitis; and a finding of hypoalbuminemia (which can cause a pure transudate) should prompt for an assessment of urine protein : creatinine ratio to assess if there is renal protein loss. imaging may be required to confirm the presence of fluid as well as to aid in diagnosis of the underlying cause. radiographic findings can vary greatly depending on the amount of abdominal fluid present and the underlying etiology. loss of normal detail or presence of a "ground glass" appearance to the abdominal cavity is suggestive of the presence of fluid (figures - and - ). very young, thin or dehydrated cats may also have a loss of detail that can mimic the presence of fluid. ultrasonography of the abdomen (figures - and if a large volume effusion causes discomfort because of abdominal distention, a three-way stopcock may be used so large volumes can be drained from one puncture (figure - ) . however, removal of large volumes of ascitic fluid can be detrimental, because it may prevent the subsequent reabsorption of valuable protein and/or red blood cells; the resulting reduction in intraabdominal pressure may encourage further accumulation of fluid; and rapid removal of large volumes can lead to fluid shifts causing cardiovascular collapse. fluid can be collected into ethylenediaminetetraacetic acid (edta) tubes (for total nucleated cell count, packed cell volume, total protein, and cytology), serum tubes (for biochemistry, such as albumin, bilirubin, creatinine, potassium, triglyceride, glucose, lactate, and lipase), sterile tubes for culture, and/or other tubes for effusion-specific tests such as pcr. samples should be prioritized according to the volume of fluid available and to the suspected underlying disease process. initial assessment of fluid retrieved is made on the basis of color and protein concentration, and much information can be gleaned from this simple assessment, even before cell numbers and types are assessed. although this brief, initial assessment is useful to refine the differential diagnoses, a thorough assessment based on underlying etiology and pathophysiology is required for definitive diagnosis and therefore appropriate management (table - ) . ascitic fluid, classified according to its pathophysiologic cause, can be divided into transudates, modified transudates, exudates (septic or nonseptic), or effusions (chylous or hemorrhagic). , can allow the detection of even very small volumes of fluid. it also enables evaluation of the size and structure of intraabdominal organs, such as the liver and spleen, which can help determine the underlying cause of ascites. abdominocentesis confirms the presence of abdominal fluid (in cases of low-volume effusion) and assessment of the fluid is required to diagnose the underlying cause of ascites. most cats tolerate abdominocentesis without sedation and the cat can be held in a standing position or in lateral recumbency (whichever is more comfortable for the cat and familiar to the clinician). the abdomen is clipped and aseptically prepared. a -to -gauge butterfly needle may be used with a -to -ml syringe. in cases with low-volume effusion, ultrasonography can help to guide fine needle aspiration from small pockets of abdominal fluid. diagnostic peritoneal lavage can be used if ultrasound-guided aspiration is unsuccessful. for this procedure, to ml/kg of warmed, sterile fluid is infused into the abdomen over to minutes after aseptic preparation of the site. the cat is gently rolled from side to side or allowed to stand; gentle massage of the abdomen also helps distribute the fluid. the fluid is allowed to dwell for a minimum of to minutes before aseptic preparation is repeated before paracentesis. no attempt is made to remove all the fluid. it must be remembered that, since the recovered fluid has been diluted by this procedure, cell counts and biochemical analyses will be affected. transudates are a consequence of altered fluid dynamics. protein-poor transudates (commonly referred to as pure transudates) form predominantly as a result of severe hypoalbuminemia, which causes a lowered colloid osmotic pressure. since there is no change in endothelial or mesothelial permeability, as fluid accumulates, there is no concurrent cell leakage; so, there is a decrease in the cell count through a dilutional effect. consequently, transudative effusions are typically clear and colorless. , , other pathologic causes of proteinpoor transudates include cirrhosis, lymphatic obstruction, and noncirrhotic portal hypertension (presinusoidal and sinusoidal). since hypoalbuminemia is the most common cause of transudates, serum albumin concentrations must be measured to guide further diagnostics. if the serum albumin concentration is normal (or only minimally decreased), then radiographs, abdominal ultrasonography, and/or echocardiography are indicated to assess cardiac function and for urinary bladder rupture. one review of feline ascitic cases found % of effusions were protein-poor transudates, of which % were the result of hepatic failure or primary renal disease. modified transudates can result from increased hydrostatic pressure within the postsinusoidal vessels of the liver secondary to right-sided congestive heart failure (e.g., tricuspid insufficiency) or potentially from mass lesions (such as neoplastic masses) obstructing blood flow from the hepatic vein or caudal vena cava into the right side of the heart. the increase in hydrostatic pressure within the vessels of the liver causes a protein-rich fluid to leach out of the liver into the abdominal cavity. since cell membrane permeability does not change, cells do not accumulate in the effusion. modified transudates can also result from increased vascular permeability in the early stages of an inflammatory process, in which case cellularity will be increased. modified transudates were described as the most common type of ascitic effusion identified in cats in one study, with most being resulting from neoplasia and congestive cardiac failure; however, this study partially included cases prior to , when right-sided heart failure associated with dilated cardiomyopathy (dcm) was prevalent. the recognition of the role of taurine deficiency in this condition and the subsequent addition of this amino acid to feline diets now means that right-sided heart failure is only rarely encountered as a cause of ascites in cats. exudates are a consequence of altered mesothelial and/ or endothelial permeability. this permeability results from a cytokine-mediated inflammatory response of any underlying cause (e.g., infectious, neoplastic, immune mediated). exudates have high protein and moderate to high cell concentrations and are classified as nonseptic or septic. exudates are often primarily composed of neutrophils. nondegenerate neutrophils (and the absence of organisms) points to a nonseptic exudate (mostly fip but also neoplasia). fip is the most common cause of exudative effusion in cats and was the most common cause of feline ascites diagnosed over a -year period at the feline centre at the university of bristol. the presence of neoplastic cells rules in neoplasia, but the absence of such cells does not rule out this diagnosis since many cases of neoplastic ascites are not associated with exfoliated neoplastic cells. other causes of nonseptic exudates include pancreatitis, lymphocytic cholangitis, and viscus rupture, such as the gall bladder or urinary bladder. degenerate neutrophils typify septic exudates (i.e., septic peritonitis), and their presence should instigate investigation for causes of infection (mostly leakage of gastrointestinal contents). chylous effusions appear as milky or pink opaque fluid, and small mature lymphocytes initially predominate in cell counts. after drainage, more macrophages and nondegenerate neutrophils may be found. chyle is typically classified as an exudate, but its physical characteristics can be consistent with a modified transudate (protein content between and g/l); biochemical analysis of triglyceride and cholesterol levels in the fluid are required to confirm the diagnosis. pseudochylous effusions resemble true chyle both in appearance and cytology but do not contain fat. similar conditions result in both chylous and pseudochylous effusions. chylous abdominal effusions are rarely reported in the cat and only accounted for % of cases of ascites in one study. the described causes of chylous ascites in cats are predominantly neoplastic. in a series of nine cats, chylous ascites was associated with nonresectable abdominal neoplasia in four cases (i.e., hemangiosarcoma and paraganglioma), with intestinal and mesenteric lymphoma in two cases and lymphangiosarcoma of the abdominal wall in another. one described case in a -year-old cat was thought to be because of fip. figure - shows an ultrasonographic image of a cat with chylous abdominal effusion associated with pancreatitis. other potential causes include right-sided congestive cardiac failure, steatitis (inflammation of fat), biliary cirrhosis, and lymphangiectasia. hemoperitoneum in companion animals is categorized as traumatic or spontaneous. traumatic hemoperitoneum is further divided into blunt causes of trauma (i.e., motor vehicle accidents and high-rise falls) and penetrating trauma (i.e., gunshot wounds and bite wounds). , inadvertent splenic aspiration, venipuncture, or acute severe hemorrhage should be suspected if the cytology is consistent with peripheral blood including platelets but without erythrophagocytosis or if the blood clots readily. when there is no history of trauma, coagulopathy or spontaneous rupture of a vascular neoplasm should be considered. in one study of feline cases of spontaneous hemoperitoneum, cases ( %) were associated with hepatic pathology such as neoplasia, necrosis, and amyloidosis. in another study of cases of spontaneous hemoperitoneum, % ( of ) of cats had abdominal neoplasia, and % ( of ) had non-neoplastic conditions. cats with neoplasia were significantly older and had significantly lower packed cell volumes (pcvs) than cats with non-neoplastic disease. hemangiosarcoma was the most often diagnosed neoplasm ( of , %), and the spleen was the most common location for neoplasia ( of , %). coagulopathies ( of , %) and hepatic necrosis ( of , %) were the most common causes of non-neoplastic hemoperitoneum. other nonneoplastic causes of hemoperitoneum include ruptured bladder, hepatic rupture secondary to hepatic amyloidosis, gastric/duodenal ulcer, hepatic hematoma, hepatitis, perinephric pseudocyst, feline infectious peritonitisinduced liver rupture, and feline infectious peritonitisinduced nephritis. , the prognosis of cats with spontaneous hemoperitoneum is poor. in two studies, only approximately % of cases survived to be discharged from hospital. , median survival time for cats that were discharged in one of those studies was days (range, to days). feline infectious peritonitis (fip) comprised % of cats with recognized ascites over a -year period at the feline centre at the university of bristol, and, as a rule of thumb, when ascites is recognized in a younger cat, fip should be considered the major rule-out. the abdominal effusion found with fip is typically straw to golden yellow (although the color can be very variable, for example, chyle may be present), may contain fibrin clots, and has a high protein concentration. the total protein content is greater than g/l and often greater than g/l, with globulins comprising % or more. one study described an effusion with total protein greater than g/l as % specific, % sensitive, and having a . positive predictive value to diagnose fip. the rivalta test evaluates the fluid's globulin content, and was found to be very sensitive but only % specific; this test is performed by adding one drop of acetic acid ( %) to ml of distilled water. this fluid is mixed thoroughly, and then one drop of effusion is gently placed on the surface of the mixture. if the drop stays at the top of the fluid or slowly floats to the bottom, the test is considered to be positive. this test can give inaccurate results if inappropriate technique is used or if there is a significant temperature difference between the fluid sample and the acetic acid solution. a positive rivalta test can result from lymphosarcoma, septic, or fip effusions; these can be distinguished by cytology and culture. immunofluorescence staining of coronavirus antigen in macrophages had a positive predictive value of . but a negative predictive value of . . the potential clinical presentations, diagnosis, and management of fip are covered in detail in chapter . one study found neoplasia to be the most common cause of ascites in cats, and neoplasia should be considered the major rule-out in older cats with ascites. the effusion from cats with ascites resulting from neoplasia may be a modified transudate, resulting from compression of hepatic veins or the caudal vena cava, or metastases to the peritoneum; hemorrhage from neoplasia can cause hemoperitoneum; chylous effusions may result from reduced lymphatic drainage or rupture of lymphatic vessels; and raised vascular permeability caused by neoplastic infiltration can result in an exudative effusion. carcinomas, mesotheliomas, and discrete (round) cell neoplasms (e.g., lymphoma, mast cell tumors, malignant histiocytosis) exfoliate cells into effusions more readily than sarcomas, and of these, lymphosarcoma is the most common malignancy of cats. cytology of ascitic fluid reveals neoplastic cells in less than a quarter of cases; so the absence of such cells does not rule out a diagnosis of neoplasia. in these circumstances, the diagnosis may be achieved by ultrasound-guided fine-needle aspiration of affected organs, or even biopsy samples obtained at laparotomy. the specific approaches will depend on the specific neoplasia diagnosed. exudates caused by septic inflammation usually result from bacterial contamination of the peritoneal cavity secondary to gastrointestinal tract leakage or penetrating wounds associated with trauma. gastrointestinal tract leakage may occur as a result of ulceration associated with neoplasia or inflammatory disease or as a result of penetration of a sharp object ingested (such as a toothpick), it can also occur subsequent to prior abdominal surgery. , , , primary septic peritonitis in which no apparent cause can be identified has also been described in cats. septic exudates are usually yellow to tan in color, with yellow particulate matter and are foul-smelling. microscopically, the fluid is characterized by the presence of degenerate neutrophils and bacteria. bacteria are often seen intracellularly within neutrophils. the condition is associated with high morbidity and mortality rates, with survival rates reported between % and %. , , , , the history and clinical signs are often vague and nonspecific but can include abdominal pain, vomiting, lethargy/depression, and anorexia. abdominal pain is an inconsistent finding, being recognized in only % of cats in one study and % in another. some cats may have an inappropriately low heart rate. , hematologic and serum biochemistry findings are also inconsistent; neutrophilia with a left shift may be present, as may neutropenia or a normal neutrophil count. similarly, cats may be hypoglycemic, hyperglycemic, or normoglycemic, and they may be hypoalbuminemic. , , one study recognized ionized hypocalcemia in % of cats with septic peritonitis at the time of diagnosis, and another suggested hyperlactatemia, when present, may be associated with a poorer prognosis. radiographic findings are usually typical of ascites of any cause, but presence of pneumoperitoneum in a cat that has not undergone recent surgery may suggest the presence of gas-forming bacteria or rupture of an abdominal viscus and warrants immediate surgical intervention. ultrasonography does not directly aid the diagnosis of septic peritonitis. exploratory laparotomy to determine and correct an underlying problem, such as full-thickness gastrointestinal perforation (often requiring partial resection) is required, as is copious abdominal lavage with sterile, warmed fluids ( figure - ). there are no statistically significant survival differences between postsurgical primary closure, open peritoneal drainage, or closed suction drainage postsurgical lavage; however, a trend toward a higher survival rate has been seen in cats treated with primary closure. , treatment also involves antibiotics, initially parenterally, based on culture and sensitivity findings. consistent with intestinal contents, most bacteria recognized are gram-negative aerobes, such as e. coli or enterobacter spp., but mixed infections are usually found. , anaerobes seem more common in cats with primary septic peritonitis, which perhaps suggests these cases may result from healed over-bite wounds into the abdomen. amoxicillin/ clavulanate would be an appropriate empirical choice of figure - fulminant peritonitis associated with gastrointestinal perforation. in this case, the effusion volume was low but the high degree of serosal inflammation is evident. antibiotics while awaiting sensitivity results. there are no definitive guidelines for duration of antibiotic treatment; the author uses extended treatment courses of to weeks. supportive care with intravenous fluids to maintain fluid and electrolyte balances is also required perioperatively. bile peritonitis is infrequently reported in cats but has been recognized in association with gunshot or motor vehicle trauma, with biliary obstruction from gall stones , and subsequent to percutaneous ultrasoundguided cholecystocentesis in a cat with infectious cholangitis. concurrent bacterial infection was recognized in each case; this increases severity of inflammation and worsens the prognosis, although full recovery was achieved in most reported cases. , , bile peritonitis has the potential to result in small-volume effusions; so, if abdominocentesis does not yield a sample of effusion but bile peritonitis is high on the differential list, then diagnostic peritoneal lavage is appropriate. since repair of or removal of the gall bladder and abdominal lavage are required, exploratory laparotomy is an appropriate means to diagnose this condition. management should be considered as for septic peritonitis of other causes. trauma, including blunt abdominal trauma, urethral catheterization, and bladder expression, is the most common cause of uroperitoneum in cats. it is also recognized as a complication of ureteral surgery. the bladder is the most frequent site of urine leakage after blunt abdominal trauma, whereas the urethra is most commonly injured following catheterization. cats with ruptured bladders may still have a palpable bladder and the ability to urinate. common historical complaints are anuria ( . %) and vomiting ( %). azotemia is a common finding, and hyperkalemia is seen in around % of cases. drainage of urine from the peritoneal cavity seems to improve patient stabilization. morbidity and mortality depended largely on the severity of associated injuries. regardless of the site of injury or the cause of uroabdomen, the first goal of treatment is patient stabilization. isotonic replacement fluids are used for initial resuscitation. treatment of hypovolemic shock, if present, is the first order of fluid therapy. after fluid resuscitation, drainage of urine from the abdomen should be established. continuous passive drainage of the urine is necessary for stabilization and allows effective diuresis to occur. indwelling catheterization of the urinary bladder is recommended to keep the bladder decompressed and reduce urine flow into the abdominal cavity in patients with bladder and proximal urethral injury. if the urethra is traumatized and a catheter cannot be placed, prepubic tube cystostomy may be necessary to achieve temporary urinary diversion. the decision to treat the uroabdomen patient surgically or conservatively should be based on the location and severity of the underlying injury, the condition of the patient at presentation, and the patient's response to initial stabilization. , congestive heart failure has become an uncommon cause of ascites in cats since the late s/early s, from which time dilated cardiomyopathy has been largely eradicated. , ascites does still result from rightsided congestive heart failure in conditions such as tricuspid insufficiency, arrhythmogenic right ventricular cardiomyopathy, myocardial fibrofatty infiltration, or restrictive cardiomyopathy. , concurrent pleural effusion or pulmonary edema is often, but not necessarily, present with cardiac induced ascites. a heart murmur is not necessarily noted. noting a jugular pulse or thrill is helpful diagnostically, if present. the ascitic fluid is typically a modified transudate, but a chylous effusion is also possible. cardiac diseases are covered in chapter . in some cases, hepatic lipidosis has been reported to cause ascites, particularly in association with pancreatitis. these cats are often hypoalbuminemic, with the possibility of intravenous fluid therapy contributing to the ascites by raising hydrostatic pressure. other liver diseases which can result in ascites include lymphocytic cholangitis, , neutrophilic cholangitis, cirrhosis, necrosis, neoplasia, and suppurative cholangiohepatitis. portosystemic shunts in cats rarely result in ascites, compared with dogs. hypoalbuminemia and hepatic failure result in transudates; portal hypertension and cirrhosis cause higher protein ascites because of raised capillary hydrostatic pressure causing leakage of high protein lymph. hepatopathies are covered in detail elsewhere in this chapter. gold references . adamama-moraitou kk, rallis ts, prassinos nn et al: benign esophageal stricture in the dog and cat: a retrospective study of cases chronic oesophageal foreign body in a cat use of a biodegradable self-expanding stent in the management of a benign oesophageal stricture in a cat suspected clindamycinassociated oesophageal injury in cats: five cases feline gastrointestinal foreign bodies a comparative study evaluating the esophageal transit time of eight healthy cats when pilled with the flavorx pill glide versus pill delivery treats primary esophageal squamous cell carcinoma in a cat risk factors and outcome of bougienage for treatment of benign esophageal strictures in dogs and cats: cases hiatus hernia in a cat megaesophagus in a -monthold cat secondary to a nasopharyngeal polyp post anaesthetic oesophageal stricture in the cat helicobacter species detection and histopathological changes in stray cats from niterói, brazil gastric emptying in cats using foods varying in fiber content and kibble shapes intestinal obstruction by trichobezoars in five cats feline gastrointestinal foreign bodies ultrasonographic findings references association of intestinal disorders in cats with findings of abdominal radiography single enterotomy removal of gastrointestinal linear foreign bodies sacrococcygeal agenesis association and anal atresia in mixed breed cats quinolone antibiotics in the treatment of salmonella infections radiographic, ultrasonographic, and endoscopic findings in cats with inflammatory bowel disease of the stomach and small intestine: cases ( - ) laparotomy for gastro-intestinal biopsies hepatic small cell lymphosarcoma in four cats intestinal leiomyosarcoma in a cat intestinal obstruction by trichobezoars in five cats conservative versus surgical management of gastrointestinal linear foreign bodies in the cat bacterial overgrowth associated with a naturally occurring enteropathy in the german shepherd dog feline immunodeficiency virus integration in b-cell lymphoma identifies a candidate tumor suppressor gene on human chromosome q beaver bv: disorders of behavior feline gastrointestinal foreign bodies intussusception in cats rapid detection of enterohemorrhagic escherichia coli by real-time pcr with fluorescent hybridization probes chlorambucilinduced myoclonus in a cat with lymphoma megacolon in the cat recto-vaginal fistula and anal imperforation in a cat: surgical treatment a systematic overview of chemotherapy effects in indolent non-hodgkin's lymphoma bright rm: gi surgery surgery of the digestive system atresia ani and urethrorectal fistula in a kitten idiopathic inflammatory bowel disease in dogs and cats: cases an unexpected bacterial flora in the proximal small intestine of normal cats effects of oral administration of metronidazole on small intestinal bacteria and nutrients of cats comparison of the bacterial flora of the duodenum in healthy cats and cats with signs of gastrointestinal tract disease tyzzer's disease a systematic overview of chemotherapy effects in b-cell chronic lymphocytic leukaemia outcome of cats with low-grade lymphocytic lymphoma: cases chronic inflammatory and non-inflammatory diseases of the gastrointestinal tract in cats: diagnostic advantages of full-thickness intestinal and extraintestinal biopsies radiology and sonography of the digestive system small intestinal adenocarcinoma in cats: cases ( - ) gram-negative bacterial infections radiological identification of nonopaque intestinal foreign bodies chronic inflammatory bowel diseases, etiopathogeny, diagnosis, bull acad vét contribution to the study of feline inflammatory bowel disease: cases ( - ) normal parameters in abdominal radiology of the dog and cat pharmacokinetics and suggested oral dosing regimen of cisapride: a study in healthy cats intussusception in dogs and cats: a review of thirty-six cases low-grade alimentary lymphoma: clinicopathological findings and response to treatment in cases characterisation of the signalment, clinical and survival characteristics of cats with mast cell neoplasia feline lymphoma in the post-feline leukemia virus era adenomatous polyps of the duodenum in cats: cases gastrointestinal obstruction vaccine site-associated sarcoma and malignant lymphoma in cats: a report of six cases strongyloides species diagnostic parasitology georgis' parasitology for veterinarians georgis' parasitology for veterinarians demonstration of ollulanus tricuspis in the stomach of domestic cats by biopsy comparison of conventional coproscopical methods and commercial coproantigen elisa kits for the detection of giardia and cryptosporidium infections in dogs and cats recent investigation on the prevalence of gastrointestinal nematodes in cats from france and germany cestodes of dogs and cats in north america first record of natural tritrichomonas foetus infection of the feline uterus gastric adenocarcinoma and chronic gastritis in two related persian cats helminth and noncoccidial protozoan parasites of the gastrointestinal tract toxoplasmosis and neosporosis toxoplasmosis and other intestinal coccidial infections in cats and dogs intestinal nematodes: biology and control kirk's current veterinary therapy xiv single-tube nested pcr for detection of tritrichomonas foetus in feline feces efficacy of ronidazole for treatment of feline tritrichomonas foetus infection use of a commercially available culture system for diagnosis of tritrichomonas foetus infection in cats experimental infection of cats with tritrichomonas foetus host responses to cryptosporidium infection acute renal failure in four cats treated with paromomycin documentation of in vivo and in vitro aerobic resistance of feline tritrichomonas foetus isolates to ronidazole prevalence of and risk factors for feline tritrichomonas foetus and giardia infection assessment of reproductive tract disease in cats at risk for tritrichomonas foetus infection prevalence of tritrichomonas foetus infection in cats with diarrhoea in the uk diseases of the small intestine prevalence of enteric zoonotic organisms in cats giardia infection in cats evaluation of fenbendazole for treatment of giardia infection in cats concurrently infected with cryptosporidium parvum kirk's current veterinary therapy xiv update on the diagnosis and management of toxoplasma gondii infection in cats cryptosporidium infections in cats and dogs activity of toltrazuril and diclazuril against isospora species in kittens and puppies comparison of direct immunofluorescence, modified acid-fast staining, and enzyme immunoassay techniques for detection of cryptosporidium spp in naturally exposed kittens diarrhea in kittens comparison of direct immunofluorescence, immunoassays, and fecal flotation for detection of cryptosporidium spp. and giardia spp. in naturally exposed cats in northern california animal shelters determining the zoonotic significance of giardia and cryptosporidium in australian dogs and cats the biology and control of giardia spp and tritrichomonas foetus neurotoxicosis in cats receiving ronidazole gastrointestinal protozoal infections diseases of the intestines diseases of the stomach prevalence of enteric zoonotic agents in cats less than year old in central new york state survey of cats for strongyloides felis efficacy of giardia vaccination in the treatment of giardiasis in cats tritrichomonas foetus infections in surveyed pet cats the cat: diseases and clinical management tritrichomonas foetus: a new agent of feline diarrhea prevalence of cryptosporidium, giardia and isospora species infections in pet cats with clinical signs of gastrointestinal disease histologic features associated with tritrichomonas foetus-induced colitis in domestic cats acute pancreatitis in cats with hepatic lipidosis pancreatic atrophy and fibrosis associated with eurytrema procyonis in a domestic cat comparisons between cats with normal and increased fpli concentrations in cats diagnosed with inflammatory bowel disease pancreatolithiasis and pancreatic pseudobladder associated with pancreatitis in a cat acinar cell carcinoma of the pancreas in a cat laparotomy for gastro-intestinal biopsies enteric bacteria: friend or foe? serum feline pancreatic lipase immunoreactivity concentration and seroprevalences of antibodies against toxoplasma gondii and bartonella species in client-owned cats polycystic kidney and liver disease in cats multiple recurrent pancreatic cysts with associated pancreatic inflammation and atrophy in a cat pancreatic paraneoplastic alopecia in three cats exocrine pancreatic insufficiency in a cat cholecystoenterostomy for treatment of extrahepatic biliary tract obstruction in cats: cases retrospective evaluation of partial parenteral nutrition in dogs and cats pancreatic cyst in a cat prospective evaluation of laparoscopic pancreatic biopsies in healthy cats prevalence and histopathologic characteristics of pancreatitis in cats differential effects of experimentally induced chronic pancreatitis on neuropeptide immunoreactivities in the feline pancreas relations between pancreatic enzyme outputs and malabsorption in severe pancreatic insufficiency histologically confirmed clinical toxoplasmosis in cats: cases ( - ) some aspects of pancreatic disease in the cat clinical differentiation of acute necrotizing from chronic nonsuppurative pancreatitis in cats: cases determination of serum fpli concentrations in cats with diabetes mellitus evaluation of serum feline pancreatic lipase immunoreactivity and helical computed tomography versus conventional testing for the diagnosis of feline pancreatitis evaluation of feline pancreas-specific lipase (spec fpltm) for the diagnosis of feline pancreatitis [abstract pancreatic function in domestic cats with pancreatic fluke infection feline intrinsic factor (if) is pancreatic in origin and mediates ileal cobalamin (cbl) absorption [abstract feline pancreatic disease comparison of the sensitivity of different diagnostic tests for pancreatitis in cats a case of feline paraneoplastic alopecia with secondary malassezia-associated dermatitis response to insulin treatment and survival in cats with diabetes mellitus ( - ) imaging findings in pancreatic neoplasia and nodular hyperplasia in cats acute necrotizing pancreatitis and acute suppurative pancreatitis in the cat pancreatic pseudocyst associated with chronic-active necrotizing pancreatitis in a cat pancreatic insufficiency and diabetes mellitus in a cat feline pancreatic insufficiency an outbreak of virulent systemic feline calicivirus disease jaundice in the cat associated with inflammation of the biliary tract and pancreas placement of jejunal feeding tubes for post-gastric feeding diabetes mellitus and exocrine pancreatic neoplasia in two cats with hyperadrenocorticism clinical and pathologic changes in experimentally induced acute pancreatitis in cats nasogastric tube feeding in cats with suspected acute pancreatitis: cases pancreatic biopsy in normal cats review of feline pancreatitis part one: the normal feline pancreas, the pathophysiology, classification, prevalence and aetiologies of pancreatitis extraperitoneal lesions in feline infectious peritonitis pancreatic disease in the cat d-lactic acidosis secondary to exocrine pancreatic insufficiency in a cat paraneoplastic alopecia associated with internal malignancies in the cat an isolated epizootic of hemorrhagic-like fever in cats caused by a novel and highly virulent strain of feline calicivirus carriage of malassezia spp. yeasts in cats with diabetes mellitus, hyperthyroidism and neoplasia exocrine pancreatic insufficiency with associated coagulopathy in a cat data from eleven united states and canadian colleges of veterinary medicine on pancreatic carcinoma in domestic animals evaluation of complications and prognostic factors associated with administration of total parenteral nutrition in cats: cases contrast-enhanced power and color doppler ultrasonography of the pancreas in healthy and diseased cats diagnostic standards for acute pancreatitis ethanol-mediated neutrophil extravasation in feline pancreas diabetes mellitus associated with pancreatic endocrine insufficiency in a kitten liver cirrhosis and pancreatitis in a cat infected with amphimerus pseudofelineus early biochemical and clinical responses to cobalamin supplementation in cats with signs of gastrointestinal disease and severe hypocobalaminemia ultrasonographic findings in cats with clinical, gross pathologic, and histologic evidence of acute pancreatic necrosis: cases evaluation of endosonography as a new diagnostic tool for feline pancreatitis exocrine pancreatic neoplasia in the cat: a case series ante mortem diagnosis of pancreatitis in four cats retrospective study: surgical intervention in the management of severe acute pancreatitis in cats: cases exocrine pancreatic insufficiency feline exocrine pancreatic disorders serum feline trypsin-like immunoreactivity in cats with exocrine pancreatic insufficiency purification and partial characterization of feline classical pancreatic lipase development and analytical validation of a radioimmunoassay for the measurement of feline pancreatic lipase immunoreactivity in serum evaluation of serum feline trypsin-like immunoreactivity for the diagnosis of pancreatitis in cats complications associated with proximal duodenal resection and cholecystoduodenostomy in two cats resolution of paraneoplastic alopecia following surgical removal of a pancreatic carcinoma in a cat feline exocrine pancreatic insufficiency: cases pancreatitis associated with a feline herpesvirus infection pancreatic pseudocysts in dogs and cats: ultrasonographic and clinicopathologic findings eurytrema procyonis and pancreatitis in a cat acute necrotizing pancreatitis laparoscopic diagnosis of pancreatic disease in dogs and cats relationship between inflammatory hepatic disease and inflammatory bowel disease, pancreatitis, and nephritis in cats traumatic pancreatic injury in a cat: a case history management of prolonged food deprivation, hypothermia, and refeeding syndrome in a cat hepatic lipidosis in cats evaluation of prophylactic and therapeutic effects of silymarin and n-acetylcysteine in acetaminophen-induced hepatotoxicity in cats hepatobiliary neoplasia in dogs and cats spontaneous hepatic rupture in six cats with systemic amyloidosis portosystemic vascular anomalies acquired portal collateral circulation in dogs and cats correlation between coagulation profile findings and bleeding complications after ultrasound guided biopsy: cases ( - ) effect of weight gain and subsequent weight loss on glucose tolerance and insulin response in healthy cats effects of protein, lipid or carbohydrate supplementation on hepatic lipid accumulation during rapid weight loss in obese cats dietary l-carnitine supplementation in obese cats alters carnitine metabolism and decreases ketosis during fasting and induced hepatic lipidosis generalized amyloidosis and acute liver haemorrhage in four cats metabolic and hormonal alterations in cats with hepatic lipidosis effects of nutritional support on hospital outcomes in dogs and cats metabolic, antioxidant, nutraceutical, probiotic, and herbal therapy relating to the management of hepatobiliary disorders current considerations for evaluating liver function in the cat diseases of the gallbladder and biliary tree diagnostic value of serum gamma glutamyl transferase and alkaline phosphatase in hepatobiliary disease in the cat: - retrospective study of cats with severe hepatic lipidosis: - measurement of serum bile acids concentrations for diagnosis of hepatobiliary disease in cats role of i- serum amyloid protein in the detection of familial amyloidosis in oriental cats high complication rate of an automatic tru-cut biopsy gun device for liver biopsy in cats important clinical syndromes associated with liver disease liver biopsy techniques arterial and venous ammonia concentrations in the diagnosis of canine hepatoencephalopathy portal vein thrombosis in cats: cases hepatic abscesses in cats: cases subnormal concentrations of serum cobalamin (vitamin b ) in cats with gastrointestinal disease urine sulfated and nonsulfated bile acids as a diagnostic test for liver disease in cats mosby, p . a. van der linde-sipman j, niewold t, tooten p et al: generalized aa-amyloidosis in siamese and oriental cats bacterial culture results from liver, gallbladder, or bile in dogs and cats evaluated for hepatobiliary disease accuracy of ultrasound guided fine needle aspirate of the liver and cytologic finding in dogs and cats: cases acute necrotizing pancreatitis feline cholangitis syndrome oxidative stress and neutrophil function following oral supplementation of a silibininphophatidylcholine complex in cats laparoscopic diagnosis of pancreatic disease in dogs and cats s-adenosylmethionine in a feline acetominophen model of oxidative injury therapeutic use of cytoprotective agents in canine and feline hepatobiliary disease liver cytology inflammatory liver disease relationship between feline inflammatory liver disease and inflammatory bowel disease, pancreatitis, and nephritis fine needle aspiration cytology suggests hepatic lipidosis in cats with infiltrative hepatic disease wsava standards for clinical and histological diagnosis of canine and feline liver diseases nutrition for anorectic, critically ill or injured cats the effects of s-adenosylmethionine on clinical pathology and redox potential in the red blood cell, liver and bile of normal cats proteins invoked by vitamin k absence in clotting times in clinically ill cats liver glutathione concentrations in dogs and cats with naturally occurring liver disease critical care nutrition extrahepatic biliary tract obstruction, a retrospective study of cases ( - ) statistical relevance of ultrasound criteria in the assessment of different liver diseases in dogs and cats clinical differentiation of acute and chronic feline pancreatitis milk thistle (silybum marianum) for the therapy of liver disease surgery of the liver clinical features of inflammatory liver disease in cats: cases ( - ) update on hepatobiliary imaging sensitivity of tru-cut and fine needle aspirate biopsies of liver and kidney for the diagnosis of feline infectious peritonitis generalised amyloidosis in two siamese cats: spontaneous liver haemorrhage and chronic renal failure severe cholestatic liver disease secondary to liver fluke (platynosomum concinnum) infection in three cats safety, pharmacokinetics and use of the novel nk- antagonist maropitant (cerenia) for the prevention of emesis and motion sickness in cats pancreatic ductal and interstitial pressures in cats with chronic pancreatitis infectious hepatopathies in dogs and cats abdominal ultrasonographic findings associated with feline infectious peritonitis: a retrospective review of cases complications and longterm outcomes of the ligation of congenital portosystemic shunts in cats clinical, clinicopathological, and histological changes observed in cats treated with glucocorticoids hepatic encephalopathy. current concepts of the pathogenesis effects of oral ursodeoxycholic acids in healthy cats on clinicopathological parameters, serum bile acids and light microscopic and ultrastructural features of the liver uroperitoneum in cats: cases ( - ) extrahepatic biliary tract surgery in the cat: a case series and review congenital portosystemic shunts in the cat: a report of nine cases feline cholecystitis and acute neutrophilic cholangitis: clinical findings, bacterial isolates and response to treatment in six cases diagnosis and management of peritonitis in small animals traumatic tricuspid insufficiency in a kitten underlying cause, pathophysiologic abnormalities, and response to treatment in cats with septic peritonitis: cases spontaneous hemoperitoneum in cats: cases primary bacterial peritonitis in dogs and cats: cases a review of the pathophysiology, classification, and analysis of canine and feline cavitary effusions feline hepatic lipidosis uroabdomen in dogs and cats chylous ascites in cats: nine cases congestive heart failure and atrial fibrillation in a cat with myocardial fibrofatty infiltration comparison of different tests to diagnose feline infectious peritonitis arrhythmogenic right ventricular cardiomyopathy in two cats retrospective study: ionized calcium concentrations in cats with septic peritonitis: cases management and outcome of cats with ureteral calculi: cases progressive lymphocytic cholangitis in the cat surgical treatment of bile peritonitis in dogs and cats: a retrospective study feline hemoperitoneum cases ( - ) duplex gall bladder associated with choledocholithiasis, cholecystitis, gall bladder rupture and septic peritonitis in a cat the cytologic examination of body cavity fluids a retrospective study of surgically treated cases of septic peritonitis in the cat chronic lymphocytic cholangitis in three cats primary bacterial septic peritonitis in cats: cases pneumoperitoneum in dogs and cats: cases chylous abdominal effusion in a cat with feline infectious peritonitis restrictive cardiomyopathy in a cat with hypereosinophilic syndrome kirk's current veterinary therapy xii: small animal practice feline infectious peritonitis: a review of clinicopathological changes in cases, and a critical assessment of their diagnostic value differential diagnosis of ascites in cats abdominal paracentesis and diagnostic peritoneal lavage peritoneal effusion in cats: cases ( - ) *when present for any length of time, a pure transudate will become modified. this is particularly true of transudates that develop slowly, such as those associated with congestive heart failure or portal hypertension. modified transudates are therefore more common than pure transudates. adapted from tasker s, gunn-moore d: differential diagnosis of ascites in cats, in practice : , . key: cord- -ys n authors: whary, mark t.; baumgarth, nicole; fox, james g.; barthold, stephen w. title: biology and diseases of mice date: - - journal: laboratory animal medicine doi: . /b - - - - . - sha: doc_id: cord_uid: ys n today’s laboratory mouse, mus musculus, has its origins as the ‘house mouse’ of north america and europe. beginning with mice bred by mouse fanciers, laboratory stocks (outbred) derived from m. musculus musculus from eastern europe and m. m. domesticus from western europe were developed into inbred strains. since the mid- s, additional strains have been developed from asian mice (m. m. castaneus from thailand and m. m. molossinus from japan) and from m. spretus which originated from the western mediterranean region. laboratory animal medicine development of the 'modern' laboratory mouse. research use of mice has grown exponentially during the past and current century with the recognition of the power of the mouse for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized mammal on earth (silver, ; lyon et al., ; morse, a) . the current ability to create highly sophisticated, genetically engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal. historical reviews have documented the origins of the laboratory mouse, which extend thousands of years into antiquity (keeler, ; morse, ; silver, ) . the laboratory mouse belongs within the genus mus, subfamily murinae, family muridae, superfamily muroidea, order rodentia, and within the m. musculus clade collectively called the 'house mouse' (lundrigan et al., ) . anatomic features of molar teeth and cranial bones were traditionally used by zoologists to identify over different species within the genus, and to differentiate them from other murids. because of considerable phenotypic variation within a single mus species, this approach has proven to be inaccurate, and given way to contemporary genetic analysis. the native range of the genus mus is eurasia and north africa. members of this genus are generally classified as aboriginal, consisting of species that live independent of humans, or commensal, which includes taxa that have coevolved and geographically radiated with human civilization since the dawn of agriculture , years before present (bp). this close association with human agrarian society gave rise to the genus name, derived from sanskrit, mush: to steal. the commensal group is known as the 'house mouse' clade, consisting of several subspecies of mus musculus, including m. m. domesticus, m. m. musculus, m. m. castaneus, m. m. bactrianus , and a lesser known lineage, m. m. gentilulus (prager et al., ) . the japanese house mouse, m. m. molossinus, is a natural hybrid of m. m. musculus and m. m. castaneus. the progenitor of the m. musculus clade arose in the northern indian subcontinent and diverged into genetically isolated and distinct species or subspecies due to geographic barriers (mountain ranges). there is debate whether these taxa are species or subspecies, and some have referred to them as 'incipient species,' but their genetic divergence is now blurring as they colonize the world and hybridize. the native ranges of these taxa are important for understanding the origins of various laboratory mice, whose genomes are mosaics derived from m.m. domesticus (~ %), m.m. musculus (~ %), and m.m. castaneus (~ %) (wade and daly, ; wade et al., ) . it is now apparent that the m.m. musculus and m.m. castaneus contributions to the laboratory mouse genome were primarily derived from m.m. molossinus japanese fancy mice (takada et al., ) . mus m. domesticus is indigenous to western europe and southwest asia, m.m. musculus to eastern europe and northern asia, m.m. castaneus to southeast asia, and m.m. molossinus to japan and the korean peninsula. the cohabitation of humans with commensal mice gave rise to captive breeding for coat color and behavioral variants in china over years bp. by the s, mouse 'fanciers' in asia had created many varieties of fancy mice, as did european fanciers, who subsequently acquired asian stocks, particularly japanese fancy mice (m.m. molossinus) , to mix with european (m.m. domesticus) fancy mouse varieties. this genetic mixing for fancy variants was also occurring in the united states, and these mouse lines contributed to many of the major laboratory mice used today. meanwhile, the european colonial expansion era contributed to the worldwide dissemination of m.m. domesticus, which now occupies every continent of the world. it is well documented that wild-caught m.m. domesticus also contributed to the genetic composition of fancy and laboratory mice on multiple occasions. despite their diverse genetic origins and phenotypic differences, most laboratory mouse strains are closely related, since many were derived from a genetically mixed but small number of fancy mice from a single mouse breeder (abbie lathrop's granby mouse farm, massachusetts) at the beginning of the th century. most inbred laboratory mice share a common maternal mitochondrial genome derived from m.m. domesticus (ferris et al., ; yu et al., ) , and a common y chromosome contributed by m.m. musculus (bishop et al., ) through its contribution to the genome of m.m. molossinus (nagamine et al., ) . thus, the most inclusive name that can be assigned to the genetically mosaic laboratory mouse is m. musculus, the over-arching name for the entire commensal clade. there are exceptions, however. c bl/ mice contain minor genetic elements derived from m. spretus (hardies et al., ) , and a number of wild aboriginal species that are not members of the m. musculus clade, including m. spretus, m. caroli, and others, have been established as inbred lines of mice. genetic mapping in mice began in the early s with a focus on inheritance of coat color. the first autosomal genes, albino and pink-eyed dilution, were linked in (haldane et al., ) . extensive linkage maps and an impressive array of inbred strains are now available to expedite genetic research (table . ) (lyon et al., ) . mice have pairs of telocentric chromosomes that are differentiated by their size and patterns of transverse bands. the chromosomes are designated by arabic numbers in order of decreasing size. during the s, chromosome rearrangements were used to assign known genetic linkage groups -identified by roman numerals -to specific chromosomes and for determining locus order with respect to the centromere. genes can be located physically on chromosomes by fluorescent in situ hybridization (fish). development of quantitative trait loci (qtl) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (moore and nagle, ) . for more information on comparative genomics, see chapter animal models in biomedical research, subsection c. one of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. histocompatibility (h) loci control expression of cell surface molecules that modulate critical immune responses, such as the recognition of foreign tissue. for example, the time, onset, and speed of skin graft rejection are controlled by two groups of h loci. the major group is located in the major histocompatibility complex (mhc, h ) on chromosome . the h complex contains several loci, including k, d, l, i-a, and i-e. inbred strains of mice, being homozygous, each have unique sets of h alleles, termed h haplotypes. for example, the balb h haplotype is h d and the c bl h haplotype is h b . the international immunogenetics (imgt) information system provides details on h haplotypes for various inbred mice (www.imgt.org/imgtrepertoiremhc/polymorphism/ haplotypes/mouse/mhc/mu_haplotypes.html). minor h loci groups are scattered throughout the genome and are responsible for delayed graft rejection. genes associated with the h complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. immune-mediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the h complex (stuart, ) . non-mhc or minor histocompatibility systems also are under active study (roopenian et al., ) . mouse genomics have accelerated tremendously in the last two decades, heralded by the development of a robust physical map and high-quality genome sequence of the c bl/ j mouse in by the international mouse genome sequencing consortium (waterston et al., ) . the mouse genomes project/wellcome trust sanger institute is extending this effort to include the genomic sequences of key mouse strains. completed and evolving sequence data are available through the european nucleotide archive (www.ebi.ac.uk/ena/home). the burgeoning numbers of inbred mouse strains, natural mutants, induced mutants, transgenic lines, and targeted mutant lines of mice are cataloged in the mouse genome informatics (mgi) database: http://www.informatics.jax.org/mgihome). the growing number of mutant mice has fostered the development of a number of mouse repositories, from which specific mice can be located and acquired. in the united states, there are four regional national institutes of health (nih)-supported mutant mouse regional resource centers (http://www.mmrrc.org), which link to international repositories in europe, japan, china, australia, and canada, as well as additional resource programs in the united states through the international mouse strain resource (imsr; http://www.informatics.jax.org/ imsr/index.jsp) for depositing, archiving, and distributing mutant mouse and embryonic stem cell lines to the scientific community. in addition to numerous mutant mice produced independently by scientists in various academic institutions, three major targeted gene knockout programs, all utilizing c bl/ n embryonic stem cells, are under way internationally, and funded by the nih, the european community, and genome canada (collins et al., ; skarnes et al., ) . these include the knock out mouse project (komp; http://www.knockoutmouse.org), the european conditional mouse mutagenesis program (eucomm; http://www.eucomm.org), and the north american conditional mouse mutagenesis project (norcomm; http://norcomm.phenogenomics.ca/index. htm). these mouse lines will be available through laboratory animal medicine three distribution centers: the german resource center for genome research (rzpd; http://www.rzpd.de), the komp repository (https://komp.org), and the canadian mouse consortium (cmc; http://www.mousecanada. ca/index.htm). the repositories are all linked to the imsr, and provide access to mice, germplasm, genomic detail, and phenotypic data. genetic, genomic, and biological data are also available through the international mouse phenotyping consortium (impc; www.mousephenotype. org) and the mouse genome database (mgd; http://www. informatics.jax.org) (eppig et al., ) . inbreeding is a fundamental genetic tool applied to the laboratory mouse and detailed information is available on the web (table . ). the first inbred strain (dba) was developed by c.c. little in , with the subsequent creation of over inbred strains and stocks of mice (festing, ) . genetic origins, basic characteristics, references, and breeding performance of inbred strains of mice are available through michael festing's online version of inbred strain characteristics (http:// www.informatics.jax.org/external/festing/mouse/ strains.shtml). overviews of genetic manipulation for the creation of different types of mice are available (lyon et al., ; silver, ) . inbred mouse lines are termed strains, and are achieved by or more brother × sister (filial; f) generations (table . ). mice within an inbred strain, for practical purposes, are genetically identical (syngeneic or isogenic) to other mice of the same strain and sex. because of residual heterozygosity, a strain is not fully inbred until after f generations. most commonly used inbred mouse strains represent or more f generations, providing a high degree of experimental reproducibility. the mouse genome is not static, so when branches of an inbred strain are separated, spontaneous mutations, residual heterozygosity, and retroelement integrations result in genetic differences. therefore, if branches of an inbred strain are separated before f , if branches have separated for generations, or if genetic differences arise, the different branches become substrains. the same holds true if branches of a substrain diverge, resulting in substrains of the inbred substrain. when two inbred mouse strains are crossed, the f hybrids are genetically identical to one another (isogenic), but maximally heterozygous (with chromosomes of each chromosomal pair separately contributed by each parental strain), whereas f hybrids are maximally genetically diverse from one another (with chromosomes of both chromosomal pairs containing a mixture of contributions from each parental strain). with each subsequent f generation, mice once again approach inbred status. this technique is used for creating recombinant inbred (ri) strains. ri strains are sets of inbred strains of mice derived from crossing two inbred strains, and developed by singlepair random matings of sibling mice from the f generation, thereby creating separate breeding lines. each line created is maintained separately, and then propagated by brother-sister matings for generations, with each line becoming a separate inbred strain, but belonging to a set of ri strains. ri mice are useful for mapping phenotypic or quantitative traits that differ between the progenitor strains (bailey, ) . ri sets are generally limited to two parental strains. an ongoing international effort has been undertaken to increase allelic diversity among ri strains by creating the collaborative cross (cc) in which a panel of ri strains are being generated mixing the genomes from eight disparately related inbred (octo-parental) mouse strains, including a/j, c bl/ j, s /svimj, nonobese diabetic (nod)/shiltj, nzo/ hlltj, cast/eij, pwk/phj, and wsb/eij. these eight strains capture nearly % of the known genetic variation present among laboratory mice. future applications of the cc will utilize ri intercrosses of pairs of ri cc lines (threadgill and churchill, ; welsh et al., ) . recombinant congenic strains are sets of inbred strains derived in a manner similar to that for ri sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the f generation, before inbreeding is begun. the other parental strain is designated as the donor strain. the proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (demant and hart, ) . advanced intercross lines (ails) are another type of ri lines. they are made by producing an f generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. the purpose is to increase the possibility of recombination between tightly linked genes. when a mutation arises spontaneously or is induced within an inbred strain, that mutant mouse becomes co-isogenic with the parental inbred strain, being virtually identical except for the single mutant allele. frequently, a mutation that arose in one inbred strain may be desired within the genetic background of another inbred strain. this can be accomplished by backcrossing, in which an f hybrid is created by mating the donor mutant strain to the desired background strain, with subsequent matings to the background strain while retaining the mutant locus. after backcross generations (n generations), the mutant mouse line is now congenic to the background inbred strain. backcrossing to create congenic strains of mice has been used extensively when targeted mutations have been induced in embryonic stem cells, with backcrossing onto c bl/ inbred mice. congenic mice are never co-isogenic, as the preserved locus in a congenic mouse is invariably surrounded by flanking dna, which may significantly influence phenotype (linder, ) . in contrast to inbred mice, outbred mice are genetically heterogeneous and are maintained by breeding systems that intentionally minimize inbreeding. outbred mice are called stocks, which are defined as a closed population (for at least four generations) of genetically variable mice that are bred to maintain maximal heterozygosity. outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. outbreeding can be achieved only in a large breeding population using a systematic breeding scheme, or randomized selection of breeders from the population. a small breeding population or passage through the genetic 'bottleneck' of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. in a population of breeding pairs, e.g., heterozygosity will decrease at % per generation with standard randomization techniques. random breeding involves the statistically random selection of breeders by using a random numbers table or computer program. an outbreeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. another 'circular' set of cages serves as the breeding nucleus for the n + generation. each mated pair in the nth generation contributes one female and one male to the n + generation. outbreeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + generation. most outbred mouse stocks are of 'swiss' origin, derived from nine mice imported to the united states in , and are therefore quite homogeneous genetically (chia et al., ) . various lines of these mice have been maintained at different institutions, giving rise to numerous closely related stocks. although considered outbred, they have a high degree of homozygosity, exemplified by the fact that many swiss mouse stocks are blind due to the homozygous recessive rd allele (serfilippi et al., b) . it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. in that regard, the diversity outbred mouse has been developed, which is a heterogeneous stock derived from the same eight founder inbred strains of the cc . additional types of inbred mice are utilized in research, including consomic and conplastic strains. consomic strains, also known as chromosome substitution strains, are inbred mice that are congenic for entire chromosomes, and are useful for studying polygenic traits (singer et al., ) . conplastic mice are inbred mice that are congenic for different mitochondrial genomes (mtdna) contributed by other inbred strains, other subspecies, or other species of mus (yu et al., ). in addition to spontaneously occurring mutations that are maintained as co-isogenic strains (such as the c bl/ beige mouse), mutant lines of mice have been created by radiation mutagenesis, chemical mutagenesis, or transgenesis. radiation was one of the earlier methods for in vivo mutagenesis (silver, ) , but in vitro radiation of embryonic stem (es) cells is also performed (thomas et al., ) . chemical mutagenesis involves in vivo treatment of male mice or in vitro treatment of es cells with mutagenic chemicals such as ethylmethanesulphonate (ems) or n-ethyl-n-nitrosourea (enu) , which induce point mutations in dna (o'brien and frankel, ; justice et al., justice et al., , . technically, a transgenic mouse is any mouse in which foreign dna has been integrated into its genome, regardless of method. however, the term transgenic commonly refers to mice that are genetically altered by additive transgenesis through microinjection of foreign dna into the pronucleus of a fertilized egg. each ensuing embryo results in a genetically different founder mouse, since the transgene is integrated in random sites of the genome of each founder mouse. since the injected dna is not homologous to the mouse genome and is not an allele, transgenic founders are hemizygous (rather than heterozygous) for the transgene until the mice carrying the transgene are bred into homozygosity for the transgene. transgenes typically integrate as tandem repeats, copy numbers affect phenotype of each founder, and may be lost in subsequent generations, thereby changing the phenotype of the mouse line (tinkle and jay, ) . transgenes are often constructed with an upstream promoter, which confers widespread (ubiquitous) or tissuespecific expression of the cdna, so that the transgene expression pattern reflects the expression pattern of the promoter. transcriptional regulation of the transgene can be inducible by drug-dependent regulatory control, such as the widely used tetracycline (tet) regulatory system, in which treatment of mice with tetracycline or doxycycline induces up-or down-regulation of the transgene (jaisser, ) . es cells are used for the less efficient integration of genetic material by homologous dna recombination, but allow large-scale screening of es cell clones for transformation. integration can be achieved in a random fashion by gene trapping, or by targeted mutation. both methods involve homologous dna recombination. gene trapping is a high-throughput approach that randomly introduces insertional mutations within the genome. vectors contain a gene trapping cassette with a promoter-less reporter gene and/or selectable genetic marker flanked by an upstream ′ splice site and a downstream termination sequence. when inserted into an laboratory animal medicine intron of an expressed gene, the gene trap is transcribed from the endogenous promoter of that gene. gene traps simultaneously inactivate and report the expression of the trapped gene at the insertion site, and provide a dna tag for the rapid identification of the disrupted gene (skarnes et al., ) . targeted gene mutations are achieved by homologous recombination of specific sites within the genome of es cells. homologous sequences flank the upstream and downstream regions of the targeted gene, and the construct between the flanking sequences may inactivate (knock out) or replace (knock in) a gene, and typically contains a reporter gene to track the integration. a variation on this approach is site-specific recombinase (ssr) technology. two of the most common recombinases are cre from the coliphage p and flp from saccharomyces cerevisiae. cre and flp mediate recombination between target sites, termed loxp and frt, respectively. for example, cre loxp target sites are engineered to flank the gene target, which can be used in different ways to achieve different outcomes (conditional mutations), depending upon the orientation and location of the flanking loxp sites. if the loxp sites are oriented in opposite directions, cre recombinase mediates inversion of the floxed segment. if the loxp sites are on different chromosomes (trans), cre recombinase mediates a chromosomal translocation. if the loxp sites are oriented in the same direction on the same chromosome (cis), cre recombinase mediates deletion of the floxed segment. once the floxed mutation is created in es cells, the transformed es cells are developed into a mouse with the conditional mutation. the conditional mutant mouse is then genetically crossed with a cre transgenic mouse, in which cre recombinase is under the control of a ubiquitous or tissue-specific promoter. wherever and whenever cre is expressed, cre recombinase will recognize and recombine the loxp sites. this approach can include insertion of reporter genes and selectable markers, and can be under the control of inducible gene expression systems (http:// www.eucomm.org/docs/protocols/mouse_protocol_ _ sanger) (nagy, ) . es cells are pluripotent with the full genetic capacity to develop into mice when implanted into the blastocyst of a developing embryo. interest in 'embryonal carcinomas' (teratomas) that arose in relatively high frequency in the testes of mice and early gene transfer experiments in the late s and early s led to the development of es cell lines derived from several different strains. this early emphasis on teratomas prompted creation of 'better' mouse lines that were more prone to development of testicular teratomas, resulting in genetic corruption of the mouse (simpson et al., ; threadgill et al., ) . this realization gave rise to the need to revise mouse nomenclature (festing et al., ) . this was necessary because genetic variation significantly impacts homologous recombination in order to match genome sequence of the es cell line with the mouse from which it was derived. es cells can be created from any mouse strain or hybrid, but es cell lines have been commonly used. recent international knockout mouse program efforts use c bl/ n es cells. transformed es cells are microinjected into the inner cell mass of recipient blastocysts, which are then implanted into the uteri of pseudopregnant surrogate mothers. the pups that are born are composed of a mixture of cells derived from recipient blastocysts and the transformed es cells (chimeras). the goal is for male chimeric progeny to produce spermatozoa of es cell origin (containing the mutation), in order to create f progeny by mating the chimera with the desired background strain (http://www.eucomm.org/docs/protocols/mouse_protocol_ _sanger). for this reason, most es cell lines are xy, which favors male chimerism. if the es cells are of (or other) strain origin, the chimeras are often bred to a desired background mouse strain (commonly c bl/ ) and backcrossed for n generations, thereby creating congenic inbred mouse lines. recent international knockout mouse efforts utilize c bl/ n es cells, so that chimeric males are bred directly with c bl/ mice, thereby creating co-isogenic lines. the latter approach saves time and money, and creates a more genetically refined mutant mouse. an alternate approach is to allow es cells to aggregate with a developing embryo to form blastocysts in culture (aggregation chimera), then implant the chimeric blastocysts (tanaka et al., ) . rna interference (rnai), which functions through short double-stranded rna (dsrna), has also been utilized to produce transgenic mice, known as gene knockdown mice (gao and zhang, ; peng et al., ) . the dsrna is enzymatically processed into small molecules, termed small interfering rna (sirna), which find homologous target mrnas, resulting in interference. this phenomenon is believed to be a self-defense mechanism against viral infection. in order to adapt this approach to generation of transgenic mice, small hairpin rna (shrna) can be expressed in the same way as other transgenes in mice, resulting in processing of the shrna into sirna with gene-silencing effects. constructs are introduced into mouse es cells by electroporation or lentiviral infection. this method can be embellished conditionally, as with other transgenes. although rnai knockdown mice are genetically stable, rnai-mediated transgenesis is never complete, has variable tissue expression, and cannot induce point mutations (peng et al., ) . recent advances in engineered endonuclease (ee) technology, including zinc finger nucleases (zfns), laboratory animal medicine transcription activator-like effector nucleases (talens), and rna-guided endonucleases (rgens), have revolutionized the field of transgenics (sung et al., ; wijshake et al., ; gaj et al., ) . zfns and talens consist of engineered proteins that target dna fused to the nonspecific endonuclease, fok (cathomen and joung, ; joung and sander, ) . zfns are comprised of three to six tandem zinc finger proteins, each of which targets a specific bp nucleotide sequence. paired zfns are generated, with each half of the pair targeting opposite dna strands, allowing dimerization of fok which is required for introduction of double-stranded breaks (dsbs) in the dna of interest (cathomen and joung, ) . talens function similarly, but are composed of tandem repeats of - amino acids, each with nucleotide specificity occurring in two hypervariable amino acids, the 'repeat variable di-residue (rvd)', at positions and (joung and sander, ) . in contrast to zfns and talens, clustered regularly interspaced short palindromic repeats (crisprs) paired with crispr-associated (crispr/cas) systems are rgen systems that target specific dna sequences. cas proteins, rather than fok , produce dsb (hsu et al., ) . dsb generated by ee are repaired by host cells by either nonhomologous end joining (nhej) or, less commonly, by homologous recombination (hr). nhej is an error-prone repair system and results in insertions or deletions (indels) with a relatively high frequency, which can result in gene disruption. hr is a less common repair pathway, but certain manipulations of the engineered nucleases can increase hr efficiency. for example, nucleases can be engineered to generate a break in a single strand of dna rather than inducing dsb, and the resulting nickases increase the incidence of hr with high fidelity (gaj et al., ; wijshake et al., ) . hr allows for the introduction of donor dna to generate knock-ins, specific point mutations, or for the generation of larger modifications such as insertions of loxp sites (brown et al., ; wijshake et al., ) . vectors encoding the ee can be injected into mouse embryos by pronuclear injection of dna, intracytoplasmic injection of rna, or transfection of mouse es cells (sung et al., ; wijshake et al., ) . one advantage of ee technologies over more traditional transgenic methods is the ability to target dna and induce mutations in any background strain of mouse negating the need to backcross onto the desired strain. multiple genes can be targeted with crisprs simultaneously, thus avoiding the need to cross single knockout animals (zhou et al., ) . in addition, it is possible to obtain bi-allelic mutations in some cases, allowing for the generation of functional gene knockout animals in a single generation (zhou et al., ; wijshake et al., ) . vectors for generating ee are available through plasmid repositories; websites are available to assist in identifying appropriate dna sequences to target; and multiple websites post protocols for generating the various types of engineered endonucleases (xie et al., ; sander et al., ; bae et al., ; reyon et al., ; herscovitch et al., ; wolfson, ) . crisprs tend to be particularly cost effective and easy to design, with minimal restrictions for targeting specific dna sequences. there are currently more than separate outbred stocks and traditional inbred strains, often with multiple substrains (table . ). in addition, there are thousands of induced mutant strains. therefore, it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion, and to ensure reproducibility of research results. as an example of substrain variation that makes precise nomenclature important, cba/j mice are homozygous for the retinal degeneration allele (rd ), whereas cba/caj mice do not carry this allele. the international committee on standardized genetic nomenclature for mice and rats, established in the early s, is responsible for genetic nomenclature rules. the rules are available online at the mgi website (http:// www.informatics.jax.org/mgihome/nomen). inbred mouse strains are designated by a series of capital letters and/or numbers, which often provide a shorthand description of the origin and history of the strain. the c bl/ j mouse serves as an example. the inbred strain c bl originated from abbie lathrop's female (and male ) at the cold spring harbor laboratory (c), and was the black (bl) line from this female. early in their history, inbred c bl mice split into major substrains, e.g., c bl/ and c bl/ . substrains are identified by appending a forward slash (/) after the inbred strain name. since , uniform international nomenclature has been built upon these historical names, so that substrains of an inbred strain are now designated using lab codes that are registered in the international laboratory code registry maintained at the institute for laboratory animal research (ilar) of the national academies (dels. nas.edu/global/ilar/lab-codes). laboratory codes are composed of one to five letters that identify an institute, laboratory, or investigator. each lab code starts with an uppercase letter, followed by lowercase letters if more than one letter is used (such as n, j, jci, crl, and tac). the j in c bl/ j means it is a substrain maintained at the jackson laboratory (j). another common substrain of c bl/ mice is c bl/ n, which is maintained at nih (n). substrains can be cumulative, reflecting the genetic history of the mouse strain. for example, there are a number of c bl/ j substrains (such as c bl/ jjci and c bl/ jjmsslc), and a number of c bl/ n substrains (such as c bl/ njci, c bl/ ncrlcrlj, dba/ j inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before generations of brother × sister breeding and is the subline maintained at the jackson laboratory (j) c h/hesn-ash/+ co-isogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on c h/hesn c bl/ j-tyrc- j /+ co-isogenic segregating inbred mutant strain carrying the albino j mutant allele of the cloned tyrosinase gene (tyr) aej/gnj-a e /a w-j inbred strain segregating for two alleles at the agouti gene congenic inbred strain in which the b haplotype at the h complex was transferred from c bl/ j (b ) to the akr background b .cba-d mit -d mit congenic strain in which the chromosomal segment between d mit and d mit was transferred from cba to b b .cg m lepr db /++ congenic inbred strain in which the linked mutant genes misty (m) and diabetes (lepr db ) were transferred from multiple, mixed, or unknown genetic backgrounds to b and are carried in coupling, i.e., on the same chromosome b .cg-m +/+ lepr db congenic inbred strain in which the m and lepr db mutations are carried in repulsion bxd- /ty recombinant inbred (ri) strain number in a set of ri strains derived from a c bl/ j (b) female mated to a dba/ j (d) male and made by taylor (ty) recombinant congenic (rc) strain number in a set made by crossing the balb/c (c) and sts (s) strains, backcrossing one or two times to balb/c and then inbreeding as with ri strains and c bl/ ntac). significant differences may exist among these substrains (mekada et al., ). thus, a string of substrain designations indicate the genetic progression of the substrain, which can be identified when reading the entire strain name. this nomenclature is highly nuanced, as c bl/ ncrlcrlj mice, whose last letter is a lowercase j, are not a substrain maintained at the jackson laboratory (j), but rather at charles river japan (crlj), underscoring the importance of upper-and lowercase lettering in rodent nomenclature. balb/c mice are another popular inbred strain with numerous substrains. like the ' ' in c bl/ , the 'c' that follows laboratory animal medicine of the mutational event as a superscript. for example, cftr tm unc is a targeted mutation (tm), first line ( ) congenic mice are often derived from es cells, backcrossed onto a background strain, such as c bl/ . under such circumstances, when the backcross generation is at n , the '.' symbol is used between the background inbred strain and the donor strain (e.g., c bl/ n. p /olahsd-abc tm zzz , abbreviated as b . -abc tm zzz . when backcrossing is incomplete but at the n generation, the mouse is an incipient congenic, designated with a ';' in lieu of a '.': b ; -abc tm zzz . if the background strain is mixed genetic origin, it is designated stock. -abc tm zzz . if the donor strain is mixed origin, it is designated 'cg'. for example, b .cg-abc tm zzz outbred stock that meets specific criteria is designated by placing the lab code before the stock symbol, separated by a full colon (':'). for example, hsd:icr designates an icr (swiss) outbred stock maintained by harlan sprague dawley (hsd). the above overview covers the nomenclature of commonly encountered types of mice. there are numerous additional specifications for nomenclature of mice. details are available at the mgi website (http://www. informatics.jax.org/mgihome/nomen). optimum housing conditions and husbandry practices for research mice should be guided by program requirements to ensure biosecurity, occupational health, efficient use of equipment, labor and financial resources, behavioral needs of mice, and investigator needs for consistent colony maintenance, including standardized husbandry practices and nutrition. the emerging interest in the mouse microbiome in combination with the immune competency of diverse genetically engineered mouse strains demands high standards of mouse care. mouse colonies are optimally maintained as specificpathogen-free (spf) which obligates veterinary and facility management to exclude specific organisms. housing options for spf immunocompetent mice typically include static or individually ventilated microisolator cages, which differ significantly in cost and labor required to maintain. severely immunodeficient strains such as nod.cg-prkdcscid il rgtm wjl/szj (nsg) mice require staff training, caging systems and husbandry practices that minimize risk for opportunistic infections the '/' in balb/c is a lowercase letter because of historical precedent. subsequent substrains follow accepted nomenclature, e.g., balb/cbyj and balb/cann. hybrids of two inbred strains are often used in research, and are particularly common with engineered mutations that are created in -derived es cells, followed by intercrossing the chimeric mice with c bl/ or other background strains of mouse. when an f hybrid is created, the female partner is listed first, e.g., a c bl/ j × s /svpas hybrid would be designated: c bl/ j s /svpasf . ri strain sets that are derived from two parental inbred strains are identified by an x between the two parental strains followed by a hyphen designating the specific ri line, e.g., c bl/ jxdba/ j- , c bl/ jxdba/ j- , etc. cc ri strains do not use the x between the parental strains because they are derived from eight parental strains, so they are designated cc- , cc- , etc. in order to simplify the complexity of this nomenclature, abbreviations are used for common inbred strains and substrains of mice (table . ), but it is important to include the full genetic nomenclature in publications. using the abbreviated nomenclature, c bl/ j s /svpasf mice would be b f and c bl/ jxdba/ j- ri mice would be bxd- . parental order is an important consideration in nomenclature, as a b mouse is genetically different from a b mouse due to mitochondrial dna (from the female) and y chromosome (from the male) differences. mutant genes are designated by a brief abbreviation for the mutation (e.g., bg for beige which arose at the jackson laboratory, j). the symbol for the parent gene is noted in italics, starting with an uppercase letter (e.g., lyst) and the mutant allele is designated in superscript (e.g., lyst bgj ). thus, the beige mutation arose in c bl/ j mice, so that c bl/ j beige mice, which are co-isogenic with c bl/ j mice, are designated c bl/ j-lyst bgj . a transgenic strain is designated by the strain and substrain name, followed by a symbol for the transgene. transgene symbols take the form tg(yyy)#zzz, where 'tg' indicates transgenic, yyy defines the transgene as a brief description of the inserted dna (such as a gene symbol), '#' is the assigned number in the series of events generated using a given construct, and 'zzz' is the lab code. for example, fvb/n-tg(mmtv-erb ) led mice are inbred fvb/n mice in which the rat erb gene was introduced under control of the mouse mammary tumor virus (mmtv) ltr promoter (mmtv-erb ), the first line ( ) created in the laboratory of phil leder (lab code led). when a transgene causes an insertional mutation in an identified endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (-abc tg zzz ). a targeted mutation, or knockout, is designated by the mutated gene with the identification laboratory animal medicine (foreman et al., ) . barrier practices and microisolator techniques may include autoclaved or irradiated feed and bedding, autoclaved or acidified water, cage-tocage transfer of mice using disinfected forceps, positive displacement change hoods, and verified sanitation of caging and equipment through tunnel or rack washers to prevent fomite transmission of infectious agents (compton et al., ) . in addition to husbandry staff, it is critical to maintenance of colony health status that investigators who handle cages are also trained in these techniques. the microenvironment for mice is the cage which will vary in design, size, and composition. vendors often successfully house production colonies in open-top cages to expedite detection of pathogen transmission should a break occur. end-users usually prefer filter-top microisolator cages which prevent (at least) gross contamination between cages by fecal contamination and aerosolized debris. the objective is to keep mice in an uncrowded, socially compatible, low-odor, dry and clean environment. ambient temperature should minimize any confounding impact on the animal model and energy expenditure for the mice, while also being suitable for staff and investigators. shoebox static cages made of polycarbonate, polypropylene, or polystyrene plastic (in order of decreasing cost and durability) with filtered microisolator tops continue to be used for housing and breeding mice. older cage designs are being rapidly supplanted by individually ventilated caging systems that promote the advantages of increasing housing capacity, decreasing labor costs, and mitigating exposure of mice to noxious gases such as ammonia and exposure of humans to allergens. as more advanced caging systems are developed, the level of biosecurity may be increased but at the cost of increased health surveillance efforts to detect the source of an infectious outbreak (shek, ) . disposable, recyclable polyethylene caging is a recent innovation, particularly for facilities not equipped with a cage wash facility. animal care programs should carefully consider the necessity for housing mice on wire-mesh flooring because of injury risk to limbs and thermoregulation issues in neonates and hairless mice which are more difficult to maintain without nesting material. solidbottom cages should contain sanitary bedding, such as hardwood chips, paper products, or ground corn cob. criteria for selecting bedding vary with experimental and husbandry needs. it may be preferable to irradiate or autoclave bedding, but if this is not done, the bedding should be used only after its origin and microbial content have been evaluated (table . ). germfree and gnotobiotic mice require positive pressure isolators, most usually flexible film, with additional protection provided by sterile air through high-efficiency particulate air (hepa) filters. this equipment can be negatively pressurized when the objective is to contain known or unknown pathogens. animal care programs should establish enrichment policies which for mice should include social housing when mice are compatible and experiments do not require single housing. species-specific behaviors are encouraged by nesting material and hiding places such as tubes or shacks. nutrient requirements for the mouse are influenced by genetic background, disease status, growth rate, pregnancy, lactation, and environmental factors such as ambient temperature. the best current estimate of nutritional requirements is shown in table . . nutritional requirements for laboratory mice are also published periodically by the national research council and have been reviewed by knapka and coworkers (knapka et al., ; knapka, ) . feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. mice consume about - g of feed per day after weaning, and maintain this intake throughout life. outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (figs. . and . ). diet is often neglected as a variable in animal-related research. diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. therefore, diet must provide a balance of essential kraft ( ) . knapka ( ) . b linoleic acid: . % is adequate. c john and bell ( ) . d theuer ( ) . e knapka et al. ( ). f nutrition ( . g hurley and bell ( ) . h pleasants et al. ( ) . nutrients, and contaminants must be kept to a minimum (see also chapter ). natural-product commercial diets for mice are usually satisfactory for breeding and maintenance. animal care programs should avoid using fresh produce, grains, fish meal, or other supplements to minimize exposure of colonies to pathogens or harmful chemicals such as pesticide residues or phytoestrogens (guerrero-bosagna et al., ) . mouse diets can be purchased as open-formula, fixedformula, constant nutrition, and closed-formula which laboratory animal medicine are designed to reduce variation in experimental data attributable to diet (reviewed in barnard et al. ( ) ). diets are supplied in standard, irradiated, or autoclavable formulations. irradiated diets will be virtually free of live microorganisms but have the risk of residual, radio-resistant bacteria. autoclavable diets are higher in heat-labile nutrient content. many programs use sterilized mouse chow exclusively to minimize risk of opportunistic infections. because commercial diets vary in nutrient content, diets should be selected for optimal maintenance of adult mice or for growth and reproduction in breeding colonies. mice should have continuous access to potable water even if a high-moisture diet is fed. water is needed for lubrication of dry food and for hydration. adult mice drink - ml of water per day. decreased water intake will decrease food consumption. water imbalance may occur immediately post weaning and weanlings on automatic watering systems need extra attention. water intake will decrease in sick mice. therefore, dosing mice with medicated water requires careful assessment of hydration and clinical or experimental efficacy of the compound administered. the main reference used to update this section of the rd edition is volume iii; normative biology, husbandry and models in the mouse in biomedical research, nd edition, aclam series published by academic press. normative data on the mouse are presented in table . , and clinical chemistry reference ranges are summarized in table . . mice have a relatively large surface area per gram of body weight. this results in dramatic physiologic changes in response to fluctuations in the ambient temperature (t a ). the mouse responds to cold exposure, e.g., by nonshivering thermogenesis. a resting mouse acclimated to cold can generate heat equivalent to triple the basal metabolic rate, a change that is greater than for any other animal. a mouse must generate about kcal/m per h to maintain body temperature for each °c drop in t a below the thermoneutral zone. mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. therefore, it is not advisable to conserve energy in animal quarters at night by lowering t a . because of the ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. its biological half-time for turnover of water ( . days) is more rapid than for larger mammals. water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. the conservation of water can preempt thermal stability. if the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. the mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. mice can partially compensate for changes in t a increases from °c to °c. it adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. its primary means of cooling in the wild is behavioral -retreat into a burrow. in the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of °c or higher. thus, the mouse is not a true endotherm. in fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before days of age. the thermoneutral zone for mice varies with strain and with conditioning but is about . - . °c, narrower than that of any other mammal measured thus far. thermoneutrality should not be equated with comfort or physiological economy. recent data have suggested that mice housed under routine vivarium conditions are chronically cold-stressed. mice maintained at °c were shown to expend more energy compared with mice housed at intermediate ( °c) and a higher temperature ( °c) with an increase in glucose utilization and activation of brown adipose tissue (david et al., ) . in contrast, other studies report that mice in a t a range of - °c grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone. the respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharnyx; the intermediate section consists of larynx, trachea, and bronchi, all of which have cartilaginous support; and the posterior portion of the respiratory tract consists of the lungs. the left lung is a single lobe. the right lung is divided into four lobes: superior, middle, inferior, and postcaval (cook, ) (fig. . loeb and quimby ( ) . a mouse at rest uses about . ml o /g/h, which is about times more o /g/h than is used by an elephant. to accommodate for this high metabolic rate, the mouse has a high alveolar p o ; a rapid respiratory rate; a short air passage; a moderately high erythrocyte (rbc) concentration; high rbc hemoglobin and carbonic anhydrase concentrations; a high blood o capacity; a slight shift in the o -dissociation curve, enabling o to be unloaded in the tissue capillaries at a high p o ; a more pronounced bohr effect, i.e., the hemoglobin affinity for o with changes in ph is more pronounced; a high capillary density; and a high blood sugar concentration. the kidneys, ureters, urinary bladder, and urethra form the urinary system. the paired kidneys lie against the dorsal body wall of the abdomen on either side of the midline. the right kidney is normally located anterior to the left kidney. kidneys from males of many inbred strains are consistently heavier than kidneys from females. the glomeruli of mice are small, about μm in diameter, or about half the size of glomeruli in rats. there are, however, . times as many glomeruli in the mouse, and the filtering surface per gram of tissue is twice that of the rat. mice excrete only a drop or two of urine at a time, and it is highly concentrated (table . ). the high concentration is made possible by long loops of henle and by the organization of giant vascular bundles (vasa recta) associated with the loops of henle in the medulla. the mouse can concentrate urine to mosm/l, whereas humans can concentrate to a maximum of mosm/l. mice normally excrete large amounts of protein in the urine. taurine is always present in mouse urine, whereas tryptophan is always absent. creatinine is also excreted in mouse urine, a trait in which mice differ from other mammals. the creatinine/creatine ratio for fasting mice is about : . . mice excrete much more allantoin than uric acid. the submaxillary salivary gland, a mixed gland in most animals, secretes only one type of saliva (seromucoid) in the mouse. the tubular portion of the gastrointestinal (gi) tract consists of esophagus, stomach, small intestine, cecum, and colon. the esophagus of the mouse is lined by a thick cornified squamous epithelium, making gavage a relatively simple procedure. the proximal portion of the stomach is also keratinized, whereas the distal part of the stomach is glandular. gastric secretion continues whether or not food is present. the gastrointestinal flora consists of (at least) species of bacteria that begin to colonize the alimentary canal selectively shortly after birth. the ceca of normal mice contain up to bacteria/g of feces. the bacteria throughout the gastrointestinal tract form a complex ecosystem that provides beneficial effects, such as an increase in resistance to certain intestinal pathogens, production of essential vitamins, and homeostasis of important physiological functions. gnotobiotic animals colonized with known microbiota have been used to great advantage as models for biomedical research (see chapter ). for certain studies, it is desirable to colonize germfree mice with a defined microbiota. in the mid- s, schaedler was the first to colonize germfree mice with selected bacteria isolated from normal mice (schaedler and orcutt, ) . he subsequently supplied animal breeders with this group of microorganisms. these defined bacteria included aerobic bacteria and some less oxygen-sensitive anaerobic organisms. the so-called extremely oxygen-sensitive (eos) fusiform bacteria, which make up the majority of the normal microbiota of rodents, were not included, because of technical difficulties in isolation and cultivation. of the defined microbiotas later used for gnotobiotic studies, the one known as the 'schaedler flora' was the most popular. in , the national cancer institute (nci) decided to revise the schaedler flora, or 'cocktail' consisting of eight bacteria, in order to standardize the microbiota used to colonize germfree rodents. the new defined microbiota, now known as the 'altered schaedler flora' (asf), consisted of four members of the original schaedler flora (two lactobacilli, bacteroides distasonis, and the eos fusiform bacterium), a spiral-shaped bacterium, and three new fusiform eos bacteria. studies have quantified the regional colonization of the asf strains along the gastrointestinal tract (sarma-rupavtarm et al., ) (fig. . ) . individual strain abundance was dependent on oxygen sensitivity, with microaerotolerant lactobacillus murinus asf present at - cells/g of tissue in the upper gastrointestinal tract and obligate anaerobic asf strains being predominant in the cecal and colonic flora at - cells/g of tissue. it is difficult to monitor a gnotobiotic mouse colony with a defined microbiota. it is necessary to demonstrate that microorganisms of the specified microbiota are present and that adventitious microorganisms are absent. in the past, monitoring relied on bacterial morphology, limited evaluation of biochemical traits, and growth characteristics. with the advent of polymerase chain reaction (pcr) technology, the eight asf strains were identified taxonomically by s rrna sequence analysis . three strains were previously identified as lactobacillus acidophilus (strain asf ), l. salivarius (strain asf ), and bacteroides distasonis (strain asf ), based on phenotypic criteria. s rrna analysis and genome sequencing indicated that each of the strains differed from its presumptive identity (wannemuehler et al., ) . the s rrna sequence of strain asf is essentially identical to the s rrna sequences of the type strains of l. murinus and l. animalis (both isolated laboratory animal medicine from mice), and all of these strains probably belong to a single species. strain asf is a novel lactobacillus that clusters with l. acidophilus and l. lactis. strain asf is a parabacteroides sp. the spiral-shaped strain, strain asf , is in the flexistipes phylum, exhibits sequence identity with rodent isolates of robertson, and has been formally named, mucispirillum schaedleri (robertson et al., ) . the remaining four asf strains, which are eos fusiform bacteria, group phylogenetically with the low-g + c content gram-positive bacteria (firmicutes, bacillus-clostridium group) (asf -eubacterium plexicaudatium; asf -firmicutes bacterium; asf and asf -clostridium sp.) (fig. . ) . the s rrna sequence information was determined by dewhirst et al. ( ) and draft genome sequences for each member of asf were recently published (wannemuehler et al., ) . this genetic data will permit detailed analysis of the interactions of asf organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms. the lymphatic system consists of lymph vessels, thymus, lymph nodes, spleen, solitary peripheral nodes ( fig. . ) , and intestinal peyer's patches. mouse lymph nodes are numerous but typically are small, reaching only a few millimeters. the typical lymph node is beanshaped and consists of a cortex and a medulla. the cortex is divided into b lymphocyte domains, called primary follicles, and t lymphocyte domains, known s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l l e s s i i i i i i c c l l section of the gi tract bacteroides sp. asf the sections are taken from the esophagus (esop.) (section e ), stomach (sections s and s ), small intestine (sections i to i ), ileocecal junction and apical cecum (sections c and c , respectively), and colon (sections l to l ). from sarma-rupavtarm et al. ( ). as the diffuse cortex. the mouse does not have palatine or pharyngeal tonsils. the spleen lies adjacent to the greater curvature of the stomach. different strains of mice have varying degrees of accessory splenic tissue. age, strain, sex, and health status can affect the size, shape, and appearance of the spleen. male spleens, e.g., may be % larger than those of females. most lymphocytes enter and leave the spleen in the bloodstream. the so-called white pulp of the spleen is organized along the central arteriole and is subdivided into t-and b-cell zones. the periarteriolar sheath is composed mainly of cd + and cd + t cells, and lymph follicles, which often contain germinal centers, are located at the periphery. the red pulp consists of sinusoids and hemoreticular tissue. cellular and humoral components of immunity are distributed to the bloodstream and tissues by efferent lymphatic vessels and lymphatic ducts, which empty into the venous system. the thymus is a bilobed lymphoid organ lying in the anterior mediastinum. it reaches maximum size around the time of sexual maturity and involutes between and days of age. the thymus plays a major role in maturation and differentiation of t lymphocytes. this function is not complete in newborn mice. thymectomy is routinely performed in immunological research for experimental manipulation of the immune system. thymectomy of newborn mice causes a decrease in circulating lymphocytes and marked impairment of certain immune responses, particularly cellular immune responses. thymectomy in adult mice produces no immediate effect, but several months later mice may develop a progressive decline of circulating lymphocytes and impaired cellular immune responses. the mutant athymic nude mouse is a powerful experimental tool in the study of the thymus in immune regulation (fogh, ) . the mucosa-associated lymph tissue (malt) contains more lymphoid cells and produces greater amounts of immunoglobulin than both the spleen and the lymph nodes. the term malt designates all peripheral lymphoid tissues connecting to cavities communicating with the external milieu. they include the peyer's patches, the cecal lymphoid tissue, and the lymphoid tissue in upper and lower respiratory tract, as well as the respiratory and genitourinary system. lymphatics drain these lymphoid-rich areas, thus providing a direct link with lymph nodes and the bloodstream. bone marrow and splenic red pulp produce erythrocytic, granulocytic, and megakaryocytic precursors over the life of the mouse. bone marrow is located in the protected matrix of cancellous bone and is sustained by reticular tissue rich in blood vessels and adipose cells (pastoret et al., ) . normal hematologic values are listed in table . . bone marrow-derived mononuclear phagocytes remove particulate antigens and act as antigen-presenting cells for lymphocytes. tissue macrophages, which often function in a similar way, are found in many tissues, including peripheral lymphoid tissues, lung, liver, intestine, and skin. the cardiovascular system of mice is reviewed extensively by hoyt et al. in the nd edition of volume iii; normative biology, husbandry and models in the aclam series the mouse in biomedical research (hoyt, ) . the heart consists of four chambers, the thin-walled atria and the thick-walled ventricles ( fig. . ) . mice conditioned to a recording apparatus have mean systolic blood pressures ranging from to mmhg. an increase in body temperature does not lead to an increase in blood pressure. heart rate, cardiac output, and the width of cardiac myofibers are related to the size of the animal. heart rates from to /min have been recorded for mice, and there are wide variations in rates and blood pressure among strains. the skeleton is composed of two parts: the axial skeleton, which consists of the skull, vertebrae, ribs, and sternum, and the appendicular skeleton, which consists of the pectoral and pelvic girdles and the paired limbs. the normal vertebral formula for the mouse is c t l s c , with some variations among strains, especially in the thoracic and lumbar regions. normal mouse dentition consists of an incisor and three molars in each quadrant. these develop and erupt in sequence from front to rear. the third molar is the smallest tooth in both jaws; the upper and lower third molar may be missing in wild mice and in some inbred strains. the incisors grow continuously and are worn down during mastication. the mouse brain has a typical mammalian structure as documented by a detailed study of the neuroanatomy of the c bl/ j mouse (sidman et al., ) . more recently, gene expression patterns have been used to study the functional anatomy of the mouse brain (bohland et al., ) . use of wild-type and genetically modified mice in behavior, learning, and memory paradigms has exponentially increased over the last decade. the male reproductive organs consist of paired testes, urethra, penis, prostate and associated ducts and glands ( fig. . ) . the female reproductive organs consist of paired ovaries and oviducts, uterus, cervix, vagina, clitoris, and paired clitoral glands ( fig. . ). the clitoral glands are homologous to the male preputial glands and secrete a sebaceous substance through ducts entering the lateral wall of the clitoral fossa. the female mouse normally has five pairs of mammary glands, three in the cervicothoracic region and two in the inguinoabdominal region ( fig. . ). the mammary glands are often not appreciated for how far they extend over the cervical, axillary, and inguinoabdominal flank regions which become the following section summarizes normal reproduction in the mouse. the reader is referred to a more comprehensive text in the aclam series (pritchett and taft, ) and online resources such as the jackson laboratories publication of the biology of the laboratory mouse (http://jaxmice.jax.org/jaxnotes/ / j.html). external influences, such as noise, vibration, diet, light cycle, and cage density, and intrinsic factors, such as health status, genetics, and parity impact reproductive success by directly or indirectly influencing the hypothalamic-pituitary axis for hormonal control of ovarian and testicular function. genotype also dramatically affects the reproductive performance of the mouse. coincident with the explosion in the number of mouse strains, each with unique induced or spontaneous mutations, a sound breeding program must include training of care staff to recognize anticipated and unanticipated breeding performance and strain or stock characteristics. in the new age of genomics, older methods of confirming genetic purity of mouse lines are being replaced with formal genetic monitoring by comparing strain-specific panels of single-nucleotide polymorphisms (snps). follicle-stimulating hormone promotes gametogenesis in both sexes. luteinizing hormone promotes the secretion of estrogen and progesterone in the female and androgen in the male. prolactin promotes lactation and development of the ovary during pregnancy. these gonadal hormones also ensure proper maintenance of the reproductive tract and modulate behavior to promote successful mating. the hypophysis is usually responsive to hormonal influence by day in the male and day in the female. ovarian follicle development begins at weeks of age and matures by days. rising levels of gonadotropins evoke signs of sexual maturity at about the same age. in the female, estrogen-dependent changes such as cornification of vaginal epithelium at the vaginal opening can occur as early as - days. puberty is slightly later in the male (up to weeks). sexual maturation varies among strains and stocks of mice and is subject to seasonal and environmental influences. mating behavior and the ability to conceive and carry fetuses to parturition are under complex hormonal control mediated by the anterior pituitary. the mouse is polyestrous and cycles every - days. in the first two phases (proestrus and estrus), active epithelial growth in the genital tract culminates in ovulation. degenerative epithelial changes occur during the third phase, followed by diestrus, a period of quiescence or slow cell growth. the cycle can be followed by changes in the vaginal epithelium that are often used to determine optimum receptivity of the female for mating and fertilization (table . ). patency of the vaginal orifice and swelling of the vulva are useful signs of proestrus and estrus ( fig. . ) . irregularities of the estrous cycle occur during aging. seasonal and dietary factors, such as estrogenic substances found in a variety of feeds, and genetic backgrounds also influence estrous cycles. estrus is routinely observed in mice at about - h after parturition (postpartum estrus). however, cornification of the vagina is not complete, and fertile matings are not as frequent compared with normal estrus. mice are spontaneous ovulators. ovulation does not accompany every estrus, and estrus may not coincide with every ovulation, because estrus is dependent on gonadal hormones, whereas ovulation is responsive to gonadotropin. the cyclicity of estrus and ovulation is controlled by the diurnal rhythm of the photoperiod. mating, estrus, and ovulation most often occur during the dark phase of the photoperiod. reversing the timing cook ( ) . laboratory animal medicine of the light-dark cycle reverses the time of estrus, ovulation, and mating. pheromones (table . ) and social environment also affect the estrous cycle. for example, estrus may be suppressed in group-housed female mice and reentry into estrus can be synchronized by exposure to pheromones in male mouse urine ('whitten effect'). once exposed to male urine, most female mice will be in estrus within days with a second estrus in about days. hence, estrus can be synchronized by group-housing females prior to pairing with males. in contrast, pheromones from a strange male mouse, particularly of a different strain, may prevent implantation or pseudopregnancy in recently bred females and is known as the 'bruce effect'. see section ii.c on behavior for more detail on the effect of pheromones on mouse reproductive behavior. mating is normally detected by formation of a vaginal plug (a mixture of the secretions of the vesicular and coagulating glands of the male) whose prevalence is highly strain dependent. the plug usually fills the vagina from cervix to vulva (fig. . ). plug detection is often coupled with vaginal cytology to evaluate fertility and conception. when the cervix and vagina are stimulated physically during estrus, prolactin is released from the anterior pituitary to enable the corpus luteum to secrete progesterone. secretion continues for about days. if fertilization has occurred, the placenta takes over progesterone production. if fertilization does not occur, a pseudopregnant period ensues, during which estrus and ovulation do not occur. fertilization usually takes place ( ) testis, ( ) head of epididymitis, ( ) caudal epididymitis, ( ) vas deferens, ( ) testicular vein, ( ) ampullary gland, ( ) seminal vesicle, ( ) anterior prostate, ( ) ureter, ( ) bladder, ( ) ventral prostate, ( ') dorsal prostate, ( ) urethra, ( ) bulbourethral muscle, ( ) ischiocavernosus, ( ) bulbourethral gland, ( ) diverticulum of bulbourethral gland, ( ) penis, ( ) preputial gland, ( ) glans penis, ( ) prepuce, ( ) testicular artery, and ( ) vas deferens artery. adapted from (komarek, ) . fallopian tube, ( ) uterine horn, ( ) endometrium, ( ) cervix, ( ) vagina, ( ) vaginal vestibulum, ( ) clitoris, ( ) clitoral gland, ( ) urethra, ( ) bladder, ( ) medial ligament of bladder, ( ) lateral ligament of bladder, ( ) left ureter, ( ') right ureter, ( ) mesovarium, ( ) mesometrium, ( ) ovarian artery, ( ) uterine horn artery, and ( ) ovarian artery and vein. adapted from (komarek, ) . adapted from komarek ( ) . in the ampulla or the upper portion of the oviduct. ova can be fertilized to produce normal embryos for - h after ovulation. gestation is usually - days. because of postpartum estrus, lactation and gestation can occur simultaneously. lactation can delay gestation because of delayed implantation. this may cause prolongation of gestation for up to - days in certain inbred strains. the effective reproductive life of some inbred strains approaches years where optimum environmental conditions are maintained, but litter size usually decreases as the female ages. therefore, females are usually retired by months of age. average litter size is strain dependent and commonly ranges from to pups. maternal care can account for about % of the variation in body weight of neonatal mice. nursing females usually lactate for weeks. milk production increases up to days postpartum and then declines until weaning at days. interestingly, oxytocin is required for nursing but is not essential for parturition or reproductive behavior (nishimori et al., ) . some transmission of humoral immunity from dam to progeny occurs in utero, but the majority of antibody is transferred through colostrum. transmission of passive immunity by colostral antibodies has been demonstrated to a wide variety of antigens, including viruses, bacteria, and parasites. antibodies continue to be secreted in the milk throughout lactation. decay of maternally acquired immunity occurs within several months after weaning. loss of maternal immunity increases susceptibility to infection and warrants continued care of weaned mice under barrier conditions. mice are socially gregarious animals with strong family bonds who communicate through complex olfactory, auditory, tactile, and visual signals. wild mice aggregate into groups called demes with low exchange of individuals between different groups. each deme consists of kinrelated members with a high degree of natural inbreeding, higher mutation rates compared to other mammals, and a wide range of developmental flexibility based on early life experience, which all contribute to their remarkably successful environmental adaptability. the deme is composed of a dominant breeding male, a hierarchy of females, subordinate males, and juveniles. wild mice occupy territories measuring just a few square meters when food is abundant to several square kilometers. mice are crepuscular (active during the twilight hours of dawn and dusk), strongly territorial, and omnivorous. coprophagy contributes to approximately one-third of their ingesta as an essential nutritional activity. aside from territoriality, social interactions, breeding, burrowing (when conducive substrates are available), and nest building are major activities. in managing laboratory mice, it is important to understand the complex behavioral biology of their free-living counterparts (latham and mason, ) . chemo-olfactory communication is mediated through extremely diverse chemical factors that trigger innate (non-learned) social responses among conspecifics, known as pheromones (table . ). pheromones have been traditionally divided into two broad categories: releaser pheromones, which elicit an immediate behavioral response, and primer pheromones, which mediate a slowly developing and longer-lasting endocrine response. this original definition of pheromone categories has been expanded to another category, termed signaler pheromones, which convey individual or group identity, as well as mediating parent-offspring recognition and mate choice. the biology and genetics of pheromone signaling is being extensively studied in the mouse as a model of mammalian pheromone communication (brennan and zufall, ; rodriguez and boehm, ) . mouse pheromones are excreted in the urine, as well as plantar, salivary, lacrimal, preputial, and mammary glands. in the urine, major urinary proteins (mups), small peptides, mhc class i peptides, volatile chemicals, and sex hormones all contribute to chemosignals that communicate dominance, kinship, diversity, and gender. wild mice possess a great deal of individual variations of roberts et al. ( ) . c ferrero et al. ( ) . laboratory animal medicine these elements, providing a 'bar code' that distinguishes individuals. inbreeding of laboratory mice has reduced individual variation, but each inbred strain possesses a characteristic array of signals, and to a certain extent, unique signals exist among individuals within a strain (sharrow et al., ; sturm et al., ) . pheromones are detected by sensory neurons in the vomeronasal organ, the olfactory epithelium, and the lesser known septal organ of masera within the olfactory epithelium, and the gruenberg ganglion, which is located at the anterior end of the nasal cavity (breer et al., ; chamero et al., ; liberles and buck, ; restrepo et al., ) . neuronal signals are transmitted to the ganglion layer of the olfactory bulb, and thence to the brain. mups are important components of chemosensory communication in mice, and also an important occupational hazard to human handlers. chromosome contains a cluster of mup genes, plus a number of pseudogenes. mups are small soluble proteins known as lipocalins, which bind small organic chemicals (pheromones) with high affinity, and function as pheromone transporters and stabilizers (thereby contributing to slow release), but also act as protein pheromones themselves. they are synthesized in the liver and excreted in the urine, as well as nasal mucosa, lacrimal glands, and salivary glands. their endogenous role on metabolic activity is not yet understood. male mice excrete significantly more mups in the urine than females. one wellcharacterized mup is 'darcin', named after fitzwilliam darcy, the romantic hero in pride and prejudice. as its name implies, it is a female attractant. mups also act as kairomones, which function as chemical signals between species. for example, cat and rat mups invoke fear in mice. mups are important in the laboratory animal management context, as they are excreted in copious amounts ( - mg/ml in urine) and are potent allergens for humans, particularly mus m (ag or ma ), which is encoded by the mup gene (sharrow et al., ) . chemosensory communication has numerous behavioral effects that influence mouse social interactions. one of the most studied behavioral effects is the bruce effect, or pregnancy block, which is a complex physiologic response in which recently conceived females resorb fetuses during early pregnancy in the presence of an unrelated male, particularly a dominant male. the continued presence of the original mate protects the female from this effect (bruce, ) . the vandenbergh effect results in acceleration of puberty of juvenile females in response to male urine (vandenbergh, ) . the lee-boot effect occurs among group-housed females that are isolated from males, in which there is suppression of estrus cyclicity (van der lee and boot, ) . the whitten effect results in synchronization of estrus among a group of females in response to a male (whitten et al., ) . the lee-boot and whitten effects are utilized in the laboratory to assist in induction of synchronized timed pregnancy, but the bruce effect can have deleterious consequences on breeding colonies when foreign males are introduced to a breeding colony, as pheromone communication can occur in the absence of direct contact. the above effects are well-defined pheromone-driven behavioral responses, but chemosensory communication has a myriad of other effects. estrus, pregnant, or lactating females also accelerate puberty among juvenile females. females use odor cues to avoid parasite-laden males, males prefer odors of estrus females, and estrus females prefer odors of dominant males. mice have strong mating and social preferences based upon mhc proteins, which indicate genetic relatedness. maternal recognition of young is also mhc-related, and pups prefer nest odors of maternal and sibling pups based upon mhc relatedness. male aggression against unrelated males is also a strong mhc-related phenomenon. mhc haplotypes determine not only mhc proteins in the urine, and mhc-specific olfactory receptors, but also the composition of volatile chemicals in the urine (kelliher and wersinger, ) . the complexity of social communication extends to auditory stimuli as well. male mice utilize ultrasonic 'birdsong' to vocally communicate and attract females. mouse vocalization patterns are largely genetically innate and unique to each strain of mouse, but they can also be modified, or learned, to a limited extent (arriaga et al., ) . the behavioral biology of the mouse is highly complex, and depends upon genetic, physiologic, social, and environmental variables, which all impact on how laboratory mice can best be managed in captivity. it is clear that this rich complexity cannot be fully addressed under laboratory conditions, but that does not mean that basic needs, such as nest building, burrowing, foraging, and olfactory environments, cannot be provided. for example, intermale aggression, which is particularly apparent in some strains of mice such as balb/c and swiss-origin stocks and strains, can be minimized by maintaining males from infancy as sibling groups, since adult siblings tend not be aggressive to one another. this sibling bond, however, can be easily broken by short-term separation. environmental enrichment often features provision of plastic houses, which may make vivarium managers feel good, but maximal enrichment can be provided by provision of nesting material, which includes structural scaffolding, such as crinkled cardboard, which facilitates construction of three-dimensional nests. mouse nests are replete with 'appeasement' pheromones, thereby contributing to harmony within the cage, whereas introduction of dirty bedding has the opposite effect. frequent cage changing, including removal of established nests, is highly stressful and disruptive to social harmony within a cage. provision of appropriate and adequate amounts of bedding material that is conducive to burrowing is desirable. it is important to remember that mice are socially gregarious, and that mouse welfare is optimally enriched by other mice within a socially harmonious deme (latham and mason, ; van loo et al., ) . a laboratory mouse ethogram, defined as an operationalized list of mouse behaviors, arranged by their adaptive meaning to the animal, is available on the web: www. mousebehavior.org. behavioral phenotyping, particularly of transgenic mice, is used extensively in genomic research. a wide variety of standardized test batteries and approaches are used, depending upon the focus of research (reviewed in crawley ) . initial behavioral evaluations include general health, body weight, body temperature, appearance of the fur and whiskers, and neurological reflexes assessment. specific tests include observations of home cage behaviors, righting reflex, acoustic startle, eye blink, pupil constriction, vibrissae reflex, pinna reflex, digiscan open field locomotion, rotarod motor coordination, hanging wire, footprint pathway, visual cliff, auditory threshold, pain threshold, and olfactory acuity. novel and complex environmental enrichment in animal housing conditions facilitates enhanced sensory and cognitive stimulation as well as physical activity. environmental enrichment and exercise have beneficial effects such as cognitive enhancement, delayed disease onset, enhanced cellular plasticity, and associated molecular processes in animal models of brain disorders (pang and hannan, ) . the immune system of the mouse is very similar to that of humans. the availability of inbred mouse strains, in which each individual animal expresses identical mhc alleles so that tissues and cells can be transplanted without tissue rejection, greatly simplifies and indeed enables functional analyses of immune system components not possible with any other outbred mammalian species. in addition, the ability to genetically manipulate the mouse genome, adding to, altering, and deleting existing genes, enables unprecedented in vivo analysis of immune cell functions. it is for these reasons that the mouse is the primary animal model for immunology research. the immune system is an unusual organ system in that it consists of both solid tissues and various migrating cell populations. the bone marrow and thymus are considered primary lymphoid organs, as sites of hematopoiesis and b-and t-lymphocyte development, respectively. lymph nodes, spleen, and intestinal peyer's patches are considered secondary lymphoid tissues, as sites of immune response initiation. lymph nodes and spleen are analyzed frequently for studies of immune responses and as organs for immune cell isolation. tertiary lymphoid tissue sites are those that form in other solid organs in response to an insult or microbial exposure. among them are the lymphoid cell aggregates of the gastrointestinal and respiratory tract, also called 'gut-associated lymphoid tissue' (galt) and bronchusassociated lymphoid tissues (balt). leukocytes are classified as belonging to the innate or adaptive immune system. the innate immune system responds rapidly to an antigen insult via recognition of pathogen-associated molecular patterns (pamps), such as lipopolysaccharide, bacterial flagellin, single (s)-and double-stranded (ds) rna, and non-methylated dna, via extracellular or intracellular pattern recognition receptors (prrs). receptors include the toll-like receptors (tlrs), such as tlr (recognizing lps), tlr / (ss and dsrna) and tlr (dna), nod-like receptors (nod / ), and rig-like receptors (rig-i, mda- ) among others (takeuchi and akira, ) . cells of the innate immune system are monocytes/macrophages, granulocytes and dendritic cells as well as innate-like lymphocyte populations (ilc) , and , which include natural killer (nk) cells (spits et al., ) . cells of the adaptive immune system (t and b lymphocytes) express a highly antigen-specific receptor that has arisen through gene rearrangement (t-cell and b-cell receptors, respectively). b cells of the b- lineage and γδ t cells are regarded as innate-like cells, as they express a rearranged antigen receptor but seem to respond in an innate-like manner. leukocytes are identified and classified by sets of monoclonal antibodies (mab) against uniquely expressed surface receptors, typically measured by flow cytometry. identification of a unique receptor by one or more mab of the same specificity leads to the assignment of a receptor name, as a 'cluster of differentiation (cd)'. for example, t cells are differentiated into two subsets based on their expression of either cd or cd . cd + t cells (t helper cells) recognize peptides presented in mhc class ii and promote b-lymphocyte activation and activate and regulate cellular immune responses via secretion of differing cytokines (see below). cd + t cells recognize antigenic peptides presented in mhc class i and serve as cytotoxic cells during the cell-mediated immune response where they can destroy infected cells (e.g., against cells containing infectious agents). the major function of b cells is to respond to an encounter with an antigen/pathogen with the production of highly antigen-specific immunoglobulins (ig; antibodies), which can bind to and inactivate pathogens and toxins. activation of b cells can lead to their differentiation to plasma cells, which produce large amounts of ig. five laboratory animal medicine classes or ig 'isotypes' can be distinguished, which differ in effector function: igm, igg, iga, ige, and igd. the latter is expressed only on the surface of b cells in mice. the igg class, the most abundant antibody class in the serum, is further divided into subtypes: igg , igg a/c , igg b , and igg . polymorphisms exist on the ig locus such that some strains of mice produce the igg a subtype (e.g., balb/c), whereas others produce igg c (e.g., c bl/ ) (zhang et al., ) . additional allelic polymorphisms of the locus also exist. for example, balb/c and sv mice express the igh-a allotype, whereas c bl/ mice express the igh-b allotype. recombinant inbred strains of mice exist for both balb/c and c bl/ , which harbor the reciprocal igh locus (i.e., igh-b for balb/c and igh-a for c bl/ mice). these mice are useful tools for tracking b cells following adaptive cell transfer via allotype-specific mab (see below). immunoglobulin isotype production varies according to the type of immunogen used to evoke the response. igm is secreted short term after initial exposure to an antigen, followed by the other ig isotypes. in viral and intracellular bacterial infections, igg a/c is dominant, whereas in extracellular bacterial infections igg dominates the response. igg b and igg are usually induced to carbohydrate or lipid antigens. ige is linked to parasitic infections and to allergy. serum antibodies specific for an immunogen can often be measured for the life of the animal. while serum iga levels are low, iga is the highest produced ig in mice. iga production, however, occurs in plasma cells lodged in the lamina propria of mucosal tissues, from where the iga is actively transported in dimeric form onto the luminal surface of mucosal tissues as 'secretory' iga (brandtzaeg, ). cytokines are secreted signaling molecules involved in cell-cell communication in a complex biological system (table . ). these include the large family of interleukins (ils, currently il- to il- ), tumor necrosis factors (tnfs), interferons (type i, ii, and iii) and growth factors such as granulocyte-macrophage colonystimulating factor (gm-csf) and stem cell factor (scf). cytokine secretion often occurs in response to recognition of antigen via prr or tcr. because of their importance in modulating immunity to antigenic stimuli, mice with specific deletions or overexpression of individual cytokines have been made and have contributed to a detailed understanding of many of their often pleotropic functions (akdis et al., ) . chemokines are a similarly large group of small, secreted molecules that regulate cell trafficking to sites of antigen encounter but also facilitate cell-cell contact by acting as chemoattractants. chemokines are grouped according to the number of cysteines and disulfide bonds in the molecule into c-x-c-, c-c, c, and cx cl chemokine ligands (l) and receptors (r) and designated accordingly as cxcr - /cxcl - and ccr - /ccl - (allen et al., ) . immune responses must be coordinated to provide the most appropriate effector functions for the type of pathogen/antigen encountered. immune effector responses differ depending on the life cycle (facultative or obligate intracellular, extracellular, localized, systemic, etc.) and antigen types displayed by the encountered antigen/ pathogen, because this affects the type of prr engaged and activated. prr engagement leads to cytokine and chemokine responses by the first responders, i.e., epithelial cells, local macrophage populations and other innate cells. the type of cytokines and chemokines produced then dictates the types of cells recruited to the site of infection and their subsequent differentiation and functions. the prr engagement also leads to antigen uptake, activation and migration of dendritic cells (dcs) from the site of insult to the regional lymph nodes, where dcs present antigen peptides on mhc molecules to t cells. in addition, the dcs secrete cytokines induced by the initial prr activation, which cause the differentiation of cd t cells towards a particular effector response. for example, secretion of il- in response to activation of tlr or will result in the induction of interferongamma (ifn-γ) production by cd t cells, whereas il- and tgf-β production by dc will induce cd t cells to secrete il- (kara et al., ) . because the dc translates signals from prr at the site of infection into differentiation signals for t cells in the lymph tissues, these cells are regarded as a 'bridge' between the innate and adaptive immune systems. the specific ig isotype secreted in response to a pathogen depends to a large degree on the type of cytokine produced by cd t cells that provide 't-cell help' for b cells. t cells that interact with b cells are identified as a discrete subset termed 't follicular helper cells (t fh )' and it is their cytokine profile that directs b cells to secrete a particular ig isotype (kara et al., ) . the classic t h /t h dichotomy outlined above was in part shaped by the observation that ifn-γ production will lead to switching of b cells to secrete igg a/c, whereas production of il- leads to the secretion of igg . interestingly, it appears that the cytokine profile induced by the effector t-cell population is mirrored by the innate immune response. innate-like lymphocytes also have effector phenotypes that correspond to those of cd t cells and are induced by the same signals and transcriptional regulators (spits et al., ) and the same appears to be true also for macrophages and other innate immune cells (sica and mantovani, ) . while initial studies identified two particular antagonistic effector response types (termed t h and t h and classified by t-cell production of ifn-γ and il- , respectively), more recent studies now demonstrate a much wider array of effector responses in which innate and adaptive immunity acts together to reinforce an immune response phenotype as well as modulate its size by induction of t regulatory cells (t regs ) that generate inhibitory cytokines (kara et al., ; sica and mantovani, ; spits et al., ) . the use of cytokine-deficient and reporter mice that enabled the identification of cytokine-producing cells via expression of a fluorescent reporter was particularly valuable for the development of this more nuanced view of the quality of immune responses. spontaneous mouse models of immune deficiencies have been used extensively in research. their use, plus the expanding number of knockout, transgenic, and dominant negative mouse mutants, has advanced understanding of human immune deficiency diseases as well as basic understanding of the immune system (table . ). interbreeding of multiple immune-deficient mice has allowed the development of 'humanized' mice in which immune cells of the mouse are replaced with those of humans. while many challenges remain to fully replenish mice with components of the human immune system, the use of immune-deficient nod/severe combined immunodeficient (scid)/il- rγ -/recipients for transfer of human peripheral blood lymphocytes, cordblood or bone marrow-derived cd + stem cells with human liver and thymus (blt-mice) is yielding promising results (akkina, ) . investigators using genetically engineered mice are constantly reminded that phenotypic analysis of these animals must be done cautiously because the immune system may be profoundly affected and in ways that are not always anticipated. this may make it difficult to determine whether a given gene product is directly involved or may be secondary to a more global dysregulation of the immune system. as with other biological systems, compensation mechanisms also may mask the phenotype. experimental approaches are being increasingly used to refine the knockout technology by restricting a specific genetic deficiency to a particular tissue of interest using the cre-lox system, in which tissue-specific or temporal restricted expression of the cre recombinase induces the deletion of a 'floxed' gene (mak et al., ) . transgenic mice are available that restrict cre expression to various hematopoietic cells or tissue or drive cre recombination following injection of tamoxifen. other approaches are the generation of 'bone marrow irradiation' chimeras. here, inbred wild-type mice or mice deficient in certain immune cells (table . ) are lethally irradiated by exposure to a gamma-irradiation source to deplete the hematopoietic stem cells. these are then replaced by transfer of bone marrow cells to the irradiated mice. reconstitution of the hematopoietic system is usually achieved within about weeks, during which time mice are provided with antibiotic-containing drinking water to avoid infections of these temporarily immune-compromised animals. transfer of bone marrow from a congenic knockout restricts the genetic defect to the hematopoietic system. a mix of bone marrow from two sources is also often used to generate tissue-specific knockouts. for example, mixing a bone marrow from t-cell-deficient mice ( %) with that of a gene knockout ( %) generates 'mixed bone marrow chimeras' in which all t cells only develop from the knockout, thus lack the gene of interest, whereas most of the other cells t from the wild-type source, effectively constraining the genetic defect to the t-cell population. sets of congenic mice with defined allotypic differences are often used to confirm the source of individual cells. such markers include the gene locus cd . /cdc . or cd . /cd . (thy . /thy . ). alternatively, cells may express a fluorescent transgene, such as green-fluorescent protein (gfp). identification is usually performed by flow cytometry, or less commonly by immunofluorescence or immunohistochemistry. generation of bone marrow chimeras circumvents the time-consuming breeding of cre recombinase-expressing flx/flx mice. however, numerous controls are needed to exclude off-target effects due to irradiation damage. repeat injection of antibodies targeting specific cell populations is another rapid approach that avoids the potential for irradiation damage and allows short-term depletion of individual cell subsets. its main disadvantage is the need to identify mab that bind to surface receptors uniquely expressed by a cell subset of interest and the verification of the efficacy of the depletion. frequently used is antibody treatment for the short-term depletion of t-cell subsets using mab against cd or cd as well as individual cytokines. contemporary knowledge about diseases of laboratory mice has developed primarily from examining the effects of disease on traditional strains and stocks. the widespread use of genetically engineered mice is likely to modify current concepts because of novel or unpredictable interactions among genetic alterations, the genetic backgrounds on which they are expressed, and exogenous factors, such as infectious agents. because the number of combinations is extraordinarily high, clinical and laboratory diagnosticians should be alert to the potential for altered disease expression in genetically engineered mice and not be misled by unexpected signs, lesions, and epizootiology. many microbial agents have the potential to cause disease in mice or interfere with mouse-based research. housing and husbandry in microbiologically sheltered environments are designed to reduce the risks of disruptive infection, especially among immunologically dysfunctional mice, but must be accompanied by effective microbiological surveillance. surveillance should encompass resident mice and mouse products (serum, cell lines, transplantable tumors) procured from external sources. because surveillance strategies will vary with research needs and operating conditions, it is prudent to consult a number of sources, such as the federation of european laboratory animal science associations (felasa) (nicklas et al., ) and commercial laboratories, for guidance. detailed discussion of microbial quality control is provided in chapter . there are also recommendations regarding specific agents in following sections. diagnostic methods involve gross and microscopic pathology, parasitology, microbial isolation and culture, serology, and pcr. serology is particularly important for viral surveillance, and now relies principally on enzyme-linked immunosorbent assay (elisa), multiplex fluorescent immunoassay (mfi) for simultaneous detection of antibodies to multiple agents (hsu et al., ) , indirect fluorescent antibody (ifa) assay, or hemagglutination inhibition (hai), with the latter two methods generally used for confirmation (livingston and riley, ; pritchett-corning et al., ) . mouse antibody production (map) testing has been historically used for testing biological materials for contamination by infectious agents. pcr panels for murine infectious agents are now commercially available and have cost and time-saving advantages as well as improved assay sensitivity and specificity. beyond the classic bacterial and viral murine infections, pcr assays are now available for endo-and ectoparasites (see chapter ). etiology mousepox is caused by ectromelia virus (ectv), an orthopoxvirus that is antigenically and genetically closely related to a number of other poxviruses, including vaccinia, variola, and cowpox viruses. the original isolate of ectv, known as the hampstead strain, was discovered by j. marchal in (marchal, as the cause of epizootic disease among laboratory mice in england. the disease featured amputation of extremities, which marchal termed ectromelia (from the greek, ectro, amputation and melia, limb). other strains of the virus include moscow, nih- , washington university, st. louis , beijing , ishibahsi i-iii, and naval (nav) strains, which vary in virulence, but are essentially indistinguishable genetically and serologically, suggesting a common origin. virus can be isolated from infected tissues by inoculation of cell cultures (bs-c- , hela, l cells) or embryonated eggs. the natural host (and original source of infection of laboratory mice) of ectv remains unknown. clinical signs the expression of clinical signs reflects an interplay among virus-related factors, including virus strain, dose and portal of entry, and host-related factors, including age, genotype, immunological competence, and gender (brownstein et al., a) . during natural epizootics, it was observed that a, bc, dba/ , dba/ , and cba strains developed acute fatal infections, whereas c bl/ mice were resistant to severe disease (briody, ) . experimental studies have shown that all strains of mice are susceptible to infection, but balb/c, a, dba/ , and c h/he mice were highly susceptible, akr and sjl mice were moderately susceptible, and c bl/ mice were highly resistant to lethal infection (bhatt and jacoby, c; wallace and buller, ) . the mechanisms of genetic resistance are not fully understood but appear to reflect multiple genes, some of which appear to be expressed through lymphoreticular cells, including nk cells (brownstein et al., a; jacoby et al., ) . the nuances of cytokine and cellular immune responses to ectv infection have received recent attention (reviewed in buller and fenner ( ) and esteban and buller ( ) ). outbreaks among susceptible mice are often volatile, with variable morbidity and high mortality in susceptible strains of mice. clinical signs such as ruffled fur or prostration may occur for only a few hours before death. mice that survive acute infection may develop chronic disease characterized by a focal or generalized rash anywhere on the body ( fig. . ). conjunctivitis also may occur. skin lesions usually recede within several weeks, but hairless scars may remain. additionally, severe viral infection of the feet and tail during the rash syndrome can lead to necrosis and amputation. epizootiology mousepox is not a common disease. outbreaks occur sporadically and recent outbreaks have been traced to the importation of contaminated mice or mouse products. for example, contaminated mouse serum was responsible for recent outbreaks in the united states (dick et al., ; . natural exposure is thought to occur through direct contact and skin abrasions. cage-to-cage transmission is low and can be virtually nil if filter-topped cages are used (bhatt and jacoby, b) . ectromelia virus is highly stable at room temperature, especially under dry conditions, leading to the potential for prolonged environmental contamination in infected colonies (bhatt and jacoby, d) . aerogenic exposure is not a major factor in natural outbreaks, and arthropod-borne transmission does not appear to occur. virus-free progeny can be obtained laboratory animal medicine from immune dams (bhatt and jacoby, b) . however, intrauterine infection and fetal deaths, albeit rare, have been reported. natural transmission is facilitated by intermediately resistant mice, which survive long enough to develop skin lesions that can shed virus for relatively long periods of time. the risks for transmission are further increased by persistence of infectious virus in excreta and exfoliated scabs. although virus excretion typically lasts for about weeks, virus has been found in scabs and/or feces for up to weeks. resistant mouse strains also are dangerous because they can shed virus during subclinical infections. however, infections in resistant mice tend to be short-lived. highly susceptible mice are a relatively small hazard for dissemination of infection, if properly discarded, because they die before virus shedding becomes prominent. thus, juxtaposition of resistant or intermediately resistant infected mice with highly susceptible mice can provoke explosive outbreaks. infant and aged mice are usually more susceptible to lethal infection than young adult mice. maternal immunity among enzootically infected breeding mice may perpetuate infection by protecting young mice from death, but not from infection. such mice may subsequently transmit infection by contact exposure. pathology the classic descriptions of ectv pathogenesis by fenner remain timely, including the frequently cited and reproduced figure summarizing the pathogenesis of infection ( fig. . ) (fenner, b) . interest in smallpox has renewed the interest in ectv as a model of host response to infection (esteban and buller, ) . ectv multiplies in the cell cytoplasm and produces two types of inclusion bodies. the a type (marchal body) is well demarcated and acidophilic in histological sections. it is found primarily in epithelial cells of skin ( fig. . ) or mucous membranes and can also be found in intestinal mucosa. the b type of inclusion is basophilic and can be found in all ectromelia-infected cells. however, it is difficult to visualize unless cells are stained intensely with hematoxylin. ectv antigen can be readily visualized by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections (esteban and buller, ; jacoby and bhatt, ) . following skin invasion, viral multiplication occurs in the draining lymph node and a primary viremia ensues. splenic and hepatic involvement begin within - days, whereupon larger quantities of virus are disseminated in blood to the skin. this sequence takes approximately week and, unless mice die of acute hepatosplenic infection, ends with the development of a primary skin lesion at the original site of viral entry. the primary lesion is due to the development of antiviral cellular immunity. severe hepatocellular necrosis occurs in susceptible mice during acute stages of mousepox. white spots indicative of necrosis develop throughout the liver ( fig. . ). in nonfatal cases, regeneration begins at the margins of necrotic areas, but inflammation is variable. splenic necrosis in acute disease commonly precedes hepatic necrosis but is equally or more severe. necrosis and scarring of red and white pulp can produce a macroscopic 'mosaic' pattern of white and red-brown the primary skin lesion, which occurs - days after exposure, is a localized swelling that enlarges from inflammatory edema. necrosis of dermal epithelium provokes a surface scab and heals as a deep, hairless scar. secondary skin lesions (rash) develop - days later as the result of viremia. they are often multiple and widespread and can be associated with conjunctivitis, with blepharitis, and, in severe cases, with buccal and lingual ulcers. the skin lesions also can ulcerate and scab before scarring. diagnosis mousepox can be diagnosed from clinical signs, lesions, serological tests, and demonstration of virus or viral antigen in tissues. observation of characteristic intracytoplasmic eosinophilic inclusions aids detection of infection. several serological tests are available to detect mousepox. historically, the standard test was hai, using vaccinia antigen as a source of hemagglutinin. elisa is more sensitive and specific and has replaced hai for serological monitoring among nonvaccinated mice (buller et al., ) . ectv infection also can be detected by ifa (buller et al., ) and pcr. serological differentiation of mousepox from vaccinia infection in vaccinated mice is based on the lack of hemagglutinin in the vaccine strain of virus. thus, serum from vaccinated mice may react by elisa but should not react by hai. differential diagnosis mousepox must be differentiated from other infectious diseases associated with high morbidity and high mortality. these include sendai pneumonia, mouse hepatitis, and tyzzer's disease. the latter two can be expressed by acute necrosis in parenchymal organs, but they can be differentiated by morphological, serological, and virological criteria. the skin lesions of chronic mousepox must be differentiated from other skin diseases caused by opportunistic or pathogenic bacteria, ascariasis, and bite wounds. prevention and control mousepox is a dangerous disease because of its virulence for susceptible mice. therefore, infected colonies should be quarantined immediately. depopulation has been used as a primary means for control, but confirmation of infection should be obtained before exposed mice are destroyed. tissues, supplies, instruments, or other items that have had potential contact with infected mice should be disinfected by heat or chemicals such as formalin, sodium hypochlorite, or chlorine dioxide. materials should be autoclaved or, preferably, incinerated. disinfected rooms should be challenged with susceptible sentinel animals that are observed for clinical signs and tested for seroconversion after several weeks. depopulation and disinfection must be carried out vigorously. because modern housing and husbandry methods based on the use of microbarrier caging are effective for containing infection, testing and culling properly isolated mice is a potential alternative, especially for irreplaceable breeding mice. such mice can be quarantined along with cessation of breeding to permit resolution of infection (bhatt and jacoby, b) . sequential testing with contact-exposed sentinels should be employed with this option. additionally, maternal immunity from fully recovered dams can protect mice from infection, thereby enhancing opportunities to derive virus-free mice from previously infected dams, with the caveat that progeny will be transiently seropositive with maternally derived antibody. vaccination can control or prevent clinically apparent mousepox. the hemagglutinin-deficient strain of vaccinia virus (ihd-t) is used to scarify skin on the dorsum of the tail. 'takes' should occur in previously uninfected mice by - days, but not in infected mice (bhatt and jacoby, a) (fig. . ). infected mice should be quarantined separately or eliminated. vaccination may not prevent infection, although infection in vaccinated mice is often transient. furthermore, vaccinia virus can be shed from scarification sites for at least several days. therefore, other preventive measures, such as strict controls on the entry of mice or mouse products, combined with periodic serological monitoring, should not be relaxed until diagnostic testing has confirmed the elimination of vaccinia and ectromelia virus. additionally, seroconversion evoked by vaccination must be taken into account in serological monitoring of vaccinated colonies. finally, vaccinia virus is a human pathogen, so vaccination procedures should include personnel protective measures to prevent exposure. research complications the primary threat from mousepox is mortality in susceptible mice. the loss of time, animals, and financial resources can be substantial. (shellam, , ) mice are naturally susceptible to two herpesviruses from the subfamily betaherpesvirinae and in the genus muromegalovirus, the two species murid herpesvirus (of which one of the members is mouse cytomegalovirus (mcmv)) and murid herpesvirus (of which one of the members is mouse thymic virus (mtv)). they are species-specific viruses and distinct from each other and from other rodent herpesviruses. mctv has received considerable attention as a model of human cmv infection. etiology mouse cytomegalovirus (mcmv) is a mouse-specific betaherpesvirus. it can, however, replicate in cell cultures from several species, including mouse (fibroblasts and t cells), hamster, rabbit, sheep, and nonhuman primate. cocultivation may be required to rescue latent virus. clinical signs mcmv causes subclinical infection in adult immunocompetent mice, but experimental inoculation of neonates can cause lethal disease due to multisystemic necrosis and inflammation. epizootiology the prevalence of mcmv in laboratory mice is probably uncommon but undefined, since infection is clinically silent and serological surveillance is not widely practiced. wild mice are commonly infected and serve as a natural reservoir for infection, which implies that the entry of virus into a modern vivarium is most likely to occur from contaminated animal products. persistence is a central feature of nonlethal infection. persistently infected mice excrete virus in saliva, urine, and tears for many months, resulting in horizontal transmission through mouse-to-mouse contact. virus also can infect prostate, testicle, and pancreas, implicating other modes of excretion. vertical transmission does not appear to be a common factor in natural infection. further, maternal immunity protects sucklings from infection. pathology mouse cytomegalovirus can replicate in many tissues, and viremia commonly occurs. lesions are not remarkable during natural infection and may be limited to occasional enlarged cells (megalocytosis) containing eosinophilic intranuclear and/or cytoplasmic inclusions associated with lymphoplasmacytic interstitial inflammation, especially in the cervical salivary glands. susceptibility to experimental infection varies with age, dose, route, virus strain, and host genotype. infection can occur in young and adult mice. however, the pathogenicity of mcmv for mice decreases with age. neonates are highly susceptible to lethal infection, but resistance to disease develops by the time mice are weaned. immunodeficient mice, however, remain susceptible to pathogenic infection as adults. persistent infection often affects the salivary glands and pancreas. the persistence of salivary gland infection appears to be dose dependent. there is experimental evidence that mcmv can produce latent infection of b cells, probably t cells as well as aforementioned tissues. persistent infection may lead to immune complex glomerulonephritis. latent persistent infection can be reactivated by lymphoproliferative stimuli and by immunosuppression. diagnosis mcmv antigens appear to be weak stimuli for humoral antibody production, which is consistent with the fact that cellular immunity is critical for protection against infection. neutralizing antibody titers are low during acute infection and difficult to find during chronic infection. serology and pcr-based diagnosis are available, but neither is widely used because of assumptions that infection has a very low prevalence. detection of enlarged cells with intranuclear inclusions, especially in salivary glands, is diagnostic, if they are present. in situ hybridization can be used as an adjunct to routine histopathology. differential diagnosis mcmv infection must be differentiated from infection with mtv. the latter virus can produce necrosis of thymic and peripheral lymphoid tissue when infant mice are experimentally inoculated. lytic lesions of lymphoid tissues are not a hallmark of mcmv. the viruses can also be distinguished from each other serologically. sialoadenitis with inclusions can occur during infection with mouse polyoma virus. like mcmv, mtv infects the salivary gland as its primary target organ. prevention and control control measures for mcmv have not been established, because it has not been considered an important infection of laboratory mice. cage-to-cage transmission has not been demonstrated, but horizontal infection from contaminated saliva must be considered. the exclusion of wild mice is essential. research complications mcmv can suppress immune responses. apart from the potential for interfering with immunology research, it can exacerbate the pathogenicity of opportunistic organisms such as pseudomonas aeruginosa. etiology mouse thymic virus (mtv) is a herpesvirus (murid herpesvirus ) that is antigenically distinct from mcmv. no suitable in vitro method for cultivation has been developed; therefore, viral propagation depends on mouse inoculation. clinical signs natural infections are subclinical. epizootiology the prevalence of mtv is thought to be low. mice can be infected at any age, although lesions develop only in mice infected perinatally. mice infected as infants or adults can develop persistent infection of the salivary glands lasting several months or more. excretion of virus in saliva is considered the primary factor in transmission. seroconversion occurs in adults but does not eliminate infection. infection in neonates may not elicit seroconversion, rendering such mice serologically negative carriers. the mode of infection is obscure, but virus is excreted in saliva, suggesting that transmission from infected dams to neonatal mice occurs by ingestion. mtv also has been isolated from the mammary tissue of a lactating mouse, suggesting the potential for transmission during nursing. prenatal transmission has not been found. pathology mtv causes severe, diffuse necrosis of the thymus and lymphoid tissue with tropism for cd + t cells in mice inoculated within approximately week after birth. the severity of thymic and lymph node necrosis can be mouse strain-dependent. grossly, the thymus is smaller than normal. infected thymocytes display mtv-positive intranuclear inclusions. necrosis is followed by granulomatous inflammation and syncytium formation. reconstitution of lymphoid organs takes - weeks. diagnosis thymic necrosis associated with intranuclear viral inclusions is the hallmark lesion. viral antigen can be detected by immunohistochemistry. serologic detection is effective, but generally not utilized, and is potentially negative in neonatally exposed mice. suspicion of infection in seronegative mice can be tested by inoculation of virus-free neonatal mice with homogenates of salivary gland or with saliva. inoculated mice should be examined for thymic necrosis - days later. pcr or the mouse antibody production (map) test can also be used to detect infection. differential diagnosis reduction of thymus mass can occur in severe mouse coronavirus infection, during epizootic diarrhea of infant mice, or following stress. prevention and control because mtv induces persistent salivary infection, rederivation or restocking should be considered if infection cannot be tolerated as a research variable. research complications mtv transiently suppresses cellular and humoral immune responses because of its destructive effects on neonatal t lymphocytes. parvoviruses are among the most common viral infections in contemporary laboratory mouse populations (livingston et al., ) (pritchett-corning et al., ) , and pose major challenges to both detection and control. the mouse parvoviruses are composed of two antigenically and genetically distinct but related groups, including minute virus of mice (mvm) and mouse parvovirus (mpv), with each group containing a number of strains. the international committee on taxonomy of viruses classifies mpv, mvm, and several other rodent parvoviruses into one genus, protoparvovirus, and species, rodent protoparvovirus , but these viruses will be treated separately herein. etiology minute virus of mice (mvm) is a small ( -kb) single-stranded dna virus. the prototypic strain is designated mvmp. an allotropic variant with immunosuppressive properties in vitro is named mvmi, and additional laboratory animal medicine named strains include mvmc and mvmm. the genome encodes two nonstructural proteins, ns- and ns- , which are highly conserved among the rodent parvoviruses and account for prominent cross-reactivity in serological assays that utilize whole virus antigen. the viral capsid proteins, vp- and vp- , are virus-specific and form the basis for serological differentiation of mvm from mpv. mvm has a broad in vitro host range. it replicates in monolayer cultures of mouse fibroblasts (a cells), c rat glial cells, sv (simian virus )-transformed human newborn kidney ( k cells), t-cell lymphomas (el ), and rat or mouse embryo cells, producing cytopathic effects that can include the development of intranuclear inclusions. clinical signs natural mvm infections are subclinical. neonatal mice of some inbred strains are experimentally susceptible to lethal renal and/or intestinal hemorrhage during mvmi infection, but this syndrome has not been reported in natural outbreaks. experimental inoculation of adult c.b- -prkdc scid (scid) mice with mvmi results in lethal infection (lamana et al., ; segovia et al., ) , and similar severe illness has been noted in naturally infected b-cell-deficient nod. cg-h h -igh null mice (naugler et al., ) . epizootiology mvm is a common virus that naturally infects laboratory mice, but appears to be less common than mpv (besselsen et al., ; livingston et al., ) . mvm is moderately contagious for mice, its only known natural host. virus can infect the gastrointestinal tract and is excreted in feces and urine. the resistance of rodent parvoviruses to environmental inactivation increases the risks of transmission after virus is excreted. therefore, contamination of caging, bedding, food, and clothing must be considered a risk for the spread of infection. transmission occurs by oronasal exposure, but viral contamination of biologicals used for experimental inoculation, such as transplantable tumors, also can be a source of infection. continuous contact exposure to infected animals or soiled bedding usually induces a humoral immune response within weeks, but limited exposure may delay seroconversion. young mice in enzootically infected colonies are protected by maternal antibody, but actively acquired immunity develops from infection sustained after the decay of maternal immunity. mvm, in contrast to mpv, is not thought to cause persistent infection; infection in immunocompetent adult mice usually lasts less than weeks (smith, ; smith and paturzo, ). infection appears to last less than month, even in oronasally inoculated neonatal mice, but immunodeficient mice may be persistently infected. there is no evidence that mvm is transmitted in utero. pathology natural infections or experimental inoculation of adult mice appears to be nonpathogenic. contact-exposed neonates have been reported to develop cerebellar lesions, but these are very rare. experimental infection of neonatal balb/c, swr, sjl, cba, and c h mice with mvmi can cause renal hemorrhage and infarction (brownstein et al., b) . dba/ mice also developed intestinal hemorrhages and accelerated involution of hepatic hematopoiesis. c bl/ neonates are resistant to vascular disease. this lesion has been attributed to viral infection of endothelium. infection of immunodeficient mice, including scid and b-cell-deficient mice, results in lethal damage to granulomacrophagic, megakaryocytic, and erythrocytic hematopoietic tissue with severe leukopenia (lamana et al., ; naugler et al., ; segovia et al., ) . intranuclear viral inclusions and viral antigen have been observed in splenic mononuclear cells of b-cell deficient mice (naugler et al., ) . diagnosis serology is the primary method of detecting infection, which utilizes recombinant mvm and mpv major capsid viral proteins (vp ) as antigens, which discriminate between the two groups of mouse parvoviruses. in contrast, the conserved nonstructural protein, ns can be used to detect antibody to both groups, but is less sensitive than vp assays (livingston et al., ) . mvm infection also can be detected by pcr, in situ hybridization, and immunohistochemistry. pcr assays can be used to detect mvm-or mpv-specific vp or all rodent parvovirus group specific ns exons (besselsen, ; besselsen et al., ) . mvm can be isolated from the spleen, kidney, intestine, and other tissues by inoculation of the c rat glial cell line. it also can be detected by the mouse antibody production test. prevention and control because mvm does not persist in immunocompetent mice, control and elimination should exploit quarantine combined with thorough disinfection of the environment, because parvoviruses are resistant to environmental inactivation. mpv has been shown to be successfully eliminated by a cage-bycage test (serology and fecal pcr) and cull approach, although there are no published reports confirming the success of this strategy for eliminating mvm (macy et al., ) . cesarean rederivation or embryo transfer may also be used to rederive virus-free progeny. prevention of mvm infection depends on strict barrier husbandry and regular surveillance of mice and mouse products destined for use in vivo. research complications mvm contamination of transplantable neoplasms can occur; therefore, infection can be introduced to a colony through inoculation of contaminated cell lines. failure to establish long-term cell cultures from infected mice or a low incidence of tumor 'takes' should alert researchers to the possibility of mvm contamination. mvmi has the potential to inhibit the generation of cytotoxic t cells in mixed lymphocyte cultures. etiology mouse parvovirus (mpv) is among the more common viruses detected within contemporary laboratory animal medicine mouse colonies, and is more common than mvm (livingston et al., ; livingston and riley, ; pritchett-corning et al., ) . mpv was initially isolated following its detection as a lymphocytotropic contaminant in in vitro assays for cellular immunity. the virus grew lytically in a cd + t-cell clone designated l and inhibited the proliferation of cloned t cells stimulated with antigen or interleukin (il- ) (mckisic et al., ) . molecular analysis of mpv indicates that regions encoding the ns proteins are similar to those of mvm (and other rodent parvoviruses). however, they differ significantly in regions encoding the capsid proteins, accounting for their antigenic specificity. the prototype isolate was first called an 'orphan' parvovirus of mice because its biology and significance were obscure, but it has subsequently been named mouse parvovirus (mpv). immortalized t cells (l ) are the only cells found thus far to support replication of mpv. there are three genetically distinct variants of mpv, including mpv- , mpv- , and mpv- . mpv- includes a number of closely related variants, including mpv- a, mpv- b, and mpv- c. in addition, a hamster parvovirus isolate is closely related to mpv- , which is infectious to mice and likely to be of mouse origin (besselsen et al., ; christie et al., ) . clinical signs mpv infection is clinically silent in infant mice and adult immunocompetent or immunodeficient mice (besselsen et al., ) . immunologic perturbations are the most likely signs of infection (mckisic et al., ) . epizootiology mpv causes persistent infection in infant and adult mice, a property that differentiates it from mvm. in situ hybridization has identified the small intestine as a site of viral entry and early replication, but respiratory infection cannot be excluded. experimental studies following inoculation of neonatal balb/c and c.b- -prkdc scid (scid) mice revealed that balb/c mice shed high levels of virus for weeks, with transmission to sentinels exposed during the first weeks of infection. thereafter, balb/c mice shed extremely low virus intermittently. in contrast, scid mice shed high levels of virus until weaning, but lower levels at weeks of age, yet they effectively transmitted infection to sentinels at all stages of infection (besselsen et al., ) . others have shown that transmission of mpv by sencar mice inoculated as infants was intermittent up to weeks, whereas transmission by mice inoculated as weanlings occurred during the first weeks of infection . transmission to balb/c progeny from infected dams was shown to occur, but embryo transfer rederivation was found to be successful in experimentally infected scid mice (besselsen et al., ) . humoral (e.g., passively or maternally acquired) immunity can protect against mpv infection. however, immunity to mvm may not confer cross-immunity to mpv (hansen et al., ) . pathology mpv appears to enter through the intestinal mucosa, which is a site of early virus replication ( fig. . ). acute infection is widespread but mild, involving the lung, kidney, liver, and lymphoid organs. histological lesions are not discernible. lymphocytotropism is a characteristic of acute and persistent mpv infection in infant and adult mice. during acute infection, virus is dispersed within lymph nodes, but during persistent infection virus localizes in germinal centers (fig. . ) . diagnosis because infected mice do not manifest signs or lesions and the virus is very difficult to propagate in cell culture, detection and diagnosis rely on serology and molecular methods. serology that utilizes mpv vp as antigen is a sensitive and specific assay that differentiates mpv from mvm (livingston et al., ) . the map test also can be used to detect parvovirus infections but is relatively time-consuming and expensive. as noted for mvm, pcr for murine parvoviruses, using nucleoprotein gene sequences that are conserved among murine parvoviruses, can be used as a screening test. pcr also can be used to detect mpv-specific sequences in the vp gene. although diagnostic pcr is sensitive and specific, it is effective only in actively infected animals. it can be used on feces to detect virus shedding, or applied to tissues, such as mesenteric lymph nodes, obtained at necropsy. differential diagnosis mpv infection must be differentiated from mvm infection. because both viruses are enterotropic and lymphocytotropic, serology and pcr must be used to distinguish between them. prevention and control the persistence of mpv in individual mice, its potential for provoking immune dysfunction, and the resistance of murine parvoviruses to environmental inactivation favor active control and prevention of mpv infection. quarantine of infected rooms is appropriate. elimination (depopulation) of infected mice should be considered if they are an immediate threat to experimental or breeding colonies and can be replaced, but a cage-by-cage test and cull approach has been shown to be successful under natural conditions (macy et al., ) . for mice that are not easily replaced, virus persistence in the absence of transplacental transmission favors cesarean rederivation or embryo transfer as relatively rapid options to eliminate infection. control of infection also should include environmental decontamination. chemical disinfection of suspect animal rooms and heat sterilization of caging and other housing equipment are prudent steps. prevention is based on sound serological monitoring of mice and surveillance of biologicals destined for inoculation of mice. with the increasing use of mouse germplasm, it is important to note that mouse sperm, oocytes, ovarian tissue, and preimplantation embryos from enzootically mpvinfected mouse colonies may have a high prevalence of mpv contamination, based upon pcr (agca et al., ) . research complications murine parvoviruses can distort biological responses that depend on cell proliferation. for mpv, such effects are seen on immune function and include augmentation or suppression of humoral and cellular immune responses. etiology adenoviruses are nonenveloped dna viruses that produce intranuclear inclusions in vitro and in vivo. two adenovirus species in the genus mastadenovirus have been associated with mice: murine mastadenovirus a (with the representative strain being mav- or fl) and murine mastadenovirus b (with the representative strain being mav- or k ). both strains replicate in mouse kidney tissue culture but are antigenically distinct. clinical signs mav- can cause severe clinical disease after experimental inoculation of infant mice. signs include scruffiness, lethargy, stunted growth, and often death within days. mav- virus is enterotropic and is responsible for virtually all naturally occurring infections in contemporary mouse populations. infection is usually subclinical in immunocompetent mice, with the possible exception of transient runting among infant mice. wasting disease can occur in athymic mice infected with mav- . epizootiology the prevalence of adenovirus infection in mouse colonies is low, particularly mav- riley, , pritchett-corning et al., ) . transmission occurs by ingestion. adult mice experimentally infected with mav- may remain persistently infected and excrete virus in the urine for prolonged periods. adult mice experimentally infected with mav- excrete virus in feces for at least weeks but eventually recover. athymic mice can shed mav- for at least weeks and episodically for at least months. pathology infection with mav- causes multisystemic disease characterized by necrosis. infant mice are especially susceptible to rapidly fatal infection characterized by necrosis of brown fat, myocardium, adrenal cortex, salivary gland, and kidney, with the development of intranuclear inclusions. more mature mice usually develop subclinical infection leading to seroconversion; however, athymic and scid mice can develop intestinal hemorrhage and wasting, with fatal disseminated infection (lenaerts et al., ) . infection with mav- produces amphophilic, intranuclear inclusions in intestinal epithelium, especially in the distal small intestine (fig. . ) . inclusions are easier to detect in infant mice than in adults. infection of c.b- -prkdc scid mice with mav- results in enteric infection, but also hepatic lesions resembling reye's syndrome (pirofski et al., ) . diagnosis although mav strains can be isolated in tissue culture, routine diagnosis depends on detection of infection by serological assay and/or demonstration of adenoviral inclusions, most commonly in the intestinal mucosa. cross-neutralization tests have revealed that antiserum to mav- neutralizes both strains, but antiserum to mav- neutralizes mav- weakly at best. therefore, mav- antigen should be used for the serological detection of adenovirus infection irrespective of the assay employed. mav also can be detected by pcr. differential diagnosis intranuclear adenoviral inclusions in intestinal epithelium are pathognomonic laboratory animal medicine and differentiate mav- infection from other known viral infections of mice. infection may resemble rotavirus infection, with runting and abdominal bloating in infant mice. prevention and control prevention requires serological monitoring of mice and examination for contamination of animal products such as transplantable tumors. because mav- infection appears to be transient in individual mice, segregation of infected colonies may be effective for control. however, rederivation coupled with subsequent barrier housing is a more conservative approach. research complications mav infection is unlikely to affect research using immunocompetent mice. however, it has the potential for pathogenicity in immunodeficient mice. mice can incur natural infection with two polyomaviruses: polyoma virus (pyv) and k virus. these viruses belong to the family polyomaviridae. k virus belongs to the genus polyomavirus and the species murine pneumotropic virus, while the classical polyoma virus belongs to the species murine polyomavirus. etiology polyoma virus (pyv) is a small dna virus that derives its name 'polyoma' (many tumors) from its ability to experimentally induce multiple types of tumors in mice experimentally infected as neonates. its primary importance stems from use in murine models of experimental oncogenesis, with natural infection being rare. the transformative activity is mediated by 't' (tumor) antigens, encoded by large t, middle t, and small t genes, with middle t (mt) being considered the major viral oncogene, and as a result has been used extensively in transgenic constructs. clinical signs natural infections in immunocompetent mice are usually subclinical. however, tumor induction, neurological disease, and wasting can occur in naturally exposed immunodeficient mice (mccance et al., ; sebesteny et al., ) . epizootiology modern husbandry and health care have essentially eliminated natural exposure in laboratory mice. pyv is used for experimental studies and thus can inadvertently be introduced to mouse colonies. inoculation of mice with contaminated biologicals or cell cultures is a potential source of entry and spread. natural transmission occurs via the respiratory route. exposure of neonatal mice results in persistent infection and shedding of the virus in urine, feces, and saliva, thereby contaminating the environment for spread to other mice. infection of adult mice is transient, with minimal virus shedding, although pcr has revealed infection lasting up to months in cba mice inoculated with virus as adults (berke and dalianis, ) . maternal antibody is highly effective at preventing infection of newborn mice, but as maternal antibody wanes, mice are partially susceptible, with transient virus shedding. thus, the natural cycle of transmission in enzootically infected populations requires contamination of bedding and nesting material in order to infect and be inefficiently transmitted, which is readily precluded by modern husbandry. intrauterine infection also can occur, and persistent renal infection, contracted neonatally, can be reactivated during pregnancy. as in immunologically immature neonatal mice, pyv infection can persist in adult immunodeficient mice. pathology pyv-induced tumors are essentially a laboratory phenomenon, optimized by virus strain and mouse strain, with akr, c h, c , cba, swr, and others being most susceptible, and c bl/ being among the most resistant to pyv oncogenesis. intranasal inoculation of neonatal mice results in initial replication in pulmonary respiratory epithelium (gottlieb and villarreal, ) followed by viremic dissemination and acute, lethal disease. tumors appear - months after inoculation of surviving mice. tumors of both epithelial and mesenchymal origin arise in multiple organs, particularly mammary carcinomas, basal cell tumors of the skin, carcinomas of salivary glands, thymomas, and various types of sarcomas. athymic mice can develop cytolytic and inflammatory lesions, followed by multisystemic tumor formation. intranuclear inclusions may be present in cytolytic lesions. demyelinating disease and skeletal tumors have been reported in experimentally . laboratory animal medicine inoculated and naturally exposed athymic mice, and myeloproliferative disease has been reported in experimentally inoculated c bl/ -scid mice (szomolanyi-tsuda et al., ) . diagnosis pyv can be isolated in mouse fibroblast cell lines, but infection is ordinarily detected serologically. additionally, pcr and immunohistochemistry can be used. differential diagnosis wasting in athymic mice can be caused by other infectious agents, including coronaviruses, sendai virus (sv), and pneumocystis. intranuclear inclusions can occur in infections caused by mouse adenovirus, mouse cytomegalovirus, and k virus. prevention and control control depends on elimination of infected mice and material, together with prevention of airborne spread. biological material destined for mouse inoculation should be tested for pyv by the map test or molecular diagnostics. research complications pyv infection can affect experiments by inadvertent contamination of cell lines or transplantable tumors, leading to infection of inoculated mice and the potential for epizootic spread. k virus infection k virus has historical importance, and is apparently absent from contemporary mouse populations (livingston and riley, ; pritchett-corning et al., ) , but it continues to be tested for, adding to the expense of infectious disease surveillance. oral inoculation of neonatal mice results in initial infection of capillary endothelium in the intestine, followed by viremic spread. vascular endothelium is the primary target in affected tissues, which often include the lung, liver, spleen, and adrenal glands. dyspnea occurs from pulmonary infection because of edema and hemorrhage. infection of immunocompetent adult mice is subclinical and results in a vigorous immune response. however, both adults and infant mice develop persistent infection. the primary organ for persistence is the kidney, with shedding of virus from tubular epithelium, and shedding can be reactivated by immunosuppression (greenlee et al., ) . additionally, infection of athymic mice can lead to clinical signs and lesions akin to those described for neonatally inoculated mice. gross lesions are limited to pulmonary hemorrhage and edema. histologically, intranuclear inclusions, which are visualized more easily using immunohistochemistry, are present in vascular endothelium of infected tissues. mild hepatitis with hepatocyte degeneration also may develop. infection can be detected by serology or pcr. prevention and control measures, if ever to be found within a mouse population, are similar to those described for pyv. etiology lactate dehydrogenase-elevating virus (ldv) is a mouse-specific small enveloped rna virus belonging to the family arteriviridae. infected mice are persistently viremic, resulting in increased concentration of several serum enzymes, most notably lactate dehydrogenase (ldh). infection is common among wild mice, but is now rare in contemporary laboratory mouse populations. however, surveys of biologic material indicate that ldv may be a common contaminant of biologic materials (nicklas et al., ) . clinical signs infection is subclinical. however, poliomyelitis has occurred in immunosuppressed c and akr mice inoculated with ldv, and has recently been observed in icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . epizootiology the primary mode of mouse-tomouse transmission is mechanical transfer from aggressive behavior (e.g., bite wounds). inoculation of mice with contaminated animal products such as cell lines, transplantable tumors, or serum is probably the most common source of induced infection. it is important to note, with respect to mechanical transmission, that infection induces lifelong viremia. natural transmission between cagemates or between mother and young is rare even though infected mice may excrete virus in feces, urine, milk, and probably saliva. pathology viremia peaks within day after inoculation, then persists at a diminished level. the elevation of enzyme levels in blood is thought to result primarily from viral interference with clearance functions of the reticuloendothelial system. ldv selectively targets mature f /f -positive macrophages, which are continually produced by uninfected progenitor cell populations, thereby maintaining persistent infection. virus also escapes immune clearance by evolution of neutralizing antibody-resistant quasi-species. no lesions are seen in naturally infected mice. the only significant lesion that can arise from experimental infection is poliomyelitis. this syndrome requires a combination of immunosuppression (due to age, genetics or induced means), mouse strain (c , akr, c h/fg, and pl), neurotropic ldv strains, and endogenous ecotropic murine leukemia virus. the mouse strain-dependent element is homozygosity for the fv- n allele, which permits replication of endogenous n-tropic ectotropic murine leukemia virus. mice develop spongiosis, neuronal necrosis, and astrocytosis of the ventral spinal cord and brain stem, with axonal degeneration of ventral roots. lesions contain both ldv and retrovirus. although this syndrome is largely experimentally induced, a natural outbreak of poliomyelitis has been reported in fv- homozygous icr-scid mice following inoculation with contaminated biologic material (carlson-scholz et al., ) . diagnosis plasma ldh levels are elevated, a response that is used to detect and titrate ldv infectivity. of the five isoenzymes of ldh in mouse plasma, only laboratory animal medicine ldh-v is elevated. sjl/j mice in particular show spectacular increases in ldh levels ( - times normal), a response controlled by a recessive somatic gene. ldv is detected by measuring ldh levels in mouse plasma before and days after inoculation of specific pathogenfree (spf) mice with suspect material. it is important to use nonhemolyzed samples because hemolysis will produce falsely elevated readings. plasma enzyme levels are measured in conventional units/ml, conventional unit being equivalent to . international units (iu). normal plasma levels are - iu, whereas in ldv infection, levels as high as iu can occur. ldv also interferes with the clearance of other serum enzymes and results in their elevation in serum. in a recent survey, serum samples were tested by serum ldh enzyme assays, among which % were deemed potentially positive. however, pcr revealed that all were false-positives (pritchett-corning et al., ) , emphasizing the inaccuracy of traditional enzyme assays. infection provokes a modest humoral antibody response, but it is difficult to detect because of formation of virus-antibody immune complexes. molecular diagnostics also can be used to diagnose infection in mouse tissues and serum and biologic materials. however, inhibitory factors in cells and serum may cause falsenegative results in pcr testing, so appropriate quality control measures are essential if this method is used (lipman and henderson, ) . prevention and control transplantable tumors have been a common source of ldv historically. therefore, tumors or cell lines destined for mouse inoculation should be monitored for ldv contamination. although ldv can contaminate tumor cell lines, it does not replicate in the tumor cells. therefore, one can attempt to free tumors of virus by passaging them through athymic nude rats, which are not nonpermissive to ldv but are permissive to xenografts. research complications ldv has numerous potential effects on immunological function. it may reduce autoantibody production, cause transient thymic necrosis and lymphopenia, suppress cell-mediated immune responses, and enhance or suppress tumor growth. etiology the house mouse is the natural host for lymphocytic choriomeningitis virus (lcmv), an old world member of the arenaviridae family that has spread worldwide along with m. musculus. lcmv virions are pleomorphic, containing single-stranded rna, and bud from the cell membrane. disease associated with infection is due to host immune response to the otherwise non-cytolytic virus. its name is derived from the immune-mediated inflammation resulting from the intracerebral inoculation of virus into immunologically competent mice. lcmv is a zoonotic virus that may cause a variety of clinical manifestations in humans, including meningitis. it has been extensively studied as an experimental model of virus-induced immune injury, using a number of closely related strains, including ones that have been selected for their relative neurotropism or viscerotropism. lcmv can be propagated in a variety of mammalian, avian, and even tick cell lines, with minimal cytopathic effect. these characteristics favor its propensity to persistently and silently contaminate biologic products, such as tumor cell lines. clinical signs natural infection in immunocompetent adult mice is usually self-limiting and subclinical. during enzootic infection of a mouse population, lcmv is transmitted in utero from persistently infected dams to their fetuses or to neonates, which are persistently infected and immunologically tolerant to lcmv. since lcmv is non-cytolytic in and of itself is minimally pathogenic, congenitally infected mice grow into adulthood, reproduce, and therefore transmit infection to the next generation. however, with age, immune tolerance breaks down, and mice develop a syndrome known as 'late disease' in which mice will progressively lose weight and die. in utero infection results in a low level of fetal mortality and maternal cannibalism of infected pups. the immune tolerance to lcmv is virus-specific, with the mice capable of eliciting effective immune responses against other agents. clinical signs following experimental inoculation of lcmv vary with age and strain of mouse, route of inoculation, and strain of virus. when virus is inoculated intracerebrally into immunocompetent adult mice, mice develop immune-mediated lymphocytic choriomeningitis, characterized by illness beginning - days after inoculation. sudden death may result or subacute illness associated with one or more of the following signs may develop: ruffled fur, hunched posture, motionlessness, and neurological deficits. mice suspended by the tail display coarse tremors of the head and extremities, culminating in clonic convulsions and tonic extension of the rear legs. spontaneous convulsions also can occur. animals usually die or recover in several days. a visceral form of infection can occur in adult mice inoculated by peripheral routes with 'viscerotropic' strains. it can be subclinical or lead to clinical signs, including ruffled fur, conjunctivitis, ascites, somnolescence, and death. if mice survive, recovery may take several weeks. surviving mice may have immune exhaustion due to consumption of lymphoid tissue, in contrast to immune tolerance that occurs when mice are infected in utero or as neonates. runting and death from lcmv infection may occur in neonatally infected mice and can lead to transient illness or to death. clinical signs are nonspecific, recovery is slow, and survivors may remain runted. this early form of disease is attributed to endocrine dysfunction caused laboratory animal medicine by lcmv infection. late-onset disease can occur in previously subclinical carrier mice that develop immune complex glomerulonephritis. it is usually the result of prenatal or neonatal infection and occurs in persistently infected mice when they are - months old. clinical signs are nonspecific and include ruffled fur, hunched posture, weight loss, proteinuria, and ascites. epizootiology lcmv is distributed widely in wild m. musculus throughout the world. among common laboratory species, mice, hamsters, guinea pigs, and nonhuman primates are susceptible to infection, but only the mouse and the hamster are known to transmit virus. lcmv infection is rare in laboratory mice produced and maintained in modern quarters (livingston and riley, ; pritchett-corning et al., ) . infection is usually introduced through inoculation of virusinfected biologicals, such as transplantable tumors, or by feral mice. wild mice are a natural reservoir of infection and a potential threat to research colonies if they gain entry inadvertently. naturally infected carrier mice can have persistently high concentrations of virus in many organs, thereby facilitating virus excretion in saliva, nasal secretions, and urine. persistently infected neonates usually reach breeding age and can perpetuate infection in a breeding colony. thus introduction of a single lcmv carrier mouse to a breeding colony can eventually result in a high prevalence of persistently infected mice. infection in adult mice, in contrast, is often acute because of the onset of effective immunity, and the spread of virus is halted. horizontal spread of infection is enhanced by close contact, but rapid horizontal spread is not characteristic. mice can transmit lcmv to hamsters, which can remain viremic and viruric for many months, even if they contract infection as adults. infected hamsters can transmit virus to other hamsters and mice and are the primary source of human lcmv infection. persistent infection in immunodeficient mice may carry greater risks for viral excretion and zoonotic transmission. pathology lcmv disease is a prototype for virusinduced, t-lymphocyte-mediated immune injury, noncytolytic endocrine dysfunction, and immune complex disease. however, lesions comparable to experimentally induced disease are rare during natural infection. intracerebral inoculation of virus into immunocompetent adult mice induces nonsuppurative leptomeningitis, choroiditis, and focal perivascular lymphocytic infiltrates. host tissues are damaged during the course of the cellular immune response to the virus. the character of visceral lesions depends on virus strain and mouse strain; the ratio of cytolytic to proliferative responses in lymphoid organs is mouse strain-dependent. in severe infection, nonsuppurative inflammation can occur in many tissues. the severity of accompanying cytolytic lesions seems to parallel the intensity of cellular immunity. liver lesions can include hepatocyte necrosis accompanied by nodular infiltrates of lymphoid cells and kupffer cells, activated sinusoidal endothelium, an occasional granulocyte or megakaryocyte, and fatty metamorphosis. cytolysis, cell proliferation, and fibrinoid necrosis can develop in lymphoid organs. necrosis of cortical thymocytes can lead to thymic involution. lesions of late-onset disease are characterized by formation of immune complexes and associated inflammation. renal glomeruli and the choroid plexus are most severely affected, but complexes may also be trapped in synovial membranes, blood vessel walls, and skin. lymphoid nodules can form in various organs. lesions associated with early deaths in neonatally infected mice have not been thoroughly described but include hepatic necrosis. the lesions of acute and persistent lcmv infection reflect separate immunopathologic processes. in adult mice with acute lcmv infection, virus multiplies in dcs, b cells, and macrophages, whereas t cells are resistant. internal viral epitopes induce humoral immune responses, but surface epitopes elicit cell-mediated immunity and neutralizing antibodies. thus, elimination of virus and virus-associated immunological injury are both t-cell-mediated. this apparent paradox has been explained by the view that prompt cellular immunity limits viral replication and leads to host survival, whereas slower cellular immune responses permit viral spread and increase the number of virus-infected target cells subject to attack once immunity is fully developed. antibody can be detected by week after infection but does not play a significant role in eliciting acute disease. lesions of lcmv infection appear to develop from direct t-cell-mediated damage to virus-infected cells and may involve humoral factors released from immune effector t cells. lcmv also can suppress humoral and cellular immunity in acutely infected mice. persistent infection commonly evolves from exposure early in pregnancy, and virus has been demonstrated in the ovaries of carrier mice. prenatal or neonatal infection induces immunological tolerance to lcmv, which can then replicate to high titer in many tissues. nevertheless, persistently infected mice develop humoral antibody to lcmv. antibody can complex with persistent virus to elicit complement-dependent inflammation in small vessels. immune complex glomerulonephritis exemplifies this process, as noted above. diagnosis lcmv infection can be diagnosed serologically. whereas immunocompetent adult mice will normally seroconvert after exposure, carrier mice may develop poor humoral immune responses. therefore, testing must avoid false-negative results. employment of adult contact sentinel mice is a useful strategy for detecting lcmv infection by seroconversion. tissues, including biologic products and cell lines, can be tested laboratory animal medicine by pcr. a traditional method for detection involved collection of small blood samples from persistently infected live suspects, which are often viremic, and using them to inoculate cultured cells or adult and neonatal mice. intracerebral inoculation of lcmv-positive tissues should elicit neurological signs in adult mice within days, whereas infant mice should remain subclinical. histological examination of brains from affected adults may reveal nonsuppurative inflammation, but lesions may be minimal in mice infected with viscerotropic isolates. immunohistochemistry can be used to detect viral antigen in brains of suckling and adult mice. intraperitoneal inoculation of adult mice may yield short-lived infection with seroconversion, i.e., the map test. virus can be grown and quantified in several continuous cell lines, including mouse neuroblastoma (n- ) cells, bhk- cells, and l cells. application of immunofluorescence staining to detect lcmv antigen in inoculated cultured cells yields results more quickly than animal inoculation. of course, all diagnostic procedures involving potential contact with live virus should be carried out under strict containment conditions to avoid infection of laboratory personnel (see chapter ). the use of in vitro detection has the added advantage, in this regard, of reducing biohazardous exposure and the use of live animals for testing. differential diagnosis neurological signs must be differentiated from those due to mouse hepatitis virus, mouse encephalomyelitis virus, and meningoencephalitis from bacterial infection. trauma, neoplasia, and toxicities also must be ruled out in neurological disease with low prevalence. late-onset disease is associated with characteristic renal lesions, including deposition of viral antigen in tissues. early-onset disease must be differentiated from other causes of early mortality, such as mouse hepatitis virus, ectromelia virus, reovirus infection, tyzzer's disease, or husbandry-related insults. prevention and control adequate safeguards for procurement and testing of animals and animal products are essential to prevent entry. because mouse-to-mouse spread is slow, selective testing and culling for seropositive or carrier mice is possible. if mice are easily replaced, however, depopulation is a safer and more reliable option. valuable stock can be rederived, but progeny must be tested to preclude in utero transmission. because infected hamsters can excrete large quantities of virus, exposed hamsters should be destroyed and hamsters should not be housed with mice. infection of immunodeficient mice poses similar risk. lcmv can be transmitted to human beings, who can contract flu-like illness or severe cns disease. more frequently, human infection is subclinical. the zoonotic potential of lcmv infection makes it especially important to detect and eliminate carrier animals and other potentially contaminated sources, such as cell cultures, transplantable neoplasms, and vaccines to prevent human exposure. serum banking and periodic serological testing of highrisk human populations, such as those working with lcmv experimentally, are recommended. research complications lcmv may stimulate or suppress immunological responses in vivo and in vitro, and it can replicate in cells used as targets or effectors for immunological studies. introduction of immune cells to a carrier animal may elicit an immunopathological response. immune complex disease can complicate longterm experiments and morphological interpretations. illness and death in mice and zoonotic risk to humans are obvious research-related hazards. etiology sv is a paramyxovirus that is antigenically related to human parainfluenza virus . viral particles are pleomorphic, contain single-stranded rna, and have a lipid solvent-sensitive envelope that contains glycoproteins with hemagglutinating, neuraminidase, and cell fusion properties. sv grows well on embryonated hens' eggs and in several mammalian cell lines (e.g., monkey kidney, baby hamster kidney , and mouse fibroblast [l]). virus replicates in the cytoplasm and by budding through cell outer membranes. once common in laboratory rodent populations, sv is now rare or absent (livingston and riley, ; pritchett-corning et al., ) . clinical signs clinically affected adult mice often assume a hunched position and have an erect hair coat. rapid weight loss and dyspnea occur, and there may be chattering sounds and crusting of the eyes. although highly susceptible adults may die, lethal infection is more common in suckling mice. sex differences in susceptibility have not been found. genetically resistant mice usually have subclinical infection. athymic mice and immunodeficient mice are at high risk for development of a wasting syndrome. they develop illness later than their immunocompetent counterparts, since clinical signs in immunocompetent mice are related to immune-mediated destruction of respiratory epithelium. opportunistic infections can complicate the clinical presentation. for example, secondary bacterial infections of the ear can cause vestibular signs. epizootiology sv is transmitted by aerosol and is highly contagious. morbidity in infected colonies is commonly %, and mortality can vary from % to %, partly because strains of mice vary greatly in their susceptibility to lethal sv infection. for example, c bl/ mice are highly resistant to clinically apparent infection, whereas dba/ mice are highly susceptible. aerogenic infection is promoted by high relative humidity and by low air turnover. prenatal infection does not occur. enzootic infection is commonly detected in postweaned mice ( - weeks old) and is associated with seroconversion within - days and the termination of infection. therefore, entrenched infection is perpetuated by the introduction of susceptible animals. there is no evidence for persistent infection in immunocompetent mice, but prolonged infection is common in immunodeficient mice. maternally acquired immunity protects young mice from infection, and actively acquired immunity is thought to be long-lived. rats, hamsters, and guinea pigs also are susceptible to sv infection. therefore, bidirectional cross-infection is a risk during outbreaks. pathology viral replication is nominally restricted to the respiratory tract and peaks by the first week after infection. gross lesions feature partial to complete consolidation of the lungs (fig. . ) . individual lobes are meaty and plum-colored, and the cut surface may exude a frothy serosanguinous fluid. pleural adhesions or lung abscesses caused by secondary bacterial infection are seen occasionally, and fluid may accumulate in the pleural and pericardial cavities. sv targets airway epithelium and type ii pneumocytes. type i pneumocytes are less severely affected. histologically, the pattern of pneumonia is influenced by mouse genotype. susceptible mice usually have significant bronchopneumonia and interstitial pneumonia, whereas the interstitial component may be less prominent in resistant mice. typical changes begin with inflammatory edema of bronchiolar lamina propria, which may extend to alveolar ducts, alveoli, and perivascular spaces. necrosis and exfoliation of bronchiolar epithelium ensue, frequently in a segmental pattern (fig. . ). alveolar epithelium also may desquamate, especially in severe disease, and necrotic cell debris and inflammatory cells can accumulate in airways and alveolar spaces. alveolar septae are usually infiltrated by leukocytes to produce interstitial pneumonia (fig. . ) . lymphoid cells also invade peribronchiolar and perivascular spaces. the lymphocytic response to sv infection reflects the fact that cellular immunity contributes both to lesions and to recovery. local immunoglobulin synthesis by infiltrating cells also occurs. the extent of inflammatory cell infiltration corresponds to the level of genetic resistance expressed by the infected host, with clinically susceptible hosts mounting a more florid immune response than resistant hosts. additionally, strain-related differences in the severity of infection may reflect differences in airway mucociliary transport. multinucleated syncytia are occasionally seen in affected sucklings and scid mice, and inclusion bodies have been reported in infected athymic mice. regeneration and repair begin shortly after the lytic phase and are characterized by hyperplasia and squamous metaplasia of bronchial epithelium, which may extend into alveolar septae. proliferation of cuboidal laboratory animal medicine epithelium may give terminal bronchioles an adenomatoid appearance. repair of damaged lungs is relatively complete in surviving mice, but lymphocytic infiltrates, foci of atypical epithelium, and mild scarring can persist. acute phase lesions are prolonged in immunodeficient mice, which can lead to wasting and death. aged mice also have a prolonged recovery phase accompanied by focal pulmonary fibrosis (jacoby et al., ) . diagnosis sv is notable for its ability to cause epizootics of acute respiratory distress in adult genetically susceptible strains. serology is an effective means to detect infection in all strains of immunocompetent mice. antibody can be detected by days postinfection and coincides with development of clinical signs related to the immune-mediated necrotizing bronchiolitis and alveolitis. repeated serologic sampling over several weeks can help stage infection within a population. alternatively, sentinel animals can be added to seropositive colonies to detect active infection. irrespective of serologic results, histopathology, immunohistochemistry (which can be performed on formalin-fixed, paraffin-embedded sections), and, where possible, virus isolation should be used to confirm infection. virus can be isolated from the respiratory tract for up to weeks, with peak titers occurring at about days postinfection. nasopharyngeal washings or lung tissue homogenates are most reliable and should be inoculated into embryonated hens' eggs or bhk- cell monolayer cultures. sv infection of cultured cells is non-cytolytic, so erythrocyte agglutination or antigen detection methods must be used. rt-pcr also can be used to detect virus in infected lungs. differential diagnosis respiratory infection caused by pneumonia virus of mice (pvm) is generally milder or subclinical. histologically, necrosis of airway epithelium is less severe. bacterial pneumonias of mice, including murine respiratory mycoplasmosis, are sporadic and can be differentiated morphologically and by isolation of causative organisms. because sv pneumonia may predispose the lung to opportunistic bacterial infections, the presence of bacteria should not deter evaluation for a primary viral insult. control and prevention sv infection is self-limiting in surviving immunocompetent mice. suckling mice from immune dams are protected from infection by maternal antibody until after weaning. control and eradication measures must eliminate exposure of susceptible animals, so that infection can 'burn out.' this is most easily accomplished by a quarantine period of - weeks wherein no new animals are introduced either as adults or through breeding. control also is aided by the fact that sv is highly labile. barrier housing is preferred for prevention and for control of transmission. vaccination with formalin-killed virus can provide short-term protection of valuable mice but is not commonly used for prevention. research complications sv can cause immunosuppression and can inhibit growth of transplantable tumors. this effect has been attributed to virus-induced modification of tumor cell surface membranes. pulmonary changes during sv pneumonia can compromise interpretation of experimentally induced lesions and may lead to opportunistic infections by other bacteria. they also have been associated with breeding difficulties in mice. this sign is thought to be an indirect effect due to stress, fever, or related changes during acute infection. clinical signs natural pvm infection in mice is subclinical. therefore, its name is clinically misleading, being derived from pneumonic illness that occurred after serial passage of the agent in mice. however, dyspnea, listlessness, and wasting may develop in immunodeficient mice infected with pvm (weir et al., ) . pvm is used experimentally as a model to study acute respiratory infection, using highly pathogenic strains of the virus (dyer et al., ) . epizootiology pvm causes natural infections of mice, rats, hamsters, and probably other rodents and may be infectious for rabbits. serological data indicate that pvm was once common, but is now relatively uncommon (livingston and riley, ; pritchett-corning et al., ). pvm appears to spread less rapidly than sv. intimate contact between mice is probably required for effective transmission. this characteristic may reflect the fact that environmental inactivation of virus occurs rapidly. infection is acute and self-limiting in immunocompetent mice but may persist in immunodeficient mice. pathology pvm replicates exclusively in the respiratory tract and reaches peak titers in the lung - days after infection. although pulmonary consolidation can occur in experimentally infected mice, gross lesions are rare during natural infection. histological lesions can occur in the upper and lower respiratory tract. they consist of mild necrotizing rhinitis, necrotizing bronchiolitis, and interstitial pneumonia, which usually occur within weeks after exposure to virus and are largely resolved by weeks. the predominant inflammatory infiltrate is comprised of mononuclear cells, but some neutrophils laboratory animal medicine are usually present. immunohistochemistry on paraffinembedded tissues can be used to detect viral antigen in bronchiolar epithelium, alveolar macrophages, and alveolar epithelium during acute infection. residual lesions include nonsuppurative perivasculitis, which can persist for several weeks after acute infection has ceased. severe progressive pneumonia, with wasting, can occur in immunodeficient mice. it is characterized by generalized pulmonary consolidation that reflects severe interstitial pneumonia with desquamated alveolar pneumocytes and leukocytes filling alveolar spaces (fig. . ) . diagnosis diagnosis is based primarily on serological detection that can be supplemented by histopathology, immunohistochemistry, in situ hybridization, and virus isolation. virus replication in bhk- cells is detected by immunofluorescence or other antigen detection methods. virus also can be detected in tissues by rt-pcr. differential diagnosis because pvm is antigenically distinct from other murine viruses, serology is the most useful method to separate pvm infection from other respiratory infections of mice. however, in immunodeficient mice, where clinical signs and lesions are typical, it must be differentiated from other pneumonias, especially those due to sv and pneumocystis. additionally, pvm can coexist with and exacerbate pneumocystis infection in immunodeficient mice (bray et al., ) . prevention and control pvm infection is acute and self-limiting in immunocompetent mice, but persistent in immunodeficient mice. seropositive mice should be viewed as either immune or in the final stages of acute infection. therefore, control and prevention follows guidelines applicable to sv infection. research complications pvm can exacerbate pneumocystosis, as noted above. (ward et al., ) two members of the family reoviridae infect laboratory mice: reovirus per se (species: mammalian orthoreovirus) and murine rotavirus (species: rotavirus a), also known as epizootic diarrhea of infant mice (edim) virus. etiology reoviruses of mammals, although taxonomically considered one type species, have been divided into three cross-reacting prototypic serotypes: reovirus , and , which can be differentiated by cross-serum neutralization. mice can be infected with any serotype, but reovirus is emphasized because it has been associated with naturally occurring disease. natural infections in mice are usually not caused by pure serotypes, because reoviruses actively recombine. a number of wild-type and laboratory strains have been characterized, and related viruses have been recovered from virtually every mammal tested, as well as birds, reptiles, and insects. the virion contains segmented, double-stranded rna and is relatively heat stable. reoviruses replicate well in bhk- cells and other continuous cell lines, as well as in primary monolayer cultures from several mammals. clinical signs clinical disease is rare and age dependent. acute disease affects sucklings at about weeks of age, whereas adults have subclinical infection. signs in sucklings include emaciation, abdominal distension, and oily, matted hair due to steatorrhea. icterus may develop and is most easily discerned as discoloration in the feet, tail, and nose. incoordination, tremors, and paralysis occur just before death. convalescent mice are often partially alopecic and are typically runted. alopecia, runting, and icterus may persist for several weeks, even though infectious virus can no longer be recovered. infants born to immune dams are protected from disease by maternal immunity. epizootiology the prevalence of reovirus infection in contemporary mouse colonies is rare (livingston and riley, ; pritchett-corning et al., ). reoviruses are highly contagious among infant mice and can be transmitted by the oral-fecal or aerosol routes, but mechanical transmission by arthropods has also been documented. additionally, virus may be carried by transplantable neoplasms and transmitted inadvertently by injection. transmission is inefficient among adult mice. there is no evidence that vertical transmission is important or that genetic resistance or gender influence expression of disease. infection in immunocompetent mice appears to be self-limiting, lasting up to several weeks but terminating with the development of host immunity. the course of infection in immunodeficient mice should be considered prolonged, but the duration has not been determined. pathology reovirus can cause severe pantropic infection in infant mice. after parenteral inoculation, virus can be recovered from the liver, brain, heart, pancreas, spleen, lymph nodes, and blood vessels. following ingestion, reoviruses gain entry by infecting intestinal epithelial cells (m cells) that cover peyer's patches. virus can be carried to the liver in leukocytes, where it is taken up by kupffer cells prior to infecting hepatocytes. in acute disease, livers may be large and dark, with yellow foci of necrosis. the intestine may be red and distended, and, in infants, intestinal contents may be bright yellow. myocardial necrosis and pulmonary hemorrhages have been reported. myocardial edema and necrosis are especially prominent in papillary muscles of the left ventricle. the brain may be swollen and congested. central nervous system lesions have a vascular distribution, and are most prevalent in the brain stem and cerebral hemispheres. neuronal degeneration and necrosis are followed quickly by meningoencephalitis and satellitosis. severe encephalitis may evoke focal hemorrhage. in the chronic phase, wasting, alopecia, icterus, and hepatosplenomegaly may persist. orally infected suckling mice can develop multifocal hepatocyte necrosis, which may include the accumulation of dense eosinophilic structures resembling councilman bodies. hepatocytomegaly, kupffer cell hyperplasia, and intrasinusoidal infiltrates of mononuclear cells and neutrophilic leukocytes also can develop. in experimentally inoculated mice, necrotic foci can persist in the liver for at least weeks. chronic active hepatitis may develop after acute infection and result in biliary obstruction. acinar cells of the pancreas and salivary glands can undergo degeneration and necrosis. because pancreatic duct epithelium is susceptible to infection, parenchymal lesions in the pancreas may be caused by obstruction rather than by viral invasion of parenchyma. pulmonary hemorrhage and degeneration of skeletal muscles also have been observed. both humoral and cellular immunity seem to participate in host defenses, but it is unclear how host immunity may influence the course of chronic infection. oronasal inoculation of infant mice with reovirus results in a similar distribution but significantly milder lesions compared to reovirus . in contrast, reovirus is highly enterotropic, inducing mild enteritis without lesions in other tissues, similar to epizootic diarrhea of infant mice (edim) . diagnosis serology uses reovirus as antigen, which detects seroconversion to all serotypes, and viral rna can be detected by rt-pcr. a presumptive diagnosis of reovirus infection is aided clinically by detection of the oily hair effect, accompanied by jaundice and wasting. the presence histologically of multisystemic necrosis is consistent with severe reovirus infection but should be confirmed by immunohistochemistry or virus isolation. differential diagnosis reovirus infection must be differentiated from other diarrheal diseases of infant mice, including those caused by mouse coronaviruses, edim virus, salmonella spp., or clostridium piliforme. prevention and control although surviving mice appear to recover completely from infection, the potential for a carrier state is unresolved. therefore, it may be necessary, after adequate testing for the continued presence of virus by the use of sentinels, map testing, or other appropriate means, to rederive or replace infected stock. prevention depends on adequate barrier husbandry coupled with adequate serological monitoring. research complications reovirus infection can interfere with research in several ways. infections in breeding colonies can result in high mortality among sucklings from nonimmune dams. virus has been commonly recovered from transplantable neoplasms and is suspected of being oncolytic. the potential exists for interference with hepatic, pancreatic, cardiovascular, or neurological research. etiology rotaviruses are double-stranded, segmented rna viruses that have a wheel-like ultrastructural appearance. edim virus is a group a rotavirus that replicates in differentiated epithelial cells of the small intestine by budding into cisternae of endoplasmic reticulum. currently, only a single antigenic strain is recognized, but antigenically distinct variants may exist. edim virus shares an inner capsid antigen with rotaviruses of rabbits, fowl, nonhuman primates, human beings, and domestic and companion animals. these agents tend to be species-specific under natural conditions and can be differentiated by serum neutralization tests. cultivation of edim virus requires the presence of proteolytic enzymes to cleave an outer capsid polypeptide. clinical signs clinical signs occur in infant mice less than weeks old. this age-related susceptibility also applies to infection in immunodeficient mice. furthermore, clinical signs occur only in offspring of nonimmune dams, because maternal immunity protects infants until they have outgrown susceptibility to clinical disease. the cardinal signs are bloated abdomens with fecal soiling of the perineum, which may extend to the entire pelage in severe cases. despite high morbidity, mortality is low because affected mice continue to nurse. transient weight loss does occur, and there may be a delay in reaching adult weight. recovery from infection usually occurs in about weeks and, once weight is regained, is clinically complete. epizootiology edim virus appears to be infectious only for mice and occurs episodically in mouse colonies, and infection is probably widespread geographically (livingston and riley, ; pritchett-corning laboratory animal medicine et al., ) . all ages and both sexes can be infected, but genetic resistance and susceptibility have not been determined. the virus is highly contagious and is transmitted by the oral-fecal route. subclinically infected adult mice can shed virus in feces for at least days, an interval that may be extended in immunodeficient mice. after oral inoculation, virus is essentially restricted to the gastrointestinal tract, although small amounts of virus may be present in the liver, spleen, kidney, and blood. nursing dams can contract infection from their litters. transplacental transmission has not been demonstrated. pathology gross lesions occur primarily in the gastrointestinal tract, but thymic involution can result from infection-related stress. the intestine is often distended, flaccid, and filled with gray-green gaseous liquid or mucoid fecal material that soils the pelage. the stomach contains curdled milk, except in terminal cases with anal impaction due to caking of dried feces. virus preferentially infects terminally differentiated enterocytes in the small and large intestines, which accounts for the agerelated susceptibility to disease; the number of such cells decreases as the intestinal tract matures. characteristic histological lesions are often very subtle, but are most easily discerned in the small intestine in mice less than weeks old. they consist of vacuolation of villar epithelial cells with cytoplasmic swelling, which give villi a clubbed appearance (fig. . ) . the vacuoles must be differentiated from normal absorption vacuoles in nursing mice. the lamina propria may be edematous, but necrosis and inflammation are not prevalent. diagnosis edim virus infection is readily detected serologically. clinical disease is diagnosed from signs and typical histological lesions in the intestine, which can be confirmed by immunohistochemical or ultrastructural demonstration of virus in the intestine or in intestinal filtrates or smears. rotavirus antigen can be detected in feces by elisa, but certain dietary ingredients can cause false-positive reactions. infection can also be diagnosed by rt-pcr. differential diagnosis edim virus infection must be differentiated from other diarrheal diseases of suckling mice such as intestinal coronavirus (mouse hepatitis virus) infection, reovirus infection, tyzzer's disease, and salmonellosis. the presence of milk in the stomach can be helpful in differentiating edim virus infection from more severe enteric infections, such as those caused by pathogenic coronaviruses, during which cessation of nursing often occurs. the possibility of dual infections must also be considered. thymic necrosis in edim virus-infected mice, although nonspecific, must be differentiated from that due to mouse thymic virus (mtv) infection or other stressors. prevention and control the spread of edim can be controlled effectively by the use of microbarrier cages and good sanitation. because infection appears to be acute and self-limiting, cessation of breeding for - weeks to allow immunity to build in adults while preventing access to susceptible neonates also is recommended. alternatively, litters with diarrhea can be culled, in combination with the use of microbarrier cages. the duration of infection in immunodeficient mice has not been determined, but it is reasonable to assume that chronic infection occurs. therefore, such animals should be eliminated. litters from immune dams are more resistant to infection. if edim virus is allowed to become enzootic within a colony, clinical signs will disappear within the population, which may be an appropriate management approach in conventional colonies. prevention of edim virus infection depends on maintenance of sanitary barrier housing with adequate serological surveillance. research complications the research complications of edim infection pertain to clinical illness with diarrhea and retarded growth. transient thymic necrosis may perturb immunological responses. infection (barthold, a,b) etiology coronaviruses are large, pleomorphic, enveloped rna viruses with radially arranged peplomers (spikes). in mice, early clinical and laboratory investigations emphasized their potential to induce hepatitis, so their original designation, which is still used actively, is mouse hepatitis virus (mhv). during that time, enteritis in infant mice was recognized as a separate entity caused by an uncharacterized virus, known as lethal intestinal virus of infant mice (livim). subsequent studies revealed that hepatitis-causing mhv and enteritis-causing livim were closely related coronaviruses, now collectively termed mhv. mhv isolates differ in biologic behavior according to their organ tropism into two biotypes: enterotropic strains, which infect primarily the intestinal tract, and polytropic strains, which initially infect the respiratory tract but may progress to multisystemic dissemination, including the liver and brain. these differences are often reflected in their cell tropism in vitro. however, natural isolates may contain features of both biotypes. several prototype polytropic strains have been extensively studied as experimental models of hepatitis and encephalitis. they include jhm (mhv ), mhv- , mhv- , mhv-s, and mhv-a . numerous additional strains have been identified that differ in virulence, tissue tropism, and antigenicity. differentiation by strain, particularly under natural conditions, is irrelevant, since mutation is common among coronaviruses, and even named prototype strains differ significantly depending upon passage history. although mhv isolates and strains share internal antigens (m and n), they can be distinguished by neutralization tests that detect strainspecific spike (s) antigens. mhv shares antigens with the coronaviruses of rats, a finding that has been exploited to develop heterologous antigens for serological tests. mhv also is related to human coronavirus oc . a number of established cell lines may be used for propagating polytropic mhv strains in vitro. however, field isolates are difficult to maintain in vitro. nctc mouse liver cells are useful for growing many polytropic strains. mhv can also be grown in mouse macrophages, cells that have been used for genetic studies of resistance and susceptibility to infection. enterotropic strains, because of their tendency to be strictly enterotropic, have been grown in cmt- cells derived from a rectal carcinoma in a c bl mouse, but are generally difficult to propagate in cell culture. irrespective of cellular substrate used for isolation or propagation, syncytium formation is emblematic of mhv infection (fig. . ) . clinical signs clinical signs depend primarily on the age, strain, and immunological status of infected mouse and strain and tropism of virus. as with many murine viruses, infection is often clinically silent among immunologically competent mature mice. clinical morbidity is most often associated with suckling mice less than weeks old or with immunodeficient mice. suckling mice infected with enterotropic mhv develop inappetence, diarrhea, and dehydration, often terminating in death (fig. . ) . epizootics of enterotropic mhv have been known to result in % mortality among neonatal mice in a breeding colony. older mice ( - weeks of age) may have ruffled pelage and runting. neurotropic strains such as mhv-jhm may induce flaccid paralysis of the hindlimb, but this sign is rarely encountered alone during natural infection. conjunctivitis, convulsions, and circling may be seen occasionally. enterotropic strains may not cause acute disease in athymic mice when exposed as adults, whereas mildly pathogenic polytropic strains can cause a progressive wasting syndrome that may be accompanied by progressive paralysis. epizootiology mhv infection, despite constant surveillance and preventive programs, continues to be a common threat to laboratory mouse populations (livingston and riley, ; pritchett-corning et al., ). there are no reports of natural transmission from mice to other species, but suckling rats have been found to develop necrotizing rhinitis after intranasal inoculation with mhv-s. mhv is highly contagious, with natural transmission occurring by respiratory or oral routes. mouse appears normal and has a milk-filled stomach. lower mouse is runted and dehydrated and has an empty stomach. from barthold et al. ( ) . enterotropic biotypes predominate in natural infections in contemporary laboratory animal facilities, since they tend to be the most contagious due to copious excretion of virus in feces, whereas polytropic strains generally spread by direct respiratory contact. natural vertical transmission has not been demonstrated. introduction of mhv through injection of contaminated biologicals can be an important factor in epizootics, especially because some isolates infect b lymphocytes and, by implication, hybridomas nonlytically. infection in immunocompetent mice is self-limiting. immune-mediated clearance of virus associated with seroconversion usually begins about a week after infection, and mice recover fully within - weeks. humoral and cellular immunity participate in host defenses to infection, and t-cell-dependent immunity is an absolute requirement. thus, age-related resistance to mhv correlates with maturation of lymphoreticular tissues, but intestinal proliferative kinetics are critical determinants of disease susceptibility with enterotropic mhv. enzootic infection had been construed to include persistent infection in individual mice. current evidence suggests, however, that enzootic infection results either from the fresh and continuous introduction of immunologically naive or deficient mice or from the recurrent infection of immune mice with mhv variants that arise by natural mutation. mutation is favored by immune pressure in enzootically infected colonies as well as missteps during natural replication, which include copying errors and recombination. thus, mice that have developed immunity to one strain of mhv can remain susceptible to one or more genetically and antigenically divergent strains, resulting in reinfection. this caveat has practical importance for breeding colonies. maternal immunity protects suckling mice against homologous mhv strains but not against antigenically variant strains. however, maternal immunity, even to homologous strains, depends on the presence of maternally acquired antibody in the lumen of the intestine. therefore, the susceptibility of young mice to infection increases significantly at weaning. strain differences in resistance and susceptibility to polytropic mhv can be inherited as an autosomal dominant trait. for example, dba/ mice are highly susceptible to mhv- and die acutely even as adults, whereas a/j mice develop resistance to lethal infection shortly after weaning. however, genetic resistance is also virus strain-dependent. therefore, mice resistant to one strain of mhv may be susceptible to another strain. it also is worth noting that the expanded use of genetically altered mice with novel or unanticipated deficits in antiviral responses may alter the outcome of virus-host interactions unpredictably. this pertains to mhv as well as other agents. for example, mhv infection has presented as granulomatous peritonitis and pleuritis in interferongamma (ifn-γ) knockout mice (france et al., ) . pathology polytropic strains replicate initially in the nasal mucosa, where necrotizing rhinitis may occur. viremic dissemination can follow if virus gains access to regional blood vessels and lymphatics. thus, viremia leads to secondary infection of vascular endothelium and parenchymal tissues in multiple organs including liver, brain, lymphoid organs, and other sites. mice also may develop central nervous system disease by direct extension of infection from the olfactory mucosa along olfactory tracts. at necropsy, yellow-white foci indicative of necrosis can occur in multiple tissues, with the involvement of the liver as the classical lesion. liver involvement may be accompanied by icterus and peritonitis. histologically, necrosis can be focal or confluent and may be infiltrated by inflammatory cells (fig. . ) . syncytia commonly form at the margin of necrotic areas and, in mild infections, may develop in the absence of frank necrosis. syncytia formation is a hallmark of infection in many tissues, including the intestine (fig. . ) , lung, liver, lymph nodes, spleen, thymus, brain, and bone marrow and in vascular endothelium in general. although syncytia are transient in immunocompetent mice, they are a persistent feature in chronically infected, immunodeficient mice ( fig. . ) . neurotropic variants cause acute necrotizing encephalitis or meningoencephalitis in suckling mice, with demyelination in the brain stem and in peri-ependymal areas secondary to viral invasion of oligodendroglia. convalescent mice may have residual mononuclear cell infiltrates around vessels or as focal lesions in the liver. immunodeficient mice can develop progressive necrotic lesions in the liver and elsewhere. compensatory splenomegaly may occur because of expansion of hematopoietic tissue. enterotropic strains infect primarily the intestine and associated lymphoid tissues, although some may also figure . necrosis, inflammation, and syncytium in the liver of a mouse infected with mhv. courtesy of s.w. barthold. cause systemic lesions, especially in the liver and brain. the most common sites are terminal ileum, cecum, and proximal colon. the severity of disease is age-related, and dependent upon intestinal proliferative kinetics, similar to edim, with young infants being at highest risk for lethal infection. pathogenic strains can cause lesions ranging from villus attenuation to fulminant necrotizing enterotyphlocolitis, which can kill suckling mice within a few days (fig. . ) . the stomach is often empty, and the intestine is filled with watery to mucoid yellowish, sometimes gaseous contents. syncytia are a consistent feature in viable mucosa (fig. . ) and not only are formed in intestine but also may be present in mesenteric lymph nodes and endothelium of mesenteric vessels. enterocytes may contain intracytoplasmic inclusions, but they are not diagnostic. surviving mice develop compensatory mucosal hyperplasia, which eventually recedes, but may contribute to clinical signs due to osmotic, secretory, and malabsorptive diarrhea. older mice are equally susceptible to infection, but are resistant to severe disease due to their mature (more rapid) intestinal proliferative kinetics. pathology may be subtle, consisting of transient syncytia without necrotic lesions. in adult mice, syncytia can be found most often in the surface mucosal epithelium of the ascending colon. the exception occurs in immunodeficient mice, such as athymic and scid mice, which can develop chronic proliferative bowel disease of varying severity with mhv antigen in mucosal epithelium (figs. . and . ). this may not always be present, as athymic nude mice exposed as adults may only manifest a few enterocytic syncytia without hyperplasia. diagnosis because mhv infection is often subclinical, serological testing is the most reliable diagnostic tool. many animal resources rely on sentinel mouse protocols for continuous serological surveillance. serology is well established, sensitive, and reliable. neutralization tests are used to differentiate individual virus strains in the research laboratory but are inappropriate for routine use, because of cost, technical complexity, and serologic identification per se does not predict biological behavior, including virulence or tissue tropism. serology also can be used in the context of map testing in which adult mice are inoculated with suspect tissues to elicit seroconversion. rt-pcr protocols to detect virus in tissues or excreta are available. the detection of syncytia augmented, when possible, by immunohistochemistry to laboratory animal medicine detect mhv antigen is a useful and practical means to confirm infection. this strategy should attempt to select mice that are in early stages of infection, because necrosis in infant mice or seroconversion in older mice may reduce the chances of detecting syncytia or viral antigens. the option of using immunodeficient mice as sentinels can be considered, because they sustain prolonged infection. however, they should be securely confined because they also amplify virus loads. if properly controlled, amplification in immunodeficient mice can, however, facilitate subsequent virus isolation in tissue culture. differential diagnosis mhv infection must be differentiated from other infectious diseases that cause diarrheal illness, runting, or death in suckling mice and wasting disease in immunodeficient mice. these include edim, mousepox, reovirus infection, tyzzer's disease, and salmonellosis. neurological signs or demyelinating lesions must be differentiated from mouse encephalomyelitis virus infection or noninfectious cns lesions, such as neoplasms, including polyoma virus-induced tumors in athymic mice. prevention and control control and prevention of mhv infection can be difficult because of the numerous variables that influence its expression. perhaps the most important factor is the duration of infection in individual mice and in mouse colonies. there is evidence that infection in an individual immunocompetent mouse is acute and self-limiting. such mice can be expected to develop immunity and eliminate virus within days. therefore, selective quarantine at the cage (not room) level with the temporary cessation of breeding can be used effectively to eliminate infection. quarantine at the room basis is likely to fail, since mutations arise and continually reinfect the mouse population. additionally, maternally derived immunity can protect infant mice from infection until they are weaned and moved to uncontaminated quarters. careful testing with sentinel mice should be used to assess the effectiveness of quarantine or 'natural rederivation,' as just described. immunodeficient mice, in contrast, are susceptible to chronic infection and viral excretion. mice with unrecognized or unanticipated immune dysfunction or with selective immune dysfunction may impact on mhv infection and its control. such colonies, which may contain highly valuable or irreplaceable mice, may be rescued by cesarean rederivation or embryo transfer if vertical transmission of mhv infection is subsequently ruled out. although rodent coronaviruses are not viable for extended periods in the environment, excreted virus may remain infectious for up to several days, so proper sanitation and disinfection of caging and animal quarters as well as stringent personal sanitation are essential to eliminate infection. the prevention of mhv requires procurement of animals from virus-free sources and maintenance under effective barrier conditions monitored by a well-designed quality assurance program. control of feral mouse populations, proper husbandry and sanitation, and strict monitoring of biological materials that may harbor virus (e.g., transplantable neoplasms, cell lines) are also important strategies to prevent adventitious infection. research complications numerous research complications have been attributed to mhv, and the unpredictable outcome of infection in genetically altered mice is likely to lengthen the list. for example, apart from its clinical impact, mhv may stimulate or suppress immune responses, contaminate transplantable neoplasms, and be reactivated by treatment of subclinically infected animals with several classes of drugs, including immunosuppressive agents, and by intercurrent infections. it also can alter tissue enzyme levels. additionally, the ubiquitous threat of mhv infection and uncertainty about its potential effects on a given research project provoke concerns that may exceed its true impact. for example, transient infection with a mild enterotropic strain is unlikely to disrupt systemic immune responses, whereas infection with a polytropic strain may be highly disruptive. this is not to say that subclinical or strictly enterotropic infection should be taken lightly but simply to caution against overreaction in assessing the impact of an outbreak. etiology mice are susceptible to infection by two members of the cardiovirus genus within the theilovirus. emcv has a less selective host range and can infect wild mice, but is not known to infect laboratory mice. tmev is a small, nonenveloped, rna virus that was discovered by max theiler during experimental studies of yellow fever virus in mice. established prototype strains include to (theiler's original), fa, da, and gd vii, the last of which is named after george martine (george's disease), an assistant in theiler's laboratory. tmev is rapidly destroyed by temperatures over °c and by alcohol but not by ether. it can be cultivated in vitro in several continuous cell lines, but bhk- cells are routinely used for isolation and propagation. tmev is antigenically related to emcv. as with other nonenveloped viruses, tmev is resistant to environmental inactivation, a factor that must be considered in control and prevention of infection. clinical signs the development of clinical disease depends on virus strain, mouse strain, and route of exposure, but natural disease is exceedingly rare (estimated at . - . % of infected mice). when clinical signs occur, they are expressed as neurological disease. the characteristic sign is flaccid posterior paralysis, which may be preceded by weakness in the forelimbs or hindlimbs, but in mice that are otherwise alert (fig. . ) . some mice may recover, but death frequently ensues, often because of failure to obtain food or water. furthermore, mice that recover from the paralytic syndrome are disposed to a chronic demyelinating phase, which is expressed as a gait disturbance. epizootiology infection occurs primarily in laboratory mice with the exception of the mgh strain, which has been isolated from laboratory rats and is pathogenic in mice and rats after experimental inoculation. the prevalence of tmev in mouse colonies is low, a reflection of the slow rate at which virus is transmitted from mouse to mouse, but it continues to be among the more common viral contaminants of mouse colonies (livingston and riley, ; pritchett-corning et al., ) . tmev infection is acquired by ingestion and replicates primarily in the intestinal mucosa. enteric infection can persist after the development of host immunity and can result in chronic or intermittent excretion of virus in feces over several months . mice often become infected shortly after weaning, but virus is seldom recovered in mice over months of age. however, neurologic infection can persist in the brain and spinal cord for at least year. immunity to one strain of tmev provides cross-protection to other strains. there are no reports of differences in mice with respect to susceptibility to infection under natural conditions. prenatal transmission has not been found. pathology intestinal tmev infection does not cause lesions, but virus can be detected in enterocytes by immunohistochemistry or in situ hybridization. poliomyelitis-like disease, the syndrome that may be encountered during natural infections, is characterized by acute necrosis of ganglion cells and neurons, neuronophagia, and perivascular inflammation, which occur particularly in the ventral horn of the spinal cord gray matter but also can involve higher centers such as the hippocampus, thalamus, and brain stem. during the subsequent demyelinating phase, mononuclear cell inflammation develops in the leptomeninges and white matter of the spinal cord, accompanied by patchy demyelination. the white-matter lesions are due to immune injury. spontaneous demyelinating myelopathy, affecting the thoracic spinal cord and associated with mev infection, has also been reported in aged mice. virulent strains may cause acute encephalitis after experimental inoculation, whereas less virulent isolates produce acute poliomyelitis followed by chronic demyelinating disease. diagnosis infection is usually detected serologically or by pcr of feces, but virus shedding from infected mice may be intermittent. clinical signs are striking, if they occur, but are too rare to rely on for routine diagnosis. histological lesions in the cns and especially the spinal cord are characteristic when present. differential diagnosis neurotropic variants of mhv may, on occasion, cause similar neurological signs. injury or neoplasia affecting the spinal cord can also produce posterior paralysis. polyoma virus infection in athymic mice can induce tumors or demyelination in the cns, which may result in clinical signs resembling those of tmev infection. prevention and control disease-free stocks were originally developed by foster-nursing infant mice. this technique, cesarean rederivation, or embryo transfer can be used successfully to eliminate infection. in either case, foster mothers should be surveyed in advance to ensure their mev-free status. selective culling can be considered as an option to eliminate infection, because laboratory animal medicine infection spreads slowly. however, the virus is hardy in the environment and resists chemical inactivation, so it may be prudent to depopulate and disinfect rooms if the presence of infection is unacceptable. research complications the principal hazard from tmev for research relates to its potential effects on the cns. noroviruses are nonenveloped rna viruses that belong to the family caliciviridae. they are notoriously resistant to environmental inactivation, and cause significant gastrointestinal morbidity in humans. noroviruses are species-specific, including mnv, which exclusively infects mice. until the discovery of mnv, replication of noroviruses in vitro has not been possible. for this reason, mnv has emerged as an important small animal model of norovirus pathogenesis. mnv was relatively recently discovered in , and subsequent surveillance has revealed that it is the most common adventitious virus infection in laboratory mice (hsu et al., ; pritchett-corning et al., ) . over mnv isolates have been found in mouse research colonies around the world, which display nearly % genetic identity, comprising a single genetic cluster. although genetically homogeneous, significant biological differences exist among mnv strains (thackray et al., ) . mnv effectively replicates in macrophages and dendritic cells, including the mouse macrophage-like raw . cell line, as well as a microglial cell line (wobus et al., ) . clinical signs clinical signs of infection in immunocompetent mice are usually absent, but infection leads to systemic disease with high mortality in interferon αβγ receptor and stat null mice. affected mice have loss of body weight, ruffled fur, and hunched posture (ward et al., ) . experimental infection of and c h mice with mnv- caused mild diarrhea (kahan et al., ) . epizootiology mnv is transmitted by the fecaloral route, and contaminates the environment as an environmentally resistant virus. for this reason, it can efficiently infect sentinel mice with soiled bedding (manuel et al., ) . duration of infection varies with mnv strain, mouse immunocompetence and mouse genotype. experimental studies have revealed that several mnv strains persist in various tissues of c bl/ j, hsd:icr, and jcl:icr and c.b- -prkdc scid mice, with fecal shedding for at least - days (goto et al., ; hsu et al., ; thackray et al., ) . although not clinically ill, rag null mice are unable to clear infection . comparative studies with mnv- and mnv- have shown differences in virus replication and shedding (kahan et al., ) . mnv has a tropism for macrophages and dendritic cells, and virus can be detected in the intestine, intestinal lymphoid tissue, liver, and spleen (hsu et al., ; kahan et al., ; wobus et al., ) . pathology naturally and experimentally infected stat or ifnγr null mice may develop splenomegaly and multifocal pale spots on the liver. microscopic findings include varying degrees of hepatitis, focal interstitial pneumonia, vasculitis, peritonitis, and pleuritis (karst et al., ; ward et al., ) . encephalitis, cerebral vasculitis, pneumonia, and hepatitis have also been described in intracerebrally infected stat null mice (karst et al., ) . infection of immunocompetent mice may be associated with mild inflammation of the intestine, splenic hypertrophy, and lymphoid hyperplasia of spleen and lymph nodes (mumphrey et al., ) . diagnosis mnv infection can be detected by serology or rt-pcr. sentinel mouse surveillance, using soiled bedding, is an effective strategy for detecting mnv (manuel et al., ) differential diagnosis the mild change in fecal consistency associated with mnv in adult mice may mimic rotavirus, coronavirus, helicobacter spp., citrobacter rodentium, or other enteric diseases. disseminated lesions in stat or ifnγr null mice must be differentiated from other polytropic viral diseases in immunodeficient mice, including mhv. prevention and control depopulation and decontamination has been shown to be effective at eliminating mnv from an enzootically infected colony, whereas testand-removal of positive mice was found to be ineffective (kastenmayer et al., ) . embryo transfer and cesarean rederivation are also effective (goto et al., ; perdue et al., ) . neonatal mice are resistant to infection, so that cross-fostering neonates onto uninfected dams is another effective means of rederivation mnv-free mice (artwohl et al., ; compton, ) . research complications the tropism of mnv for macrophages and dendritic cells is likely to modify immune responses, and mnv infection may interfere with studies involving enteric disease. hantaviruses are rna viruses belonging to the very large bunyaviridae family. they differ from other members of this family by not being arthropod-borne. each hantavirus is antigenically distinct and maintained within single or at most a few rodent or insectivore hosts, but are infectious for other hosts. infection is lifelong, and virus is transmitted by shedding of virus in urine, feces, and saliva. several hantaviruses are zoonotic and may cause severe disease in humans. although there is overlap, hantaviruses in asia and europe cause hemorrhagic fever with renal syndrome (hfrs) in humans, a multisystem disease with significant renal involvement, and hantaviruses that are endemic in the americas cause laboratory animal medicine hantavirus pulmonary syndrome (hps) in humans, which is a multisystem disease with pulmonary involvement. among the better-known old world hfrs hantaviruses are hantaan, seoul, puumala, and dobrava-belgrade viruses. sin nombre virus is the best known new world phs hantavirus, among many others. most notably from the perspective of laboratory animal medicine, the norway rat, rattus norvegicus, serves as a reservoir host for hantavirus in the wild, but infection has also been associated with laboratory rats. in addition to being endemic in wild rats in asia, it has been found to be endemic in wild rats in the eastern united states and associated with human cases of hfrs (childs et al., ; leduc et al., ; tsai et al., ) . over cases of hantavirus infection have been transmitted to humans from laboratory rats in japan, belgium, and the united kingdom (desmyter et al., ; kawamata et al., ; lloyd et al., ; umenai et al., ) . m. musculus is not considered to be a primary reservoir host, but hantavirus infection has been documented serologically in conventional and barrier-maintained laboratory mice and rats in korea (won et al., ) , infection of wild m. musculus has been documented in the united states (baek et al., ) , and infection of wild mice in europe has been associated with human exposure (diglisic et al., ) . hantaviruses are difficult to culture in vitro. infection in rodents is subclinical and is detected by serology or rt-pcr. the main research complication from natural infection is the zoonotic risk and potentially subclinical effects on the immune response associated with viral defenses such as cd + t cell (taruishi et al., ) and nk function as demonstrated in human studies (braun et al., ) . the mouse is host to a number of enveloped rna viruses of the family retroviridae, subfamily orthovirinae, including the two type species (and their variants) mouse mammary tumor virus (mmtv) and murine leukemia virus (mlv). these viruses belong to a diverse assemblage of related mobile dna elements that are integrated into the host genome, and collectively termed 'retroelements', which include retrovirus-related elements and nonviral elements. during cell division, retroelements are transcribed into rna, and subsequently reverse-transcribed into dna copies that become integrated into a new location within the genome. this process utilizes reverse transcriptase, which is encoded by the retroelement. over millennia, retroelements have been repeatedly integrated within the genome in large numbers, comprising approximately % of the mammalian genome. various families of mouse retroelements share sequence similarity, despite their random distribution throughout the mouse genome, and the majority of them are truncated, mutated, and methylated to become incapable of infectivity. nevertheless, many of them continue to be mobile within the genome. noninfectious retrovirus-related retroelements include iap, vl , musd, and etn elements. replication-competent retroviruses represent the pinnacle of the retroelement constellation and are best considered as the most evolutionarily recent members. these include mmtv and mlv. mtv and mlv share similar genetic structure, except that the long terminal repeat (ltr) region of the mmtv genome encodes an additional superantigen (sag). both mmtv and mlv include exogenous viruses, which are horizontally transmitted, replication-competent viruses, and endogenous viruses, which are closely related to exogenous viruses, encoded within the mouse genome, and transmitted by mendelian inheritance. exogenous mmtv and mlv exist in wild mouse populations, but have been eliminated from contemporary laboratory mice. however, they may continue to be used experimentally, including bittner mmtv, and gross, friend, moloney, and rauscher mlvs. in particular, mouse colonies may be purposely infected with mmtv for mammary cancer research and are termed 'mmtv-positive', reflecting their exogenous virus status, even though the mice may also carry endogenous mmtv. the genomes of all inbred strains of mice encode one or more (over in some mouse strains) endogenous mmtv loci, the distribution of which is unique to each inbred strain of mouse. most mmtv genomic loci do not encode infectious virus or are transcriptionally inactive, except for mouse strains (dba, c h, grs) that carry mtv or mtv loci. these loci encode infectious virus, which can be visualized as b-type particles by electron microscopy. likewise, all mouse strains carry endogenous mlv loci within their genome, but not all mice carry replication-competent mlv sequences. some endogenous mlvs encode infectious virus, which can be visualized as c-type particles by electron microscopy. mice have often evolved mechanisms to counter the deleterious effects of retroviruses by preventing reentry or replication of virus into other cells. if an endogenous retrovirus is still infectious to other mouse cell targets, it is termed ecotropic, whereas if it is no longer infectious for mouse cells, but can infect cells of other species, it is termed xenotropic. viruses capable of infecting cells of mice as well as other species are termed polytropic. the combinations of endogenous replication-competent mlvs and cell tropism factors are a reflection of selective breeding of mouse strains for susceptibility to various types of cancer. clinical signs mice were originally inbred for specific phenotypes, including mammary tumors and lymphomas. thus, some strains of mice were genetically selected for unique combinations of endogenous mmtv and mlv in concert with susceptibility factors laboratory animal medicine that favored their expression and disease manifestations. in addition, noninfectious retroelements continue to reintegrate randomly within the genome during cell division as retro transposons. these ongoing integrations contribute to genetic drift, spontaneous mutations, and well-recognized mouse strain phenotypes, including the athymic nude allele, the hairless allele, and the rodless retina allele, among others. epizootiology exogenous mmtv and mlv are horizontally transmissible, primarily through the milk of lactating females. endogenous retroviruses and retroelements are inherited through the genome. replicationcompetent endogenous mmtvs and mlvs are also transmissible like their exogenous counterparts, but differ by being integrated within the genome of the mouse. pathology replication-competent mmtv and mlv, regardless of their exogenous or endogenous origin, are usually clinically silent. their ability to cause neoplasia is a reflection of genetic selection for susceptibility factors that are genetically encoded within individual mouse strain genomes. mmtv derives its name from its association with induction of mammary carcinomas in mammary cancer-susceptible strains of mice. mlv is associated with lymphomas, the pattern of which is mouse strain specific. for example, akr mice develop % prevalence of thymic lymphoma between and months of age, whereas aging balb/c mice commonly develop multicentric lymphoma. in these strains of mice, multiple endogenous mlvs are coexpressed in tissues and undergo recombination events that allow them to target and transform cells into neoplasia. despite its name, mmtv can induce lymphomas in some strains of mice, such as sjl mice which develop lymphomas arising from enteric lymphoid tissue and mesenteric lymph nodes. diagnosis exogenous retroviruses have been eliminated from contemporary mouse populations, unless purposely introduced for experimental purposes. because endogenous retroviruses and retroelements are encoded within the genome, and reflect the unique genetic composition of each strain of mouse, they are not targets of diagnostic pursuit. differential diagnosis patterns of some types of neoplasia within individual inbred strains of mice are a reflection of their endogenous retroviral integration. prevention and control exogenous retroviruses have been eliminated from laboratory mice by cesarean rederivation and foster nursing. mmtv-s, the 'bittner agent', continues to be purposely maintained in some mouse breeding populations, but can be eliminated by foster-nursing or other means. caution is advised when re-deriving such mouse colonies for other purposes, as elimination of exogenous mmtv will be an unintended consequence. research complications endogenous retroviruses and retroelements influence the life span of individual strains of mice, and random integrations during cell division can give rise to spontaneous mutations and genetic drift. it is estimated that significant mutations may arise due to mobile retroelement integrations every generations. astroviruses are small, nonenveloped, singlestranded rna viruses that have been associated with human gastroenteritis and detected in association with other enteric pathogens. the viral family astroviridae is split into two genuses: avastrovirus for those astroviruses infecting avians and mamastrovirus for those infecting mammals. astrovirus infection has been detected in research mice (muastv) using metagenomic analyses and appears to have a wide geographical, institutional, and host strain distribution. clinical signs none reported. epizootiology pcr screening has found muastv infection in up to % of a variety of mouse strains housed in vendor and academic facilities in the united states and japan. the virus has been detected most commonly in immunocompromised mice (nsg, nod-scid, nsg- gs, c bl -timp- −/− , and upa-nog), but also in immuncompetent strains (b j, icr, bash , and balb/c). both immunodeficient and immunocompetent mice are susceptible to muastv, but adaptive immunity is required to clear the virus. based on human epidemiology indicating children are at highest risk for infection, the virus may preferentially infect young mice. pathology immunodeficient mice showed no sign of pathology based on histopathology. diagnosis pcr data has indicated that muastv causes a systemic, chronic infection in immunocompromised mice, indicating samples from most tissues will be pcr positive. yokoyama et al. ( ) detected high viral load (up to genome copies) per fecal pellet from immunocompetent mice. differential diagnosis none, in the absence of lesions and clinical disease. prevention and control because immunocompetent mice clear the infection, quarantine may be successful but lack of routine screening for muastv in laboratory mice will allow for uncontrolled spread of the infection. research complications based on limited surveys, muastv may have a high prevalence in laboratory mice. the impact of infection on both innate and adaptive immune responses warrants further investigation to assess the potential for confounding research data. this section briefly describes the etiology, clinical signs, epizootiology, pathology, diagnosis, differential diagnoses, prevention and control, and research complications of the most common bacterial diseases encountered in research colonies of mice. as sequencing technology becomes more available, the number and genus/species classification of bacteria potentially responsible for infections, in particular, opportunistic infections, will grow (benga et al., ) . potential candidates include members of pasteurellacaeae, bordetella hinzii, streptococcus danielae, acinetobacter spp., and others, for which little is currently known about their pathogenic potential. etiology lawsonia intracellularis, an obligate intracellular bacterium and the causative agent of proliferative enteropathy, is not a pathogen encountered in research colonies of mice but has been reported to infect wild mice and rats in close contact with infected livestock (collins et al., ) . clinical signs none reported but should consider lawsonia as a differential in necropsy cases with gross or histologic evidence of proliferative lower bowel lesions. epizootiology although mice are experimentally susceptible to infection and develop classic lesions of hyperplastic ileitis and typhlocolitis (murakata et al., ) , susceptibility varied with mouse strain and source of inoculum from rabbits or swine, suggesting important differences in l. intracellularis strains. pathology lawsonia infection may result in hyperplastic ileitis, typhlitis and/or colitis, and hemorrhagic intestines may be noted (percy and barthold, ) . diagnosis lawsonia spp. has been diagnosed using a variety of techniques, including pcr, immunohistochemistry, in situ hybridization, and warthin-starry silver stains. differential diagnosis bacterial infections associated with hyperplastic intestinal epithelium, including c. rodentium and enterohepatic helicobacter species in susceptible (typically immunodeficient) mouse strains. prevention and control species separation from hosts more commonly associated with natural infection (hamsters, ferrets, pigs). research complications none reported. the following section describes infection due to mycoplasma pulmonis and summarizes infections associated with other murine mycoplasmas including m. arthriditis, m. neurolyticum, m. collis, and m. muris. antigenic cross-reactivity among these species, and especially between m. pulmonis and m. arthriditis, mandates that reliable diagnostic strategies incremental to serology (elisa, ifa, mfia) such as culture (often false negative) and pcr be employed to distinguish potentially pathogenic infections. when screening cell lines for opportunistic pathogens, pcr is the most efficient method to discriminate between m. pulmonis and mycplasma contaminants associated with cell culture. etiology m. pulmonis is a pleomorphic, gram-negative bacterium that lacks a cell wall and has a single outer limiting membrane. it causes murine respiratory mycoplasmosis (mrm). clinical signs mice are relatively resistant to florid mrm; thus, subclinical infection is more common. when clinical signs occur, they reflect suppurative rhinitis, otitis media, and chronic pneumonia. affected mice may display inactivity, weight loss, and ruffled hair coat, but the most prominent signs are 'chattering' and dyspnea, due to rhinitis and purulent exudate in nasal passages. otitis media may cause a head tilt, whereas suppurative inflammation in the brain and spinal cord, although rare, can cause flaccid paralysis. experimental infection of the genital tract can cause oophoritis, salpingitis, and metritis, which may lead to infertility or fetal deaths. experimental inoculation of scid mice has caused systemic infection accompanied by severe arthritis (evengard et al., ) . epizootiology mrm historically was a common infectious disease of mice, but improved housing, husbandry, and health surveillance have reduced its prevalence dramatically. serologic data from a large diagnostic laboratory indicated m. pulmonis infection affects about . % of conventionally housed mouse colonies in the united states and . % in europe (pritchett-corning et al., ) . m. pulmonis infection is contracted by inhalation and can occur in suckling and adult mice. therefore, infection should be considered highly contagious. mice injected with cells harvested from m. pulmonis contaminated cell cultures may develop disease. m. pulmonis can also be transmitted venerally; in utero infection has been demonstrated in rats but not in mice. because transplacental infection occurs in rats, the same route may be possible in mice, particularly immunocompromised strains. concomitant viral pneumonia (sv, mouse coronavirus) or elevated environmental ammonia concentrations may increase susceptibility to mrm. m. pulmonis also infects rats, hamsters, guinea pigs, and rabbits. among these species, only rats are significant reservoirs of infection for mice. pathology m. pulmonis is an extracellular organism that colonizes the apical cell membranes of respiratory epithelium. attachment occurs anywhere from the anterior nasal passages to the alveoli and may be mediated by surface glycoproteins. the organism may injure host cells through competition for metabolites such as carbohydrates and nucleic acids or by release of toxic substances such as peroxides. ciliostasis, reduction in the number of cilia, and ultrastructural changes leading to laboratory animal medicine cell death have also been described. detrimental effects on ciliated epithelium can lead to disrupted mucociliary transport, which exacerbates pulmonary disease. experimental infection of mrm is dose dependent. doses of colony-forming units (cfus) or less cause mild, transient disease involving the upper respiratory tract and middle ears, whereas higher doses often lead to acute, lethal pneumonia. additionally, m. pulmonis strains can differ in virulence. survivors of severe infection may develop chronic bronchopneumonia with bronchiectasis and spread infection to other mice. intravenous inoculation of m. pulmonis can cause arthritis in mice, but arthritis is not a significant feature of natural infection. host genotype also is a major factor in the outcome of infection, with resistance being expressed phenotypically through the bactericidal efficiency of alveolar macrophages. strains derived from a c bl background appear to be resistant to pathogenic infection, whereas balb/c, c h, dba/ , swr, akr, cba, sjl, and other strains have varying degrees of increased susceptibility (cartner et al., ; lai et al., ) . the initial lesion of mrm is suppurative rhinitis, which may involve the trachea and major airways. early inflammatory lesions, if not quickly resolved, progress to prominent squamous metaplasia. transient hyperplasia of submucosal glands may occur, and lymphoid infiltration of the submucosa can persist for weeks. syncytia can sometimes be found in nasal passages, in association with purulent exudate (fig. . ) . affected mice also develop suppurative otitis media and chronic laryngotracheitis with mucosal hyperplasia and lymphoid cell infiltrates. pulmonary lesions are typified by bronchopneumonia, which spreads from the hilus. lymphoid cells and plasma cells accumulate around bronchi which often contain neutrophils in their lumen. chronic lung disease features suppurative bronchitis, bronchiolitis, and alveolitis (fig. . ) . chronicity also increases the prevalence of bronchiectasis and abcessation. diagnosis accurate diagnosis should exploit the complementary use of clinical, serological, microbiological, molecular, and morphological methods. clinical signs are variable but can be characteristic when they occur. serology is sensitive but although antibodies do not clear the infection, seroconversion may be weak or take months and may not accurately differentiate between m. pulmonis infection and m. arthriditis infection (cassell et al., ) . therefore samples for culture and pcr of the upper respiratory tract should be obtained to confirm diagnosis. buffered saline or mycoplasma broth should be used to lavage the trachea, larynx, pharynx, and nasal passages. specimens for culture from the genital tract are warranted if this site is suspected. mycoplasma spp. may be difficult to grow, so it is prudent to confirm that the relevant expertise and quality control exist in the diagnostic laboratory. speciation can be accomplished by immunofluorescence or immunoperoxidase staining or by growth inhibition. immunohistochemistry should be considered to supplement basic histopathologic examination. immunofluorescence and immunoperoxidase techniques are available to identify mycoplasma antigens in tissue sections or in cytological preparations of tracheobronchial or genital tract lavages (brunnert et al., ) . pcr assays for m. pulmonis at veterinary diagnostic laboratories and pcr kits to screen cell culures for mycoplasma are readily available. differential diagnosis mrm must be differentiated from bronchopneumonia associated with ciliaassociated respiratory (car) bacillus. silver stains may reveal car bacilli adherent to the respiratory epithelium. sv also can cause bronchopneumonia in mice but can be detected by serology and immunohistochemistry. other causes of respiratory infection include pvm, corynebacteriosis and, in immunodeficient mice, pneumocystis murina infection. combined infections with known pathogens or secondary opportunists also must be considered. prevention and control mice mount an effective immune response to m. pulmonis, as measured by their recovery from mild infection and their resistance to infection after active or passive immunization (cartner et al., ) . antibodies of various classes are produced locally and systemically, but clearance of the infection has been attributed to innate immune responses (love et al., ; sun et al., ) . there is some evidence that antibody may facilitate phagocytosis of m. pulmonis. t-cell responses, however, appear to exacerbate m. pulmonis in mice, because immunity cannot be transferred with immune cells. in addition, athymic and neonatally thymectomized mice are not more susceptible than immunocompetent mice to m. pulmonis pneumonia. nude and scid mice develop less severe respiratory disease than immunocompetent mice but infection becomes systemic and they may develop suppurative disease in multiple organs and joints (arthritis). host immunity aside, effective control and prevention of mrm depend primarily on maintenance of mycoplasma-free colonies under barrier conditions supported by careful surveillance for infection by serology, microbiology, pcr, and histopathology. cesarean or embryo rederivation may eliminate infection, although vertical transmission may occur in immunocompromised mice. treatment with tetracycline suppresses clinical disease but does not eliminate infection. earlier interest in developing dna-based vaccines against m. pulmonis has not achieved clinical application (lai et al., ) . research complications m. pulmonis can interfere with research by causing clinical disease or death. experiments involving the respiratory tract, such as inhalation toxicology, can be compromised by chronic progressive infection. additionally, affected mice are at greater risk during general anesthesia. m. pulmonis may alter immunological responsiveness. for example, it is mitogenic for t and b lymphocytes and can increase nk cell activity. perhaps one of the most important complications of mycoplasma infection is contamination of cell lines and transplantable tumors. other murine mycoplasmas cell lines are often contaminated with mycoplasma species such as m. arginini, m. hyorhinis, m. orale, or m. fermentans that can distort the results of in vitro assays (garner et al., ) . initial evidence of a contamination is often by pcr evidence of mycoplasma at the genus level when cell lines are pcr screened for opportunistic murine pathogens prior to use in mice. other than m. pulmonis, these mycoplasmas are not normally considered mouse pathogens in immunocompetent mice. in contrast, injection of mycoplasma contaminated cells into immunodeficient mice (e.g., xenografts) may result in clinical disease or confounding effects on immune responses (peterson, ) . mycoplasma contamination of murine embryonic stem cells has adversely affected germline transmission and postnatal health of chimeric progeny (markoullis et al., ) . mycoplasma arthritidis is antigenically related to m. pulmonis. therefore, serological evidence of mycoplasma infection must be supplemented by other diagnostic tests, as outlined above, to differentiate between these agents. differentiation is important because m. arthritidis, though arthritogenic in mice after intravenous inoculation, is nonpathogenic during natural infection. mycoplasma collis has been isolated from the genital tract of mice but does not appear to cause natural disease. mycoplasma neurolyticum is the etiological agent of rolling disease, a rare syndrome which occurs within hours after intravenous inoculation of m. neurolyticum exotoxin. characteristic clinical signs include spasmodic hyperextension of the head and the raising of one foreleg followed by intermittent rolling on the long axis of the body. the rolling becomes more constant, but mice occasionally leap or move rapidly. after - h of rolling, animals become comatose and usually die within h. all published reports of rolling disease are associated with experimental inoculation of m. neurolyticum or exotoxin. large numbers of organisms are needed to produce disease, and there is no indication that, under natural conditions, organisms replicate in the brain to concentrations required for the induction of these signs. because animals are frequently inoculated with biological materials by parenteral routes, contamination with m. neurolyticum may induce rolling disease inadvertently. diagnosis can be made from the appearance of typical clinical signs, astrocytic swelling, and isolation of the causative organism. clinical signs must be differentiated from rolling associated with pseudomonas-and p. pneumotropica-caused otitis. m. pulmonis has been recovered from the brain of mice but does not seem to cause overt neurological disease. hemotropic mycoplasmas ribosomal rna sequencing has reclassified hemobartonella muris and eperythrozoon coccoides as mycoplasma hemomuris and mycoplasma coccoides, respectively (neimark et al., ; percy and barthold, ) . distinct from the mycoplasmas just discussed, these agents are trophic for red blood cells and cause anemia and hemolytic disease. these laboratory animal medicine infections could be encountered in wild mice but are rarely found in research mice. diagnosis is by morphologic assessment of blood smears and pcr. clinical signs mice infected with m. coccoides may remain clinically normal or develop febrile, hemolytic anemia and splenomegaly, which can be fatal. hepatocellular degeneration and multifocal necrosis have been recorded in acute infections. hemotropic mycoplasma infections are long-lived and are expressed clinically in one of two ways: acute febrile anemia and latent or subclinical infection that can be reactivated by splenectomy. the carrier state may be lifelong. epizootiology the primary natural vector of m. coccoides, historically, is the mouse louse, polyplax serrata. infection was associated with primitive housing and husbandry conditions that no longer occur in modern vivaria. although the risks for infection have been reduced substantially by modern animal care procedures, m. coccoides can be transmitted to mice from contaminated biological products such as transplantable tumors or blood plasma. diagnosis splenectomy or inoculation of test material into splenectomized mice is the most sensitive means of detecting m. coccoides infection. these procedures provoke mycoplasmemia, usually within - days. because mycoplasmemia may be transient, blood smears stained by the romanowsky or indirect immunofluorescence procedures of the blood should be prepared every h, beginning at h after splenectomy of index animals or inoculation of test specimens into splenectomized animals to ensure that mycoplasmemia is not missed. prevention and control treatment of m. coccoides infection is not practical. control is based on culling or rederivation of infected stock. if replacement animals are readily available, euthanasia is a more prudent course. suspect biological materials destined for animal inoculation should be screened for mycoplasma contamination by inoculation of splenectomized mice. research complications subclinical infection can be reactivated by irradiation, immunosuppressive therapy, or intercurrent disease. conversely, m. coccoides may potentiate coincident viral infections in mice. this effect has been clearly demonstrated for mouse coronavirus and has been suspected for lymphocytic choriomeningitis virus and ldv. active infection also may suppress interferon production. etiology car bacillus is a slender, gram-negative, non-spore-forming bacillus, which, in rats, produces clinical disease and lesions that closely resemble those of mrm (see chapter ). clinical signs chronic respiratory disease has been produced in mice by experimental inoculation, but natural clinical disease is rare (griffith et al., ; pritchett-corning et al., ) . furthermore, putative natural cases were reported in mice that were seropositive for sv and pneumonia virus of mice. therefore, car bacillus may exacerbate respiratory disease as an opportunist rather than as a primary pathogen. on balance, it is assumed that mice contract natural infection, but attributing severe chronic respiratory disease in mice solely to car bacillus should be supported by screening for other respiratory pathogens. epizootiology car bacillus is transmitted by direct contact; dirty bedding transfer to sentinel mice may not reflect colony infection status. pathology lung lesions are typically mild in mice and are similar to respiratory mycoplasmosis. uncomplicated car bacillus infection results in peribronchiole cuffing with lymphocytes and plasma cells. severe bronchiolitis and pneumonia are possible (fig. . ) . fatal bronchopneumonia was reported in ob/ob mice (griffith et al., ) . diagnosis an elisa for serological screening is routinely used; pcr and histology are used for definitive diagnosis. in active infection, histologic assessment using warthin-starry or similar stains will reveal argyrophilic bacilli adherent to the apical membranes of bronchial respiratory epithelium along with the presence of peribronchial lymphocytes (fig. . ) . alternatively, immunohistochemistry assays have also been used successfully to detect infection. recovery of car bacillus requires cell culture or culture in embryonated eggs. differential diagnosis respiratory mycoplasmosis, bordetella (avium, hinzii). prevention and control given car bacillus does not form spores, disinfection of the environment should be effective. treatment using sulfamerazine ( mg/l) in drinking water may eradicate infection (matsushita and suzuki, ) but culling or embryo rederivation is recommended. research complications infection is most often subclinical, but like other infectious agents for mice, may confound studies particularly when mice are immunocompromised (griffith et al., ) . etiology the causative agent of transmissible murine colonic hyperplasia, c. rodentium (formerly citrobacter freundii strain ), is a nonmotile, gramnegative rod that ferments lactose but does not utilize citrate or does so marginally (barthold, ; schauer et al., ) . clinical signs c. rodentium infection can be a selflimiting colitis with sterilizing immunity or lead to severe colitis with life-threatening dehydration. clinically apparent infection is characterized by retarded growth, ruffled fur, soft feces or diarrhea, rectal prolapse, and moderate mortality in older suckling or recently weaned mice (barthold et al., ) . epizootiology c. rodentium is not detected in the gastrointestinal flora of normal mice, and therefore, there is not a carrier state. it is thought to be introduced by contaminated mice, food, or bedding, from which it spreads by contact or additional fecal contamination. c. rodentium shares several pathogenic mechanisms, such as attaching and effacing lesions mediated by the intimin receptor, with select escherichia coli (reviewed in collins et al. ( ) ). c. rodentium is used experimentally to model colitis caused by enteropathogenic (epec) and enterohemorrhagic e. coli (ehec) in humans (mallick et al., ; collins et al., ) . host genotype can influence the course and severity of disease (barthold et al., ) . for example, dba, nih swiss, and c bl mice are relatively resistant to mortality, whereas c h/hej mice are relatively susceptible both as sucklings and as adults. interestingly, c bl mice obtained from different commercial sources have varying susceptibility to c. rodentium (ostensibly due to the presence or absence of segmented filamentous bacteria). diet also can modulate infection, but specific dietary factors responsible for this effect have not been identified. pathology c. rodentium attaches to the mucosa of the descending colon and displaces the normal flora. attachment is accompanied by effacement of the microvillus border and formation of pedestal-like structures (attaching and effacing lesions) (schauer and falkow, ; newman et al., ) . colonization results in prominent mucosal hyperplasia, by unknown mechanisms. the characteristic gross finding is severe thickening of the descending colon, which may extend to the transverse colon and lasts for - weeks in surviving animals ( fig. . ) . affected colon segments are rigid and either are empty or contain semiformed feces. histologically, accelerated mitotic activity results in a markedly hyperplastic mucosa, which may be associated with secondary inflammation and ulceration (fig. . ). lesions subside after several weeks. intestinal repair is rapid and complete in adults but slower in sucklings. diagnosis diagnosis depends on clinical signs, characteristic gross and histological lesions, and isolation of c. rodentium from the gastrointestinal tract or feces. the organism can be cultured on macconkey's agar during early phases of infection, whereas the intestine may be free of c. rodentium during later stages of the disease. c. rodentium also can be detected by molecular hybridization (schauer et al., ) . barthold et al. ( ) . diagnosis transmissible murine colonic hyperplasia must be differentiated from other diarrheal diseases of mice, including infections caused by coronavirus, rotavirus, adenovirus, reovirus, salmonella, c. piliforme, and helicobacter spp. prevention and control some success in curtailing epizootics has been achieved by adding antimicrobials to the drinking water (barthold, ; silverman et al., ) . because c. rodentium may contaminate food, bedding, or water, proper disinfection of such materials is prudent before they are used for susceptible animals. additionally, the employment of microbarrier caging can reduce transmission. surveillance for c. rodentium should be incorporated into quality-assurance programs, and the organism screened for during quarantine of incoming mice from atypical sources. research complications the potential effects on research of colonic hyperplasia as a clinically severe disease are obvious. colonic hyperplasia has been shown to increase the sensitivity of colonic mucosa to chemical carcinogens and to decrease the latent period between administration of carcinogen and the appearance of focal atypical cell growth (barthold and beck, ) . c. rodentium infection has been incriminated in immune dysfunction, poor reproductive performance, and failure to thrive in t-cell receptor transgenic mice (maggio-price et al., ) . immunocompromised mice infected with c. rodentium will die from sepsis. etiology pseudomonas aeruginosa is a motile, gramnegative rod. clinical signs p. aeruginosa infections are almost always silent, but immunologically compromised animals are prone to septicemia (brownstein, ) . p. aeruginosa can, e.g., cause severe or lethal infections in athymic and scid mice. sick mice may have equilibrium disturbances, conjunctivitis, serosanguinous nasal discharge, edema of the head, weight loss, and skin infections. immunosuppressed mice may also develop gastrointestinal ulcers. generalized infection is associated with severe leukopenia, especially neutropenia. neurologic signs are rare, but there are reports of central nervous system infection. chronic proliferative inflammation in the cochlea and vestibular apparatus with dissolution of surrounding bone may cause torticollis. epizootiology p. aeruginosa is not considered a component of the normal flora. however, it is an opportunist that inhabits moist, warm environments such as water and skin. once established in a host, it may be found chronically in the nasopharynx, oropharynx, and gastrointestinal tract, all sites from which additional environmental contamination or direct transmission to susceptible mice can occur. pathology pathogenic infection is most common in immunodeficient mice. organisms enter at the squamocolumnar junction of the upper respiratory tract and, in some cases, the periodontal gingiva. bacteremia is followed by necrosis or abscess formation in the liver, spleen, or other tissues. if otitis media occurs, the tympanic bullae may contain green suppurative exudate. the bowel may be distended with fluid, and gastrointestinal ulceration has been reported. diagnosis infection is diagnosed on the basis of history (e.g., immune dysfunction or recent immunosuppression), clinical signs, lesions, and isolation of p. aeruginosa from affected mice. carrier mice can be detected either by nasal culture or by placing bottles of sterile, nonacidified, nonchlorinated water on cages for - h and then culturing the sipper tubes. p. aeruginosa can also be cultured from feces. differential diagnosis pseudomoniasis must be differentiated from other bacterial septicemias that may occur in immunodeficient mice. these include, but are not limited to, corynebacteriosis, salmonellosis, colibacillosis, staphylococcosis, and tyzzer's disease. prevention and control infection can be prevented by acidification or hyperchlorination of the drinking water (homberger et al., ) . these procedures will not, however, eliminate established infections. entry of infected animals can be prevented by surveillance of commercially procured colonies. maintenance of pseudomonas-free animals usually requires barrierquality housing and husbandry. p. aeruginosa has a long history in the literature of antibiotic resistance and resistance to quaternary amine disinfectants. research complications p. aeruginosa infection is not a substantial threat to immunocompetent mice but can complicate experimental studies by causing fatal septicemia in immunodeficient mice. viral infections that alter host defense mechanisms, such as mcmv may enhance susceptibility to pseudomoniasis. (lindsey et al., a; percy and barthold, ) etiology pasteurella pneumotropica is a short, gramnegative rod. clinical signs many early observations concerning the pathogenicity of p. pneumotropica are questionable because they were made on colonies of mice with varying levels of bacterial and viral contamination. infection is usually subclinical. therefore, p. pneumotropica is most properly viewed as an opportunistic pathogen. studies of experimental p. pneumotropica suggest that it may complicate pneumonias due to mycoplasma pulmonis or sv. it has also been associated with suppurative or exudative lesions of the eye, conjunctiva, skin, mammary glands, and other tissues, especially in immunodeficient mice or in mice with a predisposing primary infection. epizootiology p. pneumotropica is a ubiquitous inhabitant of the skin, upper respiratory tract, and gastrointestinal tract of mice. litters from infected dams can become infected during the first week after birth. pathology infections can cause suppurative inflammation, which may include abcessation. dermatitis, conjunctivitis, dacryoadenitis, panophthalmitis, mastitis, and infections of the bulbourethral glands have been attributed to p. pneumotropica. preputial and orbital abscesses also occur, especially in athymic mice (fig. . ). its role in metritis is unclear, but it has been cultured from the uterus, and there is some evidence that it may cause abortion or infertility. cutaneous lesions can occur without systemic disease. they include suppurative lesions of the skin and subcutaneous tissues of the shoulders and trunk. diagnosis diagnosis requires isolation of the organism on standard bacteriological media. although infection can be detected serologically by elisa (wullenweber-schmidt et al., ; boot et al., a, b) , subclinical carriers often do not seroconvert. pcr assays also are available (dole et al., ) and have shown that p. pneumotropica did not transmit from infected mice to contact or dirty bedding sentinels (ouellet et al., ; dole et al., ) . differential diagnosis suppurative lesions in mice may be caused by other bacteria, including staphylococcus, streptococcus, corynebacterium, klebsiella, and mycoplasma. treatment antibiotic sensitivity testing in vitro indicated p. pneumotropica was significantly more sensitive than p. aeruginosa to enrofloxacin (sasaki et al., ) . enrofloxacin in the drinking water at mg/kg daily for days eliminated clinical signs and infection in a closed breeding colonic of transgenic mice and after days of treatment there were no detectable carriers when the colony was screened weeks later (matsumiya and lavoie, ) . prevention and control because p. pneumotropica is an opportunistic organism, it should be excluded from colonies containing immunodeficient mice and from breeding colonies. achieving this goal will normally require barrier housing supported by sound microbiological monitoring. rederivation should be considered to eliminate infection in circumstances where infection presents a potential threat to animal health or experimentation. research complications clinically severe infection in immunodeficient mice is the major complication. although clinically silent, experimental evidence has shown that p. pneumotropica infection in immunocompetent mice (c bl/ ) stimulated transcription of multiple proinflammatory cytokines for at least days with residual elevation detectable days later (patten et al., ) . pioneering studies conducted in the s first linked a novel microaerobic bacterium, helicobacter hepaticus, with chronic active hepatitis and hepatic tumors in a/jcr mice (fox et al., , ward et al., ) . the organism could be visualized by electron microscopy in the bile canaliculi of the liver in susceptible mouse strains (fig. . ). subsequently, it was associated with inflammatory bowel disease in several murine models (table . ) which were further developed to examine the role of immune cell subsets, such as t regulatory cells, in the pathogenesis of inflammatory bowel disease (ibd) and colon cancer (fig. . ). helicobacteriosis is laboratory animal medicine now appreciated to be a common infection of laboratory mice. it is caused by a growing list of helicobacter spp. that vary in clinical, pathologic, and epidemiologic significance (whary and fox, ; fox et al., ) . because recognition and investigation of helicobacteriosis continues to evolve, many important questions about the impact of this infection on mice remain unresolved. h. hepaticus infection is emphasized here, because it is among the most prevalent causes of helicobacteriosis and has been studied more extensively than other murine enterohepatic helicobacter spp. (ehs) (fox et al., , ward et al., ; suerbaum et al., ) . however, current information about other murine helicobacters is summarized in the concluding section. etiology helicobacter spp. are gram-negative, microaerophilic, curved to spiral-shaped organisms that have been isolated from the gastrointestinal mucosa of many mammals, including humans and mice whary and fox, ) . to date, the genus includes formally named helicobacter spp. assigned on the basis of s rrna analysis, complemented by biochemical, molecular, and morphological characteristics. the organisms can be grown on freshly prepared antibiotic impregnated blood agar or in broth supplemented with fetal bovine serum in a microaerobic atmosphere ( % co , % n , % h ). there are currently formally named helicobacter species have been isolated from laboratory mice, as well as several other novel helicobacter spp. awaiting formal naming. species isolated from mice include h. hepaticus, h. bilis (which also infects rats), h. muridarum, h. rappini, and h. rodentium, h. ganmani, h. mastomyrinus, h. magdeburgensis, and h. typhlonius , each of which cahill et al. ( ) tcrα, β mutants defective t-receptors typhlocolitis chin et al. ( ) scid icr-defined flora b t-and b-cell deficient typhlocolitis shomer et al. ( ), shomer et al. ( c bl/il- −/−c lacks il- typhlocolitis burich et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) , kullberg et al. ( ) c blrag mice infected with h. bilis also developed ibd (shomer et al., ) . c ibd also developed in c bl/il- −/− mice experimentally infected with a novel urease-negative helicobacter spp. now named h. typhlonius (franklin et al., ) ; also ibd produced with h. trogontum (whary et al., ) and h. cinaedi (shen et al., ) . d h. bilis produces ibd (maggio-price et al., and colon cancer (maggio-price et al., ) . have been formally named (except for h. rappini) (fox and lee, ; franklin et al., ; whary and fox, ) . most recently, helicobacter pullorum, a human pathogen, has been isolated from commercial, barriermaintained mice (boutin et al., ) . these ehs are most commonly urease-, catalase-, and oxidase-positive. however, h. rodentium, h. typhlonicus, and another novel helicobacter sp. are urease-negative. clinical signs helicobacteriosis in adult immunocompetent mice is usually asymptomatic. liver enzymes are elevated in h. hepaticus-infected a/jcr mice (fox et al., a) . infection of immune-dysregulated mice with h. hepaticus can cause inflammatory bowel disease, which may present as rectal prolapse and/or diarrhea (miller et al., ) . epizootiology recent surveys and anecdotal evidence suggest that helicobacteriosis is widespread among conventional and barrier-maintained mouse colonies (shames et al., ; fox et al., b; taylor et al., ; lofgren et al., ) . furthermore, h. hepaticus (and probably other helicobacters) can persist in the gastrointestinal tract, particularly the cecum and colon, and is readily detected in feces. these results indicate that transmission occurs primarily by the fecal-oral route and imply that carrier mice can spread infection chronically in enzootically infected colonies. pathology helicobacter spp. colonize the crypts of the lower bowel, where, depending on host genotype, the organisms can be pathogenic or nonpathogenic. h. hepaticus and h. bilis, e.g., can cause inflammation in the gastrointestinal tract, which is expressed as ibd and colon cancer in immunodeficient mice or typhlitis in a/jcr mice infected with h. hepaticus (ward et al., ; knutson et al., ; shomer et al., ; erdman et al., b; nguyen et al., ) . thickening of the cecum and large bowel develops because of proliferative typhlitis, colitis, proctitis, and lower bowel carcinoma. these lesions can occur without coincident hepatitis. indeed, helicobacter spp. induced ibd and colon carcinoma are increasingly popular models to study pathogenesis of the disease in humans (table . ). helicobacter spp. also can cause liver disease. bacterial translocation is thought to occur and results in colonization of the liver and progressive hepatitis. it is characterized by angiocentric nonsuppurative hepatitis and hepatic necrosis (fig. . ) . inflammation originates in portal triads and spreads to adjacent hepatic parenchyma. hepatic necrosis also may occur adjacent to intralobular venules, which can contain microthrombi. additionally, phlebitis may affect central veins. this lesion has been linked to the presence of organisms in bile canaliculi by silver stains and electron microscopy. age-related hepatocytic proliferation can develop in infected livers, a response that is more pronounced in male mice than in female mice (fox et al., a) . this lesion may laboratory animal medicine increase susceptibility to hepatomas and hepatocellular carcinomas among aged male a/jcr and b c f mice from infected colonies. an increased incidence of hepatic hemangiosarcoma also has been noted in h. hepaticusinfected male b c f mice. in this context, a/jcr, c h/ hencr, and sjl/ncr mice are susceptible to hepatitis, whereas c bl/ mice are resistant (ward et al., ) . the finding of severe liver disease and tumor induction in b c f mice infected with h. hepaticus infers that genetic susceptibility to h. hepaticus-induced neoplasia has a dominant pattern of inheritance. studies with h. hepaticus in recombinant inbred mice also indicate that disease susceptibility has multigenetic properties (hailey et al., ; fox and lee, ; ihrig et al., ; franklin, ; hillhouse et al., ) . diagnosis rapid generic diagnosis can be accomplished by pcr detection of the highly conserved s rrna region of the helicobacter genome in feces or tissues, using suitable oligonucleotide primers (fox et al., a; shames et al., ; beckwith et al., ) . however, genus-specific pcr does not differentiate among different helicobacter spp. molecular speciation can be accomplished by s rrna sequencing, restriction fragment length polymorphism analysis of the pcr product or use of species-specific pcr assays. this procedure requires suitable skill and experience to avoid technological pitfalls and should be performed by qualified laboratories. an igg elisa using the outer membrane protein as the antigen has been proposed for serological diagnosis, but shared antigens among ehs create lack of specificity for the assay. as noted above, helicobacters can be isolated on antibiotic-impregnated blood agar under microaerobic conditions and can then be speciated biochemically, and by helicobacter species-specific pcr. isolation of h. hepaticus and from other helicobacter spp. with spiral to curved morphology from feces should be preceded by passing slurried samples through a . -μm filter before plating. if infection with larger fusiform helicobacters (h. bilis, h. rappini) is suspected, filtration at . μm is preferred. helicobacters grow slowly and require prolonged incubation of cultures (up to weeks) before they can be deemed negative. signs (rectal prolapse) and lesions (hepatitis, typhlocolitis), depending on host genotype, can be suggestive of infection. histopathological examination should include silver stains, especially of liver, to attempt to visualize spiral or curved organisms (whary and fox, ) . differential diagnosis clinically apparent helicobacteriosis must be differentiated from other gastrointestinal or hepatic infections of mice. coronavirus infection, clostridium piliforme, and salmonella spp. can cause enterocolitis and/or hepatitis. c. rodentium also causes colonic hyperplasia, which can present as rectal prolapse. infections caused by other helicobacters of mice h. bilis has been isolated from the livers and intestines of aged mice and experimentally induces ibd in scid mice as does h. hepaticus. h. bilis also experimentally produces lower bowel cancer in immunocompromised mice (nguyen et al., ) . helicobacter muridarum colonizes the ileum, cecum, and colon. it appears to be nonpathogenic, although it can colonize the stomach of mice and induce gastritis under certain circumstances. h. 'rappini' has been isolated from the feces of mice without clinical signs. h. rodentium also colonizes the intestine and may be a component of normal flora. a dual infection of h. bilis and h. rodentium was noted in a natural outbreak of ibd in immunocompromised mice (shomer et al., ) . a novel urease negative helicobacter, which has been named h. typhlonius, causes ibd in il- −/− and scid mice (franklin et al., (franklin et al., , fox et al., ) . decreased reproductive efficiency has been reported in il knockout mice infected with h. rodentium and/or h. typhlonius (sharp et al., ) . prevention and control eradication of infection from small numbers of mice, such as quarantine groups, can be achieved by standard rederivation or intensive antibiotic therapy. the best results have been obtained by triple therapy with amoxicillin, metronidazole, and bismuth given for weeks (del carmen martino-cardona et al., ) . this strategy requires repeated daily gavage rather than administration in drinking water, but it has successfully eliminated h. hepaticus from naturally infected mice. antibiotic impregnated wafers have been used to eradicate helicobacter spp. in mouse colonies (kerton and warden, ) . wide-scale, eradication of enzootic helicobacteriosis can be expensive and time-consuming, without guarantee of success. careful husbandry procedures can limit infection within a colony (whary et al., ) . therefore, strategies have to be weighed carefully against risks of enzootic infection for the health and use of mice. in contrast, infection should be avoided in immunodeficient mice, including genetically engineered mice with targeted or serendipitous immune dysfunction. lastly, the outcome of opportunistic helicobacteriosis has not been thoroughly examined. this condition could occur during simultaneous infection with two or more helicobacter species or during combined infection with an intestinal virus (e.g., coronavirus) and helicobacter spp. if highly valuable animals are exposed, antibiotic therapy or rederivation may be warranted. research complications chronic inflammation of the liver and or gastrointestinal tract may be injurious to health. additionally, it may impede the development and assessment of noninfectious disease models, such as ibd models in mice with targeted deletions in t-lymphocyte receptors (fox et al., ) . h. hepaticus infections provoke a strong th proinflammatory response, which may perturb other immunological responses. h. hepaticus infection also has been incriminated as a cofactor or promoter in the development of hepatic neoplasia in a/ jcr, b c f , ab f , b af , and carko mice (hailey et al., ; fox et al., a; garcia et al., garcia et al., , h. salmonellosis (ganaway, ; lindsey et al., etiology the genus salmonella contains two species, s. bongori which infects mainly poikilotherms and rarely, humans, and s. enterica which includes approximately serovars and are a major cause of food-borne illness in humans (fookes et al., ) . the salmonella of historical importance in mice that are now rare include s. enterica subsp. enterica serovar typhimurium (aka s. typhimurium) and serovar enteritidis (s. enteritidis). s. enteritidis is a motile, gram-negative rod that rarely ferments lactose. the genomes of many strains have been sequenced. virulence factors carried on pathogenicity islands and plasmids include antimicrobial resistance genes, type iii secretion systems, vi antigen, lipopolysaccharide and other surface polysaccharides, flagella, and factors essential for a intracellular life cycle in macrophages (de jong et al., ) . pathogenassociated molecular patterns (pamps) unique to salmonella interact with tlrs and nod-like receptors (nlrs) which recruit neutrophils and macrophages leading to inflammasome formation and release of pro-inflammatory il- , il- β, tnf-α, and ifn-γ. clinical signs acute infection is especially severe in young mice (casebolt and schoeb, ) . it is characterized by anorexia, weight loss, lethargy, dull coat, humped posture, and occasionally conjunctivitis. gastroenteritis is a common sign, but feces may remain formed. subacute infection can produce distended abdomens from hepatomegaly and splenomegaly. chronic disease is expressed as anorexia and weight loss. enzootic salmonellosis in a breeding colony can produce episodic disease with alternating periods of quiescence and high mortality. the latter can be associated with diarrhea, anorexia, weight loss, roughened hair coat, and reduced production. epizootiology s. typhimurium is commonly used experimentally and cross-contamination in a mouse facility is a risk. modern production and husbandry methods have reduced the importance of salmonellosis as a natural infection of mice. however, the organisms are widespread in nature. therefore, cross-infection from other species or from feral mice remains a potential hazard. salmonellas are primarily intestinal microorganisms that can contaminate food and water supplies. infection occurs primarily by ingestion. salmonella have a broad host range and vermin, birds, feral rodents, and human carriers are potential sources of infection. other common laboratory species such as nonhuman primates, dogs, and cats also can serve as carriers. conversely, murine salmonellosis presents a zoonotic hazard to humans. the induction and course of infection are influenced by the virulence and dose of the organism, route of infection, host sex and genetic factors, nutrition, and intercurrent disease. suckling and weanling mice are more susceptible to disease than mature mice. immune deficiency, exposure to heavy metals, and environmental factors such as abnormal ambient temperatures can increase the severity of disease. nutritional iron deficiency has an attenuating effect on salmonella infection in mice, whereas iron overload appears to promote bacterial growth and enhance virulence. resistance to natural infection is increased by the presence of normal gastrointestinal microflora. resistance to infection also can be an inherited trait among inbred strains. among the most important considerations is that mice that recover from acute infection can become subclinical carriers and a chronic source of contamination from fecal shedding. pathology the virulence of s. enteritidis depends on its ability to penetrate intestinal walls, enter lymphatic tissue, multiply, and disseminate. organisms reach peyer's patches within h after inoculation and spread quickly to the mesenteric lymph nodes. bacteremia results in spread to other lymph nodes, spleen, and liver within several days. in chronic infections, organisms persist in the spleen and lymph nodes as well as in the liver and gallbladder and from the latter are discharged into the intestinal contents. bacteria reaching the intestine can reinvade the mucosa and can be shed intermittently in the feces for months. s. enteritidis infection also has been associated with chronic arthritis. acute deaths may occur without gross lesions, but visceral hyperemia, pale livers, and catarrhal enteritis are more common. if mice survive for up to several laboratory animal medicine weeks, the intestine may be distended and reddened, whereas the liver and spleen are enlarged and contain yellow-gray foci of necrosis. affected lymph nodes are also enlarged, red, and focally necrotic. focal inflammation can develop in many organs, including the myocardium (percy and barthold, ) . histologic lesions reflect the course of disease and the number of bacteria in affected tissues. during acute infection, necrotic foci are found in the intestine, mesenteric lymph nodes, liver, and spleen. neutrophilic leukocytes and histiocytes accumulate in lymphoid tissues. thrombosis from septic venous embolism may occur, especially in the liver. granulomatous lesions are particularly characteristic of chronic salmonellosis, especially in the liver. diagnosis diagnosis is based on isolation of salmonellas together with documentation of compatible clinical signs and lesions. in mice with systemic disease, bacteria may persist in the liver and spleen for weeks. during acute stages, bacteria can also be isolated from the blood. subclinically infected animals can be detected by fecal culture using selective enrichment media (selenite f broth plus cystine followed by streaking on brilliant green agar). culture of the mesenteric lymph nodes may be more reliable, because fecal shedding can be intermittent. isolates can be speciated with commercial serotyping reagents. alternatively, isolates can be sent to a reference laboratory for confirmation. antibodies to salmonellas can be detected in the serum of infected mice by an agglutination test. however, this method is not entirely reliable, because serological crossreactivity is common even among bacteria of different genera. pcr-based assays are also available. differential diagnosis salmonellosis must be differentiated from other bacterial diseases, including tyzzer's disease, helicobacter spp., pseudomoniasis, corynebacteriosis, c. rodentium, and pasteurellosis. viral infections that cause enteritis or hepatitis must also be considered, especially infections caused by coronavirus, ectromelia virus, and reoviruses. among noninfectious conditions, mesenteric lymphadenopathy is an agingassociated lesion in mice and is not indicative of chronic salmonellosis. prevention and control salmonellosis can be prevented by proper husbandry and sanitation. contact between mice and potential carriers, such as nonhuman primates, dogs, and cats, should be prevented. diets should be cultured periodically to check for inadvertent contamination. contaminated colonies should be replaced to eliminate infection and its zoonotic potential. research complications apart from the clinical manifestations, the zoonotic potential for salmonellosis is a major concern. this includes transmission among laboratory species, but especially between mice and the personnel working with them. i. streptobacillosis (lindsey et al., e; percy and barthold, ) etiology streptobacillus moniliformis is a nonmotile, gram-negative, pleomorphic rod that can exist as a nonpathogenic l-phase variant in vivo. however, it can revert to the virulent bacillus form. clinical signs streptobacillosis generally has an acute phase with high mortality, followed by a subacute phase and finally a chronic phase that may persist for months. signs of acute disease include a dull, damp hair coat and keratoconjunctivitis. variable signs include anemia, diarrhea, hemoglobinuria, cyanosis, and emaciation. cutaneous ulceration, arthritis, and gangrenous amputation may occur during chronic infection. the arthritis can leave joints deformed and ankylosed. hindlimb paralysis with urinary bladder distention, incontinence, kyphosis, and priapism may occur if vertebral lesions impinge on motor nerves. breeding mice may have stillbirths or abortions. epizootiology streptobacillosis has historical importance as a disease of rats and mice, but modern husbandry, production, and health surveillance strategies have reduced its impact dramatically (wullenweber, ) . subclinical, persistently infected rats are the most likely source of dissemination to mice, but mouse-tomouse transmission then ensues. transmission may occur from aerogenic exposure, bite wounds, or contaminated equipment, feed, or bedding. s. moniliformis is also pathogenic for humans, causing rat bite fever (haverhill fever). pathology during acute disease, necrotic lesions develop in thoracic and abdominal viscera, especially in the liver, spleen, and lymph nodes. histological lesions include necrosis, septic thrombosis of small vessels, acute inflammation, fibrin deposition, and abscesses. chronically infected mice may develop purulent polyarthritis because of the organism's affinity for joints. diagnosis diagnosis depends on clinical and pathological evidence of septicemia and isolation of the organism on blood agar. the organism has been recovered from joint fluid as long as months after infection. isolation from chronic lesions requires serumenriched medium. s. moniliformis as a cause of septic joints in humans has been diagnosed using pcr and electrospray-ionization followed by mass spectrometry (mackey et al., ) . differential diagnosis clinical signs must be differentiated from septicemic conditions, including mousepox, tyzzer's disease, corynebacteriosis, salmonellosis, mycoplasmosis, pseudomoniasis, and traumatic lesions. prevention and control control is based on exclusion of wild rodents or carrier animals such as latently infected laboratory rats. bacterins and antibiotic therapy are not adequately effective. the potential laboratory animal medicine for cross-infection is a reason not to house rats and mice in the same room. research complications infection can be disabling or lethal in mice and has zoonotic potential for humans. j. corynebacteriosis (lindsey et al., ; weisbroth, ; percy and barthold, ) etiology corynebacteria are short gram-positive rods. corynebacterium kutscheri is the cause of pseudotuberculosis in mice and rats. corynebacterium bovis has been associated with hyperkeratosis, especially in immunodeficient mice (clifford et al., ; scanziani et al., ; dole et al., ) . clinical signs c. kutscheri infection is often subclinical in otherwise healthy mice. active disease is precipitated by immunosuppression or environmental stresses and is expressed as an acute illness with high mortality or a chronic syndrome with low mortality. clinical signs include inappetence, emaciation, rough hair coat, hunched posture, hyperpnea, nasal and ocular discharge, cutaneous ulceration, and arthritis. c. bovis infection causes hyperkeratotic dermatitis characterized by scaly skin, which is accompanied by alopecia in haired mice. severe infection may cause death. corynebacterial keratoconjunctivitis has been reported in aged c bl/ mice (mcwilliams et al., ) . epizootiology subclinically infected animals harbor c. kutscheri in the upper alimentary tract, colon, respiratory tract, regional lymph nodes, middle ear, and preputial gland. c. bovis colonizes skin and is shed in feces. therefore, transmission is by direct contact, fecaloral contact, and aerosol. resistance to infection appears to be under genetic control in some mouse strains. rats are susceptible to c. kutscheri, so cross-infection to mice may occur. pathology lesions caused by c. kutscheri develop from hematogenous spread to various internal organs and appear as gray-white nodules in the kidney, liver, lung, and other sites. cervical lymphadenopathy and arthritis of the carpometacarpal and tarsometatarsal joints also may occur. septic, necrotic lesions often contain caseous material or liquefied exudate. histologic lesions are characterized by coagulative or caseous necrosis bordered by intense neutrophilic infiltration. colonies of gram-positive organisms with 'chinese letter' configurations can usually be demonstrated using tissue gram stains of caseous lesions. mucopurulent arthritis of carpal, metacarpal, tarsal, and metatarsal joints are related to bacterial colonization of synovium accompanied by necrosis, cartilage erosion, ulceration, and eventually ankylosing pan arthritis. c. kutscheri is not a primary skin pathogen, but skin ulcers or fistulas follow bacterial embolization and infarction of dermal vessels. subcutaneous abscesses have also been reported. hyperkeratotic dermatitis caused by c. bovis is characterized grossly by skin scaliness and alopecia. microscopically, skin lesions consist of prominent acanthosis and moderate hyperkeratosis accompanied by mild nonsuppurative inflammation (fig. . ) . hyperkeratosis is typically more severe in glabrous athymic mice than in haired mice. organisms can be demonstrated in hyperkeratotic layers by gram stain. diagnosis c. kutscheri is usually diagnosed by culture and tissue gram stains on lesions from clinically apparent cases. agglutination serology is available, and immunofluorescence, immunodiffusion, and elisa tests have been reported (boot et al., a) . pcr of skin swabs or feces is a sensitive and specific method for the detection of c. bovis infection in mice (dole et al., ) . differential diagnosis the caseous nature of c. kutscheri-induced lesions helps separate them from necrotic changes or abscesses caused by other infectious agents of mice. thus, they can be differentiated from streptococcosis, mycoplasmosis, and other septicemic bacterial infections in which caseous necrosis does not occur. because mice can sustain natural infections with mycobacterium avium, histochemical techniques for acidfast bacilli and appropriate culture methods for mycobacteria should be considered if nodular inflammatory lesions of the lung are detected. diffuse scaling dermatitis in athymic nude mice is classic for c. bovis infection; however, in one case report staphylococcus xylosus was instead isolated in high numbers from the skin lesions (russo et al., ) . hyperkeratotic dermatitis caused by laboratory animal medicine c. bovis must be differentiated from scaly skin caused by low humidity in glabrous mice. prevention and control c. kutscheri infection occurs sporadically and infected colonies should be culled or rederived into an spf facility as treatment is not curative and control is difficult. c. bovis can be endemic in athymic nude mouse colonies. prevention and control are difficult because both immunocompetent and athymic mice as well as humans can carry c. bovis on the skin and in the upper respiratory system, respectively. c. bovis readily contaminates the environment as aerosolization within a class ii biosafety cabinet was shown to spread the bacterium during cage-change procedures (burr et al., ) . antibiotic treatment has been unrewarding (burr et al., ) research complications corynebacteriosis can cause morbidity and mortality, especially among immunodeficient mice. dermatologic disease in suckling mice can be fatal but is less severe and transient in weanling mice. etiology staphylococci are gram-positive organisms that commonly infect skin and mucous membranes of mice and other animals. the two most frequently encountered species are staphylococcus aureus, which can be highly pathogenic, and s. epidermidis, which is generally nonpathogenic. species subtypes are identified by phage typing and biochemistry profiles. pathogenic staphylococci are typically coagulase-positive, although s. xylosus has caused serious infections and is coagulasenegative (gozalo et al., ) . clinical signs staphylococcosis causes suppurative conjunctivitis, periorbital and retroorbital abscesses, preputial adenitis, and pyoderma in mice, particularly in immunocompromised strains such as nude mice. some evidence suggests that staphylococci can produce primary cutaneous infections, but they are more likely opportunistic organisms that induce lesions after contamination of skin wounds. eczematous dermatitis develops primarily on the face, ears, neck, shoulders, and forelegs and can progress to ulcerative dermatitis, abscessation (including botryomycotic granulomas), and cellulitis. because lesions are often pruritic, scratching causes additional trauma and autoinoculation. staphylococcal infection in the genital mucosa of males may produce preputial gland abscesses. these occur as firm, raised nodules in the inguinal region or at the base of the penis and may rupture to spread infection to surrounding tissues. male mice also may develop septic balanoposthitis secondary to penile self-mutilation. retrobulbar abscesses caused by s. aureus are frequently noted in athymic mice. sjl mice, which are nk cell deficient, are prone to necrotic dermatitis on the tail secondary to s. xylosus infection. epizootiology staphylococci are ubiquitous and can be carried on the skin and in the nasopharnyx and gastrointestinal tract. they also can be cultured from cages, room surfaces, and personnel. the prevalence of staphylococcal dermatitis appears to be influenced by host genotype, the overall health of the animal, and the degree of environmental contamination with staphylococcus spp. c bl/ , c h, dba, and balb/c mice are among the most susceptible strains. age may also influence susceptibility, with young mice being more susceptible than adults. immunodeficient mice (e.g., athymic mice) contaminated with staphylococci often develop abscesses or furunculosis (fig. . ). as noted above, behavioral dysfunction resulting in selfmutilation, including scratching and trichotillomania, is a likely predisposing factor. once virulent staphylococci contaminate the environment, colonization of the gastrointestinal tract can occur and produce a carrier state. phage typing can help to determine the source of infection. human phage types of staphylococci can infect mice, but the zoonotic importance of this connection is not clear. pathology gross lesions are typified by suppurative, ulcerative and necrotic dermatitis involving the head and neck but may extend to the shoulders and forelegs (percy and barthold, ) . superficial or deep abscesses may occur in conjunction with dermatitis or separately, as, e.g., in the external male genitalia. histologically, acute skin infections result in ulceration with neutrophils in the dermis and subcutis. chronic lesions contain lymphocytes, macrophages, and fibroblasts. deep infections appear as coalescing botryomycotic pyogranulomas with necrotic centers containing bacterial colonies. infected athymic mice may develop laboratory animal medicine furunculosis of the muzzle and face accompanied by regional lymphadenitis. diagnosis diagnosis is made by documenting gross and histological lesions, including gram staining of suspect tissues, complemented by isolation of grampositive, coagulase-positive (s. aureus), or coagulasenegative staphylococcus species. differential diagnosis staphylococcosis must be differentiated from other suppurative infections of mice, including pasteurellosis, streptococcosis, corynebacteriosis, and pseudomoniasis. ectoparasitism, fight wounds, and self-mutilation per se should also be considered. prevention, control, and treatment removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. in affected animals, nail trimming can reduce self-inflicted trauma. conditions that facilitate aggressive or self-mutilating behavior should be avoided. research complications staphylococcosis can cause illness and disfigurement in mice. immunodeficient mice are at increased risk. etiology streptococci are ubiquitous commensal gram-positive organisms and in some cases, primary pathogens. pathogenic streptococcal infections in laboratory mice are caused by β-hemolytic organisms in lancefield's group c, but epizootics caused by group a streptococci have occurred, and group g organisms have been isolated occasionally. group d has been reclassified as an enterococcus. alpha-hemolytic streptococci can cause systemic disease in scid mice, and group b streptococcus sp. infection has been reported to cause meningoencephalitis in athymic mice (schenkman et al., ) . additionally, streptococcus dysgalactiae subsp. equisimilis has lancefield group g or c antigens and was isolated from visceral abscesses of immunocompetent mice (greenstein et al., ) . clinical signs cutaneous infections can cause ulcerative dermatitis over the trunk, which may appear gangrenous, whereas systemic infections may be expressed as conjunctivitis, rough hair coat, hyperpnea, somnolescence, and emaciation. epizootiology mice can carry streptococci subclinically in their upper respiratory tracts. lethal epizootics can occur, but factors leading to clinical disease are unknown, although some infections may be secondary to wound contamination. pathology systemic lesions reflect hematogenous dissemination and include abscessation, endocarditis, splenomegaly, and lymphadenopathy (percy and barthold, ) . streptococcal cervical lymphadenitis can lead to fistulous drainage to the neck complicated by ulcerative dermatitis. infection with α-hemolytic streptococci can cause inflammatory lesions affecting kidney and heart. diagnosis diagnosis and differential diagnosis depend on isolation of organisms from infected tissues, combined with histopathologic confirmation. differential diagnosis streptococcosis must be differentiated from other suppurative infections of mice, including staphylococcosis, pasteurellosis, corynebacteriosis, and pseudomoniasis. prevention and control removal of affected animals, sterilization of food and bedding, and frequent changing of bedding may limit or reduce transmission. research complications immunodeficient mice are at increased risk for streptococcosis. etiology e. coli is a small gram-negative rod that is a normal inhabitant of the mouse intestine. epizootiology infection is considered nonpathogenic in immunocompetent mice. however, hyperplastic typhlocolitis resembling transmissible murine colonic hyperplasia has been reported in scid mice infected with a non-lactose-fermenting e. coli (waggie et al., ; arthur et al., ) . clinical signs affected mice develop lethargy and fecal staining. pathology gross lesions consist of segmental thickening of the colon or cecum, which may contain blood-tinged feces. microscopically, affected mucosa is hyperplastic and may be inflamed and eroded. diagnosis diagnosis depends on demonstrating lesions and isolating non-lactose-fermenting e. coli. differential diagnosis this condition must be differentiated from proliferative and inflammatory intestinal disease caused by lawsonia intracellularis, c. rodentium, or enterotropic mouse hepatitis virus, especially in immunodeficient mice. colibacillosis provides an example of the morbidity associated with a nominally innocuous organism when it affects an immunocompromised host. prevention and control removal of affected animals and disinfection of caging and equipment will limit or reduce transmission. research complications clinical illness may develop in immunodeficient mice. historically, klebsiella pneumoniae is a ubiquitous gram-negative organism that is a natural inhabitant of the mouse alimentary tract. most commercial vendors have excluded it from their barriers. it can be pathogenic for the respiratory and urinary tract of mice after experimental inoculation but is not a significant cause of naturally occurring disease. etiology klebsiella oxytoca is an opportunistic pathogen implicated in various clinical diseases in animals and humans. epizootiology k. oxytoca also is purported to be an etiological agent of antibiotic-associated hemorrhagic colitis (aahc) in adult humans and adolescents. in animals, k. oxytoca has been isolated from apparently healthy sentinel rodents being monitored for pathogens in health surveillance programs and from utero-ovarian infections including suppurative endometritis, salpingitis, perioophoritis, and peritonitis in aged b c f mice (davis et al., ; rao et al., ) . a model of aahc has been developed in rats by administering amoxicillinclavulanate followed by orally infecting rats with a strain of k. oxytoca cultured from a patient with aahc. studies in humans suggest that k. oxytoca exerts its pathogenicity in part through a cytotoxin. recently, authors have showed that several animal isolates of k. oxytoca, including clinical isolates, produced secreted products in bacterial culture supernatant that display cytotoxicity on hep- and hela cells, indicating the ability to produce cytotoxin. using mass spectroscopy techniques, they also confirmed tilivalline as the cytotoxin present in animal k. oxytoca strains. tilivalline may serve as a biomarker for k. oxytoca-induced cytotoxicity (darby et al., ) . clinical signs k. oxytoca has been cultured from cases of suppurative otitis media, urogenital tract infections, and pneumonia in c h/hej and nmri-foxn (nu) mice (bleich et al., ) . additionally, k. oxytoca was recently cultured from three breeding colonies of nod. cg-prkdc scid il rg tm wjl /szj (nsg) mice with chronic renal inflammation and ascending urinary tract infections (foreman et al., ) . differential diagnosis other bacterial infections capable of causing suppurative lesions, including staphylococci, streptococci, pasteurella sp., and e. coli, among others are considered a differential diagnosis. research complications morbidity and mortality from spontaneous infections can affect ongoing research. etiology clostridium difficile was identified as the etiology of antimicrobial-associated pseudomembranous colitis in humans and currently a considerable cause of morbidity in hospitalized patients who acquire nosocomial infections. in the early s, an increased interest in c. difficile infection (cdi) resulted from the emergence of a hyper-virulent strain (nap /bi/ ) associated with frequent recurrences and more severe clinical disease (abou chakra et al., ; mcfarland, ; kuijper et al., ) . c. difficile has also been implicated in antibioticassociated colitis in syrian hamsters (bartlett et al., ) , guinea pigs (lowe et al., ) , rabbits (thilsted et al., ; ryden et al., ) , prairie dogs (muller et al., ) , ostriches (frazier et al., ) , and horses (diab et al., ) . c. difficile is a rod-shaped strict anaerobe. cycloserinecefoxitin-fructose agar (ccfa) is a commonly used selective medium for c. difficile. cultures are incubated under anaerobic conditions at - °c. when grown on blood agar, c. difficile colonies are nonhemolytic and gray, and have a slightly raised umbonate profile with filamentous edges and a ground-glass appearance. colonies grown on blood agar have fluorescence under ultraviolet light. c. difficile forms acid from glucose and fructose, but is negative on lactose, maltose, and sucrose. two closely related exotoxins, toxin a and toxin b, are produced by c. difficile. recent taxonomic classification support placement of c. difficile and its close relatives within the family peptostreptococcaceae. the authors suggested renaming it peptoclostridium difficile (yutin and galperin, ) . epizootiology it is estimated that c. difficile spores germinate and establish infection less than h after ingestion. spores rapidly transit through the upper gastrointestinal tract and colonize the colon and cecum. spore shedding begins less than h postingestion. when c bl mice were challenged with cfu of c. difficile spores, severe cdi signs developed and all mice were clinically affected by h postchallenge (chen et al., ) . specific methods to control and prevent c. difficile infections in mice have not been described. given the method of transmission of c. difficile and c. perfringens are via ingestion or spores, these clostridia can probably be excluded from mouse colonies by maintaining strict husbandry practices, robust sanitation, and use of autoclaved feed, bedding, cages, and cage accessories. sudden dietary changes should be avoided and antibiotics should be used judiciously to minimize disruption of the normal gut microbiota of mice. diagnosis of c. difficile-associated disease is generally based on detection of cytotoxin using a tissue culture cytotoxicity assay. pcr assays for detection of both c. difficile and its cytotoxins have been developed (eastwood et al., ) . there are no published regimens specifically for the treatment of natural c. difficile infections in mice. oral doses given twice daily of mg vancomycin for days to experimentally infected gnotobiotic mice caused a -to -log decrease in vegetative bacterial cell count and no detectable cytotoxin. bacterial counts and cytotoxin levels returned to previous levels after treatment was discontinued. clinical signs untreated mice are relatively resistant to infection with c. difficile and do not develop fatal infections, although these mice can become asymptomatic carriers that persistently shed low numbers of spores (lawley et al., ) . susceptibility of mice to infection must be induced by disrupting the microbiota through antibiotic treatment. brief exposure to environmental laboratory animal medicine spore contamination is sufficient for transmission of c. difficile to naïve but susceptible mice. the cdi transmission model has been used to demonstrate that clindamycin treatment of asymptomatic carriers of c. difficile can inadvertently trigger the excretion of high levels of spores (lawley et al., ) . a c bl mouse model of recurrence/relapse cdi has been reported (sun et al., ) . the primary bout of cdi induced little or no protective antibody response against c. difficile toxins and mice continued shedding c. difficile spores. antibiotic treatment of surviving mice induced a second episode of diarrhea. a simultaneous reexposure of mice to c. difficile bacteria or spores elicited a full clinical spectrum of cdi similar to that of the primary infection. immunosuppressive agents resulted in more severe and fulminant recurrent disease. vancomycin treatment only delayed disease recurrence; however, neutralizing polysera against both tcda and tcdb completely protected mice against cdi relapse (sun et al., ) . a recent study in c bl mice demonstrated that antibiotic-mediated alteration of the gut microbiome favors a global metabolic profile, and therefore increases susceptibility to c. difficile clinical diseases (theriot et al., ) . c. difficile is not tissue invasive and only toxigenic strains are associated with disease. experimental c. difficile infections include diarrhea, cecitis, polymorphonuclear cell infiltration of the lamina propria, inflammation, pseudomembrane formation, and death. differential diagnosis c. difficile-induced diarrhea is most often associated with antibiotic treatment. other clostridial diseases in mice must be ruled out as well as other enteric pathogens in mice causing diarrhea and mortality. salmonella spp. and c. rodentium should be considered in the differential diagnosis. etiology clostridium perfringens is associated with a number of diseases in domestic animals and humans. c. perfringens is a nonmotile, rod-shaped, encapsulated, anaerobic bacterium measuring - µm in length and . - . µm in diameter (murray et al., ) . c. perfringens grow rapidly on blood agar, and colonies are smooth, round, and grayish in color, and are surrounded by a double zone of hemolysis. c. perfringens is grouped into five types based on the production and secretion of four major toxins. c. perfringens produces a number of other virulence-enhancing toxins and hydrolytic enzymes. the most significant of these is probably enterotoxin, released with the bacterial spore after cell lysis. epizootiology c. perfringens is most likely acquired by the ingestion of spores that originated in the soil or in the intestinal tract of a carrier animal. the organism can be a member of the normal microbiota in human and domestic animals. factors that have been associated with the proliferation of the organism of these species include poor husbandry and sudden dietary changes (quinn et al., ) . methods to control and prevent c. perfringens infections have not been evaluated in mice. because the bacterium is most likely acquired by the ingestion of spores, it can probably be excluded from mouse colonies by maintaining good sanitation and sterilizing feed, bedding, cages, and cage accessories. sudden dietary changes have also been associated with proliferation of the organism and should be avoided if possible (quinn et al., ) . clinical signs only a few reports in the literature exist describing clinical disease associated with c. perfringens infection in mice (matsushita and matsumoto, ; rozengurt and sanchez) . disease has been observed in mice of both sexes, from to days old, and in female mice of breeding age. clinical signs have included hunched posture, ruffled hair coat, enlarged painful abdomen, soft or impacted feces, hindquarter paralysis, and dyspnea. sudden death without premonitory signs has also been reported. the toxin types of c. perfringens isolated from these cases were reported to be non-type a (matsushita and matsumoto, ) , type b (rozengurt and sanchez, ) , and type d (clapp and graham, ) . mucosal necrosis in both the large and small intestine is a consistent finding on microscopic examination of tissues from mice with clinically apparent c. perfringens infections. differential diagnosis c. perfringens produces a number of major and minor toxins. different types of the bacterium produce different toxins which account for different disease outcomes. c. perfringens type a is a constituent of the normal microbiota of the intestine of humans and other animal species. bacterial culture should be obtained from live or recently dead animals, and placed in anaerobic transfer medium for transport to a microbiology laboratory and should be cultured soon after their arrival. a presumptive diagnosis for c. perfringens can be based on the presence of large grampositive rods in fecal smears or in histologic sections of intestines (quinn et al., ) . definitive diagnosis is based on toxin identification. mice treated with chlortetracycline hydrochloride in drinking water at a level of mg/l for weeks have eliminated c. perfringens-associated disease (matsushita and matsumoto, ) . penicillin g in the diet or changing the diet has also been reported to be effective in disease remission. c. perfringens treatments in domestic species include ampicillin, amoxicillin-clavulanate, tylosin, clindamycin, metronidazole, and bacitracin (marks, ; mcgorum et al., ) . commercially available bacterins for use in mice were not effective in controlling the disease (clapp and graham, ) . research complications clostridia are large, rodshaped, gram-positive anaerobic bacteria. naturally occurring clostridial infection in mice is rare. epizootics of c. perfringens type d infection with high mortality laboratory animal medicine have been reported in a barrier colony where heavy mortality occurred in -to -week-old suckling mice. clinical signs included scruffy hair coats, paralysis of the hindquarters, and diarrhea or fecal impaction. however, attempts to reproduce the disease experimentally with clostridia isolated from naturally infected animals were unsuccessful. c. perfringens also has been isolated from sporadic cases of necrotizing enteritis in recently weaned mice. clostridium piliforme -tyzzer's disease (fujiwara and ganaway, ; ganaway, ; ganaway et al., ; percy and barthold, ) etiology tyzzer's disease is named for ernest tyzzer, who first described it in a colony of japanese waltzing mice. the causative organism, c. piliforme (formerly bacillus piliformis), is a long, thin, gram-negative spore-forming bacterium that appears to require living cells for in vitro growth. it has not been grown successfully on cell-free media, but it can be propagated by inoculation of susceptible vertebrates, in select cell lines, the yolk sac of embryonated eggs, or hepatocyte cell cultures obtained from mice (ganaway et al., ; kawamura et al., ) . clinical signs clinical disease occurs as unexpected deaths that may be preceded by diarrhea and inactivity. although outbreaks can be explosive and mortality is usually high, morbidity varies. additionally, subclinical infections can occur, accompanied by the development of antibodies to c. piliforme. stresses, such as overcrowding, high temperature and humidity, moist food, and immunosuppression, and young age, may predispose mice to tyzzer's disease. susceptibility and resistance also are influenced by host genotype. it has been shown, e.g., that c bl/ mice are more resistant than dba/ mice to tyzzer's disease (waggie et al., ) . resistance to severe infection appears to be due, in part, to b-lymphocyte function. the role of t cells in resistance is not clear, because susceptibility among athymic mice appears to vary (livingston et al., ) . however, the involvement of t cells can be inferred by the fact that several interleukins modulate resistance and susceptibility. depletion of neutrophils or nk cells also increases susceptibility to infection. epizootiology current prevalence rates, reservoirs of infection, carrier states, and the mechanism of spread remain speculative. tyzzer's disease occurs in many species of laboratory animals and in domestic and free-living species. some strains appear capable of cross-infecting mice, rats, and hamsters, whereas others have a more restricted host range (franklin et al., ) . therefore, the risks for cross-infection depend on the strain causing a given outbreak. although the vegetative form of c. piliforme is unstable, spores can retain infectivity at room temperature for at least year and should be viewed as the primary means of spread. natural infection is probably due to ingestion of organisms, which are subsequently shed in feces. feces-contaminated food and soiled bedding are the most likely sources of environmental contamination. prenatal infection can be induced by intravenous inoculation of pregnant mice, but its importance in the natural transmission of infection has not been determined. pathology infection begins in the gastrointestinal tract, followed by bacteremic spread to the liver and, to a smaller extent, the heart. the lesions are characterized by necrosis in these tissues and in the mesenteric lymph nodes. grossly, segments of the ileum, cecum, and colon may be red and dilated, with watery, fetid contents, whereas the liver, mesenteric lymph nodes, and heart often contain gray-white foci. histologically, intestinal lesions include necrosis of mucosal epithelium, which may be accompanied by acute inflammation and hemorrhage. in the liver, foci of coagulation necrosis are generally distributed along branches of the portal vein, a finding compatible with embolic infection from the intestine. peracute lesions are largely free of inflammation, but neutrophils and lymphocytes may infiltrate less fulminant lesions. myocardial necrosis is sporadic in natural infection. diagnosis tyzzer's disease is diagnosed most directly by the demonstration of characteristic intracellular organisms in tissue sections of liver and intestine. bundles of long, slender rods occur in the cytoplasm of viable cells bordering necrotic foci, especially in the liver (fig. . ) and intestine. they are found more easily during early stages of infection. organisms in tissue sections do not stain well with hematoxylin-eosin stain. silver stains, giemsa stains, or periodic acid-schiff stains are usually required for visualization of the organism. pcr and serologic assays are readily available at diagnostic laboratories. older supplemental procedures included inoculation of cortisonized mice or embryonated eggs laboratory animal medicine with suspect material, followed by histological or immunocytochemical demonstration of organisms in tissues. differential diagnosis the histological detection of organisms is essential for differentiating tyzzer's disease from other infections that can produce similar signs and lesions, especially mousepox, coronaviral hepatitis, reoviral hepatitis, helicobacteriosis, and salmonellosis. it also is important not to misconstrue extracellular rods as c. piliforme. prevention and control barrier housing and husbandry that incorporate sanitation measures to avoid the introduction or buildup of spores in the environment are the bases for control or prevention of tyzzer's disease. if infection occurs, spore formation will make control or elimination by antibiotic therapy problematic. therefore, strict quarantine, followed by replacement of affected or exposed stock, must be considered. rederivation by embryo transfer or cesarean section should take the potential for prenatal transmission of infection into account in housing and testing offspring. thorough decontamination of the environment with an oxidizing disinfectant must be included in any control program. additionally, procurement of food and bedding from suppliers with thorough quality assurance and vermin control programs is essential for both prevention and control. husbandry supplies should be stored in vermin-proof quarters, and the option of heat sterilization of food and bedding should be considered. research complications research complications stem from clinical morbidity and mortality. mice with immune dysfunction are at increased risk. there is recent evidence that infection causes elevations in selected cytokines (van andel et al., ) . etiology two mycobacteria are known to be pathogenic for laboratory mice: mycobacterium avium-intracellulare and m. lepraemurium. both are acid-fast, obligate intracellular bacteria. epizootiology mycobacteria are widespread in water and soil. their presence in laboratory mice would indicate a significant break in husbandry practices. infection with m. avium-intracellulare should be considered extremely rare, with the only published report describing an episode in a breeding colony of c bl/ mice . the source of the outbreak was presumed to be drinking water. mycobacterium lepraemurium has been isolated from healthy laboratory mice and can persist as a latent infection, but its significance is primarily historical, as a model for human leprosy. it is highly unlikely to encounter this infection in a modern, well-managed mouse colony. clinical signs m. avium-intracellulare infection is typically subclinical but mice have developed granulomatous pneumonia . pathology lesions are classically a chronic granulomatous disease with granulomas, langhans giant cells, and concurrent presence of acid-fast bacteria in various organs including the lungs, liver, spleen and lymph nodes. m. lepraemurium may cause alopecia, thickening of skin, subcutaneous swellings, and ulceration of the skin. disease can lead to death or clinical recovery. gross lesions are characterized by nodules in subcutaneous tissues and in reticuloendothelial tissues and organs (lung, spleen, bone marrow, thymus, and lymph nodes). lesions can also occur in the lung, skeletal muscle, myocardium, kidneys, nerves, and adrenal glands. the histologic hallmark is perivascular granulomatosis with accumulation of large, foamy epitheloid macrophages (lepra cells) packed with acid-fast bacilli. diagnosis acid-fast bacilli in lesions are the hallmark of presumptive diagnosis of mycobacteriosis. definitive diagnosis results from positive culture which takes days to weeks to rule out or positive pcr assays which are more time-efficient but require associated expertise. differential diagnosis other bacterial species that cause granulomatous lesions in mice. research complications natural infection is very rare. etiology proteus mirabilis is a ubiquitous gram-negative organism that can remain latent in the respiratory and intestinal tracts of normal mice (percy and barthold, ) . epizootiology proteus mirabilis colonizes the intestinal tract of most humans and is commonly found in research mice unless specifically excluded. clinical signs clinical disease can occur following stress or induced immunosuppression. immunodeficient mice have a heightened susceptibility to pathogenic infection. pathology proteus has been associated with ulcerative lesions in the gastrointestinal tract of immunodeficient mice. infected animals lose weight, develop diarrhea, and die within several weeks. if septicemia develops, suppurative or necrotic lesions, including septic thrombi, may be found in many organs, but the kidney is commonly affected. proteus pyelonephritis is characterized by abscessation and scarring. ascending lesions may occur following urinary stasis, but hematogenous spread cannot be ruled out. proteus mirabilis and pseudomonas aeruginosa have been isolated concomitantly from cases of suppurative nephritis or pyelonephritis. infection in immunodeficient mice is typified by splenomegaly and focal necrotizing hepatitis. pulmonary lesions include edema and macrophage activation. septic thrombi can occur, however, in many tissues. diagnosis culture recovery of proteus mirabilis as a predominant or single isolate confirms an opportunistic local or systemic infection. differential diagnosis gram-negative bacterial infections. research complications natural infections are typically isolated cases. etiology leptospirosis remains one of the most common zoonoses transmissible from rodents (desvars et al., ) but is exceedingly rare in laboratory mice. infection with leptospira interrogans serovar ballum has been reported on several occasions (see chapter ). epizootiology leptospira are gram-negative organisms that, after a septicemic phase, establish persistent infection in the renal tubules and are periodically excreted in the urine. clinical signs natural infection is subclinical and causes no significant lesions. experimental infections can result in severe vascular, hepatic and renal lesions dependent on serovar, mouse strain and immunocompetency. diagnosis diagnosis requires isolation of organisms in kidney culture. serological testing should be used with caution because neonatal exposure can lead to persistent infection without seroconversion. histologic examination of kidney using silver stains can also be attempted. pcr assays are reliable for preliminary diagnosis. differential diagnosis not applicable in research colonies. research complications persistent murine infections associated with active shedding present a zoonotic hazard for humans; therefore, infected mice should be culled. elimination of infection from highly valuable mice requires rederivation. (percy and barthold, ) etiology chlamydia trachomatis is an intracellular organism that produces glycogen-positive intracytoplasmic inclusions (elementary bodies). c. trachomatis causes ocular and urogenital disease in humans. however, at least one strain historically referred to as the 'nigg agent' after clara nigg, is most recently classified as chlamydia muridarum and is used experimentally to model human chlamydia infection. epizootiology mice are susceptible to natural infection and experimental infection with c. trachomatis and chlamydophila psittaci, especially immunodeficient mouse strains. clinical signs natural infections are typically subclinical but persistent. pathology c. muridarum is also known as the 'mouse pneumonitis agent' due to severe acute infection which is characterized by ruffled fur, hunched posture, and labored respiration due to interstitial pneumonitis and death in h. mice with more chronic infections may develop progressive emaciation and cyanosis of the ears and tail. experimental infections to model human venereal chlamydia infections will develop hydrosalpinx, cervical, and vaginal infections in female mice and urethritis in male mice. diagnosis chlamydia can be diagnosed by impression smears stained with giemsa or macchiavello stains, cell culture, or inoculation of embryonated eggs. pcr and sequencing can be used to speciate the type of chlamydia. differential diagnosis c. muridarum, c. trachomatis, and c. psittaci are included in the differential diagnosis. research complications chlamydia is a rare spontaneous infection in research mice; its potential significance is low. etiology pneumocystis murina (pm) is a common opportunistic organism of laboratory mice and other mammals. when first described by chagas in , p. carinii was misidentified as trypanosoma cruzi and was considered a protozoan (chagas, ) . it was renamed as a new species, p. carinii, when observed in a rat in (delanoë, p. and delanoë, m. ) . p. carinii, however, has now been grouped taxonomically with the fungi based on dna analysis and the homology of p. murina housekeeping genes with those found in fungi (edman et al., ; stringer et al., ; wakefield et al., ) . these dna studies and apparent differences of host susceptibility prompted a new name, p. jiroveci, for pneumocystis isolated from humans (stringer et al., ; frenkel, ) . p. carinii is now used to name the organism in rats and p. murina, the organism in mice. clinical signs pm infection is subclinical in immunocompetent mice. however, it can be clinically severe in immunodeficient mice, because an adequate complement of functional t lymphocytes is required to suppress infection (roths et al., ; shultz and sidman, ; walzer et al., ; weir et al., ) . b cells have also been shown to be critical to clearance of infection and the mechanism appears only partially related to igg and has a more important role in promoting activation and expansion of t cells (lund et al., ) . b cells may also protect early hematopoietic progenitor activity during systemic responses to pneumocystis infection (hoyt et al., ) . infection proceeds slowly, but relentlessly in immunodeficient mice leading to clinical signs of pneumonia, usually within several months. primary signs include dyspnea and hunched posture, which may laboratory animal medicine be accompanied by wasting and scaly skin. severe cases, such as those that occur in advanced disease in scid mice, may be fatal. epizootiology pm is known to infect a number of mammalian hosts, including ferrets, rats, mice, and humans. pm is a ubiquitous organism that is often present as a latent infection. although firm prevalence data are not available, because detection methods are not simple to apply, infection is assumed to be present in mouse colonies unless ruled out by extensive surveillance. although these organisms appear morphologically similar, there are antigenic and genetic differences among p. murina isolated from different hosts (weinberg and durant, ; cushion, ) . furthermore, studies indicate that p. carinii isolated from one host species is unable to survive and replicate after inoculation into a different immunodeficient host species (gigliotti et al., b) . pm infection also occurs in human beings, but transmission between rodents and human beings has not been documented. pm is transmitted by aerosol and establishes persistent, quiescent infection in the lungs of immunocompetent mice. prenatal infection has not been demonstrated. pathology pm is normally not pathogenic but can be activated by intercurrent immunosuppression. activation fills the lung with trophic and cystic forms. gross lesions occur in the lungs, which are often rubbery and fail to deflate (fig. . ). histopathological changes are characterized by interstitial alveolitis with thickening of alveolar septa from proteinaceous exudate and infiltration with mononuclear cells (fig. . ) (roths et al., ) . alveolar spaces may contain vacuolated eosinophilic material and macrophages. special stains are required to visualize pm. silver-based stains reveal round or partially flattened -to -mm cysts in affected parenchyma (fig. . ) . in florid cases, alveolar spaces may be filled with cysts, but cysts may be sparse in mild cases. disease can be especially severe when subclinically infected immunodeficient mice are reconstituted with competent immune cells that subsequently promote pneumonitis. diagnosis respiratory distress in immunodeficient mice should elicit consideration of pneumocystosis. pathologic examination of the lung, including silver laboratory animal medicine methenamine staining, is essential to confirm a presumptive clinical diagnosis. past infections of immunocompetent mice also can be detected by elisa (furuta et al., ) . pcr can be used to detect active infection (gigliotti et al., a; reddy et al., ) and is particularly useful for screening immunodeficient mice. differential diagnosis pneumocystosis must be differentiated from viral pneumonias of mice. it is worth noting, in this regard, that pneumonia virus of mice has been shown to accelerate the development of pneumocystosis in scid mice (bray et al., ; roths et al., ) . prevention and control pm infection is a significant disease threat to immunodeficient mice. its widespread distribution strongly suggests that susceptible mice should be protected by microbarrier combined, where possible, with macrobarrier housing. husbandry procedures should include proper sterilization of food, water, and housing equipment and the use of hepa-filtered change stations. infected colonies can be rederived by embryo transfer or cesarean methods, because infection does not appear to be transmitted in utero. research complications pneumonia in immunodeficient mice is the major complication of pm infection. trichophyton mentagrophytes is the most common fungal agent of mice. however, infection rarely causes clinical disease. clinical signs include sparse hair coats or well-demarcated crusty lesions, with a chalky surface on the head, tail, and legs (favus or ringworm). skin lesions are composed of exfoliated debris, exudate, mycelia, and arthrospores with underlying dermatitis. invasion of hair shafts is not characteristic. diagnosis depends on effective specimen collection. hairs should be selected from the periphery of the lesion, and hairless skin should be scraped deeply to obtain diagnostic specimens. t. mentagrophytes rarely fluoresces under ultraviolet light, and hyphae must be differentiated from bedding fibers, food particles, and epidermal debris. histological sections should be stained with a silver stain or schiff's reagent to reveal organisms. trichophyton also can be cultured on sabouraud agar. plates are incubated at room temperature ( - °c), and growth is observed at - days. ringworm is not easily eradicated from laboratory mice. the use of antifungal agents to treat individual mice is time-consuming, expensive, and variably effective. rederivation is a more prudent course. cages and equipment should be sterilized before reuse. concurrent infection with ectoparasites also must be considered during eradication steps. candida albicans and other systemic mycoses are not important causes of disease in mice, but they can be opportunistic pathogens in immunodeficient mice. etiology giardia muris is a pear-shaped, flagellated organism with an anterior sucking disk. it inhabits the duodenum of young and adult mice, rats, and hamsters. clinical signs infection is often subclinical, unless organisms proliferate extensively, and can cause weight loss, a rough hair coat, sluggish movement, and abdominal distension, usually without diarrhea. additionally, immunodeficient mice may die during heavy infestation. epizootiology the contemporary prevalence of affected mouse colonies is not well documented, but surveys during the s found the rates exceeding %. transmission occurs by the fecal-oral route. crossinfection between mice and hamsters after experimental inoculation of organisms has been demonstrated, whereas rats were resistant to isolates from mice and hamsters (kunstyr et al., ) . c h/he mice are particularly susceptible to giardiasis, whereas balb/c and c bl/ mice are more resistant. additionally, female mice appear to be more resistant to infection than male mice (daniels and belosevic, ) . c bl/ females, e.g., have lower trophozoite burdens and for a shorter interval than male mice. females also shed cysts later than male mice. these differences may be related to a more potent humoral immune response to giardia in female mice. pathology gross lesions are limited to the small intestine, which may contain yellow or white watery fluid. histopathology reveals organisms in the lumen that often adhere to microvilli of enterocytes or reside in mucosal crevices or mucus. the crypt/villus ratio may be reduced, and the lamina propria may have elevated numbers of inflammatory cells. diagnosis diagnosis is based on detection of trophozoites in the small intestine or in wet mounts of fecal material. organisms can be recognized in wet preparations by their characteristic rolling and tumbling movements. ellipsoidal cysts with four nuclei also may be detected in feces. infection also can be detected by serology (daniels and belosevic, ) and by pcr (mahbubani et al., ) . treatment, prevention, and control murine giardiasis can be treated by the addition of . % dimetridazole to drinking water for days. prevention and control depend on proper sanitation and management, including adequate disinfection of contaminated rooms. research complications accelerated cryptal cell turnover and suppression of the immune response to sheep erythrocytes have been observed in infected mice. the potential for severe or lethal infection in immunodeficient mice was noted previously. etiology spironucleus muris is an elongated, pearshaped, bilaterally symmetrical flagellated protozoan that commonly inhabits the duodenum, usually in the crypts of lieberkühn. it is smaller than giardia muris and lacks an anterior sucking disk. clinical signs s. muris infection is usually subclinical in normal adult mice. it is more pathogenic, however, for young, stressed, or immunocompromised mice (kunstyr et al., ) . additionally, clinical morbidity may indicate an underlying primary infection with an unrelated organism. clinically affected mice can have a poor hair coat, sluggish behavior, and weight loss. mice at - weeks of age are at notably higher risk for clinically evident infection. they can develop dehydration, hunched posture, abdominal distension, and diarrhea. severe infections can be lethal. epizootiology transmission occurs by the fecaloral route and can occur between hamsters and mice as well as between mice. it does not appear to be transmitted between mice and rats (schagemann et al., ) . the most recent surveys, which are somewhat dated, indicated that prevalence rates exceeded % among domestic mouse colonies in the mid- s. there is some evidence that inbred strains vary in their susceptibility to infection and their rate of recovery (baker et al., ; brett and cox, ) . pathology gross findings associated with infection include watery, red-brown, gaseous intestinal contents. however, it is essential to rule out primary or coinfection by other organisms before attributing these lesions to spironucleosis. microscopically, acute disease is associated with distension of crypts and intervillous spaces by pear-shaped trophozoites and inflammatory edema of the lamina propria. organisms can be visualized more easily with periodic acid-schiff staining, which may reveal invasion of organisms between enterocytes and in the lamina propria. chronic infection is associated with lymphoplasmacytic infiltration of the lamina propria and occasional intracryptal inflammatory exudate. diagnosis diagnosis is based on identification of trophozoites in the intestinal tract. they can be distinguished from giardia muris and tritrichomonas muris by their small size, horizontal or zigzag movements, and the absence of a sucking disk or undulating membrane. pcr-based detection also is available (rozario et al., ) . it is not clear whether duodenitis is a primary pathogenic effect of s. muris or represents opportunism secondary to a primary bacterial or viral enteritis. therefore, it is prudent to search for underlying or predisposing infections. treatment, prevention, and control treatment consists of adding . % dimetridazole to drinking water for days, as described for giardiasis. prevention and control require good husbandry and sanitation. research complications as with giardiasis, infection can accelerate enterocytic turnover in the small intestine. there is some evidence that infected mice may have activated macrophages that kill tumor cells nonspecifically and that infection can diminish responses to soluble and particulate antigens. additionally, infected mice also have increased sensitivity to irradiation. such effects should, however, be interpreted cautiously in order to rule out intercurrent viral infections. tritrichomoniasis t. muris is a nonpathogenic protozoan that occurs in the cecum, colon, and small intestine of mice, rats, and hamsters. no cysts are formed, and transmission is by ingestion of trophozoites passed in the feces. it can be detected by microscopy or by pcr (viscogliosi et al., ) . coccidiosis eimeria falciformis is a pathogenic coccidian that occurs in epithelial cells of the large intestines of mice. it was common in european mice historically but is seldom observed in the united states. heavy infection may cause diarrhea and catarrhal enteritis. klosiella muris causes renal coccidioisis in wild mice but is rare in laboratory mice. mice are infected by ingestion of sporulated sporocysts. sporozoites released from the sporocysts enter the bloodstream and infect endothelial cells lining renal arterioles and glomerular capillaries, where schizogony occurs. mature schizonts rupture into bowman's capsule to release merozoites into the lumen of renal tubules. merozoites can enter epithelial cells lining convoluted tubules, where the sexual phase of the life cycle is completed. sporocysts form in renal tubular epithelium and eventually rupture host cells and are excreted in the urine, but oocysts are not formed. infection is usually nonpathogenic and subclinical. gray spots may occur in heavily affected kidneys and are the result of necrosis, granulomatous inflammation, and focal hyperplasia. destruction of tubular epithelium may impair renal physiology. diagnosis is based on detection of organisms in tissues. prevention and control require proper sanitation and management techniques. there is no effective treatment. cryptosporidiosis cryptosporidium muris is a sporozoan that adheres to the gastric mucosa. it is uncommon in laboratory mice and is only slightly pathogenic. cryptosporidium parvum inhabits the small intestine and is usually nonpathogenic in immunocompetent and athymic mice (ozkul and aydin, ; taylor et al., ) . athymic mice may develop cholangitis and hepatitis, however, if organisms gain access to the biliary tract. entamoebiasis entamoeba muris is found in the cecum and colon of mice, rats, and hamsters throughout the world. organisms live in the lumen, where they feed on particles of food and bacteria. they are considered nonpathogenic. encephalitozoonosis encephalitozoon cuniculi is a gram-positive microsporidian that infects rabbits, mice, rats, guinea pigs, dogs, nonhuman primates, humans, and other mammals. infection is extremely rare among laboratory mice. the life cycle of the organism is direct, and animals are infected by ingesting spores or by cannibalism. spore cells are disseminated in the blood to the brain and other sites. infection can last more than year, and spores shed in the urine serve as a source of infection. vertical transmission has not been confirmed in mice. e. cuniculi is an obligate intracellular parasite, but infection usually elicits no clinical signs of disease. organisms proliferate in peritoneal macrophages by asexual binary fission. they have a capsule that accepts giemsa and goodpasture stains but is poorly stained by hematoxylin. fulminating infection can cause lymphocytic meningoencephalitis and focal granulomatous hepatitis. in contrast to encephalitozoonosis in rabbits, affected mice do not develop interstitial nephritis. infection is diagnosed by cytological examination of ascitic fluid smears, histopathologic examination of brain tissues stained with goodpasture stain, and elisa serology. no effective treatment has been reported. prevention and control require rigid testing and elimination of infected colonies and cell lines. pcr-based assays may also be useful. toxoplasmosis toxoplasma gondii is a ubiquitous gram-negative coccidian parasite for which the mouse serves as a principal intermediate host. however, the prevalence of natural infection is negligible because laboratory mice no longer have access to sporulated cysts shed by infected cats, which were historically the major source for cross-infection. toxoplasmosis can cause necrosis and granulomatous inflammation in the intestine, mesenteric lymph nodes, eyes, heart, adrenals, spleen, brain, lung, liver, placenta, and muscles. diagnosis is based on elisa serology, histopathology, and pcr. control and prevention depend largely on precluding access of mice to cat feces or to materials contaminated with cat feces. oocytes are very resistant to adverse temperatures, drying, and chemical disinfectants; therefore, thorough cleaning of infected environments is required. b. cestodiasis baker, ) etiology hymenolepis (rodentolepis) nana, the dwarf tapeworm, infects mice, rats, and humans although the zoonotic risk has been questioned (macnish et al., ) . adults are extremely small ( - mm) and have eggs with prominent polar filaments and rostellar hooks (fig. . ) . clinical signs young adult mice are most frequently infected. signs and lesions include weight loss and focal enteritis, but clinical disease is rare unless infestation is severe. epizootiology the life cycle may be direct or indirect (r. nana is the only cestode known that does not require an intermediate host). the indirect cycle utilizes arthropods as intermediate hosts. liberated oncospheres penetrate intestinal villi and develop into a cercocystis stage before reemerging into the intestinal lumen - days later. the scolex attaches to the intestinal mucosa, where the worm grows to adult size in weeks. the cycle from ingestion to patency takes - days. pathology cysticerci are found in the lamina propria of the small intestine and sporadically in the mesenteric lymph nodes, whereas adults, which have a serrated profile, are found in the lumen. inflammation is not a feature of infection. diagnosis infection can be diagnosed by demonstrating eggs in fecal flotation preparations or by opening the intestine in petri dishes containing warm tap water to facilitate detection of adults. r. nana can be differentiated from another species of rodent tapeworm, h. diminuta, by the fact that r. nana has rostellar hooks and eggs with polar filaments. however, h. diminuta requires an intermediate arthropod host, so it is rarely found in contemporary mouse colonies. treatment, prevention, and control drugs recommended for treatment and elimination include praziquantel ( . % in the diet for days), albendazole, mebendazole, and thiabendazole. although the benzimidazoles have an excellent activity against cestodes and nematodes in rats, they have not been tested extensively in mice. the potential for successful treatment is high, however, because eggs do not survive well outside the host and because the prevalence of infestation is low in caged mice kept in properly sanitized facilities. because r. nana can directly infect humans, proper precautions should be taken to avoid oral contamination during handling of rodents (see chapter ). hymenolepis microstoma is found in the bile ducts of rodents and could be confused with r. nana in the mouse. however, the location of the adult as well as the large size of h. microstoma eggs compared with those of r. nana make differential diagnosis relatively simple. the mouse and the rat are intermediate hosts of the cestode taenia taeniaformis. the definitive host is the cat. this parasite should not be found in laboratory mice housed separately from cats. c. nematodiasis (wescott, ) syphacia obvelata (mouse pinworm) infestation etiology syphacia obvelata, the common mouse pinworm, is a ubiquitous parasite of wild and laboratory mice. the rat, gerbil, and hamster are also occasionally infected. female worms range from . to . mm in length, and male worms are smaller ( . - . mm). eggs are flattened on one side and have pointed ends (fig. . ). the nucleus fills the shell and is frequently at a larval stage when eggs are laid. clinical signs infestation is usually subclinical, although heavily infested mice can occasionally sustain intestinal lesions, including rectal prolapse, intussusception, enteritis, and fecal impaction. epizootiology pinworm infestation is one of the most commonly encountered problems in laboratory mice. a national survey revealed that more than % of barrier colonies and about % of conventional colonies were affected (jacoby and lindsey, ; carty, ) . syphacia obvelata infestation can occur unexpectedly in commercial barrier murine colonies, resulting in widespread dissemination of the parasite into academic mouse colonies. the epizootiological impact of pinworm infestation is increased by the airborne dissemination of eggs, which can remain infectious even after drying. the life-cycle is direct and completed in - days. females deposit their eggs on the skin and hairs of the perianal region. ingested eggs liberate larvae in the small intestine and they migrate to the cecum within h. worms remain in the cecum for - days, where they mature and mate. the females then migrate to the large intestine to deposit their eggs as they leave the host. there is unconfirmed speculation that larvae may reenter the rectum. infestation usually begins in young mice and can recur, but adult mice tend to be more resistant. syphacia infestation often occurs in combination with aspiculuris tetraptera. because the life cycle of syphacia is much shorter than that of aspiculuris, the number of mice that are apt to be infected with s. obvelata is correspondingly greater. there is evidence that resistance to infestation may be mouse strain-specific (derothe et al., ) . pathology gross lesions are not prevalent, aside from the presence of adults in the lumen of the intestine. diagnosis infestation is diagnosed by demonstrating reniform-shaped eggs in the perianal area or adult worms in the cecum or large intestine. four-to -weekold mice should be examined because the prevalence is higher in this age group than in older mice. because most eggs are deposited outside the gastrointestinal tract, fecal examination is not reliable. eggs are usually detected by pressing cellophane tape to the perineal area and then to a glass slide that is examined by microscopy. aspiculuris tetraptera eggs are not ordinarily found in tape preparations and are easily differentiated from eggs of s. obvelata (see below). adult worms can be found in cecal or colonic contents diluted in a petri dish of warm tap water. they are readily observed with the naked eye or with a dissecting microscope. an elisa also is available to detect serum antibodies to s. obvelata somatic antigens (sato et al., ) . pcr assays are increasingly being used to augment traditional diagnostic methods and to discriminate between pinworm species (dole et al., ) . pcr panels for pinworm detection using fecal pellets are available from commercial diagnostic laboratories. treatment, prevention, and control pinworm infestation can be treated effectively by a number of regimens, which include the use of anthelmintics such as piperazine, ivermectin, and benzimidazole compounds alone or in combination (klement et al., ; le blanc et al., ; lipman et al., ; flynn et al., ; wescott, ; zenner, ) . because some of the recommended therapies have the potential for toxicity, it is prudent to keep mice under close clinical observation during treatment (davis et al., ; skopets et al., ; toth et al., ) . fenbendazole diets can be fed with week on/ week off rotation with normal chow although the potential impact on experimental data must be considered (duan et al., ; gadad et al., ; landin et al., ) . prevention of reinfestation requires strict isolation because syphacia eggs become infective as soon as h after they are laid, and they survive for weeks, even in dry conditions. strict sanitation, sterilization of feed and bedding, and periodic anthelmintic treatment are required to control infestation. the use of microbarrier cages can reduce the spread of infective eggs. syphacia muris is the common rat pinworm. it can potentially infest mice but is not found in well-managed colonies. it can be differentiated from s. obvelata because s. muris eggs are smaller. treatment is the same as for pinworms of mice. etiology aspiculuris tetraptera is the other major oxyurid of the mouse and may coinfect mice carrying s. obvelata. females are . - . mm long, and males are slightly smaller. the eggs are ellipsoidal (fig. . ) . clinical signs ingested eggs hatch, and larvae reach the middle colon, where they enter crypts and remain for - days. they move to the proximal colon about weeks after infection of the host. because the life cycle is - days longer than in s. obvelata, infestations appear in somewhat older mice; heaviest infestation is expected in - weeks after initial exposure. infection is usually subclinical, but heavy loads can produce signs similar to those discussed for s. obvelata. light to moderate loads do not produce clinical disease. epizootiology as noted under s. obvelata, pinworm infestation is highly prevalent and contagious in laboratory mice. the life cycle is direct and takes approximately - days. mature females inhabit the large intestine, where they survive from to days and lay their eggs. the eggs are deposited at night and are excreted in a mucous layer, covering fecal pellets. they require - days at °c to become infective and can survive for weeks outside the host. pathology see s. obvelata (section iii, a, ,c). diagnosis aspiculuris tetraptera eggs can be detected in the feces, and adult worms are found in the large intestine. eggs are not deposited in the perianal area; therefore, cellophane tape techniques are not useful. measures for treatment, prevention, and control are similar to those described for s. obvelata. because a. tetraptera takes longer to mature and because eggs are deposited in feces rather than on the host, adult parasites are more amenable to treatment by frequent cage rotations. immune expulsion of parasites and resistance to reinfection are hallmarks of a. tetraptera infection. research complications see s. obvelata (section iii, a, ,c). several species of mites infest laboratory mice. they include myobia musculi, radfordia affinis, myocoptes musculinus, and, less commonly, psorergates simplex. the common murine mites are described below, while less frequently encountered species are listed in table . . these include the mouse mite trichoecius romboutsi, which resembles myocoptes and ornithonyssus bacoti, the tropical rat mite, which can infect laboratory mice. characteristics of specific infestations are described after a general introductory section. clinical signs mites generally favor the dorsal anterior regions of the body, particularly the top of (jacoby and lindsey, ; carty, ) reported mite infestations in % of colonies. acarids spend their entire lives on the host. populations are limited by factors such as self-grooming, mutual grooming, the presence of hair, and immunological responses, which tend to produce hypersensitivity dermatitis. inherited resistance and susceptibility also affect clinical expression of acariasis. mite populations, e.g., vary widely among different stocks and strains of mice housed under similar conditions. pathology gross lesions include scaly skin, regional hair loss, abrasions, and ulcerations. histologically, hyperkeratosis, acanthosis, and chronic dermatitis may occur. long-standing infestation provokes chronic inflammation, fibrosis, and proliferation of granulation tissue. ulcerative dermatitis associated with acariasis may have an allergic pathogenesis but often results in secondary bacterial infections. lesions resemble allergic acariasis in other species and are associated with mast cell accumulations in the dermis. diagnosis classic methods of detection include direct observation of the hair and skin of dead or anesthetized mice. hairs are parted with pins or sticks and examined with a dissecting microscope. examination of young mice, prior to the onset of immune-mediated equilibrium, is likely to be more productive. alternatively, recently euthanized mice can be placed on a black paper, and double-sided cellophane tape can be used to line the perimeter to contain the parasites. as the carcass cools, parasites will vacate the pelage and crawl onto the paper. sealed petri dishes can also be used. cellophane tape also can be pressed against areas of the pelt of freshly euthanatized mice and examined microscopically. skin scrapings made with a scalpel blade can be macerated in % koh/glycerin or immersion oil and examined microscopically. this method has the disadvantage of missing highly motile species and low-level populations of slower moving immature forms. it is important to remember that mite infestations may be mixed, so the identification of one species does not rule out the presence of others. detecting mites in sentinels exposed to dirty bedding from colony animals has been reported to be unreliable (lindstrom et al., ) . thus, pcr assays offered by commercial diagnostic laboratories are increasingly being used to augment traditional diagnostic methods and to test individual animals or equipment using a swabbing technique; samples can be pooled to decrease cost (jensen et al., ) . gross anatomical features facilitate differentiation of intact mites. myocoptes has an oval profile with heavily chitinized body, pigmented third and fourth legs, and tarsal suckers (fig. . ) . myobia and radfordia have a similar elongated profile, with bulges between the legs. myobia has a single tarsal claw on the second pair of legs (fig. . ) , whereas radfordia has two claws of unequal size on the terminal tarsal structure of its second pair of legs (fig. . ) . histopathological examination of skin is helpful for diagnosing unique forms of acariasis, such as the keratotic cysts associated with psorergates simplex infestation. treatment, prevention, and control ivermectin can be used topically, in drinking water or as a medicated feed and often is the first-choice approach for attempting eradication although cost and potential toxicity are concerns. because of potential differences in laboratory animal medicine blood-brain barrier permeability to ivermectin, pilot treatments should be evaluated. for large facilities, ivermectin medicated feed may be an attractive option (ricart arbona et al., ) . for valuable lines of mice, rederivation may be cost-and time-effective. control and prevention programs should be carried out on a colony-wide basis, which includes thorough sanitation of housing space and equipment to remove residual eggs. research complications hypersensitivity dermatitis has the potential to confound immunological studies (jungmann et al., ) , especially those involving skin, and has been shown to elevate serum ige (morita et al., ) . heavy mite infestations can cause severe skin lesions and have been associated with weight loss, infertility, and premature deaths. chronic acariasis also may provoke secondary amyloidosis due to long-standing dermatitis. myocoptes musculinus this is the most common ectoparasite of the laboratory mouse but frequently occurs in conjunction with myobia musculi. the life cycle includes egg, larva, protonymph, tridonymph, and adult stages. eggs hatch in days and are usually attached to the middle third of the hair shaft. the life cycle may range from to days. transmission requires direct contact, for mice separated by wire screens do not contract infestations from infested hosts. bedding does not seem to serve as a vector. neonates may become infested within - days of birth, and parasites may live for - days on dead hosts. myocoptes appears to inhabit larger areas of the body than myobia and tends to displace myobia during heavy infestations. it has some predilection for the skin of the inguinal region, abdominal skin, and back, but it will also infest the head and neck. it is a surface dweller that feeds on superficial epidermis. infestation can cause patchy thinning of the hair, alopecia, or erythema. lesions can be pruritic, but ulceration has not been reported. chronic infestations induce epidermal hyperplasia and nonsuppurative dermatitis. myobia musculi this is a common mite of laboratory mice. the life cycle of myobia can be completed in days and includes an egg stage, first and second larval stages, protonymph, deutonymph, and adult. eggs attach at the base of hair shafts and hatch in - days. larval forms last about days, followed by nymphal forms on day . adults appear by day and lay eggs within h. myobia are thought to feed on skin secretions and interstitial fluid but not on blood. they are transmitted primarily by contact. mite populations increase during new infestations, followed by a decrease to equilibrium in - weeks. the equilibrated population can be carried in colonies for long periods (up to years). population fluctuations may represent waves of egg hatchings. because mites are thermotactic, they crawl to the end of hair shafts on dead hosts, where they may live for up to days. infestation may result in hypersensitivity dermatitis, to which c bl mice are highly susceptible. clinical signs vary from ruffled fur and alopecia to pruritic ulcerative dermatitis. therefore, lesions can be exacerbated by self-inflicted trauma. radfordia affinis radfordia is thought to be common in laboratory mice, but it closely resembles myobia and may occur as a mixed infestation. therefore, its true prevalence is conjectural. additionally, its life cycle has not been described. it does not appear to cause clinical morbidity. psorergates simplex this species has not been reported as a naturally occurring infection in well-managed colonies for several decades, but it is unique in that it inhabits hair follicles. its life cycle is unknown, but developmental stages from egg to adult may be found in a single dermal nodule. transmission is by direct contact. invasion of hair follicles leads to development of cyst-like nodules, which appear as small white nodules in the subcutis. histologically, they are invaginated sacs of squamous epithelium, excretory products, and keratinaceous debris. there is usually no inflammatory reaction, but healing may be accompanied by granulomatous inflammation. diagnosis is made by examining the subcuticular surface of the pelt grossly or by histological examination. sac contents also can be expressed by pressure with a scalpel blade or scraped and mounted for microscopic exam. mesostigmoid mites rarely, blood-sucking ornithonyssus bacoti and laelaps echidnina, normally limited to wild rodents, can also infect laboratory rodent colonies (watson, ; fox, ) . these mites may also transiently bite humans and can transmit zoonotic infections (see chapter ). unlike the more common rodent fur mites, mesostigmoid mites live off the host and can travel a long distance in search of a blood meal. they access research colonies via contaminated supplies or wild rats and mice gaining access to the facility. polyplax serrata, the mouse louse, is encountered in wild mice but no longer is a significant issue in research colonies. eggs are deposited at the base of hair shafts and nymph stages and adults can be found principally on the dorsum. p. serrata causes pruritus with associated dermatitis, anemia and debilitation and historically is the vector for mycoplasma coccoides. amyloidosis is caused by the deposition of insoluble (polymerized), mis-folded amyloid protein fibrils in organs and/or tissues. primary amyloidosis is a naturally occurring disease in mice, associated with the deposition of amyloid proteins consisting primarily of immunoglobulin light chains. secondary amyloidosis is associated with antecedent and often chronic inflammation. it results from a complex cascade of reactions involving release of multiple cytokines that stimulate amyloid synthesis in the liver (falk and skinner, ) . primary amyloidosis is common among aging mice (lipman et al., ) but also may occur in young mice of highly susceptible strains such as a and sjl or somewhat older c bl mice. other strains, such as balb/c and c h are highly resistant to amyloidosis (percy and barthold, ) . secondary amyloidosis is usually associated with chronic inflammatory lesions, including dermatitis resulting from prolonged acariasis. it can be induced experimentally, however, by injection of casein and may occur locally in association with neoplasia or in ovarian corpora lutea in the absence of other disease. in reactive amyloid a (aa) amyloidosis, serum aa (saa) protein forms deposits in mice, domestic and wild animals, and humans that experience chronic inflammation. aa amyloid fibrils are abnormal β-sheet-rich forms of the serum precursor saa, with conformational changes that promote fibril formation. similar to prion diseases, recent findings suggest that aa amyloidosis could be transmissible in mice and other species (murakami et al., ) . amyloid fibrils induce a seeding-nucleation process that may lead to development of aa amyloidosis. amyloidosis can shorten the life span of mice and can be accelerated by stress from intercurrent disease. amyloid appears histologically as interstitial deposition of a lightly eosinophilic, acellular material in tissues stained with hematoxylin and eosin. however, it is birefringent after staining with congo red when viewed with polarized light. deposition patterns vary with mouse strain and amyloid type. although virtually any tissue may be affected, the following sites are common: hepatic portal triads, periarteriolar lymphoid sheaths in spleen, renal glomeruli and interstitium (which can lead to papillary necrosis), intestinal lamina propria, myocardium (and in association with atrial thrombosis), nasal submucosa, pulmonary alveolar septa, gonads, endocrine tissues, and great vessels (fig. . ) . naturally occurring mineralization of the myocardium and epicardium and other soft tissues is a common finding at necropsy in some inbred strains of mice. although this condition is usually an incidental finding at necropsy, interference with organ function such as the heart cannot be ruled out if lesions are severe. it occurs in balb/c, c h, and especially dba mice (eaton et al., ; brownstein, ; brunnert et al., ) . it is found in the myocardium of the left ventricle ( fig. . ) , in the intraventricular systems, and in skeletal muscle, kidneys, arteries, and lung and may be accompanied by fibrosis and mononuclear inflammatory infiltrates. dba mice also can develop mineralization in the tongue and cornea. dietary, environmental, disease-related, and endocrine-related factors are thought to influence the prevalence of this lesion. ectopic mineralization is associated clinically with skin and vascular connective tissue conditions in humans and mouse models have been developed to study metastatic and dystrophic tissue mineralization (li and uitto, ) . pseudoxanthoma elasticum (pxe), a heritable ectopic mineralization disorder in humans, is caused by mutations in the abcc gene. knockout abcc −/− mice model the histopathologic and ultrastructural features of pxe, notably with mineralization of the vibrissae dermal sheath, serving as a biomarker of tissue mineralization (benga et al., ) . other inbred mouse strains, including kk/hlj and s /svimj, also develop vibrissae dermal mineralization and have an snp (rs ) in the abcc gene associated with low levels of abcc protein expression in the liver. dba/ j and c h/hej mice have the same polymorphism and low abcc protein levels; however, these mice only develop tissue mineralization when fed an experimental diet enriched in phosphate and low in magnesium. a reye's-like syndrome has been reported in balb/ cbyj mice (brownstein et al., ) . the etiology is unknown; however, antecedent viral infection may be involved. affected mice rapidly become lethargic and then comatose. they also tend to hyperventilate. high mortality ensues within - h, but some mice may recover. lesions are characterized grossly by swollen, pale liver and kidneys. the major histopathological findings include swollen hepatocytes with fatty change and nuclear swelling among astrocytes in the brain. hepatic lesions resembling changes in reye's syndrome have been reported in scid mice infected with madv- (pirofski et al., ) . deficiencies (tobin et al., ) vitamin deficiencies in mice have not been thoroughly described. unfortunately, much of the information that does exist reflects work carried out - years ago; thus, the reliability and specificity of some of these syndromes is questionable. vitamin a deficiency may produce tremors, diarrhea, rough hair coat, keratitis, poor growth, abscesses, hemorrhages, and sterility or abortion. vitamin a is recognized for its importance in development of the immune system (ross, ) and knockout mouse models have been used to demonstrate genetic polymorphisms in humans that negatively regulate intestinal β-carotene absorption and conversion to retinoids in response to vitamin a requirements for growth and reproduction (von lintig, ) . vitamin e deficiency can cause convulsions and heart failure, as well as muscular dystrophy and hyaline degeneration of muscles. two knockout mouse models of severe vitamin e deficiency were independently developed and lack α-tocopherol transfer protein (α-ttp), a gene that controls plasma and tissue α-tocopherol concentrations by exporting α-tocopherol from the liver. ttpa −/− mice have very low to undetectable levels of α-tocopherol and are infertile. the phenotype includes neuronal degeneration associated with progressive ataxia and age-related behavioral defects (yu and schellhorn, ) . deficiency of b complex vitamins produces nonspecific signs such as alopecia, decreased feed consumption, poor growth, poor reproduction and lactation, as well as a variety of neurological abnormalities. choline deficiency produces fatty livers and nodular hepatic hyperplasia, as well as myocardial lesions, decreased conception, and decreased viability of litters. folic acid-deficient diets cause marked decreases in red and white cell blood counts and the disappearance of megakaryocytes and nucleated cells from the spleen. pantothenic acid deficiency is characterized by nonspecific signs, such as weight loss, alopecia, achromotrichia, and posterior paralysis, as well as other neurological abnormalities. thiamin deficiency is associated with neurological signs, such as violent convulsions, cartwheel movements, and decreased food consumption. dietary requirements for ascorbic acid have not been shown in mice, and mouse diets are generally not fortified with ascorbic acid. the gulonolactone oxidase knockout mouse (gulo −/− ) on the c bl/ background requires vitamin c supplementation although the plasma ascorbate concentration of gulo −/− mice fed a vitamin c-deficient diet is maintained at % of wild-type concentrations, suggesting an uncharacterized pathway to generate a small amount of ascorbate (yu and schellhorn, ) . the gulo −/− mouse has become the model of choice in studying the role of vitamin c in complex diseases. vitamin c production has been successfully restored in gulo −/− mice using adenovirus vectors, making it possible to robustly manipulate physiological ascorbate concentrations in an inbred mouse. mineral deficiencies have been described only for several elements, and the consequences of the deficiencies are similar to those observed for other species. for example, iodine-deficient diets produce thyroid goiters; magnesium-deficient diets may cause fatal convulsions; manganese deficiency may cause congenital ataxia from abnormal development of the inner ear; and zinc deficiency may cause hair loss on the shoulders and neck, emaciation, decreased liver and kidney catalase activity, and immunosuppression. chronic essential fatty acid deficiency may cause hair loss, dermatitis with scaling and crusting of the skin, and occasional diarrhea. infertility has also been associated with this syndrome. mice have an absolute requirement for a dietary source of linoleic and/or arachidonic acid. (sundberg, ; ward, ) the significant syndrome of ulcerative dermatitis (ud) is a common idiopathic skin lesion that causes morbidity and early euthanasia losses in c bl/ and related lines of mice. significant pruritus leads to skin trauma associated with opportunistic bacterial infection and deep dermal ulcerations. initial signs include alopecia and papular dermatitis, which usually occur over the dorsal trunk (fig. . ) . progressive inflammation can be halted, sometimes reversed, by nail trimming and therapy with a wide spectrum of topical or systemic antibiotics, steroids, and other drugs such as vitamin e and aloe, all of which speak to the frustrating search for a primary etiology. treatment should be based on microbiological culture and sensitivity and screening for ectoparasites as hypersensitivity to acariasis has been proposed. seasonal fluctuation in the incidence of disease suggests that environmental factors may play a role. the incidence appears to increase during periods of significant seasonal changes in temperature and humidity, i.e., the onset of winter and early spring. there is some evidence that incidence is related to dietary fat with mice on high fat or ad libitum diets being more susceptible than those on restricted diets (neuhaus et al., ) . ileus associated with high mortality has been reported to occur in primiparous female mice during the second week of lactation (kunstyr, ) . this disorder has been described as acute intestinal pseudo-obstruction (ipo) in c bl/ mice free of known pathogens (feinstein et al., ) . lactating mice are either found dead or becoming moribund. segments of the small intestine become distended with fluid contents and histologically there is apoptosis of the villus epithelium of the small intestine and superficial epithelial cells of the large intestine. the enteric nervous system appears morphologically normal but necrotic enterocytes, mucosal erosions, and acute mucosal inflammation are commonly observed. there is no strong evidence for metabolic issues such as hypocalcemia or low blood glucose. the direct cause is unknown but death probably results from sepsis secondary to loss of barrier function reflected in apoptosis of the gut epithelium during peak lactation. environmental variables can affect responses of mice in experimental situations. changes in respiratory epithelial physiology and function from elevated levels of ammonia, effects of temperature and humidity on metabolism, effects of light on eye lesions and retinal function, and effects of noise on neurophysiology are examples of complications that can vary with the form of insult and the strain of mouse employed. mice do not easily acclimatize to sudden and dramatic changes in temperature. therefore, they are susceptible to both hypothermia and hyperthermia. mice also are susceptible to dehydration. poorly functioning automatic watering system valves or water bottles, resulting in spills (hypothermia) or obstructed sipper tubes (dehydration), are a significant cause of husbandry-related morbidity. shipping mice between facilities, irrespective of distance, warrants institutional guidelines to minimize exposure to temperature extremes. reheat coils should be designed to fail in the closed position to avoid overheating holding rooms. ringtail is a condition associated with low relative humidity. clinical signs include annular constriction of the tail and occasionally of the feet or digits, resulting in localized edema that can progress to dry gangrene ( fig. . ). it should be differentiated from dryness and gangrene that may occur in hairless mice exposed to low temperatures and perhaps other environmental or nutritional imbalances. necrosis of legs, feet, or digits also can occur in suckling mice because of disruption of circulation by wraps of stringy nesting material such as cotton wool. corneal opacities can result from acute or chronic keratitis, injury (unilateral) and developmental defects; the latter may occur in combination with inherited microphthalmia in c black mice (koch and gowen, ) . there is some evidence that the buildup of ammonia in mouse cages may contribute to inflammatory keratitis, because it can be controlled by increasing the frequency of cage cleaning. corneal opacities and anterior polar cataracts are a developmental defect in inbred c black mice (pierro and spiggle, ) . corneal opacity may be associated with keratolenticular adhesions involving a persistent epithelial stalk of the lens vesicle, which normally disappears around day of gestation (koch and gowen, ) . typically noted in runted or cachectic mice soon after weaning, malocclusion of the open-rooted, continually growing incisor teeth is an inherited trait expressed as poorly aligned incisors, especially of the lower incisors causing osteomyelitis, soft tissue abscesses, or necrosis in the lips or oral cavity. the incidence of inherited malocclusion varies with mouse strain (petznek et al., ) . malocclusion in older mice may be the result of trauma or oral neoplasia. overgrown molar teeth have been associated with trauma to developing tooth buds. skin lesions can be caused by fighting, tail biting, and overgrooming such as whisker chewing. barbering of facial hair and whiskers in subordinate mice by a dominant cagemate is common and may be solved by removing the dominant, normally haired mouse. hair or whisker chewing (barbering) has long been interpreted to be a manifestation of social dominance. apparent dominant animals retain whiskers, whereas cagemates have 'shaved faces' (fig. . ). chronic hair chewing can produce histological abnormalities such as poorly formed or pigmented club hairs. once chewing has ceased, many mice regrow previously lost hair in several weeks. both sexes may engage in this activity, and sometimes females may be dominant. barbering of whiskers and fur-plucking behavior in mice has been suggested to model human trichotillomania (compulsive hair plucking) because of similarities including elevated serotonin levels (dufour et al., ) , 'barbers' predominately pluck hair from the scalp and around the eyes and the genitals; the behavior is female biased, and begins during puberty and is impacted by genetic background (garner et al., ) . fighting is more common in male mice and more aggressive in some strains (sjl, fvb, balb/c) with bite wounds typically located on the head, neck, shoulders, perineal area, and tail. often one mouse in the cage is free of lesions and is the likely aggressor. removal of the unaffected male may end the fighting or simply reorder the dominance order. removing males for breeding and then regrouping them often results in fighting. for programs that produce sentinel mice in-house, castration is an option to reduce aggression in group-housed male sentinels (lofgren et al., ) . regional alopecia, especially around the muzzle, may result from abrasion against cage surfaces. improperly diluted disinfectants may also cause regional hair loss. ear tags used for identification may cause pruritis and self-induced trauma. hair removal products or clipping prior to imaging or application of experimental compounds to the skin may cause pruritus and can augment lesions that interfere with test results. dermatophytosis, ectoparasitism, or idiopathic hair loss must be considered in the differential diagnoses for muzzle or body alopecia. (burek et al., ; percy and barthold, ) common idiopathic lesions in aging mice include cardiomyopathy (with or without mineralization or arteritis), chronic nephropathy (frequently with mineralization), myelofibrosis (fibrotic change in the bone marrow) especially in female mice, melanosis in the meninges, ovarian atrophy (with or without hyaline material), pigment (ceroid-lipofuscin), tubular or stromal hyperplasia, cystic endometrial hyperplasia, testicular tubular degeneration or mineralization, prostate atypical epithelial hyperplasia, gastric glandular epithelial hyperplasia, pancreatic islet cell hyperplasia, dental dysplasia of incisor teeth, pituitary hyperplasia of pars intermedia and pars distalis, cataracts, increased extramedullary hematopoiesis in spleen, and lymphocytic infiltrates or other inflammatory changes in various tissues, including harderian gland, salivary gland, kidney, liver, gall bladder, nasal, trachea, thyroid, periovarian fat, epididymis, and urinary bladder. lymphoma is also very common . spontaneous atrial thrombosis is rare in mice (< % in -year-old mice) and appears to be strain-related, with a high prevalence in rfm mice. it also is more common in aged mice affected by kidney disease and amyloidosis. organizing thrombi will be found usually in an enlarged, hyperemic left atrium and auricle and may be accompanied by amyloidosis. affected mice may display signs of heart failure, particularly severe dyspnea. induction of atrial thrombosis in b c f mice has been used to assess cardiovascular risk of chemical exposures (yoshizawa et al., ) . myocardial and epicardial mineralization is described above (section iii,b, ). periarteritis, also known as arteritis, polyarteritis, or systemic arteritis, impacts older mice and lesions may be observed in multiple tissues, including the spleen, heart, tongue, uterus, testes, kidney, and urinary bladder. the media of the affected vessels is homogenous and intensely eosinophilic with hematoxylin and eosin stain. fibrosis and mononuclear cells infiltrate the vessel wall. experimental coronary arteritis with cardiac hypertrophy has been model in dba/ and other strains by intraperitoneal administration of mannoprotein-beta-glucan complex isolated from c. albicans (nagi-miura et al., ) . hyperplasia of alveolar or bronchial epithelium occurs in old mice and must be differentiated from pulmonary tumors. pulmonary histiocytosis, acidophilic macrophage pneumonia, and acidophilic crystalline pneumonia are synonymous morphologic descriptions of an idiopathic lung lesion that can be incidental or the cause of significant morbidity. incidence varies with mouse strain or stock, with c bl, s /svjae and swiss mice and older mice in general particularly susceptible. histologically, alveoli and bronchioles are filled with varying quantities of macrophages containing eosinophilic crystalline material . the crystalline material consists of ym and/or ym chitanases and can be found in other tissues including the upper respiratory tract, stomach, gall bladder, and bone marrow where it is described as hyalinosis (nio et al., ) . gastric lesions include crypt dilatation, submucosal fibrosis, adenomatous gastric hyperplasia, mineralization, and erosion or ulceration. gastric ulcers have been induced by cold stress, food restriction (rehm et al., ) , chemical injury (yadav et al., ) , and gastritis and gastric tumors by helicobacter infection (fox et al., ) . germfree mice have reduced muscle tone in the intestinal tract. cecal volvulus is a common cause of death in germfree mice and is caused by rotation of the large, thin-walled cecum. age-associated lesions are common in the livers of mice. cellular and nuclear pleomorphism, including binucleated and multinucleated cells, are detectable by months. mild focal necrosis occurs with or without inflammation, but an association of mild focal hepatitis with a specific infectious disease is often hard to confirm. other geriatric hepatic lesions include biliary hyperplasia with varying degrees of portal hepatitis, hepatocellular vacuolization, amyloid deposition (especially in periportal areas), strangulated or herniated lobes, hemosiderosis, lipofuscinosis, and fibrosis. extramedullary hematopoiesis occurs in young mice and in response to anemia. exocrine pancreatic insufficiency has been reported in cba/j mice. acinar cell atrophy is common but is strain-and sex-dependent. blood-filled mesenteric lymph nodes may occur in aged mice, especially c h mice. this condition is an incidental finding and should not be confused with infectious lymphadenopathy such as that associated with salmonellosis. aggregates, or nodules of mononuclear cells, are found in many tissues of aged mice, including the salivary gland, thymus, ovary, uterus, mesentery and mediastinum, urinary bladder, and gastrointestinal tract. these nodules should not be mistaken for lymphosarcomas. grossly observable black pigmentation in the spleen of c bl/ is normal and is melanosis caused by melanin deposition (weissman, ) . the spleen is subject to amyloidosis and hemosiderin deposition. lipofuscin deposition is common, especially in older mice. the thymus undergoes age-associated atrophy. a variety of genetic immunodeficiencies have been described in mice, many of which increase susceptibility to infectious diseases. perhaps the most widely known of these is the athymic nude mouse that lacks a significant hair coat and, more importantly, fails to develop a thymus and thus has a severe deficit of t-cellmediated immune function. additionally, scid mice, which lack both t and b lymphocytes, are used widely and are highly susceptible to opportunistic agents such as pneumocystis murina. specific immune deficits have become excellent models for studying the ontogeny and mechanisms of immune responsiveness (table . ). age-associated osteoporosis or senile osteodystrophy can occur in some mice. it is not associated with severe renal disease or parathyroid hyperplasia. nearly all strains of mice develop some form of osteoarthrosis. it is generally noninflammatory, affects articulating surfaces, and results in secondary bone degeneration. glomerulonephritis is a common kidney lesion of mice. it is more often associated with persistent viral infections or immune disorders rather than with bacterial infections. its prevalence in some strains approaches %. nzb and nzb × nzw f hybrid mice, e.g., develop immune complex glomerulonephritis as an autoimmune disease resembling human lupus erythematosus, whereas glomerular disease is relatively mild in nzb mice (nzb mice have a high incidence of autoimmune hemolytic anemia). renal changes occur as early as months of age, but clinical signs and severe disease are not present until - months. the disease is associated with wasting and proteinuria, and lesions progress until death intervenes. histologically, glomeruli have proteinaceous deposits in the capillaries and mesangium. later, tubular atrophy and proteinaceous casts occur throughout the kidney. immunofluorescence studies show deposits of immunoglobulin and the third component of complement, which lodge as immune complexes with nuclear antigens and antigens of murine leukemia virus in glomerular capillary loops. mice infected with lcmv or with retroviruses can also develop immune complex glomerulonephritis. mice also can develop chronic glomerulopathy characterized by progressive thickening of glomerular basement membrane by pas-positive material that does not stain for amyloid. this lesion can be accompanied by proliferation of mesangial cells; local, regional, or diffuse mononuclear cell infiltration; and fibrosis. advanced cases may lead to renal insufficiency or failure. interstitial nephritis can be caused by bacterial or viral infections but may also be idiopathic. typical lesions include focal, regional, or diffuse interstitial infiltration of tubular parenchyma by mononuclear cells, but glomerular regions also may be involved. severe lesions can be accompanied by fibrosis, distortion of renal parenchyma, and intratubular casts, but not by mineralization. if renal insufficiency or failure ensues, it can lead to ascites. some strains of mice, such as balb/c, can develop polycystic kidney disease, which, if severe, can compromise normal renal function. urinary tract obstruction occurs as an acute or chronic condition in male mice. clinical signs usually include wetting of the perineum from incontinence. in severe or chronic cases, wetting predisposes to cellulitis and ulceration. at necropsy, the bladder is distended, and proteinaceous plugs are often found in the neck of the bladder and proximal urethra. in chronic cases the urine may be cloudy, and calculi may develop in the bladder. additionally, cystitis, urethritis, prostatitis, laboratory animal medicine balanoposthitis, and hydronephrosis may develop. this condition must be differentiated from infectious cystitis or pyelonephritis and from the agonal release of secretions from accessory sex glands, which is not associated with an inflammatory response. hydronephrosis also may occur without urinary tract obstruction. ascending pyelitis occurs in mice secondary to urinary tract infection. parvovarian cysts are observed frequently and may be related to the fact that mouse ovaries are enclosed in membranous pouches. amyloidosis is also common in the ovaries of old mice. cystic endometrial hyperplasia may develop unilaterally or bilaterally and may be segmental. in some strains, the prevalence in mice older than months is %. endometrial hyperplasia is often associated with ovarian atrophy. mucometra is relatively common in adult female mice. the primary clinical sign is abdominal distension resembling pregnancy among mice that do not whelp. testicular atrophy, sperm granulomas, and tubular mineralization occur with varying incidence. preputial glands, especially of immunodeficient mice, can become infected with opportunistic or pathogenic bacteria. spontaneous lesions in prostate, coagulating gland (anterior prostatic lobe), seminal vesicles, and ampullary glands were described in control b c f mice from national toxicology program -year carcinogenicity and toxicity studies conducted in one of four different laboratories (suwa et al., ) . lymphocytic infiltration, inflammation, edema, epithelial hyperplasia, mucinous cyst, mucinous metaplasia, adenoma, adenocarcinoma, granular cell tumor, and glandular atrophy were variously observed in accessory sex glands. accessory adrenal cortical nodules are found in periadrenal and perirenal fat, especially in females. these nodules have little functional significance other than their potential effect on failures of surgical adrenalectomy. lipofuscinosis, subcapsular spindle cell hyperplasia, and cystic dilatation of cortical sinusoids are found in the adrenal cortices of aged mice. some inbred strains have deficiencies of thyrotropic hormone, resulting in thyroid atrophy. thyroid cysts lined by stratified squamous epithelium and generally of ultimo-branchial origin may be seen in old mice. amyloid can be deposited in the thyroid and parathyroid glands as well as in the adrenal glands. spontaneous diabetes mellitus occurs in outbred swiss mice and genetic variants of several strains such as nod mice (lemke et al., ) . high levels of estrogen in pregnancy may influence postpartum hair shedding. various endocrine effects on hair growth have also been described. abdominal and thoracic alopecia have been reported in b c f mice. symmetrical mineral deposits commonly occur in the thalamus of aged mice. they may also be found in the midbrain, cerebellum, and cerebrum and are particularly common in a/j mice. lipofuscin accumulates in the neurons of old mice. age-associated peripheral neuropathy with demyelination can be found in the nerves of the hindlimbs in c bl/ mice. deposits of melanin pigment occur in heavily pigmented strains, especially in the frontal lobe. a number of neurologically mutant mice have been described. they commonly have correlative anatomical malformations or inborn errors of metabolism. a seizure syndrome in fvb mice has been described (goelz et al., ) and can be spontaneous or associated with tail tattooing, fur clipping, and fire alarms. mice are most often female with a mean age of . months (range, - months) and can exhibit facial grimace, chewing automatism, ptyalism with matting of the fur of the ventral aspect of the neck and/or forelimbs, and clonic convulsions that may progress to tonic convulsions and death. ischemic neuronal necrosis was consistently observed in these mice and is consistent with status epilepticus in humans. unilateral and bilateral microphthalmia and anophthalmia are frequent (as high as %) developmental defects in inbred and congenic strains of c bl mice, especially impacting the right eye and female mice. these conditions may first be recognized due to ocular infections, secondary to inadequate tear drainage. other common findings include central corneal opacities, iridocorneal and corneal-lenticular adhesions, abnormal formation of the iris and ciliary body, cataracts, extrusion of lens cortical material with dispersion throughout the eye, failure of vitreous development, and retinal folding. these syndromes can be reproduced by exposure to alcohol at critical stages of embryogenesis when the optic cup and lens vesicle are developing and impacting normal development of other ocular structures, including the iris, ciliary body, vitreous, and retina . retinal degeneration can occur as either an environmental or a genetic disorder (chang et al., ) in mice. nonpigmented mice, both inbred and outbred, can develop retinal degeneration from exposure to light, with the progression of blindness being related to light intensity and duration of exposure. mouse genetics have laboratory animal medicine been shown to be more important than potential light associated tissue injury (serfilippi et al., a) . other strains such as c h, cba, and fvb are genetically predisposed to retinal degeneration because they carry the rd gene, which leads to retinal degeneration within the first few weeks of life and has been used extensively as a model for retinitis pigmentosa (farber and danciger, ) . presence of the rd gene in some mouse strains highlights that impaired vision must be a consideration when selecting strains for behavioral assays that rely on visual clues (garcia et al., ) . blindness does not interfere with health or reproduction and blind mice cannot be distinguished from non-blind mice housed in standard caging. cataracts can occur in old mice and have a higher prevalence in certain mutant strains. vestibular syndrome associated with head tilt, circling, or imbalance can result from infectious otitis or from necrotizing vasculitis of unknown etiology affecting small and medium-sized arteries in the vicinity of the middle and inner ears. (jones et al., (jones et al., - maronpot et al., ; percy and barthold, ) neoplasms of lymphoid and hematopoietic tissues are estimated to have a spontaneous prevalence of - %. there are, however, some strains of mice that have been specifically inbred and selected for susceptibility to spontaneous tumors. leukemogenesis in mice may involve viruses and chemical or physical agents. viruses associated with lymphopoietic and hematopoietic neoplasia belong to the family retroviridae (type c oncornaviruses) and contain rna-dependent dna polymerase (reverse transcriptase). these viruses are generally noncytopathogenic for infected cells, and mice appear to harbor them as normal components of their genome. although they may be involved in spontaneous leukemia, they are not consistently expressed in this disease. recombinant viruses have recently been discovered that can infect mouse cells and heterologous cells and are associated with spontaneous leukemia development in high leukemia strains such as akr mice. their phenotypic expression is controlled by mouse genotype. endogenous retroviruses are transmitted vertically through the germ line. horizontal transmission is inefficient but can occur by intrauterine infection or through saliva, sputum, urine, feces, or milk. the leukemia induced by a given endogenous virus is usually of a single histopathological type. loss of function in nucleic acid-recognizing, tlr , tlr , and tlr can result in spontaneous retroviral viremia and acute t-cell lymphoblastic leukemia (yu et al., ) . chemical carcinogens, such as polycyclic hydrocarbons, nitrosoureas, and nitrosamines, and physical agents such as x-irradiation can also induce hematological malignancies in mice. the most common hematopoietic malignancy in the mouse is lymphocytic leukemia that originates in the thymus. disease begins with unilateral atrophy and then enlargement of one lobe of thymus as tumor cells proliferate. cells can spread to the other lobe and then to other hematopoietic organs, such as the spleen, bone marrow, liver, and peripheral lymph nodes. clinical signs include dyspnea and ocular protrusion. the latter sign is due to compression of venous blood returning from the head. tumor cells spill into the circulation late in disease. most of these tumors originate from t lymphocytes or lymphoblasts, but there are leukemias of b-lymphocyte or null cell lineage. in the last two syndromes, the lymph nodes and spleen are often involved, but the thymus is generally normal. reticulum cell sarcomas are common in older mice, especially in inbred strains such as c bl/ and sjl. primary tumor cell types have been divided into several categories based on morphological and immunohistochemical features. histiocytic sarcomas correspond to the older dunn classification as type a sarcomas and are composed primarily of reticulum cells. the tumor typically causes splenomegaly and nodular lesions in other organs, including the liver, lung, kidney, and the female reproductive tract. follicular center cell lymphomas correspond to dunn type b sarcomas. they originate from b-cell regions (germinal centers) of peripheral lymphoid tissues, including the spleen, lymph nodes, and peyer's patches. typical tumor cells have large vesiculated, folded, or cleaved nuclei and ill-defined cytoplasmic borders. tumors also often contain small lymphocytes. type c reticulum cell tumors often involve one or several lymph nodes rather than assuming a wide distribution. they consist of reticulum cells with a prominent component of well-differentiated lymphocytes. myelogenous leukemia is uncommon in mice and is associated with retrovirus infection. disease begins in the spleen, resulting in marked splenomegaly, but leukemic spread results in involvement of many tissues including the liver, lung, and bone marrow. leukemic cells in various stages of differentiation can be found in peripheral blood. in older animals, affected organs may appear green because of myeloperoxidase activity, giving rise to the term chloroleukemia. the green hue fades on contact with air. affected mice are often clinically anemic and dyspneic. erythroleukemia is rare in mice. the major lesion is massive splenomegaly, which is accompanied by anemia and polycythemia. hepatomegaly can follow, but there is little change in the thymus or lymph nodes. erythroleukemia can be experimentally induced in mice by friend spleen focus-forming virus (sffv) which initially activates the erythropoietin (epo) receptor and the receptor tyrosine kinase sf-stk in erythroid cells, resulting in proliferation, differentiation, and survival. in a second stage, sffv activates the myeloid transcription factor pu. , blocking erythroid cell differentiation, and in conjunction with the loss of p tumor suppressor activity, results in the outgrowth of malignant cells (cmarik and ruscetti, ). mast cell tumors are also very rare in mice. they are found almost exclusively in old mice and grow slowly. they should not be confused with mast cell hyperplasia observed in the skin following painting with carcinogens or x-irradiation. natural plasma cell tumors are infrequent in the mouse. they can, however, be induced by intraperitoneal inoculation of granulomatogenic agents such as plastic filters, plastic shavings, or a variety of oils, particularly in balb/c mice. mineral oil-induced plasmacytomas in balb/c mice produce large amounts of endogenous retroelements such as ecotropic and polytropic murine leukemia virus and intracisternal a particles. associated inflammation may promote retroelement insertion into cancer genes, thereby promoting tumors (knittel et al., ) . similar to other spontaneous cancers, plasmacytoma development in mice is inhibited by innate immune responses of nk cells which when activated by viruses will release γinf (thirion et al., ) . mammary tumors can be induced or modulated by a variety of factors, including viruses, chemical carcinogens, radiation, hormones, genetic background, diet, and immune status. certain inbred strains of mice, such as c h, a, and dba/ , have a high natural prevalence of mammary tumors. other strains, such as balb/c, c bl, and akr, have a low prevalence. among the most important factors contributing to the development of mammary tumors are mammary tumor viruses. several major variants are known. the primary tumor virus mmtv-s (bittner virus) is highly oncogenic and is transmitted through the milk of nursing females. infected mice typically develop a precursor lesion, the hyperplastic alveolar nodule, which can be serially transplanted. spontaneous mammary tumors metastasize with high frequency, but this property is somewhat mouse strain dependent. metastases go primarily to the lung. some mammary tumors are hormone dependent, some are ovary dependent, and others are pregnancy dependent. ovary-dependent tumors contain estrogen and progesterone receptors, whereas pregnancy-dependent tumors have prolactin receptors. ovariectomy will dramatically reduce the incidence of mammary tumors in c h mice. if surgery is done in adult mice - months of age, mammary tumors will develop, but at a later age than normal. grossly, mammary tumors may occur anywhere in the mammary chain. they present as one or more firm, welldelineated masses, which are often lobular and maybe cystic (fig. . ) . histologically, mammary tumors have been categorized into three major groups: carcinomas, carcinomas with squamous cell differentiation, and carcinosarcomas. the carcinomas are divided into adenocarcinoma types a, b, c, y, l, and p. most tumors are type a or b. type a consists of adenomas, tubular carcinomas, and alveolar carcinomas. type b tumors have a variable pattern with both well-differentiated and poorly differentiated regions. they may consist of regular cords or sheets of cells or papillomatous areas. these two types are locally invasive and may metastasize to the lungs. type c tumors are rare and are characterized by multiple cysts lined by low cuboidal to squamous epithelial cells, and they have abundant stroma. type y tumors, which are also rare, are characterized by tubular branching of cuboidal epithelium and abundant stroma. adenocarcinomas with a lacelike morphology (types l and p) are hormone dependent and have a branching tubular structure. the control or prevention of mammary neoplasms depends on the fact that some strains of mammary tumor virus are transmitted horizontally, whereas others are transmitted vertically. although horizontally transmitted virus such as mmtv-s can be determined by cesarean rederivation or by foster nursing, endogenous strains of tumor virus may remain. fortunately, these latter tumor viruses have generally low oncogenicity relative to the bittner virus. mammary tumors are increased in frequency in c bl apc +/− female mice infected with h. hepaticus (rao et al., ) . mice develop an assortment of liver changes as they age, including proliferative lesions which can progress from hyperplastic foci to hepatomas to hepatocellular carcinomas. almost all strains of mice have a significant prevalence of hepatic tumors, some of which appear to result from dietary contamination or deficiency and h. hepaticus infections in susceptible strains of mice such as the a/jcr male mouse ward et al., ) . the prevalence of spontaneous liver tumors in b c f hybrids is increased by feeding choline-deficient diets or when infected with h. hepaticus (hailey et al., ) . tumors also can develop in mice exposed to environmental chemicals, many of which are carcinogenic or potentially carcinogenic (hoenerhoff et al., ) . spontaneous liver tumors in mice occur grossly as gray to tan nodules or large, poorly demarcated darkred masses. they are usually derived from hepatocytes, whereas cholangiocellular tumors are rare. hepatomas are well circumscribed and well differentiated, but they compress adjacent liver tissue as they develop. hepatocellular carcinomas are usually invasive and display histopathological patterns ranging from medullary to trabecular. large carcinomas also may contain hemorrhage and necrosis. carcinomas also may metastasize to the lungs. primary respiratory tumors of mice occur in relatively high frequency. it has been estimated that more than % of these tumors are pulmonary adenomas that arise either from type pneumocytes or from clara cells lining terminal bronchioles. pulmonary adenomas usually appear as distinct whitish nodules that are easily detected by examination of the lung surface. malignant alveologenic tumors are infrequent and consist of adenocarcinomas and squamous cell carcinomas. they invade pulmonary parenchyma and are prone to metastasize. the prevalence of spontaneous respiratory tumors is mouse straindependent. for example, the prevalence is high in aging a strain mice but low in aging c bl mice. the number of tumors per lung is also higher in susceptible mice. pulmonary tumors often occur as well-defined gray nodules. microscopically, adenomas of alveolar origin consist of dense ribbons of cuboidal to columnar cells with sparse stroma. adenomas of clara cell origin are usually associated with bronchioles. they have a tubular to papillary architecture consisting of columnar cells with basal nuclei. pulmonary adenocarcinomas, though comparatively rare, are locally invasive. they often form papillary structures and have considerable cellular pleomorphism. given the rapid development of mouse strains genetically predisposed to neoplasia, the mouse tumor biology database maintained by jackson laboratory is a valuable centralized resource for the most current tumor descriptions. the database contains information on more than strains and substrains, tissues and organs, over , tumor frequency records, and nearly histopathological images and descriptions. risk factors for recurrence, complications and mortality in clostridium difficile infection: a systematic review detection of mouse parvovirus in mus musculus gametes, embryos, and ovarian tissues by polymerase chain reaction interleukins, from to , and interferon-gamma: receptors, functions, and roles in diseases new generation humanized mice for virus research: comparative aspects and future prospects chemokine: receptor structure, interactions, and antagonism of mice, birds, and men: the mouse ultrasonic song system has some features similar to humans and song-learning birds intestinal inflammation targets cancerinducing activity of the microbiota the use of cross-foster rederivation to eliminate murine norovirus cas-offinder: a fast and versatile algorithm that searches for potential off-target sites of cas rnaguided endonucleases leaky virus: a new hantavirus isolated from mus musculus in the united states recombinant-inbred strains: an aid to finding identity, linkage, and function of histocompatibility and other genes experimental infection of inbred mouse strains with spironucleus muris open-and closed-formula laboratory animal diets and their importance to research the microbiology of transmissible murine colonic hyperplasia mouse hepatitis virus infection, intestine, mouse mouse hepatitis virus infection, liver, mouse murine rotavirus infection, intestine, mouse modification of early dimethylhydrazine carcinogenesis by colonic mucosal hyperplasia lymphocytic choriomeningitis virus dietary, bacterial, and host genetic interactions in the pathogenesis of transmissible murine colonic hyperplasia transmissible murine colonic hyperplasia epizootic coronaviral typhlocolitis in suckling mice infectivity, disease patterns, and serologic profiles of reovirus serotypes , , and in infant and weanling mice clindamycin-associated colitis due to a toxin-producing species of clostridium in hamsters fecal pcr assay for diagnosis of helicobacter infection in laboratory rodents s ribosomal dna sequence-based identification of bacteria in laboratory rodents: a practical approach in laboratory animal bacteriology diagnostics polyoma viruses persistence of polyomavirus in mice infected as adults differs from that observed in mice infected as newborns aerobic gram-positive organisms detection of rodent parvoviruses by pcr identification of novel murine parvovirus strains by epidemiological analysis of naturally infected mice temporal transmission studies of mouse parvovirus in balb/c and c.b- /icr-prkdc(scid) mice embryo transfer rederivation of c.b- /icr-prkdc(scid) mice experimentally infected with mouse parvovirus effect of vaccination on the clinical response, pathogenesis, and transmission of mousepox mousepox in inbred mice innately resistant or susceptible to lethal infeciton with ectromelia virus. iii. experimental transmission of infection and derivation of virus-free progeny from previously infected dams mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. i. clinical responses stability of ectromelia virus strain nih- under various laboratory conditions most classical mus musculus domesticus laboratory mice carry a mus musculus musculus y chromosome klebsiella oxytoca: opportunistic infections in laboratory rodents clustering of spatial gene expression patterns in the mouse brain and comparison with classical neuroanatomy serological studies of corynebacterium kutscheri and coryneform bacteria using an enzyme-linked immunosorbent assay (elisa) an enzymelinked immunosorbent assay (elisa) for monitoring rodent colonies for pasteurella pneumotropica antibodies helicobacter pullorum outbreak in c bl/ ntac and c h/hentac barrier-maintained mice mucosal immunity: induction, dissemination, and effector functions nk cell activation in human hantavirus infection explained by virus-induced il- /il ralpha expression exacerbation of pneumocystis carinii pneumonia in immunodefi-� cient (scid) mice by concurrent infection with a pneumovirus the sense of smell: multiple olfactory subsystems pheromonal communication in vertebrates immunological aspects of giardia muris and spironucleus muris infections in inbred and outbred strains of laboratory mice: a comparative study the natural history of mousepox reproduction whole-rat conditional gene knockout via genome editing pathogenesis of bacteremia due to pseudomonas aeruginosa in cyclophosphamide-treated mice and potentiation of virulence of endogenous streptococci genetics of dystrophic epicardial mineralization in dba/ mice sendai virus and pneumonia virus of mice (pvm) spontaneous reye'slike syndrome in balb/cbyj mice duration and patterns of transmission of theiler's mouse encephalomyelitis virus infection chromosomal locations and gonadal dependence of genes that mediate resistance to ectromelia (mousepox) virus-induced mortality pathogenesis of infection with a virulent allotropic variant of minute virus of mice and regulation by host genotype an exteroceptive block to pregnancy in the mouse comparison of polymerase chain reaction and immunohistochemistry for the detection of mycoplasma pulmonis in paraffin-embedded tissue chromosomal localization of the loci responsible for dystrophic cardiac calcinosis in dba/ mice evaluation of an enzymelinked immunosorbent assay for the detection of ectromelia (mousepox) antibody mousepox selected non-neoplastic diseases helicobacter-induced inflammatory bowel disease in il- -and t cell-deficient mice strategies to prevent, treat, and provoke corynebacteriumassociated hyperkeratosis in athymic nude mice corynebacterium bovis: epizootiologic features and environmental contamination in an enzootically infected rodent room inflammatory bowel disease: an immunity-mediated condition triggered by bacterial infection with helicobacter hepaticus poliomyelitis in mulv-infected icr-scid mice after injection of basement membrane matrix contaminated with lactate dehydrogenase-elevating virus resistance to mycoplasmal lung disease in mice is a complex genetic trait roles of innate and adaptive immunity in respiratory mycoplasmosis opportunistic infections of mice and rats: jacoby and lindsey revisited an outbreak in mice of salmonellosis caused by salmonella enteritidis serotype enteritidis detection of natural mycoplasma pulmonis infection in rats and mice by an enzyme linked immunosorbent assay (elisa) zinc-finger nucleases: the next generation emerges uber eine neue trypoanosomiasis des menschen from genes to social communication: molecular sensing by the vomeronasal organ two mouse retinal degenerations caused by missense mutations in the beta-subunit of rod cgmp phosphodiesterase gene a mouse model of clostridium difficileassociated disease the origins and uses of mouse outbred stocks evidence of human infection with a rat-associated hantavirus in helicobacter hepaticus infection triggers inflammatory bowel disease in t cell receptor alphabeta mutant mice experimental infection of mice with hamster parvovirus: evidence for interspecies transmission of mouse parvovirus the diversity outbred mouse population an experience with clostridium perfringens in cesarean derived barrier sustained mice hyperkeratosis in athymic nude mice caused by a coryneform bacterium: microbiology, transmission, clinical signs, and pathology friend spleen focus-forming virus activates the tyrosine kinase sf-stk and the transcription factor pu. to cause a multi-stage erythroleukemia in mice colonisation and shedding of lawsonia intracellularis in experimentally inoculated rodents and in wild rodents on pig farms a mouse for all reasons citrobacter rodentium: infection, inflammation and the microbiota prevention of murine norovirus infection in neonatal mice by fostering transmission of mouse parvovirus by fomites anatomy lactate dehydrogenase-elevating virus behavioral phenotyping strategies for mutant mice genetic heterogeneity of rat-derived pneumocystis serum antibody responses by male and female c bl/ mice infected with giardia muris comparison of the course of infection with giardia muris in male and female mice cytotoxic and pathogenic properties of klebsiella oxytoca isolated from laboratory animals the hidden cost of housing practices: using noninvasive imaging to quantify the metabolic demands of chronic cold stress of laboratory mice the role of klebsiella oxytoca in utero-ovarian infection of b c f mice behavioral effects of ivermectin in mice host-pathogen interaction in invasive salmonellosis sur les rapports des kystes de carini du poumon des rats avec le trypanosoma lewisi eradication of helicobacter spp. by using medicated diet in mice deficient in functional natural killer cells and complement factor d recombinat congenic strains -a new tool for analyzing genetic traits by more than one gene comparison between patterns of pinworm infection (aspiculuris tetraptera) in wild and laboratory strains of mice, mus musculus laboratory rat associated outbreak of haemorrhagic fever with renal syndrome due to hantaan-like virus in belgium animal leptospirosis in small tropical areas phylogeny of the defined murine microbiota: altered schaedler flora clostridium difficile infection in horses: a review mousepox outbreak in a laboratory mouse colony isolation of a puumala-like virus from mus musculus captured in yugoslavia and its association with severe hemorrhagic fever with renal syndrome assessment of rpob and s rrna genes as targets for pcrbased identification of pasteurella pneumotropica comparison of traditional and pcr methods during screening for and confirmation of aspiculuris tetraptera in a mouse facility pathogenicity and genetic variation of strains of corynebacterium bovis in immunodeficient mice use of fenbendazole-containing therapeutic diets for mice in experimental cancer therapy studies nutritional up-regulation of serotonin paradoxically induces compulsive behavior the pneumonia virus of mice (mpnv) model of acute respiratory infection comparison of nine commercially available clostridium difficile toxin detection assays, a real-time pcr assay for c. difficile tcdb, and a glutamate dehydrogenase detection assay to cytotoxin testing and cytotoxigenic culture methods dystrophic cardiac calcinosis in mice: genetic, hormonal, and dietary influences isolation and expression of the pneumocystis carinii dihydrofolate reductase gene the mouse genome database (mgd): comprehensive resource for genetics and genomics of the laboratory mouse typhlocolitis in nf-kappa b-deficient mice cd + cd + regulatory t lymphocytes inhibit microbially induced colon cancer in rag -deficient mice cd (+)cd (+) regulatory lymphocytes require interleukin to interrupt colon carcinogenesis in mice nitric oxide and tnf-alpha trigger colonic inflammation and carcinogenesis in helicobacter hepaticusinfected, rag -deficient mice ectromelia virus: the causative agent of mousepox intranasal inoculation of mycoplasma pulmonis in mice with severe combined immunodeficiency (scid) causes a wasting disease with grave arthritis the systemic amyloidoses: an overview inherited retinal degenerations in the mouse molecular detection of novel picornaviruses in chickens and turkeys fatal acute intestinal pseudoobstruction in mice the epizootic behaviour of mouse-pox (infectious ectromelia) the pathogenesis of acute exanthema. an interpretation based on experimental investigations with mouse-pox (infectious ectromelia of mice) studies in mousepox, infectious ectromelia of mice; a comparison of the virulence and infectivity of three strains of ectomelia virus studies in mousepox, infectious ectromelia of mice; the effect of age of the host upon the response to infection a juvenile mouse pheromone inhibits sexual behaviour through the vomeronasal system evidence from mtdna sequences that common laboratory strains of inbred mice are descended from a single female origins and characteristics of inbred strains of mice revised nomenclature for strain mice congenic strains treatment of syphacia obvelata in mice using ivermectin the nude mouse in experimental and clinical research opportunistic bacterial infections in breeding colonies of the nsg mouse strain outbreak of tropical rat mite dermatitis in laboratory personnel the role of helicobacter species in newly recognized gastrointestinal tract diseases of animals helicobacter hepaticus sp. nov., a microaerophilic bacterium isolated from livers and intestinal mucosal scrapings from mice chronic proliferative hepatitis in a/jcr mice associated with persistent helicobacter hepaticus infection: a model of helicobacterinduced carcinogenesis persistent hepatitis and enterocolitis in germfree mice infected with helicobacter hepaticus hepatic helicobacter species identified in bile and gallbladder tissue from chileans with chronic cholecystitis comparison of methods of identifying helicobacter hepaticus in b c f mice used in a carcinogenesis bioassay a novel urease-negative helicobacter species associated with colitis and typhlitis in il- -deficient mice inflammatory bowel disease in mouse models: role of gastrointestinal microbiota as proinflammatory modulators host and microbial constituents influence helicobacter pylori-induced cancer in a murine model of hypergastrinemia helicobacter hepaticus infection in mice: models for understanding lower bowel inflammation and cancer granulomatous peritonitis in interferon-gamma gene knockout mice naturally infected with mouse hepatitis virus microbial considerations in genetically engineered mouse research tyzzer's infection: host specificity of clostridium piliforme isolates enteric lesions in scid mice infected with 'helicobacter typhlonicus,' a novel urease-negative helicobacter species helicobacter typhlonius sp. nov., a novel murine urease-negative helicobacter species diagnosis of enteritis and enterotoxemia due to clostridium difficile in captive ostriches (struthio camelus) pneumocystis pneumonia, an immunodeficiencydependent disease (idd): a critical historical overview manual of microbiological monitoring of laboratory animals detection of antibodies to pneumocystis carinii by enzyme-linked immunosorbent assay in experimentally infected mice effect of fenbendazole on three behavioral tests in male c bl/ n mice zfn, talen, and crispr/cas-based methods for genome engineering animal transgenesis: an overview bacterial and mycotic diseases of the digestive system tyzzer's disease propagation of the etiologic agent of tyzzer's disease (bacillus piliformis) in cell culture. contribution of laboratory animal science to the welfare of man and animals: past, present, and future transgenic rna interference in mice genetic susceptibility to chronic hepatitis is inherited codominantly in helicobacter hepaticus-infected ab f and b af hybrid male mice, and progression to hepatocellular carcinoma is linked to hepatic expression of lipogenic genes and immune function-associated networks helicobacter hepaticus-induced liver tumor promotion is associated with increased serum bile acid and a persistent microbial-induced immune response the retinal degeneration (rd) gene seriously impairs spatial cognitive performance in normal and alzheimer's transgenic mice mycoplasma detection in cell cultures: a comparison of four methods barbering (fur and whisker trimming) by laboratory mice as a model of human trichotillomania and obsessive-compulsive spectrum disorders further evidence of host species-specific variation in antigens of pneumocystis carinii using the polymerase chain reaction pneumocystis carinii is not universally transmissable between mammalian species fungal diseases in laboratory mice neuropathologic findings associated with seizures in fvb mice molecular detection of murine norovirus from experimentally and spontaneously infected mice the distribution and kinetics of polyomavirus in lungs of intranasally infected neonatal mice spontaneous staphylococcus xylosus infection in mice deficient in nadph oxidase and comparison with other laboratory mouse strains genetics and probability in animal breeding experiments the major site of murine k papovavirus persistence and reactivation is the renal tubular epithelium isolation of streptococcus equisimilis from abscesses detected in specific pathogen-free mice ciliaassociated respiratory (car) bacillus infection of obese mice epigenetic and phenotypic changes result from a continuous pre and post natal dietary exposure to phytoestrogens in an experimental population of mice impact of helicobacter hepaticus infection in b c f mice from twelve national toxicology program two-year carcinogenesis studies pathology of aging b ; mice reduplication in mice humoral immunity and protection of mice challenged with homotypic or heterotypic parvovirus line- (l ) lineages in the mouse addgene provides an open forum for plasmid sharing quantitative trait loci in a bacterially induced model of inflammatory bowel disease a review of the molecular mechanisms of chemically induced neoplasia in rat and mouse models in national toxicology program bioassays and their relevance to human cancer control of pseudomonas aeruginosa infection in mice by chlorine treatment of drinking water mouse phyisology b cells modulate systemic responses to pneumocystis lung infection and protect on-demand hematopoiesis via t cell-independent, innate mechanism when type-i-ifn-signaling is absent development of a microsphere-based serologic multiplexed fluorescent immunoassay and a reverse transcriptase pcr assay to detect murine norovirus infection in mice persistent infection with and serologic cross-reactivity of three novel murine noroviruses multiplex fluorescent immunoassay for the simultaneous detection of serum antibodies to multiple rodent pathogens development and applications of crispr-cas for genome engineering genetic influence on response to dietary manganese deficiency in mice differential susceptibility to hepatic inflammation and proliferation in axb recombinant inbred mice chronically infected with helicobacter hepaticus parvoviruses mousepox in inbred mice innately resistant or susceptible to lethal infection with ectromelia virus. pathogenesis of vaccinia (ihd-t) virus infection in balb/cann mice evidence that nk cells and interferon are required for genetic resistance to infection with ectromelia virus sendai virus pneumonia in aged balb/c mice health care for research animals is essential and affordable inducible gene expression and gene modification in transgenic mice pcr testing of a ventilated caging system to detect murine fur mites amino acid requirements of the growing mouse monographs on pathology of laboratory animals talens: a widely applicable technology for targeted genome editing murine acariasis. ii. immunological dysfunction and evidence for chronic activation of th- lymphocytes mouse enu mutagenesis effects of enu dosage on mouse strains comparative murine norovirus studies reveal a lack of correlation between intestinal virus titers and enteric pathology tailored immune responses: novel effector helper t cell subsets in protective immunity stat -dependent innate immunity to a norwalk-like virus eradication of murine norovirus from a mouse barrier facility control of laboratory acquired hemorrhagic fever with renal syndrome (hfrs) in japan growth of tyzzer's organism in primary monolayer cultures of adult mouse hepatocytes the laboratory mouse. its origin, heredity, and culture olfactory regulation of the sexual behavior and reproductive physiology of the laboratory mouse: effects and neural mechanisms review of successful treatment for helicobacter species in laboratory mice on the maximum avoidance of inbreeding an oral ivermectin regimen that eradicates pinworms (syphacia spp.) in laboratory rats and mice nutrition effect of open and closed formula rations on the performance of three strains of laboratory mice insertional hypermutation in mineral oil-induced plasmacytomas chemical and cytokine features of innate immunity characterize serum and tissue profiles in inflammatory bowel disease gross anatomy the manufacture, shipping and receiving and quality control of rodent bedding materials emergence of clostridium difficile-associated disease in north america and europe helicobacter hepaticus-induced colitis in interleukin- -deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation bacteria-triggered cd (+) t regulatory cells suppress helicobacter hepaticus-induced colitis il- plays a key role in helicobacter hepaticus-induced t cell-dependent colitis paresis of peristalsis and ileus lead to death in lactating mice experimental spironucleosis (hexamitiasis) in the nude mouse as a model for immunologic and pharmacologic studies host specificity of giardia muris isolates from mouse and golden hamster genetic control of resistance to mycoplasma pulmonis infection in mice therapeutic effect of dna immunization of genetically susceptible mice infected with virulent mycoplasma pulmonis in vitro and in vivo susceptibility of mouse megakaryocytic progenitors to strain i of parvovirus minute virus of mice effects of fenbendazole on the murine humoral immune system from house mouse to mouse house: the behavioral biology of free-living mus musculus and its implications in the laboratory antibiotic treatment of clostridium difficile carrier mice triggers a supershedder state, spore-mediated transmission, and severe disease in immunocompromised hosts use of topical ivermectin treatment for syphacia obvelata in mice hantaan-like viruses from domestic rats captured in the united states obesity and non-insulin-dependent diabetes mellitus in swiss-webster mice associated with late-onset hepatocellular carcinoma mouse adenovirus type infection in scid mice: an experimental model for antiviral therapy of systemic adenovirus infections mineralization/anti-mineralization networks in the skin and vascular connective tissues a second class of olfactory chemosensory receptors in the olfactory epithelium genetic variables that influence phenotype mycoplasma and other bacterial diseases of the respiratory system pasteurella pneumotropica pseudomonas aeruginosa salmonella enteritidis staphylococcus aureus streptobacillus moniliformis streptococcus pneumoniae soiled bedding sentinels for the detection of fur mites in mice false negative results using rt-pcr for detection of lactate dehydrogenase-elevating virus in a tumor cell line eradication of pinworms (syphacia obvelata) from a large mouse breeding colony by combination oral anthelmintic therapy mousepox resulting from use of ectromelia virus-contaminated, imported mouse serum husbandry factors and the prevalence of age-related amyloidosis in mice cardioviruses: encephalomyocarditis virus and theiler's murine encephalitis virus diagnostic testing of mouse and rat colonies for infectious agents a novel presentation of clostridium piliforme infection (tyzzer's disease) in nude mice serodiagnosis of mice minute virus and mouse parvovirus infections in mice by enzyme-linked immunosorbent assay with baculovirus-expressed recombinant vp proteins hfrs outbreak associated with laboratory rats in uk the clinical chemistry of laboratory animals lack of commensal flora in helicobacter pylori-infected ins-gas mice reduces gastritis and delays intraepithelial neoplasia castration eliminates conspecific aggression in group-housed cd male surveillance mice toll-like receptor (tlr ) plays a major role in innate resistance in the lung against murine mycoplasma clostridium difficile-associated cecitis in guinea pigs exposed to penicillin clearance of pneumocystis carinii in mice is dependent on b cells but not on p-carinii-specific antibody phylogenetic relationships in the genus mus, based on paternally, maternally, and biparentally inherited characteristics genetic variants and strains of the laboratory mouse direct detection of indirect transmission of streptobacillus moniliformis rat bite fever infection fenner's veterinary virology failure to infect laboratory rodent hosts with human isolates of rodentolepis (=hymenolepis) nana detection and control of mouse parvovirus diminished reproduction, failure to thrive, and altered immunologic function in a colony of t-cell receptor transgenic mice: possible role of citrobacter rodentium helicobacter bilis infection accelerates and h. hepaticus infection delays the development of colitis in multiple drug resistance-deficient (mdr a −/−) mice helicobacter infection is required for inflammation and colon cancer in smad -deficient mice detection of giardia cysts by using the polymerase chain reaction and distinguishing live from dead cysts knockout mice: a paradigm shift in modern immunology a novel murine infection model for shiga toxin-producing escherichia coli infection-induced colitis in mice causes dynamic and tissue-specific changes in stress response and dna damage leading to colon cancer soiledbedding sentinel detection of murine norovirus infectious ectromelia. a hitherto undescribed virus disease of mice mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny clostridium perfringens and clostridium difficile-associated diarrhea pathology of the mouse from sexual attraction to maternal aggression: when pheromones change their behavioural significance an outbreak of pasteurella pneumotropica in genetically modified mice: treatment and elimination spontaneous necrotic enteritis in young rfm/ms mice prevention and treatment of ciliaassociated respiratory bacillus in mice by use of antibiotics a paralytic disease in nude mice associated with polyomavirus infection renewed interest in a difficult disease: clostridium difficile infections -epidemiology and current treatment strategies use of metronidazole in equine acute idiopathic toxaemic colitis identification and propogation of a putative immunosuppressive orphan parvovirus in cloned t cells corynebacterium species-associated keratoconjunctivitis in aged male c bl/ j mice genetic differences among c bl/ substrains isolation of helicobacter spp. from mice with rectal prolapses complex trait analysis in the mouse: the strengths, the limitations, and the promise yet to come fur mites induce dermatitis associated with ige hyperproduction in an inbred strain of mice, nc/kuj origins of inbred mice: proceedings of a workshop building a better mouse: one hundred years of genetics and biology retroelements in the mouse a mammalian herpesvirus cytolytic for cd + (l t +) t lymphocytes prairie dog model for antimicrobial agent-induced clostridium difficile diarrhea murine norovirus infection is associated with histopathological changes in immunocompetent hosts, but clinical disease is prevented by stat -dependent interferon responses transmission of systemic aa amyloidosis in animals infection of different strains of mice with lawsonia intracellularis derived from rabbit or porcine proliferative enteropathy the musculus-type y chromosome of the laboratory mouse is of asian origin lethal and severe coronary arteritis in dba/ mice induced by fungal pathogen, caws, candida albicans water-soluble fraction cre recombinase: the universal reagent for genome tailoring manual of microbiological monitoring of laboratory animals reduced fecundity and death associated with parvovirus infection in b-lymphocyte deficient mice phylogenetic analysis and description of eperythrozoon coccoides, proposal to transfer to the genus mycoplasma as mycoplasma coccoides comb. nov. and request for an opinion experimental analysis of risk factors for ulcerative dermatitis in mice citrobacter rodentium espb is necessary for signal transduction and for infection of laboratory mice colitis and colon cancer in waspdeficient mice require helicobacter species contamination of transplantable tumors, cell lines, and monoclonal antibodies with rodent viruses recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units cellular expression of murine ym and ym , chitinase family proteins, as revealed by in situ hybridization and immunohistochemistry oxytocin is required for nursing but is not essential for parturition or reproductive behavior report of the american institute of nutrition ad hoc committee on standards for nutritional studies moving forward with chemical mutagenesis in the mouse implementation of a pcr assay of pasteurella pneumotropica to accurately screen for contaminated laboratory mice natural cryptosporidium muris infection of the stomach in laboratory mice enhancement of cognitive function in models of brain disease through environmental enrichment and physical activity handbook of vertebrate immunology perturbations in cytokine gene expression after inoculation of c bl/ mice with pasteurella pneumotropica a transgenic approach for rna interference-based genetic screening in mice mouse pathology of laboratory rodents and rabbits naturally occurring murine norovirus infection in a large research institution from bench to cageside: risk assessment for rodent pathogen contamination of cells and biologics reduced body growth and excessive incisor length in insertional mutants mapping to mouse chromosome congenital eye defects in the mouse. i. corneal opacity in c black mice murine adenovirus infection of scid mice induces hepatic lesions that resemble human reye syndrome improved lactation in germfree mice following changes in the amino acid and fat components of a chemically defined diet a systematic method of breeder rotation for noninbred laboratory animal colonies genetic variation and phylogeography of central asian and other house mouse mice, including a major new mitochondrial lineage in yemen reproductive biology of the laboratory mouse helminth parasites of laboratory mice contemporary prevalence of infectious agents in laboratory mice and rats clostridium species. veterinary microbiology and microbial disease uteroovarian infection in aged b c f mice breast cancer: should gastrointestinal bacteria be on our radar screen? detection of pneumocystis carinii in a rat model of infection by polymerase chain reaction spontaneous nonneoplastic gastric lesions in female han:nmri mice, and influence of food restriction throughout life emerging views on the distinct but related roles of the main and accessory olfactory systems in responsiveness to chemosensory signals in mice zfngenome: a comprehensive resource for locating zinc finger nuclease target sites in model organisms treatment and eradication of murine fur mites: iii. treatment of a large mouse colony with ivermectin-compounded feed pheromonal induction of spatial learning in mice mucispirillum schaedleri gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents pheromone sensing in mice minor histocompatibility antigens: from the laboratory to the clinic vitamin a and retinoic acid in t cell-related immunity spontaneous pneumocystis carinii pneumonia in immunodeficient mutant scid mice. natural history and pathobiology lethal exacerbation of pneumocystis carinii pneumonia in severe combined immunodeficiency mice after infection by pneumonia virus of mice primary structure and phylogenetic relationships of glyceraldehyde- -phosphate dehydrogenase genes of free-living and parasitic diplomonad flagellates duodenal adenomas in balb/-c mice monoinfected with clostridium perfringens diffuse scaling dermatitis in an athymic nude mouse clostridium difficile typhlitis associated with cecal mucosal hyperplasia in syrian hamsters zifit (zinc finger targeter): an updated zinc finger engineering tool spatial distribution and stability of the eight microbial species of the altered schaedler flora in the mouse gastrointestinal tract comparison of the in vitro susceptibility of rodent isolates of pseudomonas aeruginosa and pasteurella pneumotropica to enrofloxacin antibody production in syphacia obvelata infected mice hyperkeratosis-associated coryneform infection in severe combined immunodeficient mice gastrointestinal microflora host specificity of cloned spironucleus muris in laboratory rodents the eae gene of citrobacter freundii biotype is necessary for colonization in transmissible murine colonic hyperplasia genetic and biochemical characterization of citrobacter rodentium sp. nov outbreak of group b streptococcal meningoencephalitis in athymic mice demylination and wasting associated wigh polyomavirus infection in nude (nu/nu) mice severe leukopenia and dysregulated erythropoiesis in scid mice persistently infected with the parvovirus minute virus of mice assessment of retinal degeneration in outbred albino mice assessment of retinal degeneration in outbred albino mice identification of widespread helicobacter hepaticus infection in feces in commercial mouse colonies by culture and pcr assay helicobacter infection decreases reproductive performance of il -deficient mice pheromone binding by polymorphic mouse major urinary proteins role of housing modalities on management and surveillance strategies for adventitious agents of rodents murine cytomegalovirus and other herpesviruses cytolethal distending toxin promotes helicobacter cinaediassociated typhlocolitis in interleukin- -deficient mice manual of microbiological monitoring of laboratory animals helicobacter bilis-induced inflammatory bowel disease in scid mice with defined flora helicobacter bilis/helicobacter rodentium co-infection associated with diarrhea in a colony of scid mice genetically determined murine models of immunodeficiency macrophage plasticity and polarization: in vivo veritas atlas of the mouse brain and spinal cord mouse genetics: concepts and applications a natural outbreak of transmissible murine colonic hyperplasia in a/j mice genetic variation among mouse substrains and its importance for targeted mutagenesis in mice genetic dissection of complex traits with chromosome substitution strains of mice a conditional knock out resource for the genome-wide study of mouse gene function ivermectin toxicity in young mice response of weanling random-bred mice to inoculation with minute virus of mice explant cultures for detection of minute virus of mice in infected mouse tissue in vivo studies with an 'orphan' parvovirus microphthalmia and associated abnormalities in inbred black mice mouse adenoviruses innate lymphoid cells -a proposal for uniform nomenclature a new name (pneumocystis jiroveci) for pneumocystis from humans major histocompatibility complex (mhc): mouse. els mouse urinary peptides provide a molecular basis for genotype discrimination by nasal sensory neurons the complete genome sequence of the carcinogenic bacterium helicobacter hepaticus mouse relapse model of clostridium difficile infection dendritic cells are the major antigen presenting cells in inflammatory lesions of murine mycoplasma respiratory disease chronic ulcerative dermatitis in black mice generation of knockout mice using engineered nucleases spontaneous lesions in control b c f mice and recommended sectioning of male accessory sex organs acute, lethal, natural killer cell-resistant myeloproliferative disease induced by polyomavirus in severe combined immunodeficient mice the ancestor of extant japanese fancy mice contributed to the mosaic genomes of classical inbred strains pattern recognition receptors and inflammation aggregation chimeras: combining es cells, diploid and tetraploid embryos analysis of the immune response of hantaan virus nucleocapsid protein-specific cd + t cells in mice the pathogenesis of experimental infections of cryptosporidium muris (strain rn ) in outbred nude mice enterohepatic helicobacter species are prevalent in mice from commercial and academic institutions in asia, europe, and north america murine noroviruses comprising a single genogroup exhibit biological diversity despite limited sequence divergence antibiotic-induced shifts in the mouse gut microbiome and metabolome increase susceptibility to clostridium difficile infection effect of essential amino acid restriction on the growth of female c bl mice and their implanted bw adenocarcinomas fatal diarrhea in rabbits resulting from the feeding of antibiotic-contaminated feed modulation of the host microenvironment by a common non-oncolytic mouse virus leads to inhibition of plasmacytoma development through nk cell activation x-ray-induced mutations in mouse embryonic stem cells ten years of the collaborative cross geneology of the inbred strains: /svj is a contaminated inbred strain molecular biology, analysis, and enabling technologies: analysis of transgene integration nutrition toxicity evaluation of prophylactic treatments for mites and pinworms in mice serological and virological evidence of hantaan virus-related enzootic in the united states korean hemorrhagic fever in staff in an animal laboratory prolonged perturbations of tumour necrosis factoralpha and interferon-gamma in mice inoculated with clostridium piliforme acceleration and inhibition of puberty in female mice by pheromones spontaneous pseudopregnancy in mice male management: coping with aggression problems in male laboratory mice phylogeny of trichomonads based on partial sequences of large subunit rrna and on cladistic analysis of morphological data il- deficiency prevents development of a non-t cell non-b cell-mediated colitis provitamin a metabolism and functions in mammalian biology genetic variation in laboratory mice the mosaic structure of variation in the laboratory mouse genome a study of mouse strains susceptibility to bacillus piliformis (tyzzer's disease): the association of b-cell function and resistance a naturally occurring outbreak of mycobacterium avium-intracellulare infections in c bl/ n mice cecocolitis in immunodeficient mice associated with an enteroinvasive lactose negative e. coli pneumocystis carinii shows dna homology with the ustomycetous red yeast fungi kinetics of ectromelia virus (mousepox) transmission and clinical response in c bl/ j, balb,cbyj and akr inbred mice outbreaks of pneumocystis carinii pneumonia in colonies of immunodeficient mice draft genome sequences of the altered schaedler flora, a defined bacterial community from gnotobiotic mice pathology of genetically engineered mice chronic active hepatitis and associated liver tumors in mice caused by a persistent bacterial infection with a novel helicobacter species inflammatory large bowel disease in immunodeficient mice naturally infected with helicobacter hepaticus hyalinosis and ym /ym gene expression in the stomach and respiratory tract of s /svjae and wild-type and cyp a -null b , mice pathology of immunodeficient mice with naturally occurring murine norovirus infection reoviridae protozoa initial sequencing and comparative analysis of the mouse genome new building, old parasite: mesostigmatid mites -an ever-present threat to barrier facilities genetic diversity of pneumocystis carinii derived from infected rats, mice, ferrets, and cell cultures spontaneous wasting disease in nude mice associated with pneumocystis carinii infection respiratory disease and wasting in athymic mice infected with pneumonia virus of mice arthropods manual of microbiological monitoring of laboratory animals genetic and histochemical studies on mouse spleen black spots status and acces to the collaborative cross population helminths natural and experimental helicobacter infections monitoring sentinel mice for helicobacter hepaticus, h rodentium, and h bilis infection by use of polymerase chain reaction analysis and serologic testing rapid onset of ulcerative typhlocolitis in b . p -il tm cgn (il- −/−) mice infected with helicobacter trogontum is associated with decreased colonization by altered schaedler's flora estrus-inducing pheromone of male mice. transport by movement of air endonucleases: new tools to edit the mouse genome replication of a norovirus in cell culture reveals a tropism for dendritic cells and macrophages murine norovirus: a model system to study norovirus biology and pathogenesis addgene: the bank that gives points for (plasmid) deposits microbiological contamination of laboratory mice and rats in korea from streptobacillus moniliformis -a zoonotic pathogen. taxonomic considerations, host species, diagnosis, therapy, geographical distribution an enzyme-linked immunosorbent assay (elisa) for the detection of antibodies to pasteurella pneumotropica in murine colonies sgrnacas : a software package for designing crispr sgrna and evaluating potential off-target cleavage sites inhibition of tnf-alpha, and nf-kappab and jnk pathways accounts for the prophylactic action of the natural phenolic, allylpyrocatechol against indomethacin gastropathy adaptive immunity restricts replication of novel murine astroviruses chemical-induced atrial thrombosis in ntp rodent studies nucleic acid-sensing toll-like receptors are essential for the control of endogenous retrovirus viremia and erv-induced tumors recent applications of engineered animal antioxidant deficiency models in human nutrition and chronic disease dissecting the effects of mtdna variations on complex traits using mouse conplastic strains a genomic update on clostridial phylogeny: gram-negative spore formers and other misplaced clostridia effective eradication of pinworms (syphacia muris, syphacia obvelata and aspiculuris tetraptera) from a rodent breeding colony by oral anthelmintic therapy possible allelic structure of igg a and igg c in mice one-step generation of different immunodeficient mice with multiple gene modifications by crispr/cas mediated genome engineering key: cord- -j kg qf authors: jones, samuel l.; blikslager, anthony t. title: disorders of the gastrointestinal system date: - - journal: equine internal medicine doi: . /b - - - / - sha: doc_id: cord_uid: j kg qf nan however, the abdominal wall is too rigid to allow effective palpation of intraabdominal structures. abdominal auscultation is particularly useful for assessing the motility of the large intestine. progressive motility of the small intestine, conversely, is difficult to distinguish by auscultation from nonprogressive motility. the distinct character of the borborygmi produced during propulsive contractions of the cecum and ascending colon allow evaluation of the frequency and strength of retropulsion and propulsion. propulsive contractions of the cecum and ventral colon occur every to minutes and give rise to prolonged rushing sounds heard over long segments of intestine. retropulsive sounds presumably are similar to propulsive sounds, but they occur less frequently. the distinction of propulsion from retropulsion is not important clinically because both types of contractions signify normal motility. inter-and intrahaustral mixing contractions produce nonspecific sounds of fluid and ingesta movement that are difficult to distinguish from other borborygmi, such as small intestinal contractions or spasmodic contractions. auscultation over the right flank and proceeding along the caudal edge of the costal margin toward the xiphoid allows evaluation of the cecal borborygmi. auscultation over a similar area on the left side allows evaluation of the pelvic flexure and ascending colon. typical progressive borborygmi heard every to minutes on both sides of the abdomen indicate normal motility of the cecum and ascending colon. less frequent progressive sounds may indicate a pathologic condition of the large intestine or may result from anorexia, nervousness (sympathetic tone), or pharmacologic inhibition of examination of patients with disease of the gastrointestinal tract must include evaluation of the metabolic and cardiovascular status of the patient, because acute conditions of the proximal or distal intestinal tract have the potential to lead to endotoxemia and sepsis. examination of the cardiovascular system (heart, peripheral pulse, and mucous membranes), lungs, and abdomen is essential to detect clinical signs of systemic inflammation from endotoxemia, coagulation disorders, dehydration, ileus, shock, and other abnormalities resulting from injury to the small or large intestine. chapter . covers clinical signs of systemic inflammation from endotoxemia and sepsis. one performs the physical examination of the abdomen primarily by auscultation, transabdominal ballottement, and transrectal palpation. abdominal distention often indicates distention of the large intestine; however, small intestinal distention also can cause visible abdominal distention if a large proportion of the small intestine is involved. one can perform abdominal palpation in neonatal foals; after several weeks of age, motility (i.e., α -adrenergic agonists such as xylazine). [ ] [ ] [ ] absolute absence of any auscultable borborygmi suggests abnormal motility and indicates ileus resulting from a serious pathologic condition but is not specific to any segment of the intestine. , if borborygmi are audible but progressive sounds are not detectable, determining whether a significant abnormality exists is difficult. borborygmi heard more frequently than normal may result from increased motility following feeding; from excessive stimulation from irritation, distention, or inflammation; or after administration of parasympathomimetic drugs such as neostigmine. large intestinal motility increases in the early stages of intestinal distention regardless of the site. mild inflammation or irritation of the large intestinal mucosa also can stimulate motility. parasympathomimetic drugs stimulate contractions and auscultable borborygmi in the large intestine; however, an increase in parasympathetic tone may result in segmental contractions, which actually inhibit progressive motility. one can detect sand or gravel in the large intestinal ingesta by auscultation behind the xiphoid process. one can hear sand or gravel particles grinding together during progressive contractions of the ascending colon. the presence of sand in the ingesta becomes clinically detectable by auscultation or fecal sedimentation before the amount of sand is enough to produce clinical signs of pain or irritation (diarrhea). if progressive contractions are audible without hearing sand sounds, clinically important quantities of sand likely are not present. if the frequency of progressive contractions is low or absent, detecting sand by auscultation is difficult. percussion of the abdomen during auscultation can reveal gas in the large intestine. the characteristic ping produced by simultaneous digital percussion and auscultation over a gas-filled viscus often is associated with abnormal accumulation of gas under pressure. this technique is particularly useful in foals, ponies, and miniature horses because of the limitations of palpation per rectum. one can use transabdominal ballottement to detect large, firm masses or an abnormal volume of peritoneal fluid. the usefulness of this technique is usually limited to animals too small to palpate per rectum. one can detect soft tissue masses or fetuses by bumping the structures with a hand or fist. if excessive peritoneal fluid is present, one can generate a fluid wave by ballottement; however, this technique is not as useful in horses older than weeks because the abdominal wall is rigid. transrectal palpation is the most specific physical examination technique for investigation of intestinal disease and is particularly valuable when evaluating obstructive diseases. , the primary objectives of transrectal palpation are to assess the size, consistency, and position of the segments of the large intestine; to determine the presence of any small intestinal distention; and to detect intraabdominal masses. evaluation of the wall thickness and texture and the mesenteric structures (blood and lymphatic vessels and lymph nodes) also may aid in diagnosis of large intestinal disease. the interpretation of transrectal palpation findings in light of clinical signs and laboratory results is an important diagnostic aid for developing appropriate treatment strategies for intestinal diseases manifested by abdominal pain. enlargement of one or more segments of large intestine detected by transrectal palpation provides evidence of obstruction at or distal to the enlarged segment. by systematically evaluating each segment, one may determine the site of obstruction. obstruction of the pelvic flexure, for instance, results in enlargement of the pelvic flexure and ventral colon, but the dorsal and descending colons are of normal size. enlargement of a segment of the large intestine usually is accompanied by abnormal consistency of the contents. one may distinguish accumulation of gas, fluid, or ingesta and may detect foreign bodies in palpable segments. accumulation of gas and fluid infers complete and acute obstruction, whereas accumulation of ingesta infers chronic and incomplete obstruction. accumulation of fluid usually indicates ileus. one must evaluate the consistency of the contents in light of the size of the segment; ingesta in the ventral colon of a dehydrated patient may be firm, but the size of the ventral colon will be normal. conversely, if the ingesta is firm because of a distal obstruction, the ventral colon will be enlarged. displacement of a segment of the large intestine may create an obstruction detectable by enlargement of the segment and accumulation of gas and fluid, even if the site of obstruction is not palpable. torsion of the ascending colon at the sternal and diaphragmatic flexures results in acute accumulation of gas and fluid proximal to the torsion, causing distention of the left dorsal and ventral colons. depending on the degree of torsion, the position of the ventral and dorsal colons may not be significantly abnormal. displacement of a segment of large intestine often results in incomplete obstruction, and the diagnosis relies solely on detection of the displaced segment in an abnormal position. the position of the displaced segment may not be palpable, and the diagnosis then relies on the inability to find the segment in a normal position. one must take care to ensure that the segment that appears to be displaced is not in a normal position but has become too small to palpate from a decrease in the volume of ingesta. the cecum, right dorsal and ventral colons, pelvic flexure, and descending colon are palpable in most horses. one should palpate the nephrosplenic space to detect the presence of intestine, usually pelvic flexure, entrapped within the ligament. small intestine is not normally palpable in the horse. distention is an indicator of ileus with gas or fluid retention, usually following a strangulating or nonstrangulating obstruction. strangulating obstructions result from conditions such as volvulus or torsion, lipoma, or entrapments. such conditions often are accompanied by severe pain, dehydration, peritoneal fluid changes, and a varying degree of gastric fluid accumulation. the small intestine in these cases is turgid and firm on palpation. one should assess the mesentery and wall thickness as for large intestinal disorders. careful palpation of the inguinal rings in stallions with small intestinal distention is crucial for determining inguinal herniation. evaluation of the wall thickness and mesenteric vessels can reveal venous congestion (mural edema and enlarged blood and lymphatic vessels) or inflammation (mural edema with normal vessels). disruption of arterial blood flow does not cause venous congestion, but the arterial pulse is not detectable. mesenteric tears may not be palpable, but the entrapped ischemic intestinal segment may be thickened with edema. one may detect acute or chronic inflammation with cellular infiltration of the intestinal wall as thickening of the wall without edema and also may note enlargement of mesenteric lymph nodes. one should interpret abnormalities in the wall or vessels in light of the size, consistency, and position of the segment of intestine and the clinical signs. several conditions involving small intestinal strangulating lesions do not necessarily cause abnormal rectal examination findings until the disease has been present for an extended time. these conditions include diaphragmatic herniae and epiploic foramen entrapments. peritoneal fluid analysis may be normal in these cases as well because the fluid is trapped in the thorax or in the cranial abdomen. surgery is usually necessary for diagnosis. nonstrangulating causes of small intestinal distention can be divided further into intraluminal and extraluminal obstructions. ileal impactions are probably the most common cause of intraluminal obstructions, and on rare occasions one can palpate the impaction in the upper right quadrant, near the ileocecal opening. intraluminal masses caused by lymphoma, eosinophilic enteritis, foreign bodies or ascarid impactions often lead to small intestinal distention and are usually indistinguishable from one another based on palpation alone. small intestine in these cases can be moderately to severely distended, depending on the degree of obstruction. extraluminal obstructions include abdominal masses, abscesses or tumors, and large colon displacement. one always should palpate the rest of the abdomen carefully to help rule out these causes. some cases of small intestinal distention result from a physiologic rather than a mechanical obstruction. ileus may result postoperatively or following inflammatory diseases of the bowel (proximal enteritis) or peritoneal cavity (peritonitis). the bowel is usually mildly to moderately distended and almost always is accompanied by significant amounts of accumulated gastric fluid. the small colon is easily distinguishable by the presence of normal fecal balls and an antimesenteric band. in cases of impaction of the small colon, a long, hard, tubelike structure is present in the caudal abdomen, and the band is palpable along the length. fluid stool is often present in the rectum in these cases, as is tenesmus. one can detect and carefully evaluate rectal tears by palpation. one can detect mural masses in palpable segments of intestine or mesentery; however, if a mass causes obstruction, one can detect the result of the obstruction in proximal segments of intestine even if the mass is unreachable. palpation of the mesenteric vessels may reveal thickening and thrombosis, which can lead to ischemia or infarction. one can perform visual inspection of the mucosa of the rectum and descending colon with a speculum or flexible endoscope and also can evaluate rectal tears or perforations, mural masses, strictures, or mucosal inflammation. one also can perform guided biopsy of the mucosa or masses. the obvious limitations are the amount of fecal material interfering with the examination and the distance of the lesion of interest from the anus. these techniques offer little advantage over palpation in many cases unless the patient is too small to palpate. examination of the oral cavity in cases of dysphagia or weight loss is a necessary part of the physical examination. one should adequately sedate the horse and should use a full-mouth speculum to allow palpation and visualization of all parts of the oral cavity. one should examine the area for abnormal dentition, foreign bodies, fractures, abscesses, and ulceration. the presence of fluid accumulation in the stomach indicates a decrease or absence in propulsive motions of the small intestine or obstruction of gastric outflow. decreased small intestinal motility may result from a functional or mechanical blockage. masses, feed impactions, or strictures in the pylorus or in the proximal duodenum may obstruct gastric outflow. one routinely assesses fluid accumulation in the stomach by siphoning off the gastric contents with a nasogastric tube and examining the fluid for amount, color, and any particular odor. normal fluid is green and may contain foamy saliva. the volume obtained by gastric lavage is usually less than l. large volumes of fluid (> to l) accumulate in the stomach of horses with proximal enteritis, and the fluid is foul smelling and often has an orange to yellow discoloration. if one suspects proximal enteritis, one can submit the fluid for culture and gram staining. salmonella sp. and clostridium sp. have been cultured in some cases. patients with postoperative ileus also frequently accumulate large amounts of gastric fluid. horses with section . examination for disorders of the gastrointestinal tract strangulating obstructions or luminal obstructions often accumulate moderate amounts of gastric fluid, but the amount is generally less than in horses with proximal enteritis or postoperative ileus. hemorrhage in the gastric fluid usually indicates devitalized small intestine, stomach wall, or severe gastric ulceration. fluid with large amounts of food material often indicates a gastric impaction, and one should lavage the stomach until obtaining no more ingesta. horses and foals with chronic gastric ulceration in the glandular mucosa of the stomach or in the duodenum may develop strictures and have fluid accumulate in the stomach. endoscopy or contrast radiography aids in diagnosing gastric outflow obstruction. evaluation of the hemogram is essential when one assesses conditions of the gastrointestinal tract. however, hematologic alterations associated with diseases of the gastrointestinal tract are often nonspecific, reflecting systemic response to inflammation, endotoxemia, or sepsis. neutrophilic leukocytosis and normochromic, normocytic anemia with or without hyperfibrinogenemia commonly are associated with chronic inflammatory conditions of the intestine. anemia from chronic blood loss occurs infrequently in adult horses because of the large iron stores and high concentrations of iron in their diet; usually anemia follows chronic inflammation, as do alterations in the leukon and plasma fibrinogen concentrations. plasma protein concentrations vary depending on gastrointestinal losses of albumin and globulin and elevation of globulin concentration from antigenic stimulation. protein-losing enteropathies may manifest predominantly as a hypoalbuminemia or may have a concurrent hypoglobulinemia. immunoglobulin quantification can be useful in selected cases; immunosuppression with low immunoglobulin m concentration has been shown to occur in some cases of lymphosarcoma. parasitic infections, especially strongylosis, may be characterized by elevated serum immunoglobulin g(t) concentration. significant alterations of the hemogram do not accompany acute disease of the intestine unless severe inflammation, dehydration, endotoxemia, or sepsis is present. during the early stages of endotoxemia, elevations in circulating concentrations of inflammatory mediators, epinephrine, and cortisol produce characteristic changes in the hemogram. leukopenia, with neutropenia and a left shift, toxic changes in the neutrophil cytoplasm, and lymphopenia occur commonly. hemoconcentration and hyperfibrinogenemia are also common. thrombocytopenia and other coagulopathies are also features of endotoxemia. indeed, thrombocytopenia may be the earliest indicator of sepsis. endotoxemia and circulating mediators of inflammation activate the coagulation cascade, causing a hypercoagulable state that can lead to consumption of coagulation factors and coagulation defects manifested as elevated prothrombin time, partial thromboplastin time, fibrin degradation products, and bleeding time, and reduced activity of antithrombin iii. [ ] [ ] [ ] neutrophilic leukocytosis occurs during the later stages of endotoxemia. the most common serum biochemical abnormalities with diseases of the large or small intestine are electrolyte imbalances. serum calcium concentrations are often low with strangulating obstructions and acute inflammatory diseases. inflammation of the mucosa can disrupt electrolyte fluxes severely. diarrhea or gastric reflux greatly exacerbates the loss of sodium, potassium, calcium, magnesium and bicarbonate. ischemia of the intestine causing hypoxia and cellular damage may be reflected by an elevated serum phosphate concentration resulting from phosphate leakage from damaged cells. ischemia and cellular hypoxia in any segment of the intestine also causes a shift in energy metabolism to anaerobic glycolysis, resulting in increased production of lactate and elevated serum lactate concentration. reduced perfusion of peripheral tissues from hypotensive shock and intestinal ischemia can cause elevations in serum lactate. however, obstruction of the intestine during ischemia may result in absorption of lactate from the lumen. , anion gap is an indirect measurement of organic acid production during states of tissue hypoxia and is a reasonable estimate of serum lactate concentration. metabolic acidosis may accompany lactic acidemia, but an inconsistent association exists between the two, especially when mixed acid-base imbalances are present. , elevations of hepatic enzymes, specifically γ-glutamyltransferase, may occur with large colon displacements, duodenal strictures, or anterior enteritis. relative polycythemia from hemoconcentration or splenic contraction and changes in red blood cell deformability from hypoxia or hypocalcemia may increase blood viscosity. blood viscosity increases in patients with acute obstructive disease. hyperviscosity reduces perfusion of capillary beds, thereby exacerbating ischemia and tissue hypoxia. hyperviscosity is one manifestation (along with lactic acidemia, coagulopathies, and clinical signs of shock) of the pathophysiologic events that take place during acute inflammatory or vascular injury to the large intestine. laboratory tests designed to reflect the systemic effects of endotoxemia, ischemia, sepsis, and shock are important to design therapeutic strategies, and monitor response to therapy. abdominocentesis and analysis of peritoneal fluid (pf) is a diagnostic technique performed on many patients with disease of the gastrointestinal tract. one can quantitate cytologic examination of pf; white blood cell and red blood cell counts; protein, fibrinogen, lactate, phosphate, and glucose concentrations; lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity; and ph. the results of pf analysis may help establish a specific diagnosis but more importantly may reflect inflammatory, vascular, or ischemic injury to the intestine requiring surgical intervention. pf reflects a sequence of events that takes place during acute vascular injury to the intestine. the pf protein concentration first increases, followed by an increase in the red blood cell count and fibrinogen concentration. a transudative process resulting from vascular congestion and increased endothelial permeability allows small macromolecules (albumin) to escape into the pf, followed by larger macromolecules (globulin and fibrinogen), and finally diapedesis of cells (red blood cells, then white blood cells). , if ischemic inflammation of the intestine and visceral peritonitis occur, an exudative process ensues. severe inflammation of the intestine and visceral peritoneum causes large quantities of protein and white blood cells, primarily neutrophils, to escape into the pf. as damage to the bowel progresses, the protein concentration and red blood cell and white blood cell counts continue to rise. as the degree of irreversible damage to the intestine increases, the pf characteristics become more exudative. , eventually, bacteria begin to translocate across the intestinal wall and appear in the pf as the mucosal barrier breaks down. neutrophils predominate, and their cytoplasm becomes granulated, and döhle bodies often are visible. if perforation occurs, bacteria and particles of ingesta appear in the pf, and the neutrophils become degenerate, that is, pyknotic, with karyorrhexis, karyolysis, and smudge cells. elevated pf protein concentration is a sensitive indicator of early inflammation, whereas elevated red blood cell counts in the presence of normal white blood cell counts suggest vascular damage without significant tissue ischemia. elevation of the white blood cell count usually indicates severe tissue inflammation or intestinal injury. the gross color of the pf can be helpful in detecting injury and necrosis of the intestine. a serosanguinous appearance indicates vascular injury, whereas orange or brown-red indicates necrosis with the release of pigments such as hemosiderin. serial samples of pf are most useful in determining the nature and extent of damage to the intestine, but in many cases of ischemia, irreversible tissue damage has occurred by the time pf abnormalities appear. tissue hypoxia and ischemia cause a rapid elevation of pf lactate dehydrogenase, creatine kinase, and alkaline phosphatase activity and lactate concentration. , , phosphate concentration increases when cellular disruption occurs. pf enzyme activities, phosphate, and lactate concentration increase faster and higher than serum activities. [ ] [ ] [ ] pf ph and glucose concentration tend to decrease during intestinal ischemia, but not as low as in septic peritonitis. although biochemical alterations may provide early indicators of intestinal ischemia and necrosis, they are nonspecific and offer no advantage over conventional methods of pf analysis in many cases. pf alkaline phosphatase has been shown to arise predominantly from degenerating white blood cells, and elevations of other enzyme activities may occur with many inflammatory diseases. thus the specificity of many tests run on pf is questionable. however, in selected cases in which conventional pf analysis and physical examination do not provide sufficient information to develop a treatment plan, biochemical analysis of the pf may be useful. cytologically examined cells of the pf may reflect chronic inflammatory conditions of the large intestine, especially eosinophilic or lymphocytic processes. infectious and inflammatory conditions often cause increases in the neutrophil count and may be indistinguishable unless bacteria are visible. one also may detect neoplastic diseases by pf examination. chronic infection and inflammation may be associated with elevated pf protein and fibrinogen concentrations. culture of pf usually is required to distinguish bacterial infections from noninfectious inflammation unless bacteria are visible on cytologic examination. however, culture of pf is often unrewarding because factors that are found in inflammatory pf inhibit bacterial growth, and leukocytes phagocytose many bacteria in the pf. decreases in pf glucose concentrations (< mg/dl) and ph (< . ) are early indicators of a septic process. the glucose concentration and ph in the pf should approximately equal the blood glucose concentration and ph. a pf fibrinogen concentration greater than mg/dl also indicates bacterial infection. gross examination of the feces can provide information about digestion and transit time in the large intestine. large fiber particles in the feces represent poor mastication or poor digestion in the large intestine. small, mucuscovered, hard fecal balls indicate prolonged transit through the descending colon, whereas increased fluidity implies decreased transit time. feces containing sand or gravel are not necessarily abnormal. however, a significant amount of sand implies that large quantities are present in the colon. frank blood indicates substantial bleeding into the distal colon (right dorsal colon and/or small colon) from mucosal damage. laboratory analysis of the feces is performed frequently in cases of diarrhea. fecal cytologic examination and tests for occult blood detect mucosal inflammation, section . examination for disorders of the gastrointestinal tract erosion, or ulceration. severe inflammatory diseases in human beings, invasive bacterial infections in particular, have been shown to increase the shedding of leukocytes in the feces. a higher percentage of horses with salmonellosis and diarrhea have fecal leukocyte counts greater than cells per high power field than horses with negative fecal cultures for salmonella. these results suggest that high fecal leukocyte counts indicate salmonellosis in horses with diarrhea. however, the specificity of this test is probably low. low fecal leukocyte counts do not rule out salmonellosis. fecal occult blood tests detect blood in the feces, presumably from erosion or ulceration of the mucosa, but do not distinguish the source of the blood. large volumes of blood ( to l) given by nasogastric tube were required to produce a positive test for occult blood in the feces, but the amount of blood originating from the large intestine required to produce a positive test is unknown. a positive test implies significant hemorrhage into the gastrointestinal tract. newer, more sensitive tests detect not only occult blood but also degraded blood and may be useful to determine the site and quantity of blood loss. a positive test implies significant hemorrhage into the gastrointestinal tract. bacteriologic examination of the fecal flora has been used to quantitate specific bacterial species in the feces of horses with diarrhea. quantitation of clostridial species may be beneficial in diagnosing clostridial infection of the large intestine. tests to detect clostridial toxins in intestinal contents or feces are important to determine whether clostridia cultured from the feces are causing disease. the most common bacterial pathogens isolated from the feces of horses are salmonella and clostridium. the number of salmonella organisms isolated from the feces of horses with clinical salmonellosis is usually higher than from horses with asymptomatic infections. however, the volume of feces in many cases of acute diarrhea is high, and the concentration of salmonella organisms may be lower than would be expected, accounting for many false-negative fecal cultures. the sensitivity of fecal cultures for detecting salmonella infection may be as low as %. culture of five consecutive daily fecal samples is recommended to increase the sensitivity of the test. because salmonellae are intracellular organisms, culture of rectal scrapings or a rectal biopsy sample, along with fecal material, may increase the sensitivity of culture for detecting salmonella infection to %. one can perform a polymerase chain reaction assay on fecal samples to detect dna from salmonella sp. the polymerase chain reaction test is more sensitive than culture and is frequently positive in clinically normal horses that continuously shed small amounts of bacteria. polymerase chain reaction or immunologic tests also may detect clostridium perfringens and c. difficile exotoxins in the feces. qualitative fecal examination is a technique to detect nematode and cestode ova, protozoan oocysts, parasitic larvae, and protozoan trophozoites. a direct smear of fecal material is a rapid method to screen feces for ova and oocysts, to detect parasite larvae and trophozoites, and to observe motility of ciliates and parasite larvae. fecal flotation is a more sensitive technique for isolating and detecting ova and oocysts because the eggs are concentrated from the sample. zinc sulfate and sucrose solutions are often used to concentrate less dense ova and oocysts. zinc sulfate produces less distortion of trophozoites and larvae than sucrose solutions. fecal sedimentation is particularly appropriate for ciliates, giardia, and trichomonads. quantitative techniques such as the cornell-mcmaster method allow one to estimate the number of eggs per gram of feces and are most appropriate in monitoring parasite control programs. survey radiography of the normal esophagus is usually unrewarding but may be useful in horses with esophageal obstructions to determine the extent and location of the obstruction. one may detect foreign bodies or soft tissue masses, and in cases of esophageal rupture, one may see free air and ingesta in the tissues surrounding the esophagus and may observe pneumomediastinum. thoracic radiographs may be necessary to detect intrathoracic esophageal obstructions, megaesophagus, or cranial mediastinal masses causing extraluminal obstruction. one may use barium swallows or double-contrast esophagrams after resolution of the obstruction to determine whether a stricture or diverticulum or other underlying disorder is present. barium sulfate is the usual contrast medium and can be administered orally via a dose syringe or by nasogastric tube ( to ml of a % barium sulfate suspension or barium paste). oral administration is preferred for evaluation of swallowing and lesions in the proximal esophagus. administration of contrast using a nasogastric tube (preferably cuffed) allows for delivery of larger volumes of barium (up to ml) but should be performed without sedation if possible. one can follow administration of contrast material with air insufflation to create a double-contrast effect. if one suspects a rupture of the esophagus or if the likelihood of aspiration of the contrast material is high, one should use iodinated organic compounds in an aqueous solution as contrast material. contrast radiography may be the most definitive method for the diagnosis of primary megaesophagus or other functional disorders such as autonomic dysautonomia (grass sickness) affecting the esophagus. one should take care when interpreting esophageal radiographs if the horse is sedated. acepromazine or detomidine administration causes esophageal dilation in normal horses, especially after passage of a nasogastric tube. radiography of the adult equine abdomen is an effective technique in detecting radiodense material in the large intestine, such as enteroliths, sand, and metallic objects. , one survey demonstrated that radiography has . % sensitivity and . % specificity for diagnosing enterolithiasis. radiography also can be a useful tool for detecting sand accumulation in the colon that causes diarrhea or impactions ( figure . - ) and for monitoring resolution in medically treated horses. the large size and density of the adult abdomen precludes evaluation of soft tissue structures because the detail and contrast of the radiographs are usually poor. one is more likely to obtain diagnostically useful abdominal radiographs from small ponies and miniature horses than from full-size adult horses. accumulation of gas is visible on radiographs of adult horses, but distinguishing normal intestinal gas from obstruction is often difficult. horses should be fasted for to hours to reduce the amount of ingesta in the large intestine before radiography. abdominal radiography is more useful in foals than in adult horses. radiographs are more detailed and contrast can be good. radiographic evidence of gas distention in the large intestine may indicate large intestinal obstruction, and radiographic signs of displacement are often diagnostic. one may diagnose impactions, intussusceptions, foreign bodies, and other disorders with the aid of radiography. functional ileus may be difficult to distinguish from mechanical obstruction. , administration of contrast (barium sulfate % at ml/kg) via nasogastric tube increases the diagnostic capabilities of radiography. gastric ulceration also is recognizable with contrast radiography in the foal, although this is not as accurate a method as endoscopy. contrast administered retrograde via a -f foley catheter inserted into the rectum at a dose of up to ml/kg has excellent potential for diagnosing disorders of the small colon, transverse colon, and large colon in foals. ultrasonographic evaluation of the abdomen can add valuable information in cases of acute or chronic gastrointestinal disease. examination of the adult horse requires a . -to . -mhz transducer at minimum. one may use sector, linear, or curved linear transducers. clipping of the hair over the area to be examined, along with the application of isopropyl alcohol and ultrasound coupling gel, enhances evaluation. to evaluate the abdomen adequately, one must know the anatomic location and normal appearance of the individual organs. in the left cranial abdomen, one can assess the greater curvature of the stomach between the eleventh and thirteenth intercostal space, and one can use the spleen and the large splenic vein as landmarks. cases of gastric dilation from gas or impaction appear as an enlargement of the viewing area to cover greater than five rib spaces. one also can evaluate the stomach for intramural or extramural masses such as abscesses or for squamous cell carcinoma. the lesser curvature is not routinely visible. assessment of the small intestine should include evaluation for changes in thickness, motility, location, and visibility. one may find small intestinal loops easily in the left lower quadrant of the abdomen, but these normally are visible in other locations. one can visualize the duodenum consistently on the right side of the abdomen deep to the liver in the tenth to twelfth intercostal space or deep to the right kidney at the fifteenth to sixteenth intercostal space. mural thickening (> mm) may occur in cases of infiltrative or proliferative diseases, postoperative cases, enteritis, and paralytic or mechanical ileus. thickening of the small intestinal wall in foals, with or without the presence of gas shadows within the wall, should raise suspicions of clostridial enteritis. one can assess motility by monitoring a specific area for contractions over time. ultrasonography is an accurate method of distinguishing strangulating versus nonstrangulating disorders of the small intestine. strangulated small intestine has thicker small intestinal walls and larger intestinal diameter than in nonstrangulating disorders. strangulating lesions have decreased motility in the incarcerated segments with normal motility elsewhere. cases of paralytic ileus or nonstrangulating obstruction have a diffusely decreased peristalsis, but not to the degree observed with strangulating lesions. , one may diagnose some specific lesions of the small intestinal tract using ultrasonography. one may see ascarids in foals in cases of ascarid impaction and epiploic foramen entrapments as edematous loops of small intestine found in the right cranial abdomen. one may note small intestinal intussusceptions as targetlike lesions when viewed in cross sections. the presence of bowel loops, stomach, or liver in the thoracic cavity indicates the presence of herniation through the diaphragm and should be confirmed using radiography or surgical exploration. evaluation of the large intestine may be difficult because of the large amounts of gas within the lumen. however, certain disorders are readily identifiable via ultrasonography. one can assess the nephrosplenic ligament area for bowel entrapment in the left paralumbar fossa. in cases of entrapment, the spleen will be pulled away from the body wall and fluid or gas shadows will be observable dorsal to the spleen, obscuring the kidney, which is normally adjacent and abaxial to the spleen. small colon, small intestine, or pneumoperitoneum also may produce a gas shadow and obscure the kidney from view. sand impactions may appear as hyperechoic bands on the ventral abdominal wall. one may see ileocecal and cecocolic intussusceptions in the upper right paralumbar fossa. in cases of colitis, large, fluid-filled colons may be visible with or without intramural edema. one can find the right dorsal colon consistently abaxial to the liver, within the right thirteenth to fifteenth intercostal space and may be thickened (> mm) in cases of right dorsal colitis. evaluation of the abdomen always should include assessment of the peritoneal space for any evidence of an increased amount of pf or increased cellularity of the fluid as indicated by an increase in echogenicity. ultrasonography also can be useful in determining the ideal location for abdominocentesis. one also should evaluate the liver, kidneys, and spleen. one may detect choleliths, nephroliths, masses, abscesses, or enlargement of any of these organs. abscesses or tumors not associated with visceral organs may be difficult to visualize and interpret via ultrasonography. although more commonly used to diagnose lameness and musculoskeletal problems, nuclear scintigraphy has several uses for evaluation of the gastrointestinal tract. scintigraphy is now available at most universities and many private referral hospitals. one must use proper isolation protocols and waste disposal techniques strictly. the procedure requires special gamma cameras and the injection of radioactive materials into the bloodstream. one can use one of two methods: injection of technetium- m methylene diphosphonate ( m tc-mdp) directly into the blood or injection of m tc-labeled leukocytes. labeling of leukocytes involves aseptically collecting heparinized blood samples, isolating the buffy coat, and mixing those leukocytes with a radioactive dye ( m tc hexamethylpropyleneamine oxime, or m tc-hmpao) in vitro. one then reinjects the labeled leukocytes and obtains images. the principle of nuclear scintigraphy then lies in increased uptake of the dye or the white blood cells into areas of inflammation. one of the most common uses of nuclear scintigraphy in evaluating the gastrointestinal tract is diagnosis of dental disease. scintigraphy using m tc-mdp proved to be more sensitive in cases of dental disease than was radiography. scintigraphy was slightly less specific, however, and therefore should be used with radiography or computed tomography for ultimate accuracy. scintigraphy using radiolabeled white blood cells can support a diagnosis of right dorsal colitis in the horse. images taken of the abdomen hours after injection showed an increased linear uptake of leukocytes in the region of the right dorsal colon in horses with right dorsal colitis compared with normal horses. this technique also may prove useful for diagnosing intraabdominal abscesses in the horse. other uses of nuclear scintigraphy include evaluation of metastasis of abdominal tumors to bony areas, assessment of biliary kinetics, and determination of gastric emptying times. [ ] [ ] [ ] endoscopy endoscopic examination of the gastrointestinal tract begins with evaluation of the pharyngeal area by examination for any signs of collapse or dysfunction. one should evaluate the ability of the horse to swallow. the floor of the pharynx should be clean and free of feed material and foreign bodies. one can examine the oral cavity with the horse under heavy sedation or anesthesia and with the help of a full-mouth speculum. one can examine the teeth for any irregularities, obvious cavities, sharp points, or hooks and the hard and soft palpate for completeness and any evidence of ulceration, masses, or foreign bodies. one should use a -m flexible fiberoptic endoscope to examine the esophagus, which is accomplished best by passing the endoscope into the stomach and viewing the esophagus as one withdraws the endoscope while dilating the lumen with air. the esophageal mucosa normally should be a glistening, light pink color. ulceration can occur with cases of choke, reflux esophagitis or in horses that have had an indwelling nasogastric tube. erosions may be punctate, linear, or circumferential. one should evaluate carefully for any ulcers to ensure that no areas of perforation through the entire thickness of the esophageal wall exist. distinguishing normal peristaltic contractions from areas of stricture requires observation of the area and its motility over time. one also may note diverticula as outpouchings of the mucosa, sometimes associated with a stricture distally. megaesophagus, although rare, appears as a generalized dilation of the esophagus. one may detect food or foreign body impactions of the esophagus via endoscopy. one always should reevaluate the esophagus after removing any obstruction to detect the presence of complications (ulceration, rupture) or initiating causes (strictures, diverticula, and masses). a -m flexible endoscope also allows examination of the stomach. the horse should be fasted for at least hours before endoscopy. one can examine the cardia and fundus easily, as well as the margo plicatus. the squamous mucosa should resemble the esophageal mucosa. the glandular mucosa should be glistening red and may have a reticulated pattern. one should carefully examine for evidence of ulceration or masses. one can obtain transendoscopic biopsy material easily from esophageal, pharyngeal, or gastric masses, and because the biopsy size will be small, one should take several samples for histopathologic examination. pharmacologic agents (bethanechol) to empty the stomach and provide complete visualization of the entire fundic region, the pylorus, and the duodenum may be useful. for a complete description of gastroscopy and evaluation of gastric and gastroduodenal ulceration, please refer to chapter . . d-glucose or d-xylose absorption tests are useful in determining malabsorption of carbohydrates from the small intestine in horses. the protocol for absorption tests using either carbohydrate is similar. the horse should be fasted for to hours before testing. increased periods of fasting actually have been shown to decrease absorption of d-xylose and interfere with results. one administers a dosage of . to g/kg of d-glucose or d-xylose via a nasogastric tube. administration of sedatives may increase the blood glucose levels falsely and interfere with gastrointestinal transit times. one then collects blood samples to measure glucose or xylose concentrations at , , , , , , , , and minutes after administration. one may take additional samples up to hours after dosing if the results are questionable. one should measure glucose in blood samples collected with sodium fluoride as an anticoagulant and measure xylose in samples collected in heparinized plasma. a normal d-glucose absorption test, also known as an oral glucose tolerance test, should have a peak between and minutes, and this peak should be greater than % above the resting glucose value. complete malabsorption is defined as a peak less than % above the resting levels, and partial malabsorption is defined as a peak between % and % above the resting level. one must keep in mind that gastric emptying, gastrointestinal transit time, length of fasting, cellular uptake and metabolism, and endocrine function influence glucose absorption curves. malabsorption demonstrated by the oral glucose tolerance test is sensitive but not specific. diseases that may cause a lowered or delayed peak include infiltrative lymphosarcoma, inflammatory bowel disease (lymphocytic-plasmacytic or eosinophilic), cyathostomiasis, chronic colitis (salmonella sp.), multisystemic eosinophilic epitheliotropic disease, food allergies, and small intestinal bacterial overgrowth. d-xylose absorption tests have some advantages over the oral glucose tolerance test because xylose is not metabolized in the small intestinal mucosa and insulin does not influence its absorption. gastric and intestinal motility, intraluminal bacterial overgrowth, and renal function still influence xylose absorption, because the kidneys clear xylose. the other main drawback to d-xylose is that it is generally available only in research settings. however, xylose measurements are available at most major universities. a normal d-xylose absorption curve should peak between and mg/dl at to minutes after dosing. decreased xylose absorption can occur in horses with inflammatory bowel disease, lymphosarcoma, multisystemic eosinophilic epitheliotropic disease, cyathostomiasis, extensive small intestinal resections, and any cause of villous atrophy. maldigestion is a common occurrence in foals with diarrhea. bacteria (especially clostridium sp.) and viruses (especially rotavirus or coronavirus) may invade and destroy the villous epithelial cells that manufacture lactase and other disaccharidases, resulting in an inability to digest lactose. in this case, continued ingestion of the mare's milk may cause an osmotic diarrhea, which may exacerbate the underlying enterocolitis. one can perform lactose tolerance testing to assess the degree of maldigestion. one administers d-lactose at g/kg as a % solution via nasogastric tube and measures glucose concentrations in the blood at , , , , , , , , and minutes. a normal curve shows doubling of glucose levels compared with baseline by minutes after administration. accurate measurement of gastric emptying can be difficult to assess. several methods are currently available. multiple diagnostic imaging techniques have been used to study gastric emptying times. one can use contrast radiography to assess gastric emptying in foals. in the normal foal, barium remains in the stomach for varying amounts of time, but a significant amount should be gone within hours. gastric emptying of solid, nondigestible, radiopaque markers also has been used in adult horses and ponies, but the results were variable and unpredictable even in the normal horse. nuclear scintigraphy is used commonly in human beings to measure gastric emptying and can be used in horses where available. this technique requires oral administration of m tc pentenate ( mci), and serial images taken of the cranial abdomen. the tracer is usually not visible minutes after administration in normal horses. alternatively, if nuclear scintigraphy is not available, one can use acetaminophen absorption testing as an indirect determination of gastric emptying. , one performs this test by administering mg/kg of acetaminophen orally and measuring subsequent blood values and calculating the time to reach maximum serum concentrations and the absorption constant. in human beings, the proximal small intestine absorbs almost all of the acetaminophen. the median time to reach peak plasma levels using acetaminophen absorption in horses was . minutes. one often requires histopathologic examination of tissues from the intestine to diagnose chronic inflammatory, infiltrative, or neoplastic conditions, and such examination can be useful in evaluating the extent of injury after obstruction or ischemia. rectal mucosal biopsies are easy to collect with few complications. however, to collect a full-thickness biopsy of the intestine requires a surgical approach (flank or ventral midline approach). laparoscopy offers a safer technique to observe the large intestine and other abdominal structures. one can obtain biopsies of masses, lymph nodes, mesentery, or intestinal serosa via laparoscopy and mucosal biopsies of the upper gastrointestinal tract via endoscopy. other diagnostics, specifically laparoscopy and computed tomography, are available but require specialized equipment and personnel with specific training. flexible or rigid endoscopes used for laparoscopic evaluation of the abdomen allow for visualization of visceral organs and potentially for collection of biopsy material from masses or organs. full-thickness biopsies of the intestines are not routinely possible through the laparoscope and usually require flank or ventral midline laparotomy. the laparoscopic procedure can be done in the standing or recumbent horse. advantages of this technique over a flank or ventral midline celiotomy include smaller incisions, less healing time, and less procedure time. disadvantages include the large amount of equipment needed, skill involved, and the limitation as a diagnostic modality, rather than a treatment. clinical applications of diagnostic laparoscopy include rectal tears, percutaneous abscess drainage, assessment of adhesions, displacements, and integrity of the serosa of various bowel segments, and biopsy of abdominal masses. computed tomography scans are available at several universities across the country. they have been used frequently to evaluate dental disease and may be useful in evaluating tumors and masses of the head, larynx, pharynx, and proximal esophagus. computed tomography also has promise for evaluating abdominal disorders in foals. most equipment can accommodate up to lb. restrictions to computed tomography as a diagnostic aid include expense, availability, expertise, and weight and size limitation. permeability increases, cells are recruited rapidly into the tissue, plasma protein cascades are activated, and a myriad of soluble products are released that coordinate the response, trigger innate and adaptive immunity, and mobilize reparative elements. although the cellular and vascular response and the secreted mediators of inflammation are important for killing pathogens and limiting invasion of injured tissues by commensal organisms, they can be damaging to host cells and proteins if not tightly regulated. thus if the inciting stimulus is not eliminated quickly, the inflammatory response itself causes significant tissue injury. the mechanism regulating inflammation has been the focus of much research to identify therapeutic targets to modulate the damage to host tissues during many gastrointestinal diseases. recent work has provided some of the molecular and cellular details of this complex physiology and has led to novel therapeutic strategies for treating inflammation. the gastrointestinal epithelium interfaces with a luminal environment inhabited by potentially hostile microbial organisms. the epithelium presents a physical barrier to invasion by the flora of the gastrointestinal tract, consisting of the apical cellular membrane, intercellular tight junctions the permeability of which is highly regulated, and a secreted layer of mucus. breaching of the mucosal barrier by invading pathogens generates potent soluble and neural signals that initiate an inflammatory response. conceptually, the epithelium can be thought of as a sensory organ detecting pathogen invasion to trigger an appropriate host defense and reparative response. noninfectious mucosal injury or invasion of epithelial cells by pathogenic organisms such as salmonella activates synthesis of proinflammatory chemokines (chemoattractants) by epithelial cells and triggers a robust influx of neutrophils into the tissue within hours of the damage. of the chemoattractants produced by epithelium, interleukin- (il- ) has a particularly important role in initiating inflammation by recruiting neutrophils from blood [ ] [ ] [ ] and regulating neutrophil migration through tissue matrix adjacent to epithelium. , bacteriaderived formylated chemotactic peptides also act as potent chemoattractants that are fully capable of stimulating a robust inflammatory response in the intestine if the epithelial barrier permits the diffusion of the peptides across the mucosa. epithelial cells activated during infection produce cytokines such as tumor necrosis factor α (tnf-α), arachidonic acid metabolites, and other proinflammatory mediators that activate recruited leukocytes. bacterial products, particularly lipopolysaccharide and other cell wall components, are potent activators of leukocytes recruited into the tissue. once the inflammatory response has been initiated, tnf-α, il- β, and other proinflammatory products of neutrophils, monocytes, mast cells, and epithelial cells amplify the inflammatory response. the enteric nervous system has a key role in sensing and regulating inflammatory responses in the intestine. for example, clostridium difficile toxin a activates a neural pathway that triggers mast cell degranulation and neutrophil influx into the tissue. , blockade of this neural pathway is sufficient to abolish the profound inflammatory response induced by toxin a and many of the effects of toxin a on enterocyte secretion. other pathogens and immune-mediated hypersensitivity reactions similarly stimulate inflammation by mechanisms that involve the enteric nervous system. thus the epithelium interacts in a highly complex manner with the intestinal milieu, the enteric nervous system, and inflammatory cells to regulate the tissue response to injury and infection. resident macrophages located in the lamina propria, submucosa, and intestinal lymphoid organs are among the first cells beyond the epithelium to respond to infection or injury. macrophages are activated by bacterial products via pattern recognition receptors and begin to produce proinflammatory molecules important for recruiting and activating neutrophils and monocytes. pattern recognition receptors recognize microbial products ranging from lipopolysaccharide to peptidoglycan and even cpg-containing bacterial dna and signal the invasion by pathogens. of the pattern recognition receptors, the lipopolysaccharide receptor complex is perhaps the best defined. lipopolysaccharide activates macrophages via the cd -toll-like receptor complex to initiate transcription of the inflammatory cytokines tnf-α and il- β, which synergize with lipopolysaccharide to amplify the macrophage response. lipopolysaccharide, particularly in concert with inflammatory cytokines, stimulates macrophages to produce copious amounts of nitric oxide, which is microbicidal and vasoactive. nitric oxide and other nitrogen radicals react with reactive oxygen intermediates (rois) to produce some of the most toxic molecules of the host defense system: the peroxynitrites. il- is produced as well to recruit neutrophils. as the response progresses, other inflammatory mediators, particularly the arachidonic acid-derived lipids, are produced. these lipids have potent vasoactive effects and are important stimuli of endothelial cells, neutrophils, and platelets. four important changes occur in the intestinal vasculature during inflammation: . alteration of blood flow . increased vascular permeability . increased adhesiveness of endothelial cells, leukocytes, and platelets . exposure of the basement membrane and activation of the complement, contact, and coagulation cascades a wide range of mediators alters blood flow during intestinal tract inflammation, from gasses such as nitric oxide (a major vasodilator of the intestinal vasculature) to lipids (prostaglandins, leukotrienes, thromboxanes, and platelet-activating factor), cytokines, bradykinin, histamine, and others. the major sources for these mediators include activated leukocytes, endothelial cells, epithelial cells, and fibroblasts. the primary determinant of blood flow early in inflammation is vascular caliber, which initially decreases in arterioles, but then quickly changes to vasodilation coincident with opening of new capillary beds, increasing net blood flow. the increase in blood flow is short lived, for the viscosity of the blood increases because of fluid loss and tissue edema through leaky capillaries. leukocyte margination, platelet adhesion to endothelial cells and exposed matrix, and areas of coagulation protein accumulation further decrease local circulation. inflammatory mediator actions on the endothelial cells initially increase vascular permeability. histamine, leukotrienes, platelet-activating factor, prostaglandins, bradykinin, and other mediators stimulate endothelial cell contraction, and interendothelial gaps form. , this stage of increased vascular permeability is readily reversible. concurrently, mediators such as the cytokines tnf-α and il- β induce a structural reorganization of the interendothelial junctions, resulting in frank discontinuities in the endothelial monolayer. cytokines also stimulate endothelial cells to express adhesion molecules that support adhesion of leukocytes and platelets, leading to the next and perhaps most devastating event. leukocytes (primarily neutrophils) and platelets adhere to exposed basement membranes and activated endothelial cells. adherent neutrophils and platelets then are exposed to the mediators of inflammation present in the surrounding milieu, which activates the cells to release oxidants and proteases (particularly elastase) that injure the endothelium and have the potential to cause irreparable harm to the microvasculature. [ ] [ ] [ ] marginated neutrophils begin to transmigrate between endothelial cells (as described in later sections), and if their numbers section . pathophysiology of gastrointestinal inflammation are large enough, they disrupt the integrity of the interendothelial junctions, worsening the vascular leakage. conceptually, these stages of enhanced vascular permeability can be thought of as a mechanism to allow plasma proteins to enter the tissues and to potentiate the critical influx of leukocytes into tissues. however, if not regulated precisely, alterations in hydrostatic and oncotic forces and irreversible damage to the vascular bed may have devastating consequences. moreover, inappropriate activation of plasma protein cascades and leukocytes by activated endothelium and exposed matrix proteins can contribute to systemic inflammation (systemic inflammatory response syndrome; see chapter . for more information) characterized by hypotension, generalized vascular leak syndrome, and multiorgan dysfunction, which may be fatal. phosphodiesterase inhibitors reduce endothelial permeability in ischemia-reperfusion injury and other models of inflammation-induced vascular leakage , by increasing endothelial tight junction integrity and thus may be a viable therapeutic strategy to prevent or reduce the permeability alterations associated with inflammation. endothelial cells respond to products of activated epithelial cells and macrophages in the intestinal tissue to recruit cells and humoral mediators of inflammation into the tissue. activated endothelial cells display a range of molecules critical for neutrophil and platelet adhesion. intercellular permeability increases to expose basement membrane proteins that trigger humoral defense systems (complement, coagulation, and contact system cascades) and to provide access for these macromolecules to the tissue. endothelial cells are an important source of inflammatory mediators that amplify the response and vasoactive substances (particularly nitric oxide) that alter blood flow. infection or injury to the gastrointestinal mucosa causes an influx of leukocytes from the blood that lay the foundation of the inflammatory response. neutrophils, being the first to arrive during inflammation, have a dominant role in the acute response. within minutes, neutrophils are recruited into the tissue where they are activated to release products that not only are proinflammatory and lethal to pathogens but also may damage host cells and tissues. not surprisingly, much attention has been paid to the role of neutrophils in the pathophysiology of many inflammatory conditions. neutrophil depletion is protective in many models of gastrointestinal inflammatory disease. of interest to clinicians, blockade of neutrophil migration into inflamed tissues prevents many of the pathophysiologic events associated with infectious enteritis, ischemia-reperfusion injury, and other gastrointestinal diseases. , [ ] [ ] [ ] [ ] [ ] neutrophil transendothelial migration is a multistep process that is temporally and spatially regulated and has a degree of cell type specificity ( figure . - ) . the predominant sites of neutrophil transendothelial migration are in the postcapillary venules and, in some tissues, the capillaries. endothelial cells in these vessels respond to cytokines and other soluble signals by expressing molecules that promote neutrophil adhesion and transmigration, including selectins and counterreceptors for integrins. as neutrophils flow through these vessels, they are first tethered to activated endothelium. tethering is mediated by selectin molecules expressed on neutrophils (l-selectin) and on activated endothelial cells (p-and e-selectins) that bind to p-selectin glycoprotein ligand- (psgl- ), e-selectin ligand- (esl- ), and other mucin counterreceptors. , tethering functions to increase the exposure of the neutrophil to activating chemokines presented on the surface of the endothelial cells. stimulation of neutrophils by il- and other chemokines activates the second step of transendothelial migration. chemokine binding to their receptors on the neutrophil generates signals that activate the binding of integrin adhesion receptors to their ligands, called intracellular adhesion molecules or vascular cell adhesion molecules expressed on endothelial cells in inflamed mucosa. integrin ligation to cellular adhesion molecules arrests the tethered neutrophils, resulting in firm adhesion to the endothelium. of the integrins expressed on neutrophils, the β integrins have a particularly important role in transendothelial migration. calves and human beings with a disorder known as leukocyte adhesion deficiency illustrate the requirement for β integrinmediated adhesion in neutrophil function. leukocyte adhesion deficiency results from an autosomal recessive trait causing the lack of the β integrin expression. the neutrophils from these individuals cannot migrate into most tissues and do not function normally, resulting in poor tissue healing and profound susceptibility to infection, especially at epithelial barriers. , other integrins also have a role in transendothelial migration. β integrins mediate transendothelial migration in some cells and seem to be particularly important for mediating emigration of monocytes into many tissues. following this firm adhesion step, neutrophils migrate through the endothelium along a chemotactic gradient of il- and other chemoattractants such as leukotriene b . , , neutrophils migrate across the endothelial monolayer at intercellular junctions via a mechanism involving a series of integrin-ligand interactions mediated by β and β integrins and other adhesion molecules that is generally capable of maintaining the integrity of the endothelial barrier. however, massive flux of neutrophils through the endothelium alters endothelial tight junctions and injures the basement membrane, resulting in increased endothelial permeability to molecules as large as plasma proteins and even endothelial cell detachment from the basement membrane. , nonintegrin molecules such as platelet/endothelial cell adhesion molecules (pecams) also are involved in transendothelial migration of neutrophils. homotypic binding of pecams on adjacent endothelial cells form part of the intercellular junction. neutrophils express an integrin of the β family that can bind pecam, and via sequential binding of β integrins to pecam, the neutrophil can , and other proinflammatory mediators. endothelial cells stimulated by inflammatory mediators produce chemoattractants (such as il- ) and display adhesion molecules that promote neutrophil emigration. the three steps of neutrophil (polymorphonuclear [pmn]) emigrationcapture/rolling (mediated by selectins), adhesion (mediated by β integrins), and transendothelial migration (mediated by integrins and platelet/endothelial cellular adhesion molecule [pecam])-occur on activated endothelium. chemoattractant molecules, such as il- trigger neutrophil emigration. in inflamed tissues, cytokines (il- β and tnf-α) and a variety of other proinflammatory mediators stimulate the neutrophil oxidase complex to produce reactive oxygen intermediates (rois; o and h o and their derivatives). activated neutrophils degranulate to release proteases and other hydrolases, cationic peptides (defensins), myeloperoxidase, and other products into the tissue. activated neutrophils synthesize a variety of inflammatory mediators, including prostaglandins (pge ) that modulate the inflammatory response. the products of activated neutrophils (rois, proteases, and mediators) stimulate epithelial secretion and alter tight junction permeability, promoting diarrhea. neutrophils eventually migrate across the infected epithelium by a mechanism that involves integrins, disrupting tight junction integrity and increasing permeability to bacterial products, thus exacerbating the inflammatory response. "unzip" the intercellular junction and migrate through, closing it behind itself. a key feature of neutrophils and other leukocytes is the requirement for integrin-mediated adhesion to extracellular matrix proteins (ecms) or other cells to achieve an optimal effector phenotype. critical components of the ecms in inflamed tissues include fibronectin, fibrinogen, and vitronectin, deposited in tissues as a result of plasma leakage and by synthesis of new proteins by stromal cells and resident macrophages in response to inflammatory mediator activation. the changing composition of the matrix proteins deposited in tissues during inflammation serves as a clue as to the nature of the tissue environment for recruited inflammatory cells as they become activated. individual gene expression studies have demonstrated that adhesion to matrix proteins induces the expression of cytokines and chemokines and their receptors, arachidonic acid-derived lipid mediator synthases, metalloproteinases, growth factors, transcription factors, and other genes that influence the differentiation and activation of inflammatory cells. roi production, phagocytosis, degranulation, and other effector functions stimulated by inflammatory mediators and bacterial products are optimal only when neutrophils adhere to the ecms. adhesion to distinct ecm proteins selectively activates signaling pathways and gene expression of neutrophils, monocytes, and other leukocytes with differing abilities to promote certain functions such that the composition of ecms in many ways controls the development of the ultimate effector phenotype. thus integrin-mediated adhesion provides a mechanism by which neutrophils and other leukocytes can sense the complex tissue environment and respond appropriately. of the activators of neutrophils at sites of inflammation, complement (c -opsonized particles), cytokines (tnf-α and il- β), platelet-activating factor, immune complexes, and bacterial products are among the most potent stimuli. other mediators produced during inflammation may modify neutrophil activity, particularly formylated bacterial peptides, chemokines, complement fragments (c a), leukotriene b , and prostaglandins. activated neutrophils are highly phagocytic; produce large amounts of rois; degranulate to release myeloperoxidase, cationic antimicrobial peptides (defensins), serine proteases (mainly elastase), and metalloproteinases; and secrete inflammatory mediators (tnf-α, il- β, prostaglandins, leukotrienes, and others) (see figure . - ). mast cells strategically reside in mucosal tissues, including the submucosa and lamina propria of the gastrointestinal tract, and constitute a crucial first line of defense at epithelial barriers. however, they are also important effector cells of the pathophysiology of inflammatory gastrointestinal diseases. experimental depletion of mast cells, genetic deficiency in the development of mast cells, or pharmacologic stabilization of mast cells to prevent degranulation have a protective effect in a variety of models of gastrointestinal inflammatory disease, including dextran-or trinitrobenzenesulfonic acid-induced colitis, , ischemia-reperfusion injury, , and immediate hypersensitivity responses. mast cells are activated by a wide variety of microbial products and host-derived mediators. among the activators of mast cells the so-called anaphylatoxins (complement fragments c a, c a, and c a), are potent stimuli causing release of mediators of inflammation. in addition, mast cells are the primary effector cells of immunoglobulin e-mediated anaphylaxis (type i hypersensitivity reactions) by virtue of their high affinity receptors for immunoglobulin e. cross-linking of receptor-bound immunoglobulin e on mast cell surface by antigens (i.e., food antigens) causes rapid degranulation, resulting in the explosive release of granule contents. neural pathways in the intestine also regulate mast cells. mast cells respond to enteric pathogen invasion via neural reflexes that stimulate the release of inflammatory mediators. activated mast cells release preformed histamine, -hydroxytryptamine, proteases, heparin, and cytokines from granules. activation also stimulates de novo synthesis of a range of inflammatory mediators, including prostaglandins, platelet-activating factor, and leukotrienes. transcription of a number of peptide mediators, such as the cytokines tnf-α and il- β among many others, also increases on stimulation of mast cells. mast cell products have profound effects on the vasculature, increasing endothelial permeability and causing vasodilation. moreover, mast cell-derived mediators greatly enhance epithelial secretion by a mechanism that activates neural pathways of epithelial secretion and directly stimulates epithelial cells. mast cell products significantly alter intestinal motility, generally increasing transit and expulsion of intestinal contents. mast cell-derived leukotrienes and tnf-α also have a crucial role in host defense against bacterial pathogens, acting to recruit and activate neutrophils. , mast cells have a newly identified role in host defense and inflammatory responses to bacterial pathogens, which in part is caused by the release of proinflammatory mediators during bacterial infection, which is critical for recruiting and activating other innate host defense cells such as neutrophils. however, mast cells are also phagocytic, have microbicidal properties, and can act as antigen-presenting cells to the adaptive immune system. although accumulating evidence was establishing the role of mast cells in innate immunity, a seminal study that unconditionally identified the importance of mast cells in host defense demonstrated that mast cell-deficient w/ w v mice have impaired responses to gram-negative bacterial peritonitis, resulting in a significant increase in mortality. the role for mast cells in host protective responses appears to be as a sensor of bacterial invasion. unlike immunoglobulin e-mediated responses, bacterial products seem to elicit a highly regulated and selective response from mast cells. the complement cascade is a fundamental part of the inflammatory response. activation of the complement cascade by immune complexes (classical pathway) or by bacteria or bacterial products, polysaccharides, viruses, fungi, or host cells (alternative pathway) results in the deposition of complement proteins on the activating surface and the release of soluble proteolytic fragments of several complement components. in particular, activation of either pathway results in the deposition of various fragments of the complement protein c , which are potent activators of neutrophils and monocytes. opsonization of particles with c fragments constitutes a major mechanism of target recognition and phagocyte activation. during the activation of the complement cascade culminating in deposition of c , soluble fragments of c (c a), c (c a), and c (c a) are liberated. these fragments, termed anaphylatoxins, have potent effects on tissues and cells during inflammation. perhaps most notably, anaphylatoxins are chemotactic for neutrophils (particularly c a), activate neutrophil and mast cell degranulation, and stimulate roi release from neutrophils. the termination of the complement cascade results in the formation of a membrane attack complex in membranes at the site of complement activation, and if this occurs on host cells such as endothelium, it may cause irreversible cell injury. although the primary source of complement is plasma, epithelial cells of the gastrointestinal tract also produce c , suggesting that local production and activation of the complement cascade during inflammation occurs in intestinal tissues. clearly, if the regulatory mechanisms of the complement cascade fail, then the inflammatory response may be inappropriate and tissue injury can occur. the role of complement in gastrointestinal inflammation has been most studied extensively in models of ischemia-reperfusion injury. activation of the complement cascades has a major role in altered endothelial and epithelial permeability in these models. several lines of evidence support the importance of complement in intestinal injury. mice deficient in c or c are protected against ischemia-reperfusion injury. moreover, administration of monoclonal antibodies against c reduced local and remote injury and inflammation during intestinal reperfusion injury in a rat model. administration of a soluble form of complement receptor , a regulatory protein that halts the complement cascade by dissociating c and c on host cell membranes, reduced mucosal permeability, neutrophil influx, and leukotriene b production during ischemia-reperfusion injury in rats and mice. , although neutrophils and mast cells mediate many of the pathophysiologic effects of the complement cascade, the membrane attack complex may have a primary role in altered vascular permeability during ischemia-reperfusion injury. four components initiate the contact system of coagulation: hageman factor (hf), prekallikrein, factor xi, and high-molecular-weight kininogen. hf is a large plasma glycoprotein that binds avidly to negatively charged surfaces. bacterial cell walls, vascular basement membranes, heparin, glycosaminoglycans, and other negatively charged surfaces in the intestine capture hf and the other three important initiators of the contact system in a large multimolecular complex. of the surfaces that bind hf, the extracellular matrix is a potent activator of the contact system. once bound, hf is converted to hf-α, which cleaves prekallikrein to kallikrein and factor xi to factor xia. the ultimate result is further cleavage of hf by kallikrein and triggering of the contact system cascade, activation of intrinsic coagulation by factor xia, activation of the alternative pathway by hf, and proteolytic cleavage of high-molecular-weight kininogen by kallikrein, releasing biologically active kinins. the products of the contact system, particularly bradykinin, have several important biologic properties that drive many of the vascular and leukocytic responses during inflammation. bradykinin induces endothelial cell contracture and intracellular tight junction alterations that increase vascular permeability to fluid and macromolecules. bradykinin also affects vascular smooth muscle contracture, resulting in vasoconstriction or vasodilation depending on the location. bradykinin also increases intestinal motility, enhances chloride secretion by the intestinal mucosa, and intensifies gastrointestinal pain. in neutrophils, kinins stimulate the release of many inflammatory mediators, including cytokines, prostaglandins, leukotrienes, and rois. kallikrein cleaves c to release c a, a potent chemotactic factor for neutrophils, and thus has a role in recruiting and activating inflammatory leukocytes. the plasma kallikrein-bradykinin system is activated in a variety of acute and chronic inflammatory diseases of the gastrointestinal tract. , recent evidence has demonstrated that blockade of the pathophysiologic effects of bradykinin has clinical applications. oral or intravenous administration of the bradykinin receptor antagonist icatibant reduces the clinical signs, onset of diarrhea, and many of the histopathologic changes in experimental models of colitis in mice. inhibition of kallikrein by oral administration of p attenuated the intestinal inflammation, clinical score, and systemic manifestations in a model of chronic granulomatous enterocolitis. thus the contact system is a viable therapeutic target for inflammatory diseases of the intestine. changes in blood flow to the mucosa and other regions of the intestine that reduce perfusion of the tissues can potentiate the initial damage caused by infection or injury. for example, reperfusion of ischemic tissues is associated with platelet and neutrophil clumping in the small vessels of the mucosa, which can impede blood flow. platelets are activated and adhere to exposed basement membrane and activated endothelial cells and provide a surface for leukocyte adhesion. the accumulation of platelets and leukocytes can reduce vessel diameter and blood flow significantly while potentiating local coagulation and thrombus formation. soluble mediators released by activated leukocytes and endothelial cells also affect blood flow. histamine and the vasoactive lipids derived from arachidonic acid (leukotrienes, prostaglandins, thromboxane, prostacycline, and platelet-activating factor) have a prominent role in regulating local perfusion during inflammation and also may have systemic effects on blood flow. procoagulant mediators released by inflammatory cells in response to the inflammatory process (i.e., tissue factor produced by macrophages or endothelial cells), exposed basement membrane proteins, and bacterial components can trigger the contact system and the coagulation and complement cascades, the products of which affect blood flow. nitric oxide, whether produced by endothelial cells or leukocytes (macrophages), is a potent regulator of blood flow and has a significant role in the control of perfusion during inflammation. many of the mediators that affect perfusion also affect endothelial permeability, altering osmotic and hydrostatic balance and tissue edema. in extreme cases, local and systemic coagulopathies initiated by vascular injury and absorption of microbial products and inflammatory mediators induce a hypercoagulable state, leading to microthrombus formation, which can reduce blood flow, or macrothrombus formation, which causes tissue infarction. the cellular mediators of inflammation have the potential to inflict severe injury to intestinal tissues. neutrophils have an important role in the pathophysiology of many intestinal diseases, including ischemia-reperfusion injury, , infectious enterocolitis, [ ] [ ] [ ] nonsteroidal antiinflammatory drug-induced mucosal ulceration, and others. depletion of neutrophils, blockade of their emigration into tissues, or inhibition of neutrophil activation reduce the severity of these and other inflammatory diseases. thus many antiinflammatory therapies are emerging that specifically target neutrophil adhesion, migration, and activation. migration of neutrophils through endothelium during emigration into inflamed tissues is remarkable in that the permeability of the endothelial monolayer is preserved under most circumstances. however, a limit exists above which neutrophil migration alters the permeability characteristics of the endothelium. the effect is in part physical in that mere movement of large numbers of neutrophils through the endothelium is sufficient to disrupt the tight junctions mechanically and is caused in part by toxic products of neutrophils that damage endothelial cells and basement membranes. , serine proteases (particularly elastase) and metalloproteinases released by degranulating neutrophils destroy tissue matrix proteins and cell-surface proteins that make up endothelial intercellular junctions. these degradative enzymes are particularly damaging to basement membranes and the cellular barriers of the endothelium, thus contributing to vascular permeability (and local tissue edema) and thrombosis. the permeability may be affected to the extent that not only water but macromolecules (albumin, matrix proteins, complement, etc.) leak into the interstitium. blockade of neutrophil adhesion to endothelium with anti-β integrin antibodies has a sparing effect on the microvasculature in experimental intestinal ischemiareperfusion injury, reducing the alterations in vascular permeability and histopathologic evidence of microvascular damage. similar to the endothelium of inflamed tissues, massive neutrophil transmigration occurs across the epithelium in response to infection or injury. neutrophil transepithelial migration increases epithelial permeability by disrupting tight junctions. like the endothelium, neutrophils disrupt the epithelial barrier mechanically as they migrate through (see figure . - ). proteases, particularly elastase, degrade basement membrane components and tight junction proteins. products of activated neutrophils (tnf-α and interferon-γ) increase tight junction permeability by direct effects on the enterocytes. prostaglandins released by activated neutrophils stimulate epithelial secretion, thus contributing to diarrhea. subepithelial accumulation of neutrophils can lead to deadhesion of the epithelial cells from the basement membrane and mild to severe ulceration. the physiologic results of the effects of neutrophils and their products on the epithelial barrier include protein-losing enteropathy and absorption of bacterial cell wall constituents, which potentiates the local and systemic inflammatory responses. neutrophils in inflamed tissues stimulated by potent host-derived activators (such as il- β and tnf-α) and bacterial products (lipopolysaccharide) release copious amounts of rois (see figure . - ). although these oxygen and oxyhalide radicals are important for killing pathogens, they are also potentially toxic to epithelial and endothelial cells and matrix proteins. reactive nitrogen intermediates produced primarily by macrophages during inflammation combine with rois to form peroxynitrites, which are particularly toxic. in addition to injury to mucosal tissues, rois also have an as yet ill-defined role in recruiting and activating neutrophils, thereby potentiating the inflammatory response. in support of the role of rois in inflammatory diseases of the gastrointestinal tract, administration of inhibitors of roi production or pharmacologic roi scavengers can be protective in many models of reperfusion injury or enterocolitis. many therapies are aimed at inhibiting neutrophil activation and effector functions in tissues have been evaluated for use in intestinal diseases. phosphodiesterase inhibitors, by causing cyclic adenosine monophosphate accumulation in neutrophils, are antiinflammatory by virtue of their ability to suppress neutrophil activation and roi production. new phosphodiesterase inhibitors selective for the predominant neutrophil isoform of phosphodiesterase hold promise for use in many inflammatory diseases. subepithelial mast cells also have an important role in altering epithelial permeability in inflamed intestine. during the intestinal hypersensitivity response, subepithelial mast cell release of mast cell protease ii by degranulation increases epithelial permeability via an effect on tight junctions. , , this alteration in tight junction permeability results in enhanced transepithelial flux of macromolecules, including proteins and bacterial products. cytokines released by mast cells and phagocytes also regulate tight junction permeability. interleukin- , a product of mast cells and macrophages, increases epithelial permeability. moreover, tnf-α and interferon-γ, products of many inflammatory cells, synergistically increase tight junction permeability. acute equine colitis causes rapid, severe debilitation and death in horses (more than % of untreated horses die or require euthanasia). since , several reports have described a number of different acute diarrheal conditions in the horse that appear to share a common characteristic clinical presentation. - diarrhea associated with acute equine colitis occurs sporadically, is highly fatal, and is characterized by intraluminal sequestration of fluid, moderate to severe colic (abdominal pain), and profuse watery diarrhea with resultant endotoxemia, leukopenia, and hypovolemia. , , the condition can affect adult horses of all ages but usually occurs in horses between and years of age. disease onset is sudden with a rapid progression and often is preceded by a stressful event. a definitive diagnosis is made in only about % to % of cases. , most ante-and postmortem diagnostic tests remain speculative. treatment of the condition in horses is costly because of the massive fluid therapy required. currently, no curative treatment is available for acute colitis in horses, human beings, or other mammals. treatment regimens provide rehydration, electrolyte and plasma protein replacement, mitigation of the effects of circulating endotoxin, and antimicrobial therapy when indicated. attempts during the past years to develop appropriate treatments (i.e., vaccines or pharmacologic agents) have been hampered by the unavailability of acceptable equine models and have been unsuccessful because of the complex pathophysiology of the intestinal epithelium. although the mechanisms responsible for the fluid losses are not known, inflammatory cells may play an integral role because this condition is characterized by large numbers of granulocytes infiltrating the large intestinal mucosa. [ ] [ ] [ ] [ ] [ ] equine cecal and colonic tissues collected during the acute stages of experimentally induced acute equine colitis (equine ehrlichial colitis, lincomycin with and without clostridium spp. inoculation, nonsteroidal antiinflammatory drug administration) reveal the presence of numerous neutrophils and eosinophils in the lamina propria and submucosa. , , , granulocytederived reactive oxygen intermediates are crucial to antimicrobial defenses in the gut and stimulate chloride and water secretion by interactions with enterocytes. , normal equine intestinal tissue is unique compared with that in most other mammalian species for a preponderance of eosinophils located in the intestinal mucosa and submucosa. , production of reactive oxygen intermediates by stimulated phagocytic granulocytes following mucosal barrier disruption may be responsible for the massive fluid secretory response that occurs during the early stages of acute equine colitis. colitis refers to inflammation and mucosal injury of the colon and cecum (typhlocolitis) that may occur in response to a number of causes. , the cause of the colonic injury may be well-defined such as in naturally occurring infectious or experimentally induced colitis. however, many cases of human and animal diarrhea have a speculative or unknown diagnosis or no diagnosis. , , irrespective of the underlying or initiating cause of colonic injury, the colon apparently has a limited repertoire of responses to damage because most forms of colitis demonstrate similarities in histopathologic appearance and clinical presentation. various degrees of mucosal erosion and ulceration, submucosal/mucosal edema, goblet cell depletion, and presence of an inflammatory cellular infiltrate within the mucosa and submucosa are common to many types of human and animal colitis. , [ ] [ ] [ ] characteristic clinical manifestations include intraluminal fluid sequestration, abdominal discomfort, hypovolemia, and most often profuse, watery diarrhea. large bowel diarrhea results from abnormal fluid and ion transport by cecal and colonic mucosa. loss of fluid by the large intestine can result from malabsorptive or hypersecretory processes and is often a combination of the two. colonic secretory processes are a function of the crypt epithelium, whereas absorptive processes are limited to surface epithelial cells. under normal baseline conditions, an underlying secretion by crypt epithelium is masked by a greater rate of surface epithelial cell absorption. abnormal forces influencing the rates of secretion and absorption can result in massive, uncontrolled secretion and malabsorption by large intestinal mucosal epithelial cells, leading to rapid dehydration and death. , two intracellular processes control colonic secretion: the cyclic nucleotide (cyclic adenosine monophosphate [camp] and cyclic guanosine monophosphate [cgmp]) and the calcium system. , agents may activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e [pge ]) or guanyl cyclase (bacterial enterotoxins) and induce increases in camp or cgmp, respectively. this reaction causes phosphorylation of specific protein kinases that induce the actual apical and basolateral membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotide-dependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. , calcium may act through calmodulin, which then can activate membranephosphorylating protein kinases. at least four central systems control intestinal secretion: ( ) the hormonal system, ( ) the enteric nervous system, ( ) bacterial enterotoxins, and ( ) the immune system. , hormonal control of colonic electrolyte transport is exerted primarily through the renin-angiotensinaldosterone axis. , the enteric nervous system controls transport through three separate components: ( ) extrinsic nerves of the parasympathetic and sympathetic pathways; ( ) intrinsic ganglia and nerves, secreting a variety of neurotransmitters including peptides; and ( ) neuroendocrine cells (intraepithelial lymphocytes) that reside in the epithelium and release messengers onto the epithelial cells in a paracrine manner. , [ ] [ ] [ ] [ ] many bacterial enterotoxins can induce intestinal secretion by camp or cgmp signal transduction. bacterial enterotoxins can stimulate enteric neurons, providing evidence for interaction between two controlling systems. preformed inflammatory mediators such as histamine, serotonin, or adenosine and newly synthesized mediators such as prostaglandins, leukotrienes, platelet-activating factor, various cytokines, the inducible form of nitric oxide, and reactive oxygen metabolites can initiate intestinal secretion by directly stimulating the enterocyte and by acting on enteric nerves indirectly to induce neurotransmitter-mediator intestinal secretion. for instance, when added to the t colonic cell line, the known mast cell mediators histamine, adenosine, and pgd induce chloride secretion. [ ] [ ] [ ] prostaglandins of the e and f series can cause an increase in chloride secretion in intact tissue and isolated colonic cells. [ ] [ ] [ ] leukotrienes, platelet-activating factor, and a number of cytokines have been shown to have no effect on t cell secretion but have a significant effect on electrolyte transport in intact tissue, suggesting that intermediate cell types may be involved in these secretory responses. [ ] [ ] [ ] the epithelial cell chloride secretory response occurs via prostaglandin-and adenosine-mediated increases in cellular camp, whereas histamine acts by h receptor induction of phosphatidylinositol turnover, production of inositol triphosphate, and mobilization of intracellular calcium stores. , lipoxygenase products (leukotrienes) are capable of activating a colonic secretory response and do not appear to involve the cyclic nucleotides or calcium ions. phagocyte-derived reactive oxygen mediators (roms) can induce colonic electrolyte secretion in vitro, suggesting that oxidants may contribute directly to the diarrhea associated with colitis. [ ] [ ] [ ] [ ] [ ] reactive oxygen species initiate the secretory response by increasing cellular camp or stimulating mesenchymal release of pge or prostacyclin, which in turn stimulates the epithelial cell or enteric neuron, respectively. [ ] [ ] [ ] [ ] [ ] [ ] sodium nitroprusside, an exogenous source of nitric oxide, stimulated an increase in chloride secretion in rat colon that was mediated by cyclooxygenase products and enteric neurons. table . - summarizes inflammatory mediator-induced epithelial cell chloride secretion. acute colitis rarely develops by a simple cause or effect phenomenon but is influenced by many extrinsic and intrinsic host and microorganism factors. inflammatory mediators released from mast cells and monocytic or granulocytic phagocytes cause intestinal chloride and water secretion and inhibit neutral sodium and chloride absorption. , , inflammatory cells, particularly the phagocytic granulocytes, play an important role in mucosal pathophysiology in cases of colitis. , large numbers of these cells are observed on histopathologic examination of tissues from human and animal cases of colitis. products of cell activation stimulate direct and indirect secretory responses in intestinal cells and tissues. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] products of phagocyte secretion may amplify the inflammatory signal or have effects on other target cells in intestine such as enterocytes and smooth muscle cells (table . - ). the nadph-oxidase system of phagocytes (neutrophils, eosinophils, monocytes/macrophages) is a potent inducer of superoxide radicals used as a host defense mechanism to kill invading microorganisms. , during inappropriate stimulation such as inflammation, trauma, or ischemia followed by reperfusion, increased levels of toxic oxygen species are produced, causing damage to host tissues. engagement of any of several receptor and nonreceptor types including phagocytosis mediators, chemotactic agents, various cytokines, and microbial products can stimulate phagocytes. resident phagocytes or those recruited to colonic mucosa early in the disease process are considered to augment mechanisms causing fluid and electrolyte secretory processes, a so-called amplification process. , activation of the respiratory burst results in the production and release of large amounts of superoxide anion (o − ) and h o . , in addition to these roms, activated phagocytes secrete peroxidase enzyme (myeloperoxidase from neutrophils and eosinophil peroxidase from eosinophils) into the extracellular space. the peroxidases catalyze the oxidation of clby h o to yield hocl, the active ingredient in household bleach products. the peroxidase-h o -halide system is the most cytotoxic system of the phagocytes; hocl is to times more toxic than o or h o . hocl is a nonspecific oxidizing and chlorinating agent that reacts rapidly with a variety of biologic compounds including dna, sulfhydryls, nucleotides, amino acids, and other nitrogen-containing compounds. hocl reacts rapidly with primary amines (rnh ) to produce the cytotoxic n-chloramines (rnhcl). the mechanisms by which hocl and rnhcl damage cells and tissue remain speculative, but possibilities include direct sulfhydryl oxidation, hemoprotein inactivation, protein and amino acid degradation, and inactivation of metabolic cofactors of dna. peroxidase-derived oxidants have been shown to degrade hyaluronic acid and collagen. in addition, luminal perfusion of specific roms increased mucosal permeability and serosal application caused increases in clsecretion in vitro. tissue myeloperoxidase activity, an index of tissue granulocyte infiltration, is used clinically and experimentally to assess degree of intestinal inflammation. , myeloperoxidase activity is elevated in acute flare-ups of human inflammatory bowel disease and various animal models of acute colitis. [ ] [ ] [ ] [ ] [ ] the acute inflammatory response in these conditions is characterized predominantly by neutrophils, the predominant source of myeloperoxidase activity. however, this assay measures total hemoprotein peroxidase, which includes monocyte and eosinophil peroxidase in addition to neutrophils. moreover, levels of peroxidase activity in equine circulating eosinophils are greater than in circulating neutrophils, and this may apply to resident tissue eosinophils as well. arachidonic acid metabolites are thought to play a role in intestinal inflammation in diarrheal disease. , , elevated levels of these intermediate metabolites have been demonstrated in natural disease and experimental models of colitis and appear to parallel increases in roms in inflamed intestine. addition of h o or hocl to rat colonic tissue in ussing chambers has been shown to induce pge release and active clsecretion. , prostaglandins can stimulate increases in clsecretion in intact intestinal tissue , , and in isolated colonic t cells. , interactions between roms and mesenchymal release of pge /pgi may be relevant to the mechanisms producing the diarrheic condition. fibroblasts co-cultured or juxtaposed to colonic t cells greatly increased the clsecretory response to h o in vitro through the release of pge . in addition, equine colonic mucosa has an increased sensitivity to endogenously released prostaglandin by exhibiting a significant secretory response under in vitro conditions. endotoxin, the lipopolysaccharide component of the outer cell wall of gram-negative bacteria, is present in large quantities in the large intestine of healthy horses. , endotoxins are released into the immediate surroundings when gram-negative bacteria undergo rapid proliferation or die. , the intact bowel forms an effective barrier to the transport of significant amounts of these highly antigenic toxins, but the diseased gut absorbs these macromolecules in large amounts, causing the subsequent adverse systemic effects that are often life threatening. disruption of the intestinal barrier (i.e., ischemia, trauma, inflammatory conditions) overwhelms the capacity of the liver to clear endotoxins, and systemic endotoxemia ensues. endotoxins have been shown to be potent activators of the inflammatory process, stimulating the production and release of numerous cytokines by activated macrophages and other immunocytes. in vitro studies suggest that endotoxin activates phagocytic granulocytes to secrete roms, increase release of lysozymes, and enhance the migratory response to chemotactic stimuli. prostacyclin and thromboxane a mediate hemodynamic dysfunction, and lipoxygenase products may induce tissue ischemia. the cytokine interleukin- causes a febrile response and initiates the acute phase response. tumor necrosis factor contributes to many of the abnormal physiologic responses, particularly hemostatic functions that potentiate coagulopathy. additional mediators include interleukin- , platelet-activating factor, procoagulant mediators, and various other speculated substances. endotoxins trigger mucosal immune cells and subsequent release of inflammatory mediators in cases of colitis. the first report of experimentally induced endotoxemia described clinical signs and hematologic findings that closely paralleled those reported for severe colitis in horses. studies in which endotoxin was administered intravenously in human beings and laboratory animals caused significant dose-related gastrointestinal changes, ranging from mild diarrhea to bloody, watery diarrhea. , in vitro studies on the effects of endotoxin on intestinal water and electrolyte transport in adult male rats showed a significant decrease in net colonic sodium absorption and increased colonic permeability. in animal models of protein energy deficiency, endotoxin-induced mortality increased compared with that of well-nourished control animals. endotoxin depresses lymphocyte responses to specific mitogens. thus the adverse effects of malnutrition and endotoxin are mutually aggravating. the importance of a normal immune system to the defense of the mucosal surface of the gastrointestinal tract is evident in the immunosuppressed state. primary immunodeficiencies affecting the gastrointestinal tract are well documented. [ ] [ ] [ ] common agammaglobulinemia is the most frequently reported gastrointestinal disease and causes b cell deficiency-associated giardiasis. interestingly, selective immunoglobulin a (iga) deficiency rarely results in intestinal disease because of a speculated increase in mucosal igm response. however, combined iga and igm deficiencies with a higher incidence of intestinal disease occur. a selective deficiency of secretory iga has been associated with intestinal candidiasis. certain mucosal pathogens may enhance their pathogenicity by producing iga proteases. defects in cell-mediated immunity are associated most commonly with intractable diarrhea, and organisms frequently involved include salmonella spp., escherichia coli, and shigella spp. acquired immunodeficiency or immunosuppression in adults can result from infectious diseases (particularly viral), nutritional deficiencies, aging phenomenon, and drugs (corticosteroids, azathioprine, cyclophosphamide). nutrition is a critical determinant of immunocompetence and risk of illness. , impaired systemic and mucosal immunity contribute to an increased frequency and severity of intestinal infections observed in cases of undernourishment. abnormalities occur in cell-mediated immunity, complement system, phagocytic function, mucosal secretory antibody response, and antibody affinity. morbidity caused by diarrheal disease is increased particularly among individuals with stunted growth rate because of malnourishment. the critical role of several vitamins and minerals in immunocompetence has been substantiated in animals deprived of one dietary element and findings in human patients with single-nutrient deficiency. tables . - and . - summarize nutritional-related immune system abnormalities. in summary, nutritional deficiency can cause increased colonization of the intestine with microorganisms, alter the symbiotic characteristics of resident intestinal bacterial populations, and impair defenses of the gastrointestinal tract, allowing increased risk of systemic spread of infection and absorption of macromolecules (in particular, endotoxin). indigenous microflora greatly impede colonization of the gastrointestinal mucosa by pathogenic organisms. the ability of a potential pathogen to initiate an infection depends on its ability to breach the mucosal epithelial barrier. one mechanism by which the normal flora inhibit establishment of pathogens is by preventing adherence of the pathogen to mucosal cells by occupying the site or by stearic hindrance. [ ] [ ] [ ] [ ] resident microbes also produce byproducts such as antibacterial factors that allow symbiosis rather than competition between them. another hindrance mechanism is production of volatile fatty acids by normal microbial digestive processes to create an environment that is toxic to many bacterial populations, particularly the enterobacteriaceae. disturbances in motility patterns occur during inflammatory diseases of the colon, but the role of motility alterations in the pathogenesis of diarrhea remains unclear. invasive bacteria cause characteristic motor patterns in the colon consisting of rapid bursts of motor activity that appear to decrease transit time through the large intestine. the result is reduced clearance of bacteria from the large intestine, which may contribute to the virulence of the organism. absorption of endotoxin and the release of inflammatory mediators such as prostaglandins disrupts the motility patterns of the large intestine, resulting in less coordinated contractions, and may contribute to the alterations in motility seen with invasive bacteria. although the effect of endotoxin and prostaglandins on transit time is not profound, the disruption of coordinated activity may play a role in causing diarrhea. thorough mixing and prolonged retention time of ingesta are important not only in microbial digestion of nutrients but also in absorption of microbial byproducts and fluid. , the ingesta is viscous and therefore must be mixed to bring luminal ingesta in contact with the mucosa for absorption. in addition, poor mixing increases the thickness of the unstirred layer, decreasing contact of ingesta with the mucosa and decreasing absorption. , progressive motility must be present, however, if a diarrheal state is to occur. , ileus may be accompanied by increased fluid in the lumen of the large intestine, pyridoxine, folic acid, impairs cell-mediated immunity and vitamin c, vitamin e reduces antibody response. vitamin b decreases lymphocyte stimulation response to specific mitogens. zinc deficiency affects humoral and cell-mediated immunity. iron inhibits bacterial multiplication. copper depresses antibody response. needed by neutrophils and lymphocytes for optimal function, which may be related to myeloperoxidase and ribonucleotidyl reductase deficiencies. but without progressive motility the fluid is not passed. frequently, acute colitis causes a period of ileus characterized by scant stool. diarrhea is apparent only when motility returns and the ingesta is passed. increased progressive motility has been suggested to cause diarrhea by decreasing transit time and is thought to play a role in irritant catharsis and in the mechanism of action of some laxatives. irritation and distention increase motility and may well decrease transit time, but increased secretion also is thought to contribute to diarrhea caused by these substances. one should direct the clinical investigation of malabsorption at ascertaining and trying to localize the source of the abnormality. in medical practice, impairment of one or more phases of the digestion and absorption of dietary constituents may precipitate clinical signs that are associated primarily with carbohydrate, protein, or fat malabsorption. this level of differentiation is not possible in the horse because of the herbivorous diet and the contribution of large intestinal functions. in human and small animal medicine, disturbances in digestive processes especially from exocrine pancreatic insufficiency or reduced intestinal bile salt concentration are principal determinants of many clinical malabsorption syndromes. the rarity of pancreatic problems in horses and the herbivorous diet makes maldigestion less of a concern and difficult to pursue diagnostically. nevertheless, maldigestion undoubtedly contributes to chronic weight loss conditions in horses, which may be significant with severe infiltrative disease of the small intestine with partial to total villous atrophy and flattened mucosa. impairment of digestive processes may exacerbate diarrhea in the suckling foal through reduced intestinal bile salt concentrations from hepatic or ileal dysfunction. malabsorption is not synonymous with diarrhea, although diarrhea may be a feature. adult horses rarely exhibit diarrhea with small intestinal problems unless large intestinal involvement is concomitant. chronic diarrhea is predominantly a large intestinal disorder that reflects an overload of water and electrolytes and thus may be considered a state of impaired absorption. primary small intestinal disease is more likely to occur in neonates and young foals. for example, acquired small intestinal brush border lactase deficiency may result in increased lactose fermentation in the large intestine and induction of osmotic diarrhea. box . - lists conditions that have been or potentially could be associated with malabsorption syndromes and maldigestion in the horse. malabsorption in horses has no pathognomonic clinical syndrome. case recognition derives from the robust investigation of horses with chronic wasting. prevalence is unknown. no strict case definition exists, even for chronic wasting. interest generated by unusual clinical test results and their related pathologic findings has stimulated publication of reports of cases considered as malabsorption in horses. pathologic description of the predominant cellular infiltrate and the pattern of intestinal distribution have resulted in the classification of many conditions as representing examples of chronic inflammatory bowel disease (cibd) (see box . - ), drawing analogies from human medicine. in the affected animal, coexistent enteric protein loss reflecting changes in mucosal integrity from extensive infiltration and inflammation in the intestinal tract is likely to be more debilitating than the malabsorption. the principal concern of the owner is the weight loss and poor condition of the horse. many clinical examination findings, except for body condition, may appear within normal limits. investigation of the weight loss, together with the clinical pathologic findings, helps to eliminate other more commonly encountered causes of wasting. box . - lists clinical signs that may be associated with malabsorption syndromes. no characteristic clinical pathologic profile of malabsorption exists. findings relate to the stage of the underlying disease process and intercurrent problems. the syndrome tends to cause anemia (normocytic, normochromic) and neutrophilia. hemolytic or macrocytic anemia and thrombocytopenia have been observed in alimentary lymphosarcoma. lymphocytosis (leukemia) rarely is encountered. eosinophilia is uncommon even with suspected immune-mediated conditions and widespread tissue eosinophilia. many animals are hypoalbuminemic and hypoproteinemic; horses with alimentary lymphosarcoma may exhibit hyperproteinemia and hypergammaglobulinemia. serum or plasma may be lipemic. the clinician may find elevated hepatic and biliary tract enzymes (γ-glutamyltransferase and alkaline phosphatase) in multisystemic conditions; for example, eosinophilic granulomatosis (multisystemic eosinophilic epitheliotropic disease; eg [meed]). abdominocentesis has been of diagnostic value in several alimentary lymphosarcoma cases, but rarely in the granulomatous conditions. ultrasonographic examination of the abdomen can yield infomation on intestinal distention, wall thickness, and unexplained masses detected on rectal palpation. rectal biopsy is easy to perform and may provide an indication of cellular infiltration that could be present at more proximal locations. however, pathologists examine few equine rectal samples, and the interpretation is frequently equivocal. adoption of standardized grading or classification would improve the diagnostic value. a proposed classification system was based on a retrospective study of rectal biopsies from horses ages to years with clinical signs of intestinal disease. necropsy results were studied from horses. biopsy specimens ( horses) and necropsy rectal tissue ( horses) from horses ages to years served as controls. simple proctitis, the presence of neutrophils in the crypt or surface epithelium, was an abnormal finding compared with mild scattered neutrophil infiltration in controls. simple proctitis was found in association with malignant lymphoma and other inflammatory disorders. inflammatory bowel disease was diagnosed from rectal biopsy specimens in of eg (meed) cases and of granulomatous enteritis cases confirmed at necropsy. rectal biopsy aided diagnosis for of horses in a series of lymphocytic-plasmacytic enterocolitis cases. eosinophils were demonstrated on impression smears of rectal mucosal biopsies from of horses with eosinophilic enterocolitis. skin biopsies or ultrasound-guided biopsies of liver, lymph node, or lung may reveal evidence of multisystemic disease. one can obtain intestinal and lymph node biopsies via a standing laparotomy. exploratory laparotomy facilitates rigorous inspection of the gastrointestinal tract and associated organs to obtain multiple biopsies from intestinal sites and lymph nodes. the procedure may provide a diagnosis, enabling one to make decisions on potential treatment and management options. cost and potential postoperative complications may limit surgical procedures for diagnosis. laparoscopy may provide an alternative means to facilitate biopsy of certain tissues. however, one should consider surgical exploration as an option early in the process rather than as a last resort. the noninvasive breath hydrogen test used to assess carbohydrate malabsorption in human beings has not proved reliable in equine studies. intestinal function tests can provide a practical and inexpensive means to assess the absorptive capability of the small intestine. for clinical practice purposes this is limited to carbohydrate absorption. abnormality of carbohydrate absorption has become an important precept on which to base a diagnosis of malabsorption in the horse. however, results of the oral glucose tolerance test (ogtt) or d-xylose absorption test require cautious interpretation. pathologic changes in the mucosa and submucosa must be extensive and widely distributed to greatly affect the peak plasma concentration and shape of the curve. the tests are easy to perform in practice and require a baseline blood sample predosing and further samples for up to hours after administration of the solution. many commercial laboratories conduct glucose and xylose assays. the immediate dietary history, gastric emptying rate, intestinal transit, age, and hormonal effects of the horse influence glucose peak and curve shape. higher glucose peaks are recorded from healthy animals eating grass or hay than from those eating concentrates. recent appetite or the level of cachexia may affect test results. maximum plasma glucose level (> % baseline) is reached by minutes in healthy animals given g glucose per kilogram body mass as a % solution. , break points below which the probability increases of carbohydrate malabsorption associated with intestinal morphology changes have been proposed. a referral population of mature horses with chronic weight loss was divided into three groups based on ogtt results to determine if any concurrence with the morphologic diagnoses existed. group (n = ) had a normal ogtt (peak glucose concentration at minutes > % baseline) and contained animals that had normal small intestinal morphology, and a few with large intestinal lesions. group (n = ) had partial malabsorption and included horses with small intestinal infiltrative disease that allowed some glucose uptake. diagnoses included lymphosarcoma, villous atrophy, granulomatous enteritis and eg (meed). seven horses had normal small intestinal histologic findings. peak glucose concentrations were less than % and greater than % of baseline at minutes. seventeen horses in the group had large intestinal pathologic conditions. group horses (n = ) had total malabsorption; the peak concentration at minutes was less than % above baseline. these horses had severe infiltration throughout most of the small intestine that was attributed predominantly to lymphosarcoma or granulomatous enteritis. however, the test is far from definitive; one cannot assume a flat curve indicates malabsorption and a poor prognosis. two horses with chronic weight loss initially diagnosed with malabsorption based on flat ogtt curves subsequently showed more normal ogtt responses. full-thickness intestinal biopsies were unremarkable. one horse had an elevated serum immunoglobulin e to oat allergen. oats and oat straw were removed from access. dexamethasone was given on a tapered protocol, and a repeat ogtt was normal at months. the other horse received oral probiotics to counter suspected small intestinal bacterial overgrowth, was clinically normal in months, and had an improved ogtt with a minute peak. therefore malabsorption, as defined by an absorption test and weight loss, may occur in the horse without significant morphologic changes in the intestine, and the condition may be transient. demonstration of carbohydrate malabsorption in of horses with chronic diarrhea showed poor diagnostic sensitivity for small intestinal involvement. impaired glucose absorption was recorded in horses with predominantly large intestinal problems, cyathostomiasis, chronic colitis, alimentary lymphosarcoma, and meed. although prior dietary history influences peak plasma xylose concentration, xylose is not confounded by hormonal effects or mucosal metabolism. gastric emptying rate, intestinal motility, intraluminal bacterial overgrowth, and renal clearance do affect curve shape. healthy mares not fed for up to hours had flatter curves and a slower decrease in plasma xylose than when deprived for to hours. hence recent appetite or the level of cachexia may influence test results. abnormal d-xylose absorption represented by a flat curve or delayed absorption is considered indicative of significant jejunal disease and has been observed with most examples of cibd, parasitism, and idiopathic villous atrophy. , ponies may have lower peak d-xylose concentrations at and minutes than horses, although the range of peak values at the test dosage ( . g d-xylose per kilogram body mass in a % solution) is wide. potentially diagnostic discriminatory cut off points for peak plasma xylose concentrations have not been determined. abnormal absorption curves have been detected in the absence of small intestinal histologic changes, and interpretation is clouded further by findings from small intestinal resection studies in healthy ponies. nine ponies with % distal small intestinal resection and four shamoperated controls were placed on interval feeding for weeks and then turned out to pasture until months after surgery. grazing was supplemented by twice daily (meal feeding) concentrate rations. all ponies gained weight and were clinically normal, and none developed diarrhea. however, the mean peak xylose concentration at minutes declined progressively (at monthly intervals) over the study period in the resection group to % of that of controls. lack of clinical malabsorption was attributed to adaptation of the residual % of healthy small intestine and of large intestinal function. bacterial overgrowth in the small bowel remnant from refluxed cecal contents (resected ponies had ileocecal valve bypass) may have contributed to the abnormal xylose assimilation. by contrast, xylose absorption decreased over months, associated with substantial weight loss, lethargy, and diarrhea, in an earlier study of extensive (≥ %) small intestinal resection in ponies. an important difference was the feeding pattern; those ponies received pelleted feed twice daily for the entire month follow-up period. consequently, horses with suspected malabsorption may adapt to an interval feeding regimen. the critical factor could be the availability of sufficient unaffected or minimally affected small intestine and large intestinal functional capacity. the outcome for animals with small intestinal disease and some unknown degree of large intestinal pathologic dysfunction may be less successful than shown in the experimental study. abnormal xylose absorption reverted to normal following days of corticosteroid therapy in an adult thoroughbred gelding with a -week history of weight loss and diarrhea for weeks; peak xylose concentration at minutes was within normal limits and the horse had gained weight. d-xylose absorption was abnormal in an adult standardbred gelding with a -month history of poor performance, weight loss, intermittent fever, mesenteric lymphadenopathy, elevated fibrinogen, and decreased albumin and globulin levels. multiple fullthickness small intestinal biopsies revealed evidence of granulomatous enteritis. the horse received antibiotics postoperatively and then corticosteroids parenterally for to months. after weeks, peak plasma xylose had increased, although absorption was delayed. five months after cessation of corticosteroid therapy, the horse had regained weight and was bright and alert, and d-xylose absorption was normal. diagnostic predictions were made retrospectively by examining d-xylose absorption in horses with granulomatous enteritis compared with those with eg. peak xylose concentrations were much lower in horses with granulomatous enteritis than those with eg, whereas in eg the absorption curve shifted to the right with the peak occurring at minutes. the small intestine is affected predominantly in granulomatous enteritis with extensive villous atrophy and more diffuse lesions in the large intestine, whereas in eg (meed) the large intestine is more involved. hence, the extent and distribution of pathologic changes in the small and large intestines may influence xylose absorption test results. the chronic wasting horse with suspected malabsorption and probable enteric protein loss has at best a guarded to poor prognosis. prognosis may be improved through early and aggressive investigation to achieve a diagnosis, and perhaps assess the stage in the natural progression of the disorder. the owner may elect euthanasia of the animal or may be willing to determine whether the condition can be improved. in the short term, intravenous infusion of plasma or colloids, with or without fluids and electrolytes, may be necessary to stabilize the condition. prognosis is much worse for the horse that is inappetent. prolonged intensive total parenteral nutrition and/or oral alimentation may not be a realistic course of action. the overall therapeutic and management plan can prove to be expensive. the owner must be cognizant from the start that the outcome may not be altered, even after protracted therapy. one cannot make predictions for outcome of therapy without meaningful data because only a few case reports of successful responses with long-term follow up exist. some level of digestive and absorptive capability remains in the diseased small intestine. interval feeding of small quantities of food may be beneficial if the horse continues to eat, and particularly for animals with ravenous appetites that seem able to maintain their reduced state of body condition without further losses. diet should include feeds with a high fiber content to favor large intestinal fermentation, including grass hay and access to pasture complemented by commercial high-fiber rations based on beet pulp and soybean hulls. energy intake can be increased through feeding high-energy dense fats that provide . times more calories than carbohydrates. most affected horses should tolerate high fat ( % to %) processed feeds containing vegetable oils or rice bran (up to % of the concentrate mix, equivalent to % vegetable oil) to achieve the higher-fat composition. changeover to a higher-fat concentrate should be gradual. even in healthy animals that can eat up to % added fat, appetite may decrease as the percentage increases, and fecal consistency may change. clearly, the objective for the horse with suspected malabsorption is to sustain, and preferably increase, dietary intake, value, and efficiency. the owner of an affected horse must be prepared to experiment with feeds, must be patient, and must keep records. no standard procedure exists. exposure to a feed component may contribute to the problem as an allergen eliciting a hypersensitivity reaction. identifying the potential allergen through immunologic testing or by stepwise removal and outcome assessment over a longer period may be difficult. the clinician should give immunosuppressive drugs early in the process. immunosuppressive agents have produced the most promising responses to ameliorate the effects of conditions associated with malabsorption, particularly cibd. short-duration, and in some cases more prolonged and sustained, improvements in body condition, weight gain, demeanor, energy and activity levels have occurred following corticosteroid administration. one should start treatment as early as possible. one should follow initial parenteral (intramuscular or intravenous) loading doses of dexamethasone (sodium phosphate) with a series of depot injections, or orally administered prednisolone or prednisone, on a tapered dose protocol over a period of months. interval low-dose therapy may be necessary if clinical signs return after treatment ends. one uses the lowest dose to control the clinical signs for alternate-day therapy. clinical benefits far outweigh concerns over potential adverse effects. chemotherapeutic agents such as vincristine, cytosine, cyclophosphamide, and hydroxyurea have been tried in a few cases of cibd or lymphosarcoma with no apparent success, probably related to the advanced stage of the disease when treatment was initiated and the dose selected. resection of a segment of intestine that is edematous, hemorrhagic, or constricted is an option in localized forms of cibd, , particularly if gross changes are not discernible in adjacent or distant parts of the intestinal tract, that is, malabsorption is not a feature. long-term outcome has been favorable. removal of a substantial proportion of the diseased small intestine may be indicated in a horse with malabsorption, considering that resection of % distal small intestine was performed in healthy animals without inducing adverse effects. however, because pathologic changes may exist in normalappearing small or large intestine that is not resected or biopsied, the prognosis remains guarded. two young horses with granulomatous enteritis had the thickened terminal small intestine resected with positive outcomes; one survived months, the other has a follow up extending more than years. anthony t. blikslager to gain an appreciation of the mechanisms whereby the mucosa is injured and subsequently repaired, one must understand how the integrity of the mucosa is regulated physiologically. regulation of mucosal integrity is referred to as mucosal barrier function, which is vital because it prevents bacteria and associated toxins from gaining access to subepithelial tissues and the circulation. however, the mucosa has two conflicting functions: it must serve as a protective barrier and continue to absorb solutes necessary to maintain well-being of the host. this conflict is most notable at the intercellular (paracellular) space, which allows passage of select solutes and water, - but which does not admit large molecules, including bacterial toxins. the paracellular space is regulated almost exclusively by the tight junction, which is the interepithelial junction at the apical-most aspect of the paracellular space. although these tight junctions originally were viewed as inert cellular adhesion sites, what has become clear in recent years is that tight junction permeability depends on tissue-specific molecular structure and is regulated by a complex array of intracellular proteins and the cytoskeleton. tight junctions consist of a group of transmembrane proteins that interdigitate from adjacent cells. although occludin originally was thought to be the predominant tight junction transmembrane protein, a group of proteins termed claudins appear to be more critical. these transmembrane proteins interact with the cytoskeleton via a series of intracellular proteins, including zonula occludens , , and ; cingulin; and others. in addition, local regulatory proteins such as the small guanosine triphosphatase-rho are also critical to tight junction function. in general the relative contractile state of the actin cytoskeleton determines the degree to which tight junctions are open or closed, but the complexities of regulation of this process are understood poorly. , the most sensitive measure of mucosal barrier function is transepithelial electric resistance, which is measured by mounting mucosa in an ex vivo system called an ussing chamber, because this measurement is largely a reflection of the permeability of mucosa to ions. , ions may follow one of two routes when traversing epithelium: transcellular and paracellular. because cell membranes have a resistance to passive flow of ions . to log units greater than that of the epithelium as a whole, measurements of transepithelial resistance largely reflect the resistance of the paracellular space, and in particular the tight junctions that regulate paracellular flow of ions. because tight junctions differ in structure from different portions of the mucosa, measurements of transepithelial resistance reflect the net resistance of epithelium of variable permeability within a given tissue. for example, tight junctions in the intestinal glandular structures called crypts are leakier than those in the surface epithelium because of fewer and less organized tight junction strands. , conversely, surface epithelium has a greater number of well-organized tight junction strands that result in epithelium with a high resistance. this correlates well with the absorptive function of epithelium located on the mucosal surface and the secretory function of crypt epithelium. structure of tight junctions also varies with the segment of intestine. for example, tight junctions have more strands in the ileum than in the jejunum, which is reflected by a higher transepithelial resistance in the ileum. the stomach has four regions based on the type of mucosal lining (in an orad to aborad order): nonglandular stratified squamous epithelium, cardiac epithelium, proper gastric mucosa, and pyloric mucosa. stratified squamous epithelium has distinct differences in terms of barrier function compared with the remainder of the gastrointestinal tract. this epithelium has baseline transepithelial resistance measurements of approximately to Ω/cm , which is an order of magnitude higher than the adjacent cardiac mucosa. , thus the stratified squamous mucosa is exceptionally impermeable. this in effect is the only mechanism this mucosa has to defend itself against injury. the stratified squamous epithelium consists of four layers: the outer stratum corneum, stratum transitionale, stratum spinosum, and the basal stratum germinativum. however, not all layers contribute equally to barrier function, the barrier being composed mostly of interepithelial tight junctions in the stratum corneum and mucosubstances secreted by the stratum spinosum. , the relative impermeability of stratified squamous mucosa can be demonstrated by the effects of hcl on this type of epithelium in vitro, which has little effect until it reaches a ph of . or lower. thus although most of the literature on equine ulceration pertains to the effects of hcl and inhibitors of hcl secretion, [ ] [ ] [ ] [ ] other factors may be critical to the development of gastric ulcer disease. the site of hcl secretion (proper gastric mucosa) also is protected from so-called back-diffusion of h + by a high transepithelial electric resistance (compared with cardiac mucosa), but a number of other critical mechanisms also exist to prevent acid injury. the gastric mucosa secretes mucus and bicarbonate, which together form a hco --containing gel that titrates acid before it reaches the lumen. , the mucus layer is formed principally by glycoproteins (mucins) secreted by goblet cells but also includes other gastric secretions and sloughed epithelial cells. mucins consist of core peptides with a series of densely packed o-linked polysaccharide side chains that, once secreted, become hydrated and form a viscoelastic gel. however, the mucus layer does not form an absolute barrier to back-diffusion of acid. thus for acid that does back-diffuse into the gastric mucosa, epithelial na + /h + exchangers are capable of expelling h + once the cell reaches a critical ph. recent studies have renewed interest in the protective mechanisms of mucus because of the discovery of a group of compounds secreted by goblet cells called trefoil peptides. the name of these peptides is derived from a highly conserved cloverleaf structural motif, which confers substantial resistance to degradation by proteases including pepsin. three members of this group are known, ps , sp, and intestinal trefoil factor, the latter of which is secreted solely by goblet cells in the small and large intestine. ps and sp are secreted by goblet cells within the stomach and are believed to intercalate with mucus glycoproteins, possibly contributing to the barrier properties of mucus. these peptides also play a critical role in repair of injured mucosa. an additional mucosal function that serves to reduce the level of injury is adaptive cytoprotection, wherein application of topical irritants to gastric mucosa results in subsequent protection of mucosa in response to repeated exposure to damaging agents. for example, pretreatment with % ethanol protected against mucosal damage in response to subsequent application of absolute ethanol, and this effect was abolished by treatment with the cyclooxygenase inhibitor indomethacin. the cytoprotective effect of prostaglandins has been demonstrated directly in studies in which preadministration of prostaglandins protected gastric mucosa from damage by agents such as concentrated hcl and hypertonic saline. prostaglandins appear to be cytoprotective in the stomach at doses less than those used to inhibit gastric acid secretion, ruling out a simple antacid mechanism. although not fully characterized, cytoprotection has been attributed in part to prostaglandin-stimulated mucus production. an associated beneficial effect of prostaglandins is the increased production of bicarbonate, which is trapped within mucus on the surface of the mucosa. , interestingly, prostaglandin e (pge ) appears to lose its cytoprotective activity in the presence of the mucolytic agent n-acetylcysteine. attention also has been directed at enhanced mucosal blood flow as a potential mechanism for prostaglandin-mediated cytoprotection. for example, pretreatment with pgi protected against ethanol-induced mucosal damage as a result of increased mucosal blood flow. although pge , which is also cytoprotective, does not increase blood flow, it may prevent vascular stasis associated with irritant-induced vascular damage resulting from inhibition of neutrophil adherence to damaged endothelium. sensory nerves also have been implicated in cytoprotective mechanisms. these nerves are distributed throughout gastrointestinal mucosa. as an example of their importance in mucosal cytoprotection, pretreatment of newborn rats with capsaicin (to which sensory nerves are sensitive) renders the mature rats more susceptible to gastric injury. alternatively, use of a low dose of capsaicin, which stimulates rather than destroys sensory nerves, protects gastric mucosa against injurious agents. , sensory nerves contain neuropeptides such as calcitonin-gene-related peptide (cgrp) and substance p, which may play a protective role via vascular mechanisms. for instance, cgrp stimulates increased gastric blood flow, which is theorized to reduce injury in much the same way as prostaglandins do. in fact, recent studies suggest that the roles of prostaglandins and cgrp in gastric cytoprotection are intertwined intimately. in particular, pgi is believed to sensitize sensory nerves following treatment with a mild irritant, with resultant increases in cgrp release and mucosal flow. similar studies have shown that antagonists of cgrp inhibit the cytoprotective action of pge . another neural mediator, nitric oxide, also has been implicated in adaptive cytoprotection. interestingly, nitric oxide has a number of actions that are similar to those of prostaglandins, including maintenance of mucosal blood flow. regulation of barrier function in the intestine is not as well characterized as that of the stomach, although mechanisms of barrier function, including secretion of mucus and regulation of mucosal blood flow, are presumed to be similar. the proximal duodenum also has to protect itself from acid damage as it receives gastric contents, and this involves secretion of mucus and bicarbonate in much the same way as the stomach. one other mechanism that helps the stomach and the intestine to maintain mucosal barrier function is the speed with which the mucosa repairs. thus for a defect to develop in the mucosal barrier, injurious factors have to outpace mucosal recovery. such recovery initially involves epithelial migration across denuded regions of basement membrane (restitution), a process so rapid that epithelial defects may be resurfaced within minutes. for example, in bile salt-injured colon, denuded surface mucosa was covered completely by restitution. in the small intestine, villi greatly amplify the surface area of the mucosal luminal surface, which in turn takes far longer to resurface with restituting epithelium once it has become denuded. however, intestinal villi are able to reduce the denuded surface area considerably by extensively contracting. these mechanisms are described in detail under mechanisms of gastrointestinal mucosal repair. although the stratified squamous epithelium is relatively impermeable to hcl, a number of factors can enhance the damaging effects of hcl in this epithelium. in particular, bile salts and short-chain fatty acids are capable of breaking down the squamous epithelial barrier at an acid ph, thereby exposing deep layers to hcl, with subsequent development of ulceration. , high concentrations of short-chain fatty acids normally exist within the equine stomach because of microbial fermentation. these weak acids penetrate squamous mucosa and appear to damage na + transport activity principally located in the stratum germinativum. bile salts also may be present in the proximal stomach because of reflux from the duodenum. although such reflux has a high ph, bile salts appear to adhere to stratified squamous epithelium, becoming lipid soluble and triggering damage once the ph falls below . diet and management (e.g., periods of fasting) also play crucial roles in the development of conditions conducive to gastric ulceration. typically, a ph gradation in horses exists from proximal to distal compartments of the stomach, with the lowest ph values in the distal stomach. however, fasting disrupts this stratification such that low ph values may be recorded in the proximal stomach. fasting conditions also increase the concentration of duodenal contents within the proximal stomach, particularly bile. proper gastric mucosa is exposed to injurious agents, including pepsin, bile, and acid. parietal cells in the horse secrete acid constantly as an adaptation to near-continuous intake of roughage, but the enterochromaffin-like cells within the proper gastric mucosa and g and d cells within the pyloric mucosa tightly regulate acid secretion. histamine released by enterochromaffin-like cells amplifies acid secretion and interacts with h receptors on parietal cells and g cells, which release the prosecretory hormone gastrin. a combination of histamine and gastrin can have a synergistic effect on parietal cell gastric secretion, because these mediators have distinct receptors and second messengers. however, d cells are sensitive to an acidic environment and release somatostatin, which inhibits acid secretion. nonetheless, gastric mucosa may be exposed to acid for prolonged periods of time, particularly in horses that are extensively meal fed and that do not have the benefit of roughage, which tends to buffer stomach contents. , aside from peptic ulceration induced by combinations of acid and pepsin, research in human medicine has revealed the tremendous importance of helicobacter pylori in inducing ulceration. infection with this organism has the effect of raising gastric ph because of disruption of gastric glands and also induces an inflammatory reaction that causes damage. however, little evidence to date indicates that this organism is involved in gastric ulcers in horses. in the absence of a known role for infectious agents in gastric ulceration in animals, ulceration likely develops from injurious factors similar to those found in the proximal stomach, including gastric acid and bile. however, some factors that are important to induction of squamous epithelial ulceration may not be important in development of proper gastric mucosal ulceration. for example, feed deprivation and intensive training reproducibly induce squamous epithelial ulceration in horses but have little effect on proper gastric mucosa in horses. gastric acid likely plays a key role, whereas other factors such as nonsteroidal antiinflammatory drugs (nsaids) serve to reduce gastric defense mechanisms. in particular, inhibition of prostaglandin production reduces mucus and bicarbonate secretion while also reducing gastric mucosal blood flow. some of the nsaids also have a topical irritant effect, although this appears to be of minor significance because the route of administration (oral or parenteral) seems to have little influence on development of ulceration. the source of prostaglandins responsible for gastric protection originally was assumed to be cyclooxygenase (cox- ), because this isoform is expressed constitutively in gastric mucosa, whereas cox- is not expressed in the stomach unless induced by inflammatory mediators. however, mice in which the cox- gene has been knocked out fail to develop spontaneous gastric lesions, possibly because of compensatory increases in prostaglandin production by cox- . this concept agrees with recent data indicating that inhibition of both cox isoforms is required to induce gastric ulceration. from a clinical perspective this data indicate that drugs selective for cox- or cox- may be less ulcerogenic in the horse. because cox- elaborates prostaglandins induced by inflammatory stimuli, selective inhibitors of cox- may be particularly useful because of their ability to serve as antiinflammatory agents that are less ulcerogenic. the most notable cause of intestinal mucosal injury in horses, particularly those suffering from colic, is ischemia. initially, that a reduction in gastrointestinal blood supply leads to mucosal injury seems intuitive. however, the anatomy of the gastrointestinal tract and the differing structure of the intestinal mucosa at various anatomic locations have a significant influence on the extent of mucosal injury. furthermore, ischemic injury may be induced by several different mechanisms, including occlusion of arterial supply by a thrombus, strangulation of intestinal vasculature, and generalized reduction in blood flow associated with various shock states. in addition, a number of seemingly distinct mechanisms of intestinal injury, such as intestinal distention, also trigger mucosal injury via an ischemic mechanism. finally, reperfusion injury also may influence the extent of mucosal injury following an ischemic episode and has been proposed as a potential site of therapeutic intervention. , thus understanding the mechanisms of ischemia-reperfusion injury is critical to developing an understanding of the severity of various clinical conditions and beginning to formulate a therapeutic approach to diseases characterized by this devastating form of injury. the intestinal circulation is capable of closely regulating blood flow during periods of low systemic perfusion pressure. , in particular, local regulation of resistance vessels within the microvasculature is particularly prominent, whereby metabolic end products of adenosine triphosphate (atp) result in continued dilation of resistance vessels despite reductions in systemic arterial pressure. dilation results in continued perfusion of gastrointestinal tissues during the early stages of shock, while other organs such as skeletal muscle undergo massive shunting of blood resulting from increased constriction of resistance vessels. the reasons for these differences in regulation are not entirely clear but may relate to the high level of energy required to fuel the intestinal mucosa and the serious systemic effects of breaches in the mucosal barrier. however, as blood flow falls below a critical level, regulatory systems are no longer effective and oxygen uptake by the gastrointestinal tissue decreases, culminating in tissue damage. the tip of the villus is the most susceptible region affected by hypoxia in the equine small intestine, largely because of the countercurrent exchange mechanism of blood flow in the small intestinal villus. this countercurrent exchange mechanism is attributable to the vascular architecture, which consists of a central arteriole that courses up the core of the villus, arborizes at the tip, and is drained by venules coursing down the periphery of the villus. as oxygenated blood flows into the central arteriole, oxygen tends to diffuse across to the adjacent venules, which flow in the opposite direction. this series of events takes place along the length of the villus, resulting in a tip of the villus that is hypoxic even under normal conditions. furthermore, reduced blood flow as occurs in shock exacerbates the countercurrent exchange of oxygen, and the tip becomes absolutely hypoxic. this mechanism might explain why the small intestinal mucosa is more susceptible to ischemic injury, compared with the colon, which has no villi. for example, the duration required to produce severe morphologic damage to the equine colon is approximately % longer than in the small intestine. intestinal mucosal epithelium is susceptible to hypoxia because of the high level of energy required to fuel the na + /k + -atpase that directly or indirectly regulates ion and nutrient flux. the first biochemical event to occur during hypoxia is a loss of oxidative phosphorylation. the resulting diminished atp concentration causes failure of the energy-dependent na + /k + -atpase resulting in accumulation of sodium, and subsequently intracellular water. the ph of the cytosol drops as lactic acid and inorganic phosphates accumulate from anaerobic glycolysis. the falling ph damages cell membranes, including lysosomal membranes, resulting in the release and activation of lysosomal enzymes into the cytosol, further damaging cellular membranes. damage to the cell membrane allows the accumulation of high concentrations of calcium in the cytosol, which activates calciumdependent degradative enzymes. these events result in cytoplasmic blebbing of the basal membrane with subsequent detachment of cells from the underlying basement membrane. recent studies on epithelial injury during ischemia suggest that most epithelial cells undergo programmed cell death (apoptosis) during ischemia and reperfusion rather than necrosis, allowing retention of reusable components of irreversibly injured cells. in one study, % of detached epithelium during small intestinal ischemia and reperfusion underwent apoptosis. although the most obvious result of apoptosis is loss of surface epithelium, a number of cells on the lower portion of the villus (in the small intestine) and cells within the crypts also may undergo apoptosis that only may become evident up to hours following reperfusion of ischemic tissue. morphologic changes observed in ischemic-injured small intestinal mucosa follow a similar sequence regardless of whether ischemia alone or ischemia and reperfusion induce injury (table . - ). initially, epithelium separates from the underlying basement membrane, forming a fluid-filled space termed grüenhagen's space ( figure . - ). the mechanism of fluid accumulation in this space is not understood entirely but may result from continued epithelial absorption of nacl and water before it has detached fully from neighboring epithelial cells. this fluid accumulation likely exacerbates epithelial separation from the basement membrane. subsequently, epithelium progressively sloughs from the tip of the villus toward the crypts, which are the last component of the intestinal mucosa to become injured. [ ] [ ] [ ] injury of crypts likely relates to the vascular architecture, because crypts receive a blood supply separate from the vasculature involved in the villous countercurrent exchange mechanism. the early morphologic changes observed in the equine large colon during ischemia are different from those described in the equine small intestine because of the lack of intestinal villi. however, as might be expected, the more superficially located surface cells are sloughed before those in crypts. , the orderly progression of tissue injury has been used by one group of investigators to predict accurately the survival of horses with large colon volvulus. the researchers took biopsies from the pelvic flexure, which has been shown previously to reflect mucosal changes along the length of the colon accurately, and examined them histologically for the width of the crypts and intercrypt interstitial space. they expressed the latter measurements as a ratio of interstitium to crypt width (i:c) and defined nonviable colon as that which has greater than % loss of crypt and an i:c ratio greater than . using this methodology, researchers correctly predicted survival in % of horses. because of the dramatic decline in strongylus vulgarisinduced colic, which was associated frequently with infarction of intestinal arterial blood supply, most ischemic lesions are associated with strangulating obstruction. therefore considering mechanisms of ischemic injury in horses with naturally occurring strangulating lesions is important. the majority of experimental work has assessed complete ischemia (complete occlusion of the arterial blood supply) or low-flow ischemia (during which arterial blood flow is reduced). , however, during intestinal strangulation, a disparity between the degree of occlusion of the veins and arteries occurs whereby veins are occluded before arteries because of differences in compliance of vascular walls. thus strangulating lesions are typically hemorrhagic (hemorrhagic strangulating obstruction) as the arteries continue to supply blood to tissues that have little or no venous drainage. the result is ischemic injury, as previously outlined, but also a tremendous congestion of the tissues. such hemorrhagic congestion has two opposing effects: it disrupts tissue architecture, including the mucosa and its epithelium, and continues to provide oxygenated blood to the tissues during much of the ischemic episode. in contrast, when strangulation results in sudden cessation of arterial blood flow (ischemic strangulating obstruction), tissues appear pale, and the mucosa rapidly degenerates because of a complete lack of oxygenated blood. because intestine that may look nonviable (dark red) may in fact have less mucosal injury than that of ischemic strangulated intestine. an additional consideration in clinical strangulating obstruction is the degree of ischemia that intestinal distention may induce. for example, experimental distention ( cm of h o for hours) and decompression ( hours) of jejunum resulted in a significant increase in microvascular permeability and a significant decrease in tissue oxygenation similar to that which would be expected with low-flow ischemia. , in particular, microscopic evaluation of vasculature revealed capillary endothelial cell damage and local edema formation. this data suggest that distended intestine proximal to an obstruction may undergo mucosal injury despite its normal appearance. indeed, in one study, intraluminal pressures greater than cm h o in naturally occurring cases of colic correlated with a poor prognosis for survival. although that reperfusion of ischemic tissues results in exacerbation of mucosal injury recently has been taken for granted, one should remember that mechanisms underlying intestinal reperfusion injury have been defined largely in laboratory animals under specific conditions. [ ] [ ] [ ] [ ] [ ] however, studies on reperfusion injury in horses have had some conflicting results. , , the conflict may be attributable to the way in which the studies have been performed. in particular, the type of ischemia used in most laboratory animal studies has been low-flow ischemia (in which the blood flow typically is reduced to % of baseline flow), whereas studies in horses have used a number of different ischemic models, including various types of strangulating obstruction. although strangulating obstruction is of great clinical relevance, this type of ischemic insult is less likely to develop reperfusion injury. , , conversely, low-flow ischemia appears to prime tissues for subsequent injury once the tissue is reperfused, and considerable evidence supports the presence of reperfusion injury in horses following low-flow ischemia. , , , nonetheless, lowflow ischemia may not be a common clinical entity. in addition to the type of ischemia, other factors are involved in priming tissues for reperfusion injury, including species and anatomic-specific variation in oxidant enzyme and neutrophil levels ( table . - ). for example, the foal appears to have low levels of small intestinal xanthine oxidase, an enzyme that has been shown to play a critical role in triggering reperfusion injury in laboratory animals, , , whereas adult levels are much greater, particularly in the proximal small intestine. in addition, horses appear to have low numbers of resident neutrophils in the intestinal mucosa, and this population of neutrophils (rather than those recruited from the circulation) appears to be most critical for induction of reperfusion injury. however, studies demonstrating reperfusion injury in the equine colon following low-flow ischemia have shown significant accumulation of neutrophils within the mucosa. therefore a complete understanding of mechanisms of neutrophilic infiltration and the mechanisms whereby they damage tissue requires further study. reperfusion injury is initiated during ischemia when the enzyme xanthine dehydrogenase is converted to xanthine oxidase and when its substrate, hypoxanthine, accumulates simultaneously because of atp use ( figure . - ). , however, little xanthine oxidase activity occurs during ischemia, because oxygen is required as an electron acceptor. during reperfusion, xanthine oxidase rapidly degrades hypoxanthine in the presence of oxygen, producing the superoxide radical as a by-product. the superoxide radical contributes to oxidative tissue damage and, most importantly, activates neutrophil chemoattractants. , thus inhibition of xanthine oxidase in feline studies of intestinal ischemiareperfusion injury prevents infiltration of neutrophils and subsequent mucosal injury. , however, inhibition of xanthine oxidase has had no effect on ischemiareperfusion injury in equine small intestine and colon, suggesting that reperfusion injury is simply a continuation of injury initiated during ischemia, as suggested in some equine studies, or that the classic reperfusion injury pathway is activated by alternate sources of reactive oxygen metabolites. the latter has been suggested by studies in feline models of ischemia-reperfusion injury in which the source of a significant proportion of reactive oxygen metabolites is unknown and is independent of xanthine oxidase and neutrophils. a veterinary review of the pathogenesis of intestinal reperfusion injury in the horse suggested the concept of a therapeutic window wherein treatment of reperfusion injury would be beneficial. the basis of this concept is that certain conditions exist under which ischemic injury is minimal and that tissues are damaged severely during reperfusion. thus under conditions of low-flow ischemia, little injury is demonstrated during hours of ischemia, but remarkable injury occurs during hour of reperfusion. [ ] [ ] [ ] however, a therapeutic window may not exist under conditions of strangulating obstruction in which severe injury occurs during ischemia and minimal injury occurs during reperfusion. this in turn greatly reduces clinicians' ability to ameliorate ischemiareperfusion injury with treatments such as antioxidants at the time of reperfusion. mechanisms of gastric repair depend greatly on the extent of injury. for instance, superficial erosions can be covered rapidly by migration of epithelium adjacent to the wound; a process termed epithelial restitution. however, ulceration (full-thickness disruption of mucosa and penetration of the muscularis mucosa) requires repair of submucosal vasculature and extracellular matrix. the formation of granulation tissue initiates repair and supplies connective tissue elements and microvasculature necessary for mucosal reconstruction. connective tissue elements include proliferating fibroblasts that accompany newly produced capillaries that form from proliferating endothelium. recent studies indicate that nitric oxide is critical to both processes, , which likely explains the reparative properties of nitric oxide in the stomach. once an adequate granulation bed has formed, newly proliferated epithelium at the edge of the wound begins to migrate across the wound. in addition, gastric glands at the base of the ulcer begin to bud and migrate across the granulation bed in a tubular fashion. repairing epithelium expresses epidermal growth factor, which appears to facilitate these processes. in addition, a mucoid cap facilitates these events and retains reparative factors and serum adjacent to the wound bed. once the ulcer crater has been filled with granulation tissue and the wound has been reepithelialized, the subepithelial tissue remodels by altering the type and amount of collagen. despite the remodeling process, ulcers tend to recur at sites of previous ulceration, and the concern is that this remodeling can result in excessive deposition of collagen and fibrosis. reparative mechanisms are similar in the intestine, except that in the small intestine, mucosal villi contribute to mucosal repair. once intestinal epithelium is disrupted, two events occur almost immediately to reduce the size of the denuded portion of the villus: contraction of the villus and epithelial restitution ( figure . - ). for example, in porcine ileum subjected to hours of ischemia, villi were % of their former height and % of the denuded villous surface area was covered in flattened epithelium within hours. enteric nerves appear to regulate villous contraction, because inhibition of enteric nerve conduction prevents villous shortening following injury. the contractile component of the villus is a network of myofibroblasts distributed throughout the lamina propria of the villus and along the central lacteal. inhibition of villous contraction results in retarded epithelial repair because of the larger denuded surface that remains to be covered by migrating epithelium compared with similarly injured villi that have contracted. pge also has been implicated in regulating villous contraction, because application of pge resulted in villous contraction when perfused through normal rat ileum. as villi contract, assuming the basement membrane is intact, epithelium from the margins of the wound migrates centripetally to resurface toward the tip of the villus. the process of restitution is similar in denuded colonic mucosa, except that it may proceed more rapidly because of the lack of villi. epithelial restitution is solely a migratory event that does not depend on provision of new enterocytes by proliferation. cellular migration is initiated by extension of cellular lamellipodia that receive signals from the basement membrane via integrins. intracellular signaling converges on the actin cytoskeleton, which is responsible for movement of lamellipodia. specific components of the basement membrane appear to be critical to the migratory process. for example, application of antibodies to collagen types iii and iv, which are important components of intestinal mucosal basement membrane, impeded epithelial restitution. , other elements of the basement membrane, including proteoglycans, hyaluronic acid and noncollagenous proteins such as fibronectin and laminin also may provide important signals. these subepithelial matrix components that facilitate restitution may form the basis for clinical treatments designed to speed up the repair process, analogous to administration of matrix components to horses with articular cartilage damage. although epithelial restitution results in gross closure of previously denuded regions of gastrointestinal mucosa, closure of interepithelial spaces ultimately is required to restore normal epithelial barrier resistance. because the tight junction is principally responsible for regulating the permeability of the interepithelial space, repair and closure of this structure likely is critical to restore intestinal barrier function. recent research indicates that prostaglandins play a vital role in recovery of tight junction resistance, indicating that administration of nonselective cox inhibitors to horses with colic, particularly those recovering from strangulating obstruction, may be deleterious. therefore judicious use of nsaids is appropriate until more selective drugs that allow continued production of reparative prostaglandins are available for use in horse. after restoration of the epithelial barrier, the epithelium must reestablish normal mucosal architecture to allow normal gut absorptive and digestive function. in porcine ileum subjected to hours of ischemia, the epithelial barrier was restored within hours, but villi were contracted and covered in epithelium with a squamous appearance. restoration of normal villous architecture required another days. newly proliferated crypt epithelium replaces the flattened villous epithelium that characterizes restitution. under normal circumstances the dividing stem cells, of which the base of each mucosal crypt has approximately four, form new enterocytes. newly divided enterocytes migrate from the crypt onto the villus. during migration, enterocytes differentiate and acquire specific absorptive and digestive functions. fully differentiated enterocytes reside on the upper third of the villus for to days and then are sloughed into the intestinal lumen. this process accelerates during mucosal repair and requires increased proliferative rates. a variety of locally available gut-derived factors, including luminal nutrients, polyamines, and growth factors, may stimulate increased proliferation within to hours. the return of the normal leaflike shape of the villus occurs following the appearance of normal columnar epithelium. although prostaglandins have been implicated in mucosal cytoprotective function, few studies have assessed their importance in mucosal repair. one study implicated prostaglandins in growth factor-stimulated restitution, but a more prominent role of prostaglandins in mucosal repair is their ability to close interepithelial tight junctions. , , for instance, ischemic-injured small intestine rapidly recovers barrier function (as measured in vitro as transepithelial resistance) in the presence of pgi and pge , despite the fact that these prostanoids had little effect on villous contraction and epithelial restitution. however, electron microscopic examination of tissues reveals dilation of tight junctions in tissues treated with nsaids, whereas those additionally treated with prostaglandins have closely apposed tight junctions (figures . - and . - ). prostaglandins stimulate closure of tight junctions via the second messengers cyclic adenosine monophosphate and ca + , which interestingly were among the first mediators found to modulate tight junction permeability. , such tight junction closure is of importance to patients with intestinal injury that are treated with nsaids, because reduced prostaglandin levels may result in increased intestinal permeability. for example, in a study on ischemic-injured porcine ileum, treatment with the nsaid indomethacin resulted in a significant increase in intestinal permeability to inulin and lipopolysaccharide compared with tissues that were treated additionally with pgi and pge . section . pathophysiology of mucosal injury and repair a b figure . - ultrastructural appearance of repairing ischemic-injured mucosa. a, restituting epithelium hours following a -hour ischemic episode in the presence of the nonselective cyclooxygenase inhibitor indomethacin. dilation of the interepithelial space and the apical tight junction (arrows) correlates with a leaky intestinal barrier, b, similar restituting epithelium had been treated additionally with prostaglandins e and i . the close apposition of the tight junction (arrows) and the interepithelial space correlate with normalization of intestinal barrier function. -cm bar = µm. the process of restitution absolutely depends on a group of compounds called polyamines. , the rate-limiting enzyme in the formation of the polyamines spermine, spermidine, and putrescine is ornithine decarboxylase. in rats with stress-induced duodenal ulcers, systemic administration of the ornithine decarboxylase inhibitor α-difluoromethylornithine significantly reduced polyamine levels and greatly reduced epithelial restitution. furthermore, intragastric treatment of these same rats with putrescine, spermidine, and spermine prevented the delayed mucosal repair induced by α-difluoromethylornithine. interestingly, gastric tissue levels of ornithine decarboxylase increased in rats with stress-induced gastric ulcers, suggesting that tissue injury enhances polyamine production, which may contribute to the normal rapid rate of epithelial restitution. the mechanisms whereby polyamines stimulate epithelial restitution are not clear. mccormack, wang, viar, et al. hypothesized that polyamines increase transglutaminase activity, an enzyme that catalyzes the cross-linking of cytoskeletal and basement membrane proteins. further investigation of the role of polyamines in iec- cell migration showed that depletion of polyamines resulted in disruption of the cytoskeleton and reduced the physical extension of lamellipodia. more recent studies have clarified this pathway. in particular, polyamines have been shown to regulate cytoskeletal cellular migration via activation of the small guanosine triphosphatase-rho-a by elevating intracellular ca + levels. these elevations in ca + result from polyamine regulation of expression of voltagegated k + channels and altered membrane electric potential. polyamines also play a role in the normal physiologic regulation of crypt cell proliferation and differentiation. , polyamines are produced by fully differentiated enterocytes at the tip of the villus and may reach the crypt within sloughed luminal epithelium or via local villous circulation. following intestinal injury, polyamines appear to stimulate enhanced proliferation by increasing the expression of protooncogenes, which control the cell cycle. the mechanism whereby polyamines influence gene expression likely relates to the cationic nature of these compounds, which may influence the tertiary structure of negatively charged dna and rna. locally produced growth factors-including epidermal growth factor (egf), transforming growth factor α (tgf-α), tgf-β, and hepatocyte growth factor-have the ability to modulate mucosal recovery. the most important of these growth factors in early mucosal repair events is tgf-β, which is a potent stimulus of epithelial restitution and modulator of the extracellular matrix. neutralization of tgf-β retards epithelial migration in vitro, and tgf-β apparently may serve as a point of convergence for mediators of restitution, because neutralizing tgf-β also inhibits the effects of other peptides. however, tgf-β paradoxically inhibits epithelial proliferation, thereby reducing the supply of new enterocytes for mucosal repair. conversely, egf, produced by the salivary glands and duodenal brunner's glands, and the related tgf-α, produced by small intestinal enterocytes, are potent stimulants of enterocyte proliferation. these growth factors share approximately % of their amino acid structure, bind to the same receptor on the basolateral surface of enterocytes, and are not related to tgf-β. the physiologic role of egf is difficult to discern because it is present in the intestinal lumen, with no apparent access to its basally located receptor. however, egf has been proposed to act as a "surveillance agent" that gains access to its receptor during epithelial injury (when the egf receptor likely would be exposed) to stimulate proliferation. tgf-α presumably has a similar role, but it is present in greater concentrations in the small intestine because it is produced by differentiated villous enterocytes. the mature peptide is cleaved from the extracellular component of the transmembrane tgf-α precursor and released into the lumen. another group of proreparative peptides produced within the gastrointestinal tract are the trefoil peptides. under physiologic conditions, trefoil peptides are secreted by mucus-producing cells at distinct anatomic sites. for example, gastric epithelium produces the trefoil peptide ps , whereas the small and large intestine mucosa produce intestinal trefoil peptide. however, any of the trefoil peptides may be upregulated within repairing epithelium regardless of anatomic site. , in addition, trefoil peptides have the ability to induce their own expression, amplifying the level of these reparative factors at sites of mucosal repair. trefoil peptides are the most potent stimulants of epithelial migration in vitro, and their effects are independent of growth factors, including tgf-β. however, recent evidence suggests that egf receptor activation is required for induction of ps and another of the trefoil peptides, termed spasmolytic peptide, in gastric epithelium in vitro. the importance of trefoil peptides to the mucosal repair response in vivo is illustrated by gene knockout studies, in which mice deficient in intestinal trefoil factor have greatly reduced ability to repair intestinal injury. in fact, detergent-induced mucosal injury was lethal because of a lack of restitution compared with wild-type mice that fully recovered from similar mucosal injury. the fact that administration of intestinal trefoil factor restored restitution has important therapeutic implications. the mechanism whereby trefoil peptides stimulate epithelial migration is yet to be characterized fully but appears to involve translocation of the adherens junction protein e-cadherin, thereby allowing cells to become untethered from neighboring cells. the principal metabolic fuel of enterocytes is glutamine and of colonocytes, butyrate. however, recent studies suggest that glutamine and butyrate have more specific proliferative actions aside from their role as nutrients. for example, in the piglet ipec-j enterocyte cell line, glutamine enhanced gene transcription by increasing mitogen-activated protein kinase activity. , similarly, butyrate stimulated mucosal growth following colonic infusion in the rat. because of such growth-promoting actions, glutamine was shown to prevent intestinal mucosal atrophy and dysfunction that accompanies starvation , and long-term total parental nutrition. , additionally, glutamine improves function of transplanted small intestine , and protects intestinal mucosa from injury if administered before chemotherapy and radiation therapy. , intestinal nutrients also may synergize with other proliferative agents. for example, administration of glutamine and tgf-α to porcine ileum that had been subjected to hours of ischemia resulted in a synergistic increase in mitogen-activated protein kinase activity, enterocyte proliferation, and villous surface area. although concern has arisen that such early return to normal surface area may result in dysfunctional mucosal digestive and absorptive function because of resurfacing denuded mucosa with immature epithelium, nutrients and growth factors also appear to promote early differentiation. in the case of glutamine and tgf-α restoration of postischemic small intestine, rapid recovery of digestive enzymes also was documented. effective gastrointestinal motility involves a complex interaction between the enteric nervous system, muscular wall, and luminal contents. additional factors that influence the net transit of digesta include gravity, the volume and viscosity of the contents, and pressure gradients created by simultaneous contraction and relaxation of adjacent segments of bowel. casual use of the term intestinal motility in veterinary medicine often underestimates the complexity of the processes involved in the transit of intestinal contents. this is particularly true when the term is used to describe the frequency and or intensity of intestinal sounds, or borborygmi. the existence of borborygmi does not always equate with progressive movement of intestinal contents. disruption to normal motility occurs commonly in horses for a variety of reasons. examples of diseases in which altered motility may be present include gastroduodenal ulceration, intraluminal obstruction or impaction, excessive wall distention, strangulating obstructions, peritonitis, and inflammatory bowel diseases such as duodenitis proximal jejunits or colitis. ineffective intestinal motility is also a feature of several neonatal diseases, including prematurity, systemic sepsis, and perinatal asphyxia. certain parasitic infections, electrolyte derangements, and endotoxemia can modify digesta transit in horses of all ages. general anesthesia and specific sedatives, such as xylazine, romifidine, or detomidine, also disturb motility. the inhibition of propulsive bowel activity usually is referred to as ileus. ileus is ascribed most frequently to the condition that occurs after laparotomy and is termed simple or uncomplicated postoperative ileus (poi). the term complicated or paralytic ileus describes intestinal motility disturbed for longer periods after surgery. poi in horses is associated most commonly with surgery of the small intestine, particularly after resection and anastomosis, , is a common complication of small intestinal surgery, and can have a negative effect on short-term postoperative survival. , motility dysfunction likely is present in all horses after laparotomy, but many are affected subclinically and require minimal or no specific intervention. in symptomatic animals, clinical signs are apparent shortly after recovery and include colic, tachycardia, dehydration, decreased borborygmi and fecal output, and sequestration of fluid within the stomach. rectal examination and ultrasound reveal small intestinal distention with rare or absent wall movement. the severity and duration of intestinal stasis varies, lasting from minutes to days. a specific motility disorder involving the cecum or ileocecocolic region occurs sporadically in horses. [ ] [ ] [ ] the condition most commonly occurs after general anesthesia and extraabdominal surgery, particularly orthopedic and upper airway procedures, and therefore often is categorized as a form of poi. anecdotally, horses at greatest risk are young male performance animals. other cases occur spontaneously, often in animals with painful primary conditions such as uveitis or septic tenosynovitis. the syndrome is frustrating in that clinical signs are often subtle unless cecal perforation has occurred. in horses with a cecal emptying defect after anesthesia, overt signs are usually apparent to days after the procedure. the earliest detectable signs include depression and a reduction in feed intake and fecal output. ineffective emptying results in overfilling of the cecum with moist contents, which is manifest by signs of mild to moderate colic. if the condition is recognized late or untreated, the cecum may rupture and result in fatal peritonitis. the inherent rhythmicity of electric activity in the intestine is controlled by the interstitial cells of cajal, specialized cells that are electrically coupled to myocytes via gap junctions. these cells are responsible for generating and propagating slow-wave activity and may be critically involved in a range of motility disorders. the enteric nervous system primarily controls and coordinates intestinal contraction. a combination of central and autonomic innervation influences events, but contraction does not require external neural input. the parasympathetic supply to the gastrointestinal tract is via the vagus and pelvic nerves, and the sympathetic supply is through postganglionic fibers of the cranial and caudal mesenteric plexuses. a complex network of interneurons within each plexus integrates and amplifies neural input; the intensity and frequency of resultant smooth muscle contractions are proportional to the amount of sympathetic and parasympathetic input. additional binding sites for a number of other endogenous chemicals, including dopamine, motilin, and serotonin exist within the enteric nervous system and on smooth muscle cells. acetylcholine is the dominant excitatory neurotransmitter in the gastrointestinal tract and exerts its action through muscurinic type receptors on smooth muscle cells. sympathetic fibers innervating the gastrointestinal tract are adrenergic, postganglionic fibers with cell bodies located in the prevertebral ganglia. activation of α adrenergic receptors on cholinergic neurons within enteric ganglia inhibits the release of acetylcholine and therefore reduces intestinal contraction. β -, β -, and β-atypical receptors are directly inhibitory to the intestinal smooth muscle. inhibitory nonadrenergic, noncholinergic neurotransmitters include adenosine triphosphate, vasoactive intestinal peptide, and nitric oxide. , these neurotransmitters are critical for mediating descending inhibition during peristalsis and receptive relaxation. substance p is a nonadrenergic, noncholinergic neurotransmitter that may be involved in contraction of the large colon. , the rate and force of intestinal contractions along the small intestine and large colon of the horse are important determinants of intestinal motility; of even greater importance to the net propulsion of digesta are the cyclical patterns of contractile activity. these patterns are known as the small intestinal and colonic migrating motility (or myoelectric) complexes (mmcs). , the colonic complex usually originates in the right ventral colon and variably traverses the ascending and descending colons. many of these complexes are related temporally to a specialized motility event of the ileum, the migrating action potential complex. local inflammation within the intestinal muscularis and inhibitory neural events are important initiators of intestinal ileus. , intestinal inflammation not only is important in primary intestinal diseases in horses, such as duodenitis-proximal jejunitis and colitis but also is induced after simple intestinal handling during laparotomy. experimental data from other species suggests that handling of the small or large intestine at the time of surgery activates resident macrophages with resultant increased expression of p-selectin and intercellular adhesion molecule on endothelial cells within the vasculature of the muscularis. the upregulation of associated ligands on leukocytes leads to sequential "sticking and rolling," followed by neutrophil migration into the interstitium. the subsequent release of neutrophil products interferes with cell signaling and results in reduced intensity of smooth muscle contraction. furthermore, the inflamed intestine fails to contract normally in response to putative prokinetic agents. another key factor in the development of intestinal stasis after inflammation is the local overproduction of nitric oxide caused by the upregulation of inducible nitric oxide synthase (inos) by resident macrophages. nitric oxide is a key inhibitory neurotransmitter of the nonadrenergic, noncholinergic system. nitric oxide synthase inhibition has been a pharmacologic target in the treatment of experimental ileus. the inhibitory effects of α -agonists such as xylazine and detomidine on cecal and large colon motility are well described. [ ] [ ] [ ] [ ] [ ] [ ] intravenously administered xylazine inhibits cecal and large colon motility for to minutes without seriously disrupting small intestinal myoelectric activity, and detomidine can reduce large intestinal myoelectric activity for up to hours. the α -antagonist yohimbine has a weak but positive effect on cecal emptying in normal ponies, suggesting that normal motility is under constant α -adrenergic tone. atropine is a postganglionic blocking agent that binds to muscarinic receptors. when administered at . mg/kg, atropine inhibits individual small intestinal, cecal, and colonic contractions for about minutes but supresses small intestinal and colonic migrating complexes for up to hours. neural reflexes also may mediate inhibition of motility associated with peritoneal inflammation. , the afferent segment is composed partly of capsaicin-sensitive visceral afferent c fibers that terminate in the dorsal horn of the spinal cord, where they can activate inhibitory sympathetic fibers or synapse directly on the sympathetic ganglia. consequently, the efferent limb of the reflex expresses increased sympathetic outflow, primarily mediated through stimulation of α -adrenoreceptors, and inhibition of acetylcholine release, which provides the rationale for α -blockade in treating ileus. intraluminal infusion of capsaicin before abdominal surgery ameliorated the severity of poi in experimental rats. this finding highlights the importance of visceral afferent fibers in the development of poi. ileus also can occur in association with intestinal obstruction or displacement. mild to moderate distention of the bowel, such as that occurring in the early stages of an intraluminal obstruction, evokes an increase in local contractile activity. , excessive distention results in inhibition of motility within the distended segment of bowel. intestinal stasis is not always detrimental and under certain conditions may be protective. endotoxemia is a clinical feature of many diseases of the equine gastrointestinal tract, and endotoxins independently can exert a negative effect on intestinal motility and transit. a variety of mediators likely are involved, but activation of α -adrenoreceptors and production of prostanoids appear to be important, for pretreatment with yohimbine or nonsteroidal antiinflammatory drugs (nsaids; phenylbutazone or flunixin), respectively, ameliorates the inhibitory effects of experimental endotoxin infusion. , endotoxin infusion induced an inflammatory response in the intestine of rats that mimicked the response induced by handling during laparotomy. the similarity of the responses were highlighted in a recent study that demonstrated that prior exposure of the muscularis to endotoxin protected the intestine from the effects of manipulation. the pathophysiology of cecal emptying defect is not known. this syndrome may best mimic poi in human beings and generally is considered a large intestinal disorder. an important difference in horses is that laparotomy is a rare predisposing factor, and most cases occur in horses undergoing routine extraabdominal surgical procedures. general anesthesia itself is a potent inhibitor of gastrointestinal motility in horses, but these effects are short-lived and reversible within hours of anesthetic withdrawal. the return of normal motility in horses after experimental ileus was most delayed in the cecum, suggesting that this may be a common site of ileus in horses. a link between routine postoperative medications, such as phenylbutazone and aminoglycoside antibiotics, has been suspected but not established. an inhibitory effect of nsaids on large colon contractility has been demonstrated using in vitro techniques. primary sympathetic overstimulation could be involved, for many of the affected animals are young, male horses or animals with painful diseases. the duration of surgery influences the development of small intestinal poi, but not cecal emptying dysfunction. , technique may have a weak influence on small intestinal poi after jejunojejunostomy. the duration of intestinal ileus was shorter in animals that received a sideto-side stapled anastomosis than those that had a hand sewn end-to-end procedure. the duration of ileus after stapled end-to-end anastomosis was not different from that after either procedure. reported risk factors for the development of poi in horses include age (> years), small intestinal resection and anastomosis, breed (arabians had a greatest risk than other breeds), and duration of surgery. interestingly, performing a pelvic flexure enterotomy and emptying the colon had a protective effect against poi. the diagnosis of ileus is based on history and physical examination findings. important tests include determination of pulse rate and rhythm, auscultation and percussion of the abdomen, rectal palpation, and passage of a nasogastric tube. a complete blood count with fibrinogen estimation and cytologic analysis of peritoneal fluid may improve the accuracy of diagnosis. affected animals may be colicky because of accumulation of fluid in the upper gastrointestinal tract (classical poi) or cecal contents (cecal emptying defect). decompression of the stomach is important diagnostically and therapeutically in horses with poi after small intestinal surgery. failure to relieve pain with gastric decompression could point toward mechanical obstruction, severe inflammation of the intestine, or peritonitis. most animals with ileus are depressed and have reduced fecal output and intestinal borborygmi. one should interpret intestinal sounds with caution, however, because the presence of borborygmi does not always equate to progressive intestinal motility and merely may reflect local, nonpropagated contractions. rectal palpation findings in cases of persistent poi or duodenitis-proximal jejunitis are usually nonspecific but may reveal dilated, fluid-filled loops of small intestine. the clinician occasionally can palpate roughened peritoneal surfaces if peritonitis is present. one can palpate cecal distention with digesta in horses with advanced cecal dysfunction. distinguishing functional ileus from mechanical obstruction is important and can be difficult, but horses with mechanical obstruction typically have sustained high volumes of gastric reflux that vary little over time. the management of intestinal ileus depends on the segment of gastrointestinal tract involved. therapy for ileus of the proximal gastrointestinal tract involves a combination of gastric decompression, fluid and electrolyte therapy, and antiinflammatory drug therapy. electrolyte therapy is critical, particularly for maintaining adequate extracellular concentrations of potassium, calcium, and magnesium. calculation of the volume of fluid to be administered should include maintenance requirements ( to ml/kg/day) plus an estimate of losses, especially those lost through gastric decompression. one should consider parenteral provision of calories when feed has been withheld for more than hours, particularly after the horse has had surgery. a combination of amino acids and dextrose with or without lipids effectively provides these calories. hand walking also may provide some benefit to these animals but is not likely to have a direct effect on intestinal motility. one should avoid drugs that may impair normal intestinal motility, including the anticholinergics such as atropine and opiate receptor agonists such as morphine and meperidine. butorphanol appears to have little or no adverse effect on small or large intestinal motility. , one should use α -agonists sparingly because of their inhibitory effects on large intestinal motility. fluid therapy is the key component in managing cecal emptying defect, usually in combination with lubricants or laxatives, such as mineral oil or magnesium sulfate, and with careful use of antiinflammatory drugs. horses with primary cecal impaction or impaction caused by an emptying defect frequently require surgery to prevent fatal rupture. the surgical management of these cases is controversial and may include typhlotomy alone, typhlotomy with a bypass procedure such as ileocolic or jejunocolic anastomosis, or a bypass without typhlotomy. most horses that undergo simple typhlotomy have an uneventful recovery, although a small number experience impaction again and require a second laparotomy. experimental and anecdotal evidence provides a strong rationale for using antiinflammatory drugs to prevent and treat gastrointestinal ileus, particularly in animals that may have endotoxemia. flunixin meglumine is used widely in equine practice as an analgesic and antiinflammatory agent, and it ameliorates many of the adverse systemic effects of endotoxin, particularly those on the cardiovascular system. a potential negative effect of nsaids on large intestinal contractility has been suggested. broad-spectrum antimicrobials are indicated when one suspects sepsis or for the compromised immune system, as in cases of moderate to severe endotoxemia. theoretical concerns have been raised regarding the use of aminoglycoside antibiotics in animals with ileus. high concentrations of aminoglycoside antimicrobials inhibited intestinal contractions in exposed sections of intestine in vitro, but this inhibitory effect is unlikely to occur at clinically relevant doses. motility-enhancing drugs have been advocated to treat gastrointestinal ileus. unfortunately, information directly pertinent to horses is limited and must be extrapolated cautiously from that of other species because of the differences in intestinal anatomy and physiology. prokinetic drugs potentially can shorten the length of hospitalization, thereby reducing the cost of treatment and the number of potential complications such as weight loss, thrombophlebitis, and laminitis. experimental evidence indicates that prokinetic drugs can minimize the development of postoperative abdominal adhesions. most prokinetic drugs require a healthy gut wall to enhance intestinal contraction. therefore one should not assume that many of these drugs would be effective in the presence of an inflammatory injury such as that which can occur after intestinal manipulation at surgery or that associated with duodenitis-proximal jejunitis. bethanechol is a parasympathomimetic agent that acts at the level of the myenteric plexus and directly on intestinal smooth cells through muscarinic receptors. bethanechol is a synthetic ester of acetylcholine and is not degraded by anticholinesterase. bethanechol has cholinergic side effects, including abdominal discomfort, sweating, and salivation, although these are minimal when the drug is administered at . mg/kg body mass subcutaneously or orally. bethanechol has efficacy in diseases that involve abnormal gastric emptying and delayed small intestinal transit and has been shown to increase gastric contractility and hasten the emptying of liquid and solid phase markers from the stomach of normal horses. [ ] [ ] bethanechol also increases the strength and duration of wall contractions in the cecum and right ventral colon and consequently speeds up cecal emptying. neostigmine increases receptor levels of acetylcholine by inhibiting cholinesterase. the drug ( . to . mg/kg intravenously) promotes cecal and colonic contractile activity and hastens the emptying of radiolabeled markers from the cecum. neostigmine has been used to manage small intestinal ileus, but it significantly delayed the emptying of -mm beads from the stomach of normal adult horses. metoclopramide acts principally as a -hydroxytryptamine -receptor ( ht- ) agonist and ht- -receptor antagonist. in contrast to newer generation benzamides, metoclopramide is also an antagonist at dopamine (da ) and (da ) receptors. antagonism of prejunctional da receptors facilitates acetylcholine release and smooth muscle contraction. metoclopramide crosses the blood-brain barrier, where its antagonist properties on central da receptors can result in extrapyramidal signs, including seizure. these signs were responsible for poor acceptance of the drug in equine practice. most investigators have failed to demonstrate significant effects of metoclopramide in experimental animals, but constant intravenous infusion ( . mg/kg/hr) in a population of postoperative horses significantly decreased the volume and duration of gastric reflux over control and intermittent drug infusion groups. infusion was well tolerated and appeared to be superior to intermittent infusion or no treatment at all. cisapride is a second-generation benzamide that acts as a ht- agonist and ht- receptor antagonist but is without antidopaminergic action. stimulation of ht- receptors within the enteric nervous system enhances release of acetylcholine from the myenteric plexus. several reports suggest the efficacy of cisapride in managing intestinal disease in horses, including the resolution of persistent large colon impaction, treatment of equine grass sickness, and as a preventative for poi in horses after small intestinal surgery ( . mg/kg body mass intramuscularly during the postoperative period). [ ] [ ] [ ] [ ] the horse erratically absorbs tablets administered rectally, but a method for preparing a parenteral form of the drug from tablets has been described. cisapride has the potential to cause adverse cardiac side effects mediated through blockage of the rapid component of the delayed rectifier potassium current that include lengthening of the qt interval and development of torsades de pointes, a potentially fatal arrhythmia. these adverse effects have resulted in withdrawal of the drug in the united states. domperidone acts as a competitive antagonist at peripheral da receptors. the drug is a therapeutic agent ( . mg/kg/day) for mares grazing endophyte-infected tall fescue, principally because of drug-enhanced prolactin release. the potential prokinetic effects of domperidone have not been studied extensively in horses, but a modest efficacy of domperidone ( . mg/kg intravenously) has been demonstrated in experimental ileus in two ponies. erythromycin is a direct motilin receptor agonist on smooth muscle cells and also may act within the enteric nervous system to facilitate the release of acetylcholine and motilin. erythromycin enhances gastric emptying in normal horses but has a more pronounced effect on the hindgut. , erythromycin lactobionate ( . mg/kg intravenously) hastens cecal emptying in normal animals and induces colonic mmc-like activity across the colon. administration often is associated with defecation and abdominal discomfort. the drug may help prevent cecal impaction in horses after anesthesia, although its effectiveness on cecal motility in the immediate postoperative period may be reduced. high doses, constant infusion, or prolonged use of erythromycin induces receptor downregulation and inhibition of activity. erythromycin can induce diarrhea in adults, therefore one should avoid dosing over many days. naloxone ( . mg/kg intravenously) induces contractile activity in the cecum and left colon. defecation commonly follows administration of naloxone within to minutes. α -adrenoreceptor antagonists such as yohimbine or tolazoline counteract increased sympathetic outflow in response to nociceptive stimulation. yohimbine infusion ( µg/kg) also may attenuate the negative effects of endotoxin on motility. intravenous infusion of lidocaine may suppress primary afferent neurons, thereby limiting reflex efferent inhibition of motility. an infusion dose of to mg/min over to hours has been recommended for horses. lidocaine infusion is associated with reversible side effects that include muscle fasciculations, ataxia, and seizure. consequently, the rate of infusion requires close monitoring. katharina l. lohmann, michelle henry barton endotoxemia is defined as the presence of endotoxin in the bloodstream. most often, however, the term is used to refer to the associated clinical manifestations caused by an overshooting inflammatory reaction. in its pathophysiologic consequences the innate immune response to endotoxin (lipopolysaccharide) is similar to the response to other stimuli; for example, overwhelming bacterial infection, viral infection, or severe trauma. the term systemic inflammatory response syndrome therefore was introduced to describe a general systemic inflammatory process independent of cause. sepsis is defined as the "systemic inflammatory response to infection," and septic shock as "sepsis-induced hypotension, persisting despite adequate fluid resuscitation, along with the presence of hypoperfusion abnormalities or organ dysfunction." according to these definitions the diagnosis of sepsis requires documentation of infection by culture in addition to two or more of the following findings: hypo-or hyperthermia, tachycardia, tachypnea or hypocapnia, and leukocytosis, leukopenia, or an increased proportion of immature leukocyte forms. organ failure is a common sequela of endotoxic or septic shock, and the term multiple organ dysfunction syndrome describes insufficiency of two or more organ systems, as evident by clinical or clinicopathologic changes. in horses one should include the laminae of the feet in the list of organs susceptible to failure. german scientist richard pfeiffer ( - ), in working with vibrio cholerae, first described endotoxin as a toxin "closely attached to, and probably integral of, the bacterial body." he observed this toxin to be distinct from the actively secreted, heat-labile, and proteinaceous bacterial exotoxins. endotoxin later was found to be a heat-stable lipopolysaccharide structure, and the terms endotoxin and lipopolysaccharide now are used interchangeably. lipopolysaccharide is a major structural cell wall component of all gram-negative bacteria, including noninfectious species (figure . - ). with to × molecules per cell, lipopolysaccharide makes up about % of the outer layer of the outer cell membrane and is a key functional molecule for the bacterial outer membrane, serving as a permeability barrier against external noxious agents. the lipopolysaccharide molecule consists of four domains, which are essential for the virulence of gram-negative bacteria. three of the domains (inner core, outer core and o-specific chain) represent the hydrophilic polysaccharide portion of the molecule, whereas the lipid a portion represents the hydrophobic lipid portion ( figure . - ). combined, these domains confer the overall amphiphilic properties of the molecule that lead to the formation of micellar aggregates in aqueous solutions. o-specific chains (also called o-antigen polysaccharides or o-chains) are characteristic of any given type of lipopolysaccharide and show enormous structural variability between bacterial serotypes. o-chains are synthesized by addition of preformed oligosaccharide blocks to a growing polymer chain and therefore have a repetitive structure. o-specific chains determine part of the immunospecificity of bacterial cells and, on interaction with the host immune system, serve as antigens for the production of species-specific antibodies. o-specific chains are further responsible for the smooth appearance of gram-negative bacterial colonies on culture plates, and lipopolysaccharide molecules containing an o-chain are termed smooth lipopolysaccharide. the inner (lipid a-proximal) and outer (o-chainproximal) core oligosaccharide portion is more conserved between different strains of gram-negative bacteria than the o-specific chain. the core of all lipopolysaccharide molecules contains the unusual sugar kdo ( -deoxy-dmanno-oct- -ulopyranosonic acid), which links the core region to the lipid a molecule. synthesis of a minimal core is essential for the survival of bacteria, and the smallest naturally occurring lipopolysaccharide structure consists of lipid a and kdo. in contrast to the s-form colonies, colonies of gram-negative bacteria with lipopolysaccharide molecules that lack the o-specific chain but contain a core region show a rough appearance on culture plates. rough lipopolysaccharide molecules are denoted further as ra, rb, etc. to indicate the length of the core region. in re-lipopolysaccharide (also called deep rough lipopolysaccharide), the core region is reduced to a kdo residue. remutants often are used to raise antibodies against the core region in an attempt to provide cross-protection against a variety of bacterial species. the lipid a portion serves to anchor the lipopolysaccharide molecule in the bacterial outer membrane and has been identified as the toxic principle of lipopolysaccharide, and its structure is highly conserved among gramnegative bacteria. the common structure shared by lipid a molecules is a , '-bisphosphorylated β , -linked d-glucosamine disaccharide backbone (lipid a backbone), which is acylated by up to six fatty acids. figure . - shows the acylation pattern for escherichia coli lipopolysaccharide. variation in the lipid a structure between gramnegative bacteria affects the number, length, and position of fatty acids and the backbone structure and the substitution of phosphate by other polar groups. according to its nature as a structural cell wall component, the presence of endotoxin implies the presence of gram-negative bacteria as a source. depending on the nature of the underlying disease, these bacteria may circulate in the bloodstream in their intact form (i.e., bacteremia), may be confined to a localized infectious process, or may be part of the endogenous bacterial flora colonizing the gastrointestinal tract. in any of these scenarios, endotoxin molecules are released as a by-product of bacterial growth and in large numbers on bacterial cell death. common infectious conditions associated with endotoxemia in horses include neonatal gram-negative sepsis, bacterial pneumonia and pleuropneumonia, endometritis, peritonitis, and infectious colitis with bacteria such as salmonella spp., that are not part of the normal intestinal flora. in one study, for example, endotoxin was detectable in plasma of % of foals evaluated for presumed sepsis. the term translocation describes entry of endogenous bacteria and bacterial products from the gastrointestinal tract into tissues and the systemic circulation. the natural intestinal flora of horses consists mainly of gramnegative, anaerobic bacteria, and thus large amounts of endotoxin normally exist in the lumen of the equine intestinal tract. even in health, small amounts of endotoxin cross the intact mucosal barrier and reach the portal circulation and the liver. these molecules are cleared, however, by the mononuclear phagocytic system in the liver and only lead to a localized and restricted activation of the host immune system. for endotoxin translocation to become detrimental, excessive amounts have to cross the intestinal barrier and overwhelm the mononuclear phagocytic system or the capacity of the liver to detoxify lipopolysaccharide must be compromised. the latter may be a concern in conditions such as hepatitis, cholangiohepatitis, or portosystemic shunting of blood. permeability of the intestinal mucosal barrier frequently increases in cases of acute gastrointestinal disease. colic patients are prime candidates to development endotoxemia, and plasma endotoxin was detectable in % to % of colic patients on admission. , a higher percentage of horses tested positive for endotoxin when only patients presented for surgical intervention were evaluated. aside from gastrointestinal rupture, increased permeability to intact bacteria or free endotoxin molecules is thought to be associated most commonly with ischemic insults such as strangulating obstruction and bowel infarction, severe inflammation as in proximal enteritis and colitis, bacterial overgrowth, and intraluminal acidosis, which occurs with grain overload. , one study, however, found no difference in plasma endotoxin detection between disease groups, therefore emphasizing the fact that any disease of the abdominal cavity can induce endotoxemia in horses. in the same study, endotoxin was approximately times more likely to be detected in peritoneal fluid as opposed to plasma samples. similarly, higher cytokine concentrations have been measured in peritoneal fluid than in plasma. the likely explanation for these findings is a local inflammatory response in the peritoneal cavity elicited by translocated bacteria and/or lipopolysaccharide molecules before their absorption into the systemic circulation. although certainly the most important factor in horses, conditions other than gastrointestinal disease may result in translocation of endotoxin and bacteria. in experimental studies using laboratory animals, entry of gutassociated bacteria into the lymphatic system was demonstrated after hypovolemic shock, burn injuries, trauma, malnutrition, and starvation. [ ] [ ] [ ] furthermore, endotoxin itself caused bacterial translocation into mesenteric lymph nodes after intraperitoneal administration to mice. these findings have received much attention in the literature concerning human patients because they serve to explain cases of endotoxic shock in the absence of demonstrable bacterial infection. one should keep in mind the possibility of translocation when evaluating cases of presumed systemic inflammatory response syndrome in horses, in which one cannot demonstrate bacterial infection or gastrointestinal disease. endotoxin translocation also may be associated with strenuous exercise, which results in reduced splanchnic blood flow, hypoxemia, and a higher body temperature. in fit racehorses a significantly increased mean plasma lipopolysaccharide concentration was found after racing, whereas antilipopolysaccharide immunoglobulin g levels were decreased. fit horses showed significantly higher antilipopolysaccharide immunoglobulin g concentrations at rest than sedentary controls, suggesting leakage of small amounts of endotoxin from the intestinal lumen during training and racing. the clinical significance of these findings requires further investigation. the initiating event in the pathophysiology of endotoxemia is the activation of lipopolysaccharideresponsive cells by endotoxin, resulting in altered cellular functions and increased expression of inflammatory mediators. immune cells such as macrophages, which are the first to encounter endotoxin, respond to minute amounts of lipopolysaccharide, which usually allows them to eliminate gram-negative bacteria and free lipopolysaccharide molecules efficiently. an important factor in the exquisite sensitivity to lipopolysaccharide is the presence of lipopolysaccharide-binding protein (lbp). lbp is an approximately -kd plasma glycoprotein synthesized by hepatocytes and belongs to the group of acute phase proteins. under the control of inflammatory agents and cytokines, lbp concentration in plasma increases approximately -fold within hours of an inflammatory stimulus. the main function of lbp is to transfer lipopolysaccharide to endotoxin-responsive cells, which include mononuclear phagocytes, neutrophils, lymphocytes, and endothelial cells. the importance of a highly sensitive response to lipopolysaccharide for protection against gram-negative bacterial infection is demonstrated in experiments using lbp "knock-out" mice (mice that lack the lbp gene and are therefore unable to synthesize lbp). although these animals are resistant to the effects of isolated lipopolysaccharide, they are unable to control bacterial infection and rapidly succumb. despite its crucial importance for an effective host defense, lbp is not essential for lipopolysaccharide-receptor interaction per se, because high concentrations of lipopolysaccharide can activate cells in the absence of lbp. aside from its role as a catalyst of cellular activation by lipopolysaccharide, lbp has opsonizing activity and participates in the phagocytosis of lipopolysaccharide by macrophages and neutrophils. , although phagocytosis of lipopolysaccharide is receptor dependent, it appears to be uncoupled from intracellular signaling events and occurs in the absence of cell activation. lbp further catalyzes transfer of lipopolysaccharide to lipoproteins such as high-density lipoprotein, which neutralizes lipopolysaccharide activity. this detoxifying effect may become important when large amounts of lipopolysaccharide are present. a protective effect of lbp against lipopolysaccharide challenge and infection has been demonstrated in a murine model. the most important lipopolysaccharide receptors known to date are cluster differentiation antigen (cd ) and toll-like receptor (tlr ). both are classified as pattern recognition receptors, which means that they recognize lipopolysaccharide as a pattern common to all gram-negative bacteria. cd is a -kd protein that in its membrane-bound form (mcd ) is inserted into the cell membrane via a glycosylphosphatidyl-inositol anchor. cd is expressed primarily on monocytes and tissue macrophages and to a lesser extent on neutrophils. cd also is found in a free, or soluble, form (scd ) that can bind to cell types lacking cd , such as endothelial cells, and make them lipopolysaccharide-responsive. in addition to this proinflammatory effect, high concentrations of scd can sequester and neutralize lipopolysaccharide. the amount of circulating scd greatly increases during inflammation, which makes it a useful marker of acute and chronic inflammation. although cd is known to be crucial for cellular activation, it cannot transmit signals to the inside of the cell because it lacks a transmembrane domain. the missing link between cd and the cytosolic environment is a toll-like receptor in association with a molecule named md- . the name toll-like receptor stems from the homology of the mammalian receptor with a receptor type in drosophila (toll) that is important for dorsoventral orientation and immune responses in the fly. a number of toll-like receptors have been identified in mammalian species so far, but tlr appears to be the receptor subtype most important for lipopolysaccharide signaling. the importance of cd and tlr in the cellular response to lipopolysaccharide has been demonstrated in a number of experiments. mice deficient in cd are incapable of mounting a normal inflammatory response to lipopolysaccharide, whereas mutation or deletion of the gene encoding for tlr causes lipopolysaccharide hyporesponsiveness. [ ] [ ] [ ] after binding of lipopolysaccharide to cellular receptors, a multitude of signaling events takes place within the cell and results in the alterations of cellular metabolism known as cell activation. signaling pathways are characterized by sequential phosphorylation and thereby activation of enzymatic activities. a typical end result of intracellular signaling is the activation of transcription factors; for example, proteins that bind to dna and promote gene transcription. translational mechanisms are activated in a similar manner. among the bestcharacterized pathways in endotoxin-induced cell signaling are the mitogen-activated protein kinase (mapk) pathways and the activation of transcription factor nuclear factor κb (nfκb) (figure . - ) . , in the nfκb pathway the intracellular domain of tlr associates with the adapter protein myeloid differentiation factor and recruits interleukin- receptor-associated kinase to the complex. activation of interleukin- receptor-associated kinase, tumor necrosis factor receptor-associated factor, nfκb-inducing kinase, and iκb-kinase follow, and lastly, iκb is phosphorylated. iκb is an inhibitor protein complex that sequesters and inactivates nfκb in the cytoplasm. on phosphorylation, iκb is ubiquinated and degraded, and nfκb is translocated to the nucleus where it unfolds its activity. nfκb is a dimeric protein complex with several isoforms of which the p /p heterodimer is the most important inducible complex in mammals. proteins of importance for the pathogenesis of septic shock, the genes of which contain promoter elements for nfκb, include cytokines, inducible nitric oxide synthase, and cyclooxygenase (cox- ). three groups of mapks known to be crucially important for lipopolysaccharide-induced signal transduction are extracellular signal-regulated kinase, c-jun-terminal kinase, and p . final effects of signaling through mapk pathways include the activation of several transcription factors, translation initiation factors, and cytosolic enzymes such as phospholipase a , as well as an increase in the expression of adhesion molecules on the cell surface. despite the characterization of seemingly separate pathways, one should recognize that interaction and synergy between pathways is likely to occur. for example, simultaneous activation of p , c-jun-terminal kinase, and extracellular signal-regulated kinase results in much higher levels of tumor necrosis factor (tnf) reporter expression than activation of a single pathway. , aside from the mechanisms described here, pathways involving atypical protein kinase c , and receptor-independent integration of lipopolysaccharide into the cell membrane and ceramide-like second messenger activity of lipopolysaccharide have been proposed. additional pathways are likely to be uncovered in the ongoing investigation of intracellular signaling mechanisms and their in vivo significance. although endotoxin can exert some direct effects, cytokines are a primary mediator of lipopolysaccharide effects. cytokines are glycoprotein molecules that regulate inflammatory and immune responses by acting as a signal between cells. cytokines of major interest in the pathogenesis of endotoxemia include tnf, the interleukins, chemokines, and growth factors such as granulocyte-monocyte colony-stimulating factor. tnf is thought of as the most "proximal" cytokine released in response to lipopolysaccharide. studies corroborate this by showing that administration of recombinant tnf mimics the effects of lipopolysaccharide, and that antibodies directed against tnf protect against the lethal effects of endotoxin. increased plasma activity of tnf is associated with increased mortality in equine patients with acute gastrointestinal disease and in septic neonates. despite being a structurally diverse group of proteins, cytokines share several characteristics that allow them to execute their complex functions in the inflammatory response. any individual cytokine generally is produced by several different cell types, can act on different cell types, and has multiple effects on any given cell. furthermore, cytokine effects are redundant, meaning that different cytokines can share the same effect. in endotoxemia, this is particularly true for the effects of interleukin- (il- ) and tnf. many of the biologic activities of cytokines in vivo result from synergistic or antagonistic actions involving two or more cytokines. within itself the cytokine response is highly regulated: cytokines induce or suppress synthesis of other cytokines including their own (feedback regulation), regulate expression of cytokine receptors, and regulate cytokine activities. additional regulatory mechanisms include the release of specific cytokine inhibitors such as soluble il- and tnf-α receptors, cytokine receptor antagonists such as il- receptor antagonist, and antiinflammatory cytokines including il- , il- , il- , and transforming growth factor β. glucocorticoids also are produced increasingly in response to endotoxin and inhibit the production of cytokines. during a controlled inflammatory response, therefore, cytokine secretion is a selflimited event, whereas excessive stimulation of cytokine release can lead to the perpetuation of the inflammatory response even after the initial stimulus has been removed. conversely, the compensatory antiinflammatory reaction can become severe enough to cause anergy of the immune system and increased susceptibility to infection, which has been termed the compensatory antiinflammatory response syndrome. overall, excessive and unbalanced stimulation of the immune system therefore may result in predominantly proinflammatory (systemic inflammatory response syndrome), antiinflammatory (compensatory antiinflammatory response syndrome), or combined (mixed antagonist response syndrome) responses. interestingly, tolerance to endotoxin develops after repeated exposure to lipopolysaccharide. tolerance can be demonstrated in vitro and in vivo and encompasses decreased production of cytokines and a diminished clinical response. , mechanisms that likely are responsible for the development of endotoxin tolerance include receptor downregulation and inhibition of intracellular signaling pathways. , cytokines such as tnf are important mediators in the development of endotoxin tolerance. the development of endotoxin tolerance in horses has been reported. , aside from cytokines, a number of other molecules function as inflammatory mediators in the pathogenesis of endotoxemia, the synthesis and release of which are stimulated by endotoxin and by cytokines. these mediators include the arachidonic acid metabolites or prostanoids, platelet-activating factor (paf), oxygenderived free radicals, nitric oxide (no), histamine, kinins, and complement components. table . - summarizes the origins, targets, and effects of the most important inflammatory mediators involved in the pathogenesis of endotoxemia. figure . - shows the pathways of arachidonic acid metabolism by cox and lipoxygenase. cox products are the prostaglandins (pgs), prostacyclin (pgi ) and thromboxanes, and the lipoxygenase produces the leukotrienes. the innate immune response to lipopolysaccharide is an efficient defense mechanism that provides maintenance of homeostasis and therefore health in the face of an almost continuous exposure to microorganisms and their products. detrimental consequences of this immune response only occur if excessive and uncontrolled mediator output results in endothelial damage, neutrophil-mediated tissue damage, and uncontrolled activation of the coagulation and fibrinolytic cascades and the complement system. ultimately, the combination of these events culminates in cardiovascular instability, impaired hemostasis, organ failure, shock, and death. the following discussion addresses the various pathophysiologic events in the development of endotoxemia and shock and the role of inflammatory mediators. normal endothelium plays an important role in regulating blood pressure and regional tissue perfusion and provides an anticoagulant surface. endothelial dysfunction and damage result in a decreased responsiveness to vasoactive agents (vasoplegia), increased vascular permeability, and a tendency for clot formation in the microvasculature. if the basement membrane and underlying matrix are compromised, further microvascular hemorrhage can occur. endothelial cell damage is primarily neutrophilmediated. more specifically, damage is caused by oxygen-derived free radicals, which are produced within endothelial cells via reactions involving neutrophil-derived elastase and hydrogen peroxide molecules, endothelial cell enzymes such as xanthine oxidase, and endothelial cytosolic iron. the hypochloric anion radical (ho˙) is thought to be responsible most directly for endothelial cell cytotoxicity. no˙produced by constitutively expressed nitric oxide synthase in endothelial cells may afford protection from oxygen radical-induced endothelial cell damage. no is able to scavenge superoxide radicals and react with them to form peroxynitrite. variations in the ability to produce no may explain why vascular beds in different organs vary in their susceptibility to neutrophilmediated damage. excessive production of no by an inducible form of nitric oxide synthase (i nos), however, contributes to tissue damage, and increased peroxynitrite concentrations may be responsible in part for paf-induced increases in vascular permeability. in addition to oxygen-derived free radicals, activated neutrophils release matrix metalloproteinases, which contribute to tissue damage. vascular endothelial cells are further susceptible to direct effects of various cytokines, most prominently tnf-α and il- . these cytokines are thought to cause damage via the induction of cox activity and production of prostanoids and through generation of free radicals. neutrophil activation by lipopolysaccharide and cytokines results in stimulation of phagocytosis and the respiratory burst, release of lysosomal enzymes and inflammatory mediators, and expression of adhesion molecules. perhaps the single most specific clinicopathologic indicator of endotoxemia is pronounced neutropenia, which temporally correlates with peak plasma concentrations of tnf. neutropenia is caused primarily by margination of neutrophils in the vasculature, whereas significant loss through active migration into peripheral tissues likely is limited to the presence of a localized source of infection. margination is made possible by adhesion molecules on endothelial cells and leukocytes that interact and allow sticking of leukocytes to the endothelial lining of blood vessels. endotoxin or cytokines such as tnf and il- can initiate expression of adhesion molecules. subsequent transmigration of cells into tissue spaces is aided by the production of leukocyte collagenase, which allows enzymatic destruction of the vascular basement membrane. margination and transmigration of neutrophils occurs in three phases. , in the first phase of tethering and rolling, endothelial cells are stimulated to express p-selectin and e-selectin, which bind to p-selectin glycoprotein ligand- and sialylated lewis-x-like structures on leukocytes, respectively. whereas p-selectin is stored preformed in weibel-palade bodies of endothelial cells, e-selectin is expressed only following stimulation by cytokines. additionally, constitutively expressed l-selectin on neutrophils can bind to endothelial glycoproteins and glycolipids. during the second phase, firm adhesion is mediated by binding of neutrophil integrins (lfa- and mac- , also known as cd a/cd and cd b/cd ) to intercellular adhesion molecule (an immunoglobulin structure) on endothelial cells. although integrins are expressed constitutively on the leukocyte surface, activation signals are necessary to induce a high-affinity state and interaction with the endothelial surface. transmigration finally requires the expression of yet another adhesion molecule, namely platelet/endothelial cell adhesion molecule , which is located at the intercellular junction of endothelial cells. chemotactic factors including activated complement factor c a and the cxc chemokines control transmigration. the latter group includes il- , which is expressed by endothelial cells in response to activation. rebound neutrophilia, which is observed frequently following episodes of endotoxemia, is caused by neutrophil release from the bone marrow reserve pool and by stimulation of myeloid cell proliferation via granulocyte-macrophage colony-stimulating factor and is mediated primarily by tnf and il- . in health, coagulation and fibrinolysis underlie stringent control mechanisms that allow appropriate clot formation and their resolution. coagulopathies frequently are observed in horses with colic , , and foals with sepsis and are likely attributable to endotoxemia. disseminated intravascular coagulation (dic) results from a widespread activation of the coagulation and fibrinolytic systems and failure of their control mechanisms. ultimately, this leads to disseminated fibrin deposition in the microvasculature, consumption of platelets and clotting factors, and accumulation of fibrin degradation products (fdps). depending on the underlying disease process and the impairment of the systems, dic can manifest as a diffuse thrombotic syndrome leading to ischemic organ failure, a fibrinolytic syndrome with uncontrolled hemorrhage, or a combination of both. a procoagulant state in which one can detect clinicopathologic abnormalities precedes dic. activation of the coagulation cascade culminates in the cleavage of fibrinogen to fibrin by the serine protease thrombin. thrombin deposition on endothelial cell surfaces leads to platelet adherence and their activation by surface-bound paf. the intrinsic and extrinsic arms of the coagulation cascade are activated in endotoxemia. the intrinsic pathway is initiated by activation of coagulation factor xii (hageman factor), prekallikrein, and highmolecular-weight kininogen, which compose the contact system. although direct activation of coagulation factor xii by endotoxin has been demonstrated, the extrinsic pathway likely is more important for the development of coagulopathy in endotoxemia and sepsis. activation of the extrinsic pathway depends on the interaction of coagulation factor vii with tissue factor, which is the only coagulation factor not constitutively present in blood. tissue factor is present in subendothelial tissues and is exposed on vascular injury but also is expressed on endothelial cells and mononuclear phagocytes in response to lipopolysaccharide. , increased expression of monocyte tissue factor (also described as increased procoagulant activity) was found to be associated significantly with coagulopathy and poor prognosis in horses with colic. furthermore, lipopolysaccharide-induced tissue factor expression by equine peritoneal macrophages may be associated with the development of intraabdominal adhesions. regulatory mechanisms of the coagulation cascade include tissue factor pathway inhibitor, antithrombin iii (at iii), and the protein c system. protein c acts as an anticoagulant by inactivating clotting factors v and viii and promotes fibrinolysis by inactivating plasminogen activator inhibitor (pai). protein c activation by thrombin-thrombomodulin complexes is important for the anticoagulative properties of normal endothelium, and downregulation of endothelial thrombomodulin expression by tnf and il- and decreased expression of at iii and tissue factor pathway inhibitor by damaged endothelial cells contribute to the procoagulant state in endotoxemia and sepsis. [ ] [ ] [ ] in addition, activation of vascular endothelial cells leads to a loss of prostacyclin and no production and an increased release of thromboxane a (txa ). as a result, platelets are stimulated to aggregate and release txa and paf, thereby further promoting clot formation. the crucial step in the fibrinolytic cascade is the conversion of plasminogen to plasmin, a fibrin-degrading enzyme. tissue-type (tpa) and urokinase-type (upa) plasminogen activator are the major initiators of fibrinolysis, whereas pai and α -antiplasmin are the main regulatory components. , tnf and il- have been shown to induce the release of upa and tpa and the synthesis of pai. activation of fibrinolysis leads to consumption of α -antiplasmin and accumulation of fdps, which if present in high concentrations can interfere with platelet aggregation, fibrin polymerization, and thrombin formation and can promote bleeding. additionally, fdps mediate an increase in vascular permeability. lipopolysaccharide infusion in rabbits and human beings resulted in an early increase in plasma tpa activity, followed by a later profound rise in pai activity and fall in tpa activity. increased plasma pai concentrations also were found in horses with colic compared with controls. , thus although fibrinolysis may compensate initially for accelerated coagulation, its subsequent inhibition contributes to clot formation. activation of the complement system in endotoxemia occurs via the alternative pathway through interaction with lipopolysaccharide. increased concentrations of plasmin and kallikrein (caused by activation of the fibrinolytic and contact system) further promote this pathway by directly activating complement factors c a and c a. aside from being key molecules in the complement cascade, c a and c a are anaphylatoxins and cause an increase in vascular permeability via mast cell degranulation. c a further activates the lipoxygenase pathway in neutrophils and monocytes, acts as a chemotaxin for leukocytes and monocytes, and promotes neutrophil adhesion to endothelial cells. in response to acute inflammation, synthesis and secretion of a number of proteins called the acute phase proteins increases in hepatocytes, whereas synthesis of albumin decreases. the primary function of this acute phase response may be to suppress and contain inflammatory responses. il- and il- are the most important cytokines that induce the acute phase response, which typically begins within a few hours of the insult and subsides within to hours, unless the initiating cause persists. in horses, fibrinogen (the most commonly evaluated), haptoglobin, transferrin, ferritin, ceruloplasmin, coagulation factor viii:c, serum amyloid a protein, c-reactive protein, α -acid glycoprotein, and phospholipase a are considered part of the acute phase response. one must consider the effect of acute inflammation on the serum concentration of several coagulation factors when evaluating coagulation profiles. serum fibrinogen concentration is determined primarily by the acute phase response, although fibrinogen is consumed increasingly on activation of the clotting cascade. shock is characterized by a loss of homeostasis attributable to breakdown of hemodynamic control mechanisms, decreases in cardiac output and the effective circulating volume, and inadequate perfusion of vital organs. shock caused by endotoxemia is classified as distributive shock and is largely initiated by vascular dysfunction in the periphery. peripheral vascular beds are of major importance for the regulation of local tissue perfusion and affect systemic blood pressure by regulating total peripheral resistance. normally, vascular smooth muscle tone is regulated by endothelin- (vasoconstriction), no, and prostacyclin (vasodilation) released from vascular endothelial cells. as mentioned before, detrimental effects of no are attributable to induction of i nos in macrophages and other cell types, rather than endothelialderived no. peripheral vasomotor effects of endotoxin manifest as vasodilation and vasoplegia and are mediated by pgi , no, and mediators such as bradykinin. widespread vasodilation leads to vascular blood pooling, decentralization of blood flow, decreased venous return, and in effect decreased effective circulating volume and cardiac output. compensatory responses in the form of an initial hyperdynamic phase include tachycardia, increased cardiac output and central venous pressure, pulmonary hypertension, peripheral vasoconstriction, and increased peripheral vascular resistance. , , the early vasoconstrictive phase corresponds to an increased serum concentration of txa , but additional vasoconstrictors such as arginine vasopressin, angiotensin ii, serotonin, endothelin, and norepinephrine likely are implicated in the pathogenesis of shock and organ failure. with progression of disease, the animal enters a stage of decompensated shock and progressive systemic hypotension, which correspond to increased plasma concentrations of prostacyclin, pge and bradykinin. , inadequate blood flow and oxygen delivery to tissues caused by hypotension is confounded by direct myocardial suppression via no, increased vascular permeability, intravascular microthrombosis, and impaired tissue oxygen extraction and results in progressive metabolic acidosis and inhibition of normal cellular metabolism. quantification of endotoxin in plasma samples is possible. the limulus amebocyte lysate assay is an activity assay based on the endotoxin-sensitive hemolymph coagulation cascade in the horseshoe crab limulus polyphemus. in limulus this reaction is thought to be a defense mechanism against gram-negative infection. although frequently used as a research tool, the assay is not convenient enough to become a routine clinical test. the clinician therefore must appreciate the primary disease processes associated with a high risk of endotoxemia and rely on clinical signs and clinicopathologic data to achieve a diagnosis. in some cases, endotoxemia may be the first indication of disease or may be the most overt of otherwise subtle clinical manifestations. with colitis or proximal enteritis, for example, one may detect signs of endotoxemia before the development of colic, diarrhea, or gastric reflux, which more specifically indicate the nature of the primary illness. diseases such as peritonitis or pleuritis, however, may show nonspecific clinical findings including fever, anorexia, and depression. findings such as neutropenia, which indicate endotoxemia, should urge the clinician to search for a septic process. in vivo experiments in horses clearly show that many of the clinical signs associated with acute gastrointestinal disease and sepsis are attributable to the activities of lipopolysaccharide and cytokines such as tnf. on administration of sublethal doses of lipopolysaccharide the clinical response can be divided into the early hyperdynamic and the later hypodynamic or shock phases. clinical signs during the first phase, which begins within to minutes after lipopolysaccharide administration, include anorexia, yawning, sweating, depression, evidence of abdominal discomfort, muscle fasciculation, and recumbency. heart and respiratory rates increase, and decreased borborygmi suggest ileus. hyperemia of the mucous membranes and an accelerated capillary refill time indicate the hyperdynamic state. if one administers large amounts of lipopolysaccharide or if exposure is ongoing, depression worsens progressively, anorexia persists, and feces develop diarrheic character. signs of colic typically abate after the initial stage. fever develops as a result of direct action of tnf on the thermoregulatory center and il- -induced local production of pge in or near the hypothalamus. , because of compromised peripheral perfusion, mucous membrane color changes to brick red or purple, a dark "toxic" line appears, and capillary refill time is prolonged. inadequate peripheral perfusion and compromised organ function finally characterize the hypodynamic shock phase. body temperature may become subnormal and the skin, especially on extremities, is cool to the touch. the arterial pulse weakens and venous fill is decreased. vascular endothelial damage and increased capillary permeability result in a muddy mucous membrane color and diffuse scleral reddening. hemostatic abnormalities can manifest in the form of thrombosis, such as of the jugular vein, or increased bleeding tendency with mucosal petechiation or ecchymoses and prolonged bleeding from venipuncture sites. bleeding also may occur in spontaneous epistaxis or prolonged hemorrhage after nasogastric intubation. additional clinical signs typically reflect the development of organ failure. renal failure and laminitis appear to be common complications of endotoxemia in horses. renal failure results from ischemic cortical necrosis and acute tubular necrosis caused by coagulopathy-induced afferent arteriolar obstruction. clinical signs may include oliguria, anuria, or hematuria caused by renal infarction. laminitis may lead to lameness, increased digital arterial pulsation, increased warmth of the hoof wall, and sensitivity to hoof tester pressure. other signs of organ failure include icterus, anorexia and depression caused by liver failure, tachypnea and dyspnea caused by pulmonary failure, colic and bleeding caused by ischemia-induced gastrointestinal ulceration and abnormal motility patterns, and persistent tachycardia or cardiac arrhythmia in cases of cardiac failure. in pregnant mares, fetal death and abortion can occur because of increased production of pgf α and decreased serum progesterone concentrations. , alterations in the hemogram and serum biochemical profile are nonspecific and mainly reflect the underlying disease process and the occurrence of organ failure. leukopenia caused by neutropenia may be the most specific indicator of acute bacterial sepsis or endotoxemia. in prolonged cases, an increased proportion of immature neutrophil forms (bands) and toxic changes are observable. toxic changes resulting from neutrophil activation include vacuolation, cytoplasmic granulation, basophilic cytoplasm, and döhle's bodies. because neutropenia occurs early in the development of endotoxemia, it also may be a useful parameter for monitoring horses at risk. on recovery, neutropenia typically is followed by a pronounced rebound neutrophilia. an elevated hematocrit and total serum protein concentration are evidence of dehydration; however, splenic contraction caused by increased sympathetic stimulation and protein losses also influence these parameters. a normal or only slightly decreased serum protein and albumin concentration in the face of an elevated hematocrit and clinical signs of dehydration should alert the clinician to the possibility of protein loss. hypoproteinemia and hypoalbuminemia can occur because of loss via the gastrointestinal or urinary tract or with pleural or peritoneal cavity effusion. increased vascular permeability and edema formation contribute to hypoproteinemia. serum electrolyte abnormalities primarily depend on the nature and duration of underlying disease processes and need to be evaluated individually. common sources of electrolyte loss are gastrointestinal secretions, urine, and sweat; however, severe effusion into body cavities may contribute. in anorexic patients, lack of dietary intake is a confounding factor that warrants consideration. in human patients, gram-negative sepsis frequently is associated with hypocalcemia, more specifically a decrease in serum ionized calcium concentration. endotoxin is thought to be a causative factor, and proposed mechanisms include acquired parathyroid gland insufficiency, dietary vitamin d deficiency, impaired calcium mobilization, and renal α-hydroxylase insufficiency leading to decreased , -hydroxylation of vitamin d. hypocalcemia in septic human patients was found to be associated with hypotension and poor outcome. in horses with surgically managed gastrointestinal disease, decreased serum ionized calcium concentration was a common finding and was most severe in patients with strangulating or nonstrangulating infarctions. in some horses, ionized calcium concentration decreased further throughout surgery. treatment with calcium gluconate resulted in normalization of serum ionized calcium concentrations in all cases. septic neonatal patients are frequently hypoglycemic. aside from decreased oral intake and generally increased metabolism, glucose use by the infecting bacteria, inhibition of gluconeogenesis by endotoxin, and insulinlike activity produced by macrophages are responsible for hypoglycemia. interestingly, experimental endotoxin administration results in transient hyperglycemia in adult horses, whereas profound hypoglycemia occurs in foals. one should evaluate coagulation parameters if coagulopathy is suspected. the most significant changes can be expected with severe inflammatory disease such as colitis, , devitalized intestine as with strangulating obstruction, , and with increased duration of disease. in horses with acute gastrointestinal disease, coagulation profiles were considered normal in only horses. although coagulation times may be shortened during the procoagulant state, commonly observed abnormalities with developing dic include an increased concentration of fdps and soluble fibrin monomer, prolonged prothrombin time indicative of factor vii consumption, prolonged activated partial thromboplastin time indicative of factor viii:c and ix consumption, prolonged thrombin time, decreased at iii activity, thrombocytopenia, and decreased protein c and plasminogen activities. fibrinogen concentration frequently increases and reflects the acute phase response rather than coagulation abnormalities. some clinicians make a diagnosis of dic if three or more coagulation parameters (specifically at iii, fdps, platelet count, prothrombin time, and activated partial thromboplastin time) are abnormal, whereas others require overt clinical signs of hemorrhage and concomitant thrombosis in addition to classic laboratory findings. the prognostic value of coagulation parameters has been evaluated. , , overall, persistence or worsening of abnormalities in the face of treatment appears to be more indicative of poor outcome than alterations in any specific parameter. in one study, decreased serum at iii concentration was the parameter most commonly associated with fatal outcome in mature horses with colic. one should further evaluate the serum biochemical profile regarding compromise or failure of specific organ systems. increases in serum creatinine and urea nitrogen concentration can have prerenal, renal, or postrenal causes. in cases of endotoxemia and sepsis, prerenal azotemia caused by dehydration and decreased renal blood flow and renal azotemia caused by organ failure are most likely to occur. one can use urine specific gravity and the response to fluid therapy to differentiate renal from prerenal causes of azotemia. although ideally one should perform urinalysis before initiating fluid therapy, one should never delay treatment to obtain a sample and instead should use the first available urine sample. with prerenal azotemia, urine specific gravity is increased, urinalysis is normal in other respects, and azotemia resolves with adequate fluid therapy. azotemia in the face of normal or decreased urine specific gravity, however, indicates compromised renal function. depending on the extent of renal damage, proteinuria and hematuria also may be present. bacteriuria and an elevated urine leukocyte count may occur if urinary tract infection is the underlying cause for the development of endotoxemia. in these cases, urine culture and sensitivity testing are indicated to aid appropriate antimicrobial therapy. increased serum activity of liver enzymes (aspartate aminotransferase, γ-glutamyltransferase, sorbitol dehydrogenase, alkaline phosphatase) are common in endotoxemic patients; however, liver failure caused by endotoxemia is rare. sorbitol dehydrogenase is the most liver-specific of the enzymes and a sensitive indicator of acute hepatocellular necrosis; however, sorbitol is unstable and routine assays may not be available. one should evaluate liver enzymes and function tests (serum indirect and direct bilirubin concentration, serum bile acids and blood ammonia) in cases of prolonged and profound depression to rule out hepatoencephalopathy. one should evaluate arterial blood gases in patients with primary respiratory disease or with clinical evidence of respiratory failure and in profoundly depressed, recumbent patients, especially neonates. hypoxemia observed in response to endotoxin infusion is thought to be caused by an increase in ventilation-perfusion mismatch rather than pulmonary edema as occurs in human patients with acute respiratory distress syndrome. the lung is not a major shock organ in horses; however, pulmonary edema may occur in patients with associated sepsis or complications such as dic. the ideal treatment for endotoxemia is prevention. if one possibly can recognize and closely monitor patients at risk, one can provide treatment proactively and may reverse the effects of endotoxin before the inflammatory response has developed a dynamic of its own. unfortunately, endotoxemia can develop rapidly, and horses are exquisitely sensitive to the effects of endotoxin; therefore, many equine patients are not evaluated until reaching more severe stages of endotoxemia or shock. prognosis and patient outcome then frequently depend on the severity of complications associated with endotoxemia. treatment of endotoxemia involves multiple aspects, and the following strategies have been proposed : • inhibition of endotoxin release into the circulation • scavenging of lipopolysaccharide molecules to prevent direct effects and interaction with inflammatory cells • inhibition of cellular activation by lipopolysaccharide • inhibition of mediator synthesis • interference with the effects of inflammatory mediators • general supportive care in addition, complications such as renal failure, one also must address liver failure, cardiac failure, laminitis, and abortion in pregnant mares. when evaluating reports concerning the efficacy of any one treatment, one should keep in mind differences in underlying disease processes and the complexity of the inflammatory cascade. a "one for all" treatment most likely will not be found, and similarly, any one treatment can only address one or few pathophysiologic aspects of endotoxemia. understanding the rationale for different treatment strategies is important to be able to tailor treatment to the needs of the patient. inhibition of endotoxin release requires identification and removal of its source. therefore whenever endotoxemia is evident, the clinician should strive to reach a diagnosis of the underlying disease and ascertain whether ischemic or inflamed bowel or a gram-negative septic process is present. aside from history, physical examination, and routine laboratory tests, evaluation may include exploratory laparotomy in colic patients, roentgenologic and ultrasonographic evaluation of the pleural and peritoneal cavity and organs, ultrasonographic evaluation of umbilical remnants in neonatal foals, evaluation of passive transfer and calculation of a sepsis score in foals, and repeated culturing of blood or other specimens. if one suspects an infectious process, one should pursue identification of the responsible microorganisms and their antimicrobial sensitivity spectrum; however, one should not delay treatment to obtain culture results. specimen containers with removal devices for antimicrobials are available and are useful in cases for which one has initiated treatment before specimen collection. once one reaches a diagnosis, one must take appropriate measures to correct the primary disease process. examples are removal of devitalized sections of bowel or infected umbilical remnants, drainage of infected pleural or peritoneal fluid, and gastric lavage followed by administration of mineral oil and/or activated charcoal in cases of grain overload to prevent further absorption of endotoxin. one must address any septic process with appropriate antimicrobial therapy. initially, broad-spectrum coverage of the most likely organisms is recommended; one then should specify therapy according to results of culture and sensitivity testing. sepsis in foals is caused predominantly by gram-negative organisms, of which e. coli, actinobacillus spp., klebsiella spp., salmonella spp. and pasteurella spp. frequently are identified. the reader is referred to other texts for review of general principles of antimicrobial therapy. regarding endotoxemia specifically, antimicrobial therapy has been suggested to increase the amount of circulating endotoxin by inducing endotoxin release on cell death of gram-negative bacteria. a recent in vitro study compared endotoxin release and inflammatory mediator activity between antimicrobials commonly used to treat e. coli septicemia in foals and specifically evaluated amikacin, ampicillin, amikacin plus ampicillin, ceftiofur, and imipenem. although these antimicrobials showed no difference in the ability to kill bacteria, amikacin and the amikacin/ampicillin combination resulted in the lowest and ceftiofur in the greatest release of endotoxin. endotoxin release appeared to be dose-dependent in that lesser amounts were released at higher antimicrobial concentrations. based on these results and clinical experience, combining antimicrobial therapy with endotoxin-binding agents such as polymyxin b may be beneficial, especially when using β-lactam antimicrobials. antimicrobials frequently are given perioperatively to colic patients to lower the risk of peritonitis, incisional infection, and generalized sepsis and endotoxemia. because antimicrobial therapy has been implicated in the development of colitis, the duration of treatment should be minimal. unless evidence of sepsis, such as fever or changes in the leukogram, is present, perioperative administration of antimicrobials should not exceed a to hours. conversely, antimicrobial therapy frequently is used in cases of infectious colitis to treat the inciting cause and to prevent sepsis from translocation of bacteria. endotoxin typically has a short plasma half-life and is removed rapidly by mononuclear phagocytes or neutralized by binding to serum proteins and lipoproteins. many conditions responsible for the development of endotoxemia in horses, however, are associated with an ongoing release of endotoxin. examples include severe gastrointestinal inflammation as in proximal enteritis or colitis, grain overload, or uncontrolled sepsis. therapy directed against endotoxin itself may be able to interrupt the continuous activation of the inflammatory cascade in these cases. further benefits of antiendotoxin treatment may be derived if large amounts of endotoxin have been released before the inciting cause can be addressed. an important consideration regarding the efficacy of immunotherapy is the region of the lipopolysaccharide molecule against which antibodies are raised. the o-chain of lipopolysaccharide acts as a potent antigen on infection with gram-negative bacteria ; however, antibodies directed against the o-chain are serotype specific and cannot afford significant cross-protection against heterologous bacterial strains. the core and lipid a region, both of which show a much higher degree of homology between lipopolysaccharide derived from different bacterial strains, offer a more promising target for immunotherapy. active immunization against endotoxin has been reported for horses. vaccination with a bacterin/ toxoid vaccine prepared from rough mutants of salmonella typhimurium or s. enteritidis protected horses against homologous and heterologous endotoxin challenge , and carbohydrate overload. despite these encouraging results and the current availability of a vaccine for use in horses (endovac-equi, immvac inc., columbus, missouri), active immunization against endotoxin does not appear to be a common practice. in comparison, passive immunization with antilipopolysaccharide antibodies is used widely. rough bacterial mutants, most commonly j of e. coli o :b and s. minnesota re , are used to immunize donor horses and subsequently prepare serum or plasma products. proposed mechanisms of action after binding of the antibodies to lipopolysaccharide include steric blockade of lipid a interaction with cellular receptors and enhanced bacterial clearance by opsonization. [ ] [ ] [ ] studies concerning the efficacy of antibody administration in equine patients vary in their results. beneficial effects have been described in experimental models of endotoxemia, acute gastrointestinal disease, and neonates with sepsis, , [ ] [ ] [ ] whereas in other studies, antibodies failed to protect foals and horses against endotoxin effects. - furthermore, administration of a s. typhimurium antiserum to foals was associated with an increased respiratory rate and higher serum activities of il- and tnf. various equine serum and plasma products are currently commercially available. an antiserum raised against the lipopolysaccharide-core of s. typhimurium (endoserum) is available for administration to endotoxemic horses at a recommended dose of . ml/kg body mass. diluting the serum ten-to twentyfold in crystalloid intravenous solutions, administering it slowly over to hours, and monitoring the patient for adverse reactions is advisable. although the product is marketed for use in foals with failure of passive transfer, adverse effects have been reported, and one should use caution when administering it to neonates. plasma from donors inoculated with j (e. coli) and s. typhimurium (re mutant) is available under a restricted license (polymune-j, vet dynamics, inc., san louis obispo, california). the manufacturer recommends administration of at least to l in cases of endotoxemia. in addition, hyperimmune plasma, which has a guaranteed minimum immunoglobulin g content but does not contain specific antiendotoxin antibodies (hi-gamm equi, lake immunogenics, inc., ontario, new york; polymune and polymune-plus, vet dynamics, inc.), is marketed for treatment of failure of passive transfer, and many clinicians use it to treat endotoxemia and sepsis. in addition to antibodies and protein, plasma contains active constituents such as complement components, fibronectin, clotting factors, and at iii and therefore may be particularly useful in patients with endotoxemia-induced coagulopathy. volumes of to ml/kg body mass of hyperimmune plasma have been recommended for use in endotoxemic patients. , polymyxin b polymyxin b is a cationic polypeptide antibiotic that binds to the anionic lipid a portion of lipopolysaccharide and neutralizes its endotoxin capacity. at dosages required for antimicrobial activity, polymyxin b carries the risk of respiratory paralysis and ototoxic, nephrotoxic, and neurotoxic side effects; however, a much lower dose is required for endotoxin-binding activity. the effects of polymyxin b in horses have been evaluated in different experimental models. , , in an in vivo study in foals, treatment with polymyxin b at a dosage of u/kg body mass before infusion with s. typhimurium lipopolysaccharide resulted in significantly less severe elevations of body temperature, respiratory rate, and serum activities of tnf and il- compared with untreated controls. similarly, polymyxin b treatment of adult horses given endotoxin significantly ameliorated clinical signs and decreased plasma tnf activity. in the latter study, benefits of treatment were also evident at lower dosages of polymyxin b ( and u/kg body mass) and administration of polymyxin b hour after the start of endotoxin infusion. conversely, polymyxin b failed to ameliorate clinical signs of endotoxemia or prevent the development of coagulopathy, acidosis, lameness, and shock in experimental carbohydrate overload. side effects suggestive of neurotoxicity appeared after repeated administration of mg/kg body mass ( , u/kg) and in milder form, . mg/kg body mass ( , u/kg) polymyxin b. nephrotoxicity was not observed. currently, use of polymyxin b in equine patients is recommended at dosages of to u/kg body mass every to hours. one should initiate treatment as early in the disease process as possible, because the beneficial effects of lipopolysaccharide scavenging are limited to the first to hours after the onset of endotoxemia, before tolerance develops. side effects in the form of neuromuscular blockade and apnea, which necessitate slow infusion of the drug in human patients, have not been observed in horses. one therefore can administer the entire dose as a slow bolus. if one uses polymyxin b in horses with hypovolemia, dehydration, or azotemia, one should attempt to improve peripheral tissue perfusion, minimize the polymyxin b dose, and closely monitor patients for nephrotoxicity. close monitoring is also important if one administers medications such as aminoglycoside antibiotics, which share a similar spectrum of potential side effects, concurrently. azotemic neonates have been reported to be more susceptible to the nephrotoxic effects of polymyxin b than adult horses. in an attempt to decrease the risk for adverse effects while preserving lipopolysaccharide-neutralizing ability, a conjugate of polymyxin b with dextran has been developed. in conjugated form, polymyxin b is prevented from extravasation into tissues, where it exerts toxic effects by interaction with cell membranes. in addition, conjugation increases the residence time of polymyxin b in the circulation and therefore should prolong the antiendotoxic effect. the polymyxin b-dextran combination was evaluated at a total dose of mg/kg body mass of polymyxin b in . g/kg body mass dextran given minutes before administration of endotoxin in horses. treatment was found to block the development of tachycardia, tachypnea, fever, and neutropenia completely and to prevent increases in serum concentrations of tnf, il- , txb (a txa metabolite), and the prostacyclin metabolite -keto-pgf α . although mild adverse effects in the form of tachypnea, sweating, and increased systolic blood pressure were observed, these were transient and could be prevented by pretreatment with ketoprofen. the polymyxin b-dextran combination is not commercially available at this time. natural endotoxin-binding proteins such as lbp, lipoproteins, and scd have been evaluated experimentally. results of these studies are controversial, and detrimental effects occurred in some cases. a protein receiving much attention regarding potential therapeutic efficacy is the bactericidal permeability-increasing protein (bpi). this protein is structurally similar to lbp but is expressed exclusively in myeloid precursors of polymorphonuclear leukocytes. bpi is stored in primary granules of mature neutrophils and during inflammation is expressed on their cell membranes and secreted into the extracellular environment. bpi has an even higher affinity for lipopolysaccharide than lbp and shows antibacterial activity specific for gram-negative bacteria. binding of bpi to the gram-negative bacterial membrane results in growth arrest and is an important factor in the antibacterial activity of intact neutrophils. furthermore, bpi binding disrupts normal membrane organization and makes bacteria more susceptible to hydrophobic substances, including antimicrobials. experimentally, recombinant bpi has been shown to protect against the toxic and lethal effects of isolated lipopolysaccharide and intact gram-negative bacteria, and clinical trials in human patients show promising results concerning its therapeutic use. the biology and potential use of bpi in horses has not been evaluated. treatments aimed at inhibiting lipopolysaccharide interaction with cells or turning off intracellular signaling pathways are under investigation. nontoxic lipopolysaccharide or lipid a structures can act as endotoxin antagonists, if they competitively inhibit binding to lbp or cellular receptors or inhibit cellular activation by other mechanisms. of the potential antagonists that have been evaluated experimentally, lipopolysaccharide and lipid a from the phototrophic bacterium rhodobacter sphaeroides, and a synthetic compound (e ) the structure of which is based on r. sphaeroides lipopolysaccharide, have been most promising. [ ] [ ] [ ] [ ] [ ] unfortunately, species differences exist regarding cellular response to these structures, and r. sphaeroides lipopolysaccharide acts as a potent inducer of cytokine expression in equine cells. based on results of receptor transfection studies in other species, , tlr is likely responsible for this phenotypic variation. additional compounds including lipopolysaccharide derived from nitrogen-fixing plant bacteria of the species rhizobium are being evaluated to reveal further insight into structural requirements for endotoxin antagonists in horses. nonsteroidal antiinflammatory drugs (nsaids) are probably the most commonly used drugs to treat endotoxemia. the rationale for their use is inhibition of prostaglandin endoperoxide h synthase, that is, cox, and thereby inhibition of prostanoid production (see figure . - ). additional beneficial effects may include scavenging of oxygen-derived free radicals and iron chelation; however, side effects may occur at dosages required to achieve these effects. prostanoids have been identified as important mediators in the inflammatory response in a number of studies, and inhibition of their synthesis is associated with beneficial effects. two cox isoforms are recognized: constitutively expressed cox- and inducible cox- . upregulation of cox- expression results from various proinflammatory stimuli, including lipopolysaccharide, tnf-α, and il- . constitutively expressed cox products are likely important for maintenance of homeostasis, whereas increased production of prostanoids by cox- is thought to be responsible for detrimental effects during inflammation and shock. research has focused on the development of selective cox- inhibitors; however, none of these products are currently available for use in horses. in horses the most commonly used nsaid to treat endotoxemia is flunixin meglumine. beneficial effects of flunixin meglumine have been described in experimental models of endotoxemia [ ] [ ] [ ] and in clinical cases. in equine colic patients, treatment with flunixin meglumine before exploratory surgery resulted in reduced plasma concentrations of txb and pge and had a favorable effect on cardiovascular parameters. flunixin meglumine was shown further to maintain cardiac output and systemic arterial blood pressure, improve blood flow to vital organs, reduce pulmonary endothelial damage, and improve survival on endotoxin challenge. [ ] [ ] [ ] [ ] nsaid use in horses carries the risk of side effects, most importantly the development of gastrointestinal ulceration and renal papillary necrosis (renal crest necrosis). in a study comparing the side effects of flunixin meglumine ( . mg/kg body mass), phenylbutazone ( . mg/kg body mass), and ketoprofen ( . mg/kg body mass) given times daily for days, lesions of the gastric glandular mucosa occurred most commonly. phenylbutazone resulted in the most severe side effects, which included small intestinal edema, erosions, and ulcers in the large colon and decreased serum albumin concentration. renal crest necrosis occurred more frequently in horses treated with phenylbutazone but also occurred with flunixin meglumine treatment. despite the higher risk of side effects, use of phenylbutazone has been suggested for certain cases. in colic patients, phenylbutazone may provide analgesia and ameliorate endotoxin-induced ileus without masking cardiovascular effects of endotoxin, which are used to determine the necessity of surgical exploration. for similar reasons and to minimize side effects a reduced dose of flunixin meglumine ( . mg/kg body mass) has been suggested and is used widely in horses. at this dosage, flunixin meglumine was shown to inhibit eicosanoid synthesis efficiently in an in vivo model of endotoxemia. reduction of clinical signs, however, was dose dependent, and therefore one should choose the appropriate dose based on the circumstances of each case. ketoprofen has been suggested to have superior effects because of a proposed dual inhibitory effect on cox and lipoxygenase and may carry a decreased risk of side effects compared with flunixin meglumine and phenylbutazone. a comparison of cytokine and eicosanoid production by lipopolysaccharide-stimulated isolated monocytes in vitro, however, showed no significant difference between horses pretreated with flunixin meglumine ( . mg/kg body mass) or ketoprofen ( . mg/kg body mass), respectively. eltenac has been evaluated in an experimental endotoxemia model in horses. given minutes before lipopolysaccharide infusion, eltenac at a dose of . mg/kg protected against changes in clinical, hemodynamic, and hematologic parameters and blunted the lipopolysaccharide-induced rise in plasma cytokine concentrations in comparison with controls. some parameters, however, including heart rate, leukocyte count, lactate concentration, and plasma tnf activity, were not improved. ibuprofen may have beneficial effects superior to the other nsaids, because it may be possible to achieve tissue concentrations safely that allow iron chelation to occur. according to a study in healthy foals, dosages of ibuprofen up to mg/kg every hours can be given safely for up to days. the use of corticosteroids for antiinflammatory therapy in sepsis and endotoxemia has been controversial in human and equine patients, and beneficial effects superior to the ones achieved by nsaids have not been demonstrated consistently overall. corticosteroids inhibit the activity of phospholipase a and the release of arachidonic acid from cell membrane phospholipids, as well as the production of tnf, il- , and il- in response to a lipopolysaccharide stimulus. experimentally, beneficial effects of dexamethasone in equine endotoxemia have been demonstrated. , to inhibit tnf production by equine peritoneal macrophages, however, the required concentration of dexamethasone was high and corresponded to an in vivo dosage (approximately mg/kg body mass) greatly exceeding current recommendations. although single doses of corticosteroids are unlikely to carry a disproportionate risk of side effects, one should consider the suggested association of laminitis with corticosteroid use in horses. in cases of sepsis, further immunosuppressive effects could be detrimental. in human patients with certain types of septic shock, dysfunction of the hypothalamic-pituitary-adrenal axis has been recognized and successfully treated with hydrocortisone replacement therapy. use of corticosteroids for this indication has not been evaluated in horses. pentoxifylline, a methylxanthine derivative and phosphodiesterase inhibitor, has been suggested for use in endotoxemia because of its effects on neutrophil function and its ability to inhibit the production of various cytokines, interferons, and thromboplastin. decreased production of tnf, il- , txb , and thromboplastin in response to endotoxin was shown in an equine ex vivo model. in horses given endotoxin followed by treatment with pentoxifylline ( . mg/kg body mass followed by continuous infusion of mg/kg/hr for hours), however, only minimal beneficial effects were observed. treatment significantly improved body temperature, respiratory rate, and whole blood recalcification time, but no effect was observed regarding heart rate, blood pressure, leukocyte count, plasma fibrinogen concentration, and serum cytokine concentrations. the conclusion was that benefits of treatment with pentoxifylline might be restricted to administration of high bolus doses or continuous infusion early in the pathophysiologic process. in an in vivo endotoxemia model in horses, combination of pentoxifylline ( mg/kg body mass) and flunixin meglumine ( . mg/kg body mass) was found to have greater benefit than each treatment on its own. the currently recommended dosage for oral administration of pentoxifylline is mg/kg every hours. because of its rheologic properties, that is, the ability to increase erythrocyte deformability and microvascular blood flow, pentoxifylline may be particularly useful in endotoxemic patients showing evidence of laminitis. an intravenous preparation of pentoxifylline is not commercially available. dimethyl sulfoxide (dmso) is used frequently in an attempt to scavenge oxygen-derived radicals. the treatment may be most appropriate in cases of ischemia-induced intestinal damage and associated reperfusion injury. however, dmso failed to show beneficial effects in an experimental model of intestinal ischemia when administered on reperfusion of the ischemic intestine. dmso at the commonly used dose of g/kg body mass was shown to increase mucosal loss after ischemia and reperfusion of the large colon, and hence a reduced dose of . g/kg body mass has been proposed for cases of intestinal ischemia. for intravenous administration, dmso needs to be diluted in polyionic solutions to a concentration not exceeding %. oral administration of a % to % solution via nasogastric intubation is also possible. aside from dmso the xanthine oxidase inhibitor allopurinol has been suggested as a treatment to prevent oxygen radical-induced tissue damage. during periods of ischemia, tissue xanthine dehydrogenase is converted to xanthine oxidase, which on reperfusion catalyzes the generation of superoxide radicals. , evaluation in horses showed beneficial effects of mg allopurinol per kilogram body mass administered hours before endotoxin challenge. in another study, mucosal damage attributable to oxygen-derived free radicals was not attenuated by allopurinol in an experimental ischemia-reperfusion model. lidocaine given intravenously has been suggested as an antiinflammatory, analgesic, and prokinetic agent, and some clinicians use it to treat colic and laminitis in horses. in an experimental endotoxemia model in rabbits, lidocaine was found to inhibit hemodynamic and cytokine responses to endotoxin profoundly if given immediately following lipopolysaccharide infusion. use of lidocaine therefore may have additional merit in endotoxemic patients. a common regimen for lidocaine use in horses is administration of an initial bolus ( . mg/kg body mass) section . endotoxemia followed by continuous infusion at a rate of . mg/ kg/min. one should monitor patients for toxic neurologic effects associated with a lidocaine overdose. high concentrations of ω- fatty acids can alter the phospholipid composition of cellular membranes toward a decreased ratio of ω- to ω- and thereby can affect membrane functions such as phagocytosis, receptor binding, and activities of membrane-bound enzymes. most importantly for the treatment of endotoxemia, ω- fatty acid incorporation into cell membranes decreases the availability of arachidonic acid (an ω- fatty acid) for eicosanoid synthesis and provides alternative substrates. metabolism of ω- fatty acids via the cox and lipoxygenase pathway leads to the production of -series prostaglandins and -series leukotrienes, which have less biologic activity than their -series and -series counterparts derived from arachidonic acid. aside from these mechanisms, ω- fatty acids prevent lipopolysaccharideinduced upregulation of cd in monocytic cells and therefore may be able to block transmembrane signaling of lipopolysaccharide. cells from horses given linseed oil (high in ω- fatty acids) for weeks before blood collection showed significantly decreased expression of procoagulant activity, txb , and tnf in response to lipopolysaccharide stimulation. , in an in vivo experimental model of endotoxemia in horses, treatment resulted in prolonged activated partial thromboplastin time and whole blood recalcification time, suggesting an anticoagulant effect; however, a significant beneficial effect on clinical response and serum eicosanoid concentrations was not observed. because dietary addition of ω- fatty acids requires several weeks of treatment, intravenous infusion was evaluated and shown to alter the composition of cell membrane phospholipids rapidly. further evaluation of this treatment for use in horses is necessary before dosage recommendations can be made. monoclonal and polyclonal antibodies against equine tnf have been evaluated. [ ] [ ] [ ] administration of a monoclonal antibody preparation before lipopolysaccharide infusion resulted in significantly reduced plasma tnf-activity, improved clinical abnormality scores, lower heart rate, and higher leukocyte count compared with controls. furthermore, plasma concentrations of lactate and -keto-pgf α were reduced significantly, whereas txa production was not affected. in another study, administration of a rabbit polyclonal antibody against recombinant human tnf was unable to improve clinical and hematologic parameters when given shortly ( minutes) after lipopolysaccharide infusion, although inhibition of tnf activity was present in vitro. , findings in horses are in agreement with studies in other species and suggest that beneficial effects of tnf inhibition may be limited to administration before lipopolysaccharide exposure. widespread clinical use therefore is unlikely to become feasible. clinical trials in human patients have not shown significant benefits of tnf antibody treatment. , the effects of selective paf receptor antagonists have been evaluated. paf is implicated in the development of systemic hypotension, lipopolysaccharide-induced platelet aggregation, ileus, and increased vascular permeability and may mediate recruitment of leukocytes to inflamed tissues. , a study in horses using the paf receptor antagonist sri - before lipopolysaccharide infusion showed significant decreases in heart rate and shorter elevation of lactate concentrations in response to the treatment. although not statistically significant, additional beneficial effects included delayed onset of fever, a shortened period of neutropenia, and reduced maximal platelet aggregation. whenever possible, the clinician should correct volume and electrolyte deficits, or at least improve them, before anesthetizing a patient for a surgical procedure. for initial resuscitation, polyionic solutions such as lactated ringer's solution given at rates of to ml/kg/hr are appropriate. patients with severe hypovolemia and shock may require higher fluid volumes. a viable alternative to large-volume resuscitation with isotonic fluids is the use of small volumes of hypertonic solutions, which transiently raise plasma osmolality, thereby causing a fluid shift from the interstitial space into the vasculature and rapidly restoring circulating volume. hypertonic saline solution ( . % sodium chloride) is the most commonly used hypertonic solution and has been shown to have beneficial effects in endotoxemic horses. a dose of ml/kg is recommended, which one should give as a bolus infusion over to minutes, followed by administration of an isotonic solution to restore total body fluid volume. one should use hypertonic saline with caution in patients with sodium and/or chloride derangements and should monitor serum electrolyte concentrations in the case of repeated administration. improvement of the cardiovascular status in response to fluid therapy is indicated by normalization of heart rate, mucous membrane color, and capillary refill time. failure of urination to occur despite appropriate fluid resuscitation should result in critical evaluation of renal function. once one has stabilized the patient, one should choose a maintenance fluid rate to maintain adequate hydration and plasma volume. for adult horses, the maintenance fluid rate is approximately ml/kg/hr, whereas neonatal foals that are not nursing may require larger volumes ( ml/kg/hr). one should monitor fluid administration carefully in endotoxemic patients, because lowered plasma oncotic pressure caused by hypoproteinemia along with an increased vascular permeability increase the risk of tissue edema formation. furthermore, a rapid increase in total body fluid volume may be detrimental in patients with compromised cardiac and peripheral vasomotor function and may increase the severity of vascular pooling in peripheral organs. in these patients, hypertonic saline or colloids may be more appropriate means of stabilization than large volumes of crystalloid solutions. plasma is an ideal colloid and should be administered to maintain a serum total protein concentration above . g/dl. to raise plasma protein concentration and colloid osmotic pressure significantly, however, horses often require large volumes of plasma ( to l or more in a -kg horse), and one should consider alternative colloids. furthermore, high-molecular-weight polymers are thought to provide superior oncotic effects in cases of sepsis and endotoxemia, when vascular permeability is increased. hetastarch, or hydroxyethyl starch, (hespan) is commercially available as a % solution in . % sodium chloride. hetastarch molecules have very high molecular weight, and degradation must occur before renal excretion. these properties result in a longer plasma half-life and prolonged oncotic effects compared with other colloids; persistence of the oncotic effect for hours was found in hypoproteinemic horses. a dosage of to ml/kg given by slow intravenous infusion along with an equal or greater volume of crystalloid fluids is recommended. , in human patients, prolonged activated partial thromboplastin time, decreased factor viii activity, and decreased serum fibrinogen concentration have been described in association with hetastarch use. in the limited number of equine studies, bleeding times were not affected , ; however, one should monitor patients treated with hetastarch for coagulopathy. one should base correction of serum electrolyte concentrations on the results of laboratory evaluation. ideally, one should evaluate serum electrolyte concentrations of patients receiving fluid therapy daily. one should take ongoing losses and lack of dietary intake into account, especially when serum concentrations, as in the case of potassium, poorly reflect total body electrolyte stores. potassium supplementation is recommended in patients experiencing prolonged (greater than hours) periods of anorexia. one can add calcium in the form of calcium gluconate, which is available as a % solution. based on a study in healthy horses, rates of administration for calcium gluconate in the range of . to . mg/kg/min are recommended, and as a guideline, one should administer . to ml/kg body mass per day of a % solution. one can add potassium in the form of potassium chloride or potassium gluconate to intravenous solutions at a dose of to meq/l given at a maintenance rate. administration of potassium should not exceed a rate of . to meq/kg/hr. metabolic acidosis in endotoxic shock is attributable to lactic acidemia and inadequate tissue perfusion. acid-base balance often improves considerably after fluid resuscitation (preferably with alkalinizing solutions such as lactated ringer's solution) alone; however, additional sodium bicarbonate may be required in cases in which serum bicarbonate concentration remains below meq/l. for adult horses, the bicarbonate deficit (in meq hco ) is calculated as . × body mass (kg) × base deficit, whereas for foals one should use a factor of . . as a general rule, one should administer half the required amount as a bolus followed by the remaining half over to hours. because endotoxemia is a dynamic process and losses are ongoing, one should reevaluate acid-base status at least once daily. foals with sepsis are frequently hypoglycemic, and % dextrose solutions are useful as initial resuscitation fluids. one should reduce the glucose concentration of intravenous solutions according to the blood glucose concentration to avoid prolonged hyperglycemia. administration of hyperimmune plasma ( to ml/kg body mass) is highly recommended in foals with evidence of partial or complete failure of passive transfer. one should consider positive inotropic and vasomotor agents in patients with persistently inadequate tissue perfusion. lower dosages of dopamine ( . to µg/kg/min) result in vasodilation of the renal, mesenteric, coronary, and intracerebral vasculature via dopaminergic effects, whereas higher dosages (up to µg/kg/min) also exert stimulation of β -adrenergic receptors, resulting in increased myocardial contractility and heart rate. dobutamine is a direct β -adrenergic agonist and does not appear to have significant vasodilator properties. dosages for dobutamine of to µg/kg/min as continuous intravenous infusion have been recommended for use in horses. in addition, norepinephrine was evaluated in hypotensive critically ill foals that were refractory to the effects of dopamine and dobutamine. at dosages up to . µg/kg/min administered concurrently with dobutamine, six out of seven foals showed an increase in mean arterial pressure, and all foals had increased urine output. because of the risk of cardiac side effects, close monitoring of heart rate and rhythm should accompany infusion of inotropes. indirect blood pressure measurements using a tail cuff may be used to monitor the effects of treatment. more frequently than overt thrombosis or bleeding attributable to dic, hemostatic abnormalities occur in the form of alterations in the coagulation profile. a procoagulant state with shortened bleeding times or prolonged bleeding times caused by consumption of clotting factors may be evident. one should address abnormalities in the coagulation profile as early as possible but especially if they persist more than hours after initiation of therapy. because of the complex interactions of coagulation and fibrinolysis during endotoxemia, one should combine anticoagulant therapy with the administration of fresh frozen plasma to replace clotting and fibrinolytic factors. heparin acts as an anticoagulant by activation of at iii and subsequent inhibition of thrombin, release of tissue factor pathway inhibitor from endothelial cells, and inhibition of platelet aggregation. because endogenous at iii levels frequently are decreased in patients with coagulopathy, addition of heparin to fresh frozen plasma may be the most effective route of administration. an initial dose of iu/kg body mass followed by to iu/kg body mass times daily has been recommended. anemia caused by erythrocyte agglutination occurs in some patients during therapy with unfractionated heparin , but typically resolves within hours if therapy is discontinued. because of the risk of microthrombosis associated with erythrocyte agglutination, use of low-molecular-weight heparin ( iu/kg body mass subcutaneously every hours) has been recommended but may be cost-prohibitive. one may give aspirin orally ( to mg/kg body mass, every hours), which irreversibly inhibits platelet cox activity, to inhibit platelet aggregation and microthrombosis. platelet hyperaggregability has been implicated in the pathogenesis of carbohydrate-induced laminitis, and heparin and aspirin have been recommended to prevent development of laminitis. in an in vitro study, however, aspirin did not inhibit endotoxin-induced platelet aggregation. luteolysis caused by increased concentrations of pgf α leads to pregnancy loss in endotoxemic mares before day of pregnancy. daily administration of altrenogest (regu-mate, hoechst-roussel agri-vet, somerville, new jersey) at a dose of mg orally consistently prevented fetal loss in mares if administered until day of pregnancy. treatment with flunixin meglumine, by blockade of pgf α release, also may contribute to the maintenance of pregnancy in endotoxemic mares. the pathogenesis of fetal loss and abortion caused by endotoxemia, surgery, or systemic disease later in gestation is not understood completely. proposed mechanisms include direct effects on the fetus, placental function, or placental progesterone production. the pathophysiology of laminitis caused by endotoxemia is understood incompletely; however, decreased digital blood flow , and intravascular microthrombosis have been implicated. decreased no production by vascular endothelial cells in response to endotoxin has been suggested as a mechanism for vasoconstriction and decreased blood flow ; however, use of no donors remains controversial. maintenance of adequate peripheral perfusion and anticoagulant and antiinflammatory therapy may be helpful in preventing and treating laminitis caused by endotoxemia. although the innate immune response to endotoxin (lipopolysaccharide) is crucially important for the preservation of homeostasis and health, large amounts of endotoxin can evoke an excessive and uncontrolled inflammatory response and result in a dysfunction of hemostatic and circulatory control mechanisms, loss of vascular integrity, and finally tissue damage. conditions commonly associated with the development of endotoxemia in horses are acute gastrointestinal diseases, especially of ischemic and severe inflammatory nature, and localized or generalized infections. although measuring endotoxin concentrations in equine plasma is possible, this is not feasible in a clinical setting, and one typically reaches a diagnosis of endotoxemia based on clinical signs and clinicopathologic data. successful treatment of endotoxemia requires resolution of the primary disease process in addition to neutralization of circulating endotoxin, interference with the activities of inflammatory mediators, and general supportive care. newer treatments, such as blockade of endotoxin-interaction with cells or interruption of cell signaling pathways, are under investigation. possible sequelae of endotoxemia include dic, multiple organ failure, circulatory failure, and death. frequently, the outcome of conditions associated with endotoxemia in horses depends on the severity of associated complications; for example, renal compromise, laminitis, and abortion. resting state the lateral margins of the vesicle, that is, the buccal mucosa, are in close contact with the cheek teeth. caudally, the external space communicates with the pharynx through the aditus pharyngis. the mucous membrane of the mouth is continuous at the margin of the lips with the skin and during life is chiefly pink but can be more or less pigmented, depending on the skin color and the breed type. the lips are two muscular membranous folds that unite at angles close to the first cheek teeth. each lip presents an outer and an inner surface. the upper lip has a shallow median furrow (philtrum); the lower lip has a rounded prominence or chin (mentum). the internal surface is covered with a thick mucous membrane that contains small, pitted surfaces that are the openings of the ducts of the labial glands. small folds of the mucous membrane called the frenula labii pass from the lips to the gum. the free border of the lip is dense and bears short, stiff hairs. the arteries of the mouth are derived from the maxillary, mandibular, labial, and sphenopalatine arteries of the major palatine artery. the veins drain chiefly to the lingual facial vein. sensory nerves originate from the trigeminal nerve (cranial nerve v) and the motor nerves from the facial nerve (vii). the cheeks spread back from the lips and form both sides of the mouth and are attached to the alveolar borders of the bones of the jaws. the cheeks are composed of skin and muscular and glandular layers and then the internal mucous membrane. the skin is thin and pliable. in contrast, the oral mucous membrane is dense and in many areas of the oral cavity is attached firmly to the periosteum so that construction of oral mucosal flaps can be achieved only by horizontal division of the periosteal attachment. such a feature is important in reconstructive techniques applied to the oral cavity. the blood supply to the cheeks comes from the facial and buccal arteries and the sensory nerves from the trigeminal and motor nerves from the facial nerve. the hard palate (palatum durum) is bounded rostrally and laterally by the alveolar arches and is continuous with the soft palate caudally. the hard palate has a central raphe that divides the surface into two equal portions. the word mouth is used commonly to signify the first part of the alimentary canal or the entrance to it. the mouth is bounded laterally by the cheeks, dorsally by the palate, and ventrally by the body of the mandible and by the mylohyoideus muscles. the caudal margin is the soft palate. the mouth of the horse is long and cylindric, and when the lips are closed, the contained structures almost fill the cavity. a small space remains between the root of the tongue and the epiglottis and is termed the oropharynx. the cavity of the mouth is subdivided into sections by the teeth. the space external to the teeth and enclosed by the lips is termed the vesicle of the mouth, and in the enter the nose from the glands of the vomeronasal duct. to what extent these secretions aid in pheromone reception is not known. that portion of the palatine mucosa immediately behind the incisor teeth frequently is swollen (lampas) during eruption of the permanent teeth. this swelling is physiologic and not pathologic. the tongue is situated on the floor of the mouth between the bodies of the mandible and is supported by the sling formed by the mylohyoideus muscles. the root of the tongue is attached to the hyoid bone, soft palate, and pharynx. the upper surface and the rostral portion of the tongue are free; the body of the tongue has three surfaces. the apex of the tongue is spatulate and has a rounded border. the mucous membrane adheres intimately to the adjacent structure and on the dorsum is dense and thick. the lingual and sublingual arteries supply the tongue from the linguofacial trunk and matching veins. the linguofacial trunk drains into the linguofacial vein. the lingual muscles are innervated by the hypoglossal nerve (xii) and the sensory supply is from the lingual and glossopharyngeal (ix) nerves. the formula for the deciduous teeth of the horse is times i - c - p - for a total of . the permanent dental formula is times i - c - p - or p - m - for a total of or . in the mare the canine teeth are usually small or do not erupt, hence reducing the number to or . the first premolar tooth (wolf tooth) is often absent and has been reported as occurring in only % of the upper dentition of thoroughbred horses. the teeth of the horse are complex in shape and are compounded of different materials (dentin, cementum, and enamel). they function as grinding blades to masticate and macerate cellulose food in the important first stage of the digestive process. the cheek teeth in the horse are a well-documented feature of the evolution of equus caballus. the first incisor is present at birth or the first week of life. the second incisor erupts at to weeks of age; the third incisor, at to months of age; the first and second premolars, at birth to weeks of age; and the third premolar, months of age. the eruption times for the permanent teeth are as follows: first incisor, / years of age; second incisor, / years of age; third incisor, / years of age; the canine tooth, to years of age; the first premolar (wolf tooth), to months of age; the second premolar, / years of age; the third premolar, years of age; the fourth premolar, years of age; the first molar, to months of age; the second molar, years of age; and the third molar, / to years of age. this eruption sequence clearly indicates that the eruption of the second and third permanent premolar teeth give the potential for dental impaction. the modern horse has six incisor teeth in each jaw that are placed close together so that the labile edges form a semicircle. the occlusal surface has a deep enamel invagination (infundibulum) that is filled only partially with cementum. as the incisor teeth wear, a characteristic pattern forms in which the infundibulum is surrounded by rings of enamel, dentin, enamel, and crown cementum in a concentric pattern. each incisor tooth tapers from a broad crown to a narrow root so that as the midportion of the incisor is exposed to wear, the cross-sectional diameters are about equal; that is, at years of age, the central incisor tooth of the horse has an occlusal surface that is an equilateral triangle. observations on the state of eruption, the angles of incidence of the incisor teeth, and the pattern of the occlusal surfaces are used as guides for aging of horses. the canine teeth are simple teeth without complex crowns and are curved. the crown is compressed and is smooth on its labial aspect but carries two ridges on its lingual aspect. no occlusal contact occurs between the upper and lower canine teeth. when erupted, the six cheek teeth of the horse function as a single unit in the mastication of food. each arcade consists of three premolar and three molar teeth. the maxillary arcade is slightly curved, and the teeth have a square occlusal surface. the occlusal surfaces of the mandibular teeth are more oblong, and each arcade is straighter. the horse is anisognathic, that is, the distance between the mandibular teeth is narrower (one-third) than the distance between the upper cheek teeth. this anatomic arrangement affects the inclination of the dental arcade as the jaws slide across each other in the food preparation process. the unworn upper cheek tooth presents a surface with two undulating and narrow ridges, one of which is lateral and the other medial. on the rostral and lingual side of the medial style is an extra hillock. the central portion of these surfaces is indented by two depressions that are comparable with, but much deeper than, the infundibula of the incisor teeth. when the teeth have been subjected to wear, the enamel that closed the ridges is worn through and the underlying dentin appears on the surface. thus after a time the chewing surface displays a complicated pattern that may be likened to the outline of an ornate letter b, the upright stroke of the b being on the lingual aspect. dentin supports the enamel internally, cementum supports the enamel lakes, and the peripheral cementum fills in the spaces between the teeth so that all six teeth may function as a single unit, that is, the dental arcade. transverse ridges cross each tooth so that the whole maxillary arcade consists of a serrated edge. the serrations are formed so that a valley is present at the area of contact with adjacent teeth. these serrations match fitting serrations on the mandibular arcade. the true roots of the cheek teeth are short compared with the total length of the tooth. cheek teeth have three roots: two small lateral roots and one large medial root. by custom, that portion of the crown embedded within the dental alveolus is referred to as the reserve crown, and the term root is confined to that area of the tooth that is comparatively short and enamel free. wear on the tooth gradually exposes the reserve crown, and the roots lengthen. in an adult -lb horse the maxillary cheek teeth are between . and . cm in length. dental wear accounts for erosion and loss of tooth substance at a rate of mm/yr. the pulp chambers of the teeth are also complex. the incisors and canines have a single pulp chamber. the mandibular cheek teeth have two roots and two separate pulp chambers. the maxillary cheek teeth, although they have three roots, have in fact five pulp chambers. as occlusal wear proceeds, deposition of secondary dentin within the pulp chambers protects the chambers (e.g., the dental star, medial to the infundibulum on the incisor teeth). in the mandibular cheek teeth the transverse folding of the enamel anlage (during morphogenesis of the tooth) does not take place, and the occlusal surface is a simple surface of central dentin surrounded by enamel. each tooth then is conformed to a single arcade by the presence of peripheral crown cementum. the oral cavity and oropharynx are subject to a variety of diseases. however, many conditions affecting the first portion of the alimentary system produce the same clinical signs, regardless of their cause. the clinical signs may include inappetance or reluctance to eat, pain on eating or swallowing, oral swelling, oral discharge, and fetid breath. affected animals may show some interest in food but hesitate to eat it. salivation may be excessive and may be contaminated with purulent exudate or blood. the occurrence of bruxism (i.e., grinding of teeth) can indicate discomfort in other areas of the alimentary tract; for example, bruxism and frothing oral saliva are characteristic features of gastric ulceration in the horse. the clinician needs to be aware that considerable weight loss can occur rapidly with inability to feed and swallow. diseases that result in denervation of the pharynx and inappropriate swallowing can have the complication of inhalation pneumonia. after a complete physical examination and ascertaining the history, the clinician should approach examination of the mouth systematically in all cases. one can examine a considerable portion of the mouth and teeth from the outside by palpation of the structures through the folds of the cheek. most horses allow an oral examination without sedation or the use of an oral speculum. in many cases, however, one best achieves the detailed oral examination by sedation and the use of an oral speculum and a light source. one should irrigate the mouth to wash out retained food material so as to be able to inspect and palpate the lips, cheeks, teeth, and gums. the classic signs of dental disease in the horse include difficulty and slowness in feeding, together with a progressive unthriftiness and loss of body condition. in some instances, the horse may quid, that is, it may drop poorly masticated food boluses from the mouth, and halitosis may be obvious. additional problems reported by owners include bitting and riding problems and headshaking or head shyness. facial or mandibular swelling may occur. nasal discharge can result from dental disease associated with maxillary sinus empyema. mandibular fistulae frequently are caused by lower cheek tooth apical infections. some correlation exists between the age of the animal and clinical signs (table . - ). ancillary aids for a complete examination of the oral cavity of the horse may include radiology, endoscopic examination, fluoroscopy, biopsy, and culture. one should take care always during endoscopic evaluation of the oral cavity using a flexible endoscope. the author recommends sedation and the use of an oral speculum to prevent inadvertent mastication of the endoscope. if one uses general anesthesia as part of the diagnostic workup, then endoscopic evaluation of the oral cavity is much easier. in selected cases, advanced imaging technologies such as computed tomography, magnetic resonance imaging, or nuclear scintigraphy may be beneficial. the lips of the horse are mobile and prehensile. in many ways they function like the tip of the elephant's trunk in that they test, manipulate, and sample the environment for potential nutritive value. consequently, loss of motor function (e.g., facial palsy) affects the efficiency of the prehensile system. the lips grasp food in grazing or browsing, and the incisor teeth section the food. with mastication and lubrication with saliva, the bolus of food forms and is manipulated from side to side across the mouth, assisted by the tight cheeks of the horse and the palatine ridges. swallowing begins as the food bolus contacts the base of the tongue and the pharyngeal walls. during swallowing, the soft palate elevates to close the nasopharynx, the base of the tongue elevates, and the hyoid bone and the larynx move rostrally following contraction of the hyoid muscles. during this process, the rima glottidis closes and the epiglottis tilts dorsally and caudally to protect the airway so that food is swept through lateral food channels around the sides of the larynx into the laryngoesophagus. fluoroscopic studies in nursing foals in the dorsoventral view showed that contact occurs between the lateral food channels in the midline so that in outline the food bolus achieves a bow tie shape. dysphagia is defined as a difficulty or inability to swallow. anatomic classifications for dysphagia include prepharyngeal, pharyngeal, and esophageal (postpharyngeal) dysphagias. the site of the cause for dysphagia influences the clinical signs. prepharyngeal dysphagia is characterized by dropping food (quidding) or water from the mouth, reluctance to chew, hypersalivation, or abnormalities in prehension. pharyngeal and esophageal dysphagias are characterized by coughing; nasal discharge containing saliva, water, or food material; gagging; anxiousness; and neck extension during attempts to swallow. the following section describes esophageal dysphagia in more detail. causes of dysphagia can be divided into four types: painful, muscular, neurologic, or obstructive (table . - ). pain and obstruction cause dysphagia by interfering with the mechanics of prehension, bolus formation and transfer to the pharynx, and deglutition. muscular and neurologic causes of dysphagia impede prehension and swallowing by affecting the motor function of the lingual or buccal musculature, muscles of mastication (temporal and masseters), and pharyngeal and cranial esophageal muscles. sensory loss to the lips, buccal mucous membranes, pharynx, or tongue also may cause dysphagia. neurologic causes of dysphagia may affect the forebrain, brainstem, or peripheral nerves that control prehension (cranial nerves vm, vs, vii, and xii), transfer of the food bolus to the pharynx (cranial nerves vs and xii) and swallowing (cranial nerves ix and x). diagnosis of the cause of dysphagia is based on physical examination including a careful oral examination, neurologic examination, clinical signs, and endoscopy of the pharynx, esophagus, and guttural pouches. radiology may be useful to assess the bony structures of the head and throat. ultrasonography is valuable for examining the retropharyngeal space and esophagus to detect and evaluate masses. one may detect pharyngeal or esophageal causes of dysphagia with routine endoscopic examination or with contrast radiography. although one also can use endoscopy to assess deglutition, one must remember that sedation adversely affects the deglutition mechanism. one may assess deglutition using fluoroscopy or manometry, but these techniques require specialized equipment. specific diagnostic procedures for nonalimentary causes of dysphagia are covered elsewhere in this text (see chapter ). specific treatments aimed at resolving the underlying disorder causing dysphagia are discussed in detail elsewhere. one should avoid feeding roughage with long fiber length (hay or grass) to most horses with dysphagia. dietary modifications that promote swallowing such as feeding slurries made from complete pelleted feeds may be sufficient to manage some cases of partial dysphagia. one must take care to prevent or avoid aspiration pneumonia in horses with pharyngeal or esophageal dysphagia. one can manage foals by feeding mare's milk or a suitable substitute through a nasogastric tube. one also may administer pellet slurries or formulated liquid diets via nasogastric tubes to older horses. prolonged nutritional management of dysphagic horses may require extraoral feeding using a tube placed through an esophagostomy. formulated pelleted diets are often easy to administer through a tube as slurry and are balanced to meet the nutritional requirements for healthy horses. one must feed sufficient quantities to deliver adequate calories ( to mcal/day for a -kg horse). adjustments may be necessary for horses that are cachectic or have extra metabolic demand (such as pregnancy). adding corn oil to the ration ( cup every or hours) is a common method of increasing fed calories. liquid diets also have been used for enteral feeding but may not be tolerated as well as pelleted diets. regardless of the method of nutritional management, one must monitor and replace salivary losses of electrolytes. saliva contains high concentrations of na, k, and cl. a group of ponies with experimental esophagostomies and a horse with esophageal squamous cell carcinoma were fed a complete pelleted diet through esophagostomy tubes but developed metabolic acidosis, hyponatremia, and hypochloremia apparently because of salivary losses. surprisingly, salivary losses of potassium did not result in hypokalemia in these cases, presumably because of replacement in the diet. however, if the diet is deficient in potassium, hypokalemia may result. one often can accomplish electrolyte replacement by adding nacl and kcl to the diet. one can maintain horses for months with frequent feedings through an esophagostomy tube. parenteral nutrition (total or partial) may be useful in the short term but is not often feasible for long-term management. tooth eruption is a complex phenomenon involving the interplay of dental morphogenesis and those vascular forces responsible for creating the eruption pathway. these changes are responsible for osteitis and bone remodeling within the maxilla and mandible. young horses frequently show symmetric bony swelling resulting from these eruption cysts. in some cases, additional clinical signs of nasal obstruction with respiratory stridor or nasal discharges may be apparent. pathologic problems associated with maleruption include a variety of dental diseases. oral trauma can displace or damage erupting teeth or the permanent tooth buds. as a result, teeth may be displaced and erupt in abnormal positions or may have abnormal shapes. supernumerary teeth, incisors and molars, can develop, as well as palatal displacement of impacted teeth (maxillary p - , or third cheek tooth). in almost all of these conditions some form of surgical treatment is necessary. significant evidence from the location of apical osteitis in diseased teeth (table . - ) confirms that dental impaction is a major cause of dental disease in the horse. in a series of extracted teeth, were p - or p - (cheek tooth or , respectively). early observations had indicated that the first molar (m , or cheek tooth ) was the most commonly diseased tooth, and an "open infundibulum" in this tooth has been suggested as the cause. studies on cementogenesis of the maxillary cheek teeth have shown, however, that in fact most maxillary cheek teeth have a greater or lesser degree of hypoplasia of cementum within the enamel lakes and that this "lesion" rarely expands into the pulp. the central infundibular hole is the site of its vascular supply to the unerupted cement lake. on those occasions in which cases one can use apicoectomy and retrograde endodontic techniques to save the diseased tooth. one must take care, however, in selection of patients. in most cases of apical osteitis in the horse that result from dental impaction, immature root structures make achieving an apical seal of the exposed pulp difficult. gingival hyperemia and inflammation occur during the eruption of the permanent teeth and are common causes of a sore mouth in young horses (particularly -year-olds as the first dental caps loosen). such periodontal changes usually resolve as the permanent dental arcade is established. during normal mastication, the shearing forces generated by the occlusal contact of the cheek teeth essentially clean the teeth of plaque and effectively inhibit deposition of dental calculus. wherever occlusal contact is ineffective, periodontal changes and calculus buildup occur; for example, the deposition of calculus on the canine teeth of mature geldings and stallions is common. routine dental prophylaxis forms an important component of maintaining normal occlusal contact, and for this reason one should remove arcade irregularities that result in enamel point formation on the buccal edges of the maxillary cheek teeth and the lingual edges of the mandibular cheek teeth. one should remove these edges annually in horses that are at grass and twice yearly in young horses, aged horses, and stabled horses. horses at grass have been shown to have a greater range of occlusal contact and therefore better periodontal hygiene than stabled horses. in stabled horses the range of occlusal contact is narrower and the formation of enamel points occurs more frequently with subsequent buccal ulceration and the initiation of a cycle of altered occlusal contact and hence irregular arcade formation. this process leads to severe forms of periodontal disease and wave mouth formation. periodontal disease occurs with abnormal occlusal contact and initiation of the cycle of irregular wear and abnormal contact. such changes progress to loss of alveolar bone, gross periodontal sepsis, and loss of tooth support. in this sense periodontal disease truly is the scourge of the equine mouth and results in tooth loss. palatine clefts may result from an inherited defect and are caused by failure of the transverse palatal folds to fuse in the oral cavity. harelip accompanies few palatine clefts in the horse. the degree of palatine clefting depends on the stage at which interruption in the fusion of the part ii disorders of specific body systems caries of cementum occurs, that is, secondary inflammatory disease and acid necrosis of the cementum, apical osteitis may develop. pulpitis is key to the pathogenesis of dental decay in the horse. the initiation of inflammatory pulp changes may be a sequela to dental impaction or dental caries or may result from fracture of a tooth. if the onset of the inflammatory process is slow, then formation of secondary dentin within the pulp chambers may protect the pulp and the tooth. secondary dentin formation occurs from stimulation of odontoblasts within the pulp chamber. such changes are the normal process of protection during dental wear and attrition as crown substances wear away and the reserve crown comes into wear. in acute disease, however, this defense mechanism is ineffective, and the changes that occur and that are sequelae to pulpitis reflect the location of each affected tooth. for example, pulpitis and apical osteitis of the third mandibular cheek tooth most commonly results in the development of a mandibular dental fistula. pulpitis of the third maxillary cheek tooth, however, results in an inflammatory disease within the rostral maxillary sinus and in development of chronic maxillary sinus empyema (figure . - ) . oblique radiographs greatly assist the diagnosis of dental decay by demonstrating sinus tract formation, sequestration of bone, mandibular osteitis, hyperplasia of cementum, and new bone formation (so-called alveolar periosteitis). the management of dental decay in the horse usually involves surgical extraction of the diseased tooth. in some palatopalatal folds occurs. toxic or teratogenic effects are documented in other species, but little data are available in the horse. in recent years, treatment for repair of uncomplicated palatine defects has been recommended but prognosis is generally poor because of the considerable nursing care required and the high incidence of surgical failures. one should emphasize early surgery and the use of mandibular symphysiotomy in affording surgical exposure. the combination of mandibular symphysiotomy and transhyoid pharyngotomy to approach the caudal margins of the soft palate affords surgical access, and one can construct mucosal flaps to repair the defects. however, the incidence of surgical breakdown is high, and healing by first intention is the exception rather than the rule. a recent surgical report documented the successful closure of a median cleft of the lower lip and mandible in a donkey. foals born with a severely deviated premaxilla and palate have a wry nose. one can achieve surgical correction of the deviated premaxilla by submucosal division of the premaxilla across the nose at the line of the first cheek tooth. circumstantial evidence indicates that such a defect has a genetic cause, and the defect occurs most frequently in the arabian breed. other developmental abnormalities are subepiglottic cysts resulting from cystic distortion of remnants of the thyroglossal duct, which may cause dyspnea and choking in foals. surgical removal of these cysts results in normal function. the most significant developmental defect of dental origin is a maxilla that is longer than the mandible, that is, the horse is parrot-mouthed. an overbite of cm in the incisor arcade may be present in a horse with a mismatch of less than cm between the first upper and lower cheek teeth. parrot mouth and monkey or sow mouth are thought to be inherited conditions. some correction of minor incisor malocclusion occurs up to years of age. recognition and detection of parrot mouth are important in the examination of potential breeding stock. surgical attempts to inhibit overgrowth of the premaxilla by wiring or by the application of dental bite plate procedures have been documented in recent years. as has been indicated, the horse is by nature a curious animal and uses its lips as a means of exploring a variety of objects. wounds of the lips, incisive bone, and the mandibular incisor area occur commonly in the horse and usually result from the horse getting the lips, jaw, or teeth caught in feeding buckets, in fence posts, or in halters or having a segment of tongue encircled with hair in tail chewing. as the horse panics and pulls away from its oral entrapment, considerable trauma can occur to the lips, teeth, and gums. most wounds repair satisfactorily, provided one finds them early and observes the basic principles of wound hygiene, excision of necrotic tissue, and wound closure. one must ensure that oral mucosal defects are closed and that effective oral seals are made before external wounds are closed. in some cases, offering specially constructed diets or even feeding the horse by nasogastric tube or esophagostomy during the healing processes may be necessary. foreign body penetration of the tongue, cheek, or palate has been reported in grazing and browsing horses and in particular in horses that have certain hay sources that contain desiccated barley awns or yellow bristle grass. other plant material and grass awns also occasionally may penetrate the tongue, gingiva, or cheek, causing inflammation or abscesses. ulcerative stomatitis also results from the toxicity of phenylbutazone therapy. vesicular stomatitis is a highly contagious viral blistering disease described in more detail elsewhere. treatment of glossitis and stomatitis primarily aims at removing the inciting cause. actinobacillus lignieresii, the causative agent of actinobacillosis, has been isolated and identified from ulcers on the free border of the soft palate and oral and laryngeal granulomata. the bacterium also was reported in a sublingual caruncle in a horse with a greatly swollen tongue. therapy with ml of % sodium iodide and g of ampicillin every to hours effected a clinical cure. saliva is important for lubricating and softening food material. the horse has paired parotid, mandibular, and polystomatic sublingual salivary glands. the parotid gland is the largest of the salivary glands in the horse and is situated in the space between the ramus of the mandible and the wing of the atlas. the parotid duct is formed at the ventral part of the gland near the facial crest by the union of three or four smaller ducts. the duct leaves the gland above the linguofacial vein, crosses the tendon of the sternocephalicus muscle, and enters the mouth obliquely in the cheek opposite the third upper cheek tooth. the parotic duct orifice is small, but some dilation of the duct and a circular mucous fold (the parotid papillae) exist at this point. the mandibular gland is smaller than the parotid gland and extends from the atlantal fossa to the basihyoid bone. for the most part, the mandibular gland is covered by the parotid gland and by the lower jaw. the mandibular duct is formed by union of a number of small duct radicles that emerge along the concave edge of the gland and run rostral to the border of the mouth opposite the canine tooth. the orifice is at the end of a sublingual caruncle. the mandibular gland possesses serous, mucous, and mixed alveolar glandular components. the parotid gland is a compound alveolar serous gland. the parotid salivary gland can secrete saliva to yield rates of ml/min, and a total daily parotid secretion can be as much as l in a -kg horse. parotid secretion only occurs during mastication, and administration of atropine or anesthesia of the oral mucosa can block secretion. parotid saliva is hypotonic compared with plasma, but at high rates of flow, concentrations of sodium, chloride, and bicarbonate ions increase. parotid saliva of the horse has a high concentration of calcium, and occasionally calculi (sialoliths) form within the duct radicles of the parotid salivary gland. congenital parotid duct atresia, acquired stricture from trauma to the duct, or obstruction by plant material (sticks or foxtails and other seeds) also may occur. the clinical signs of sialolithiasis or other forms of ductule obstruction include a fluid swelling in the form of a mucocele proximal to the stone and occasionally inflammation of the parotid gland. ultrasonography is useful to diagnose salivary mucoceles and to detect foreign bodies or sialoliths. measurement of electrolyte concentrations in aspirates from suspected mucoceles might be helpful to distinguish them from hematomas. salivary potassium and calcium concentrations are higher than plasma. treatment may require surgical removal of the stone or plant material in the case of sialolithiasis or foreign body obstructions. other causes of obstruction may require resection of the affected portion of the duct or chemical ablation of the gland. primary sialoadenitis is unusual but can occur in one or both glands. the condition is painful and may be associated with a fever and anorexia. secondary sialoadenitis is more common and usually is associated with trauma. infectious sialoadenitis from corynebacterium pseudotuberculosis or other bacterial pathogens also may occur. diagnosis is by physical examination and by finding an enlarged edematous parotid gland tissue on ultrasonographic examination. culture and cytologic examination of aspirates may be useful for diagnostic purposes. treatment in usually palliative, consisting of nonsteroidal antiinflammatory drugs. appropriate antibiotic therapy is indicated as directed by culture and sensitivity results. chemical irritation, glossitis, stomatitis, or other causes of prepharyngeal dysphagia cause ptyalism or excessive salivation in horses. specific therapy for the ptyalism usually is not required as long as salivary losses are not excessive, resulting in dehydration and electrolyte imbalances. ingestion of the fungal toxin slaframine also causes hypersalivation in horses. the fungus rhizoctonia leguminicola, which produces slaframine, causes black patch disease in red clover. slaframine is a parasympathomimetic compound that stimulates exocrine secretion in the parotid gland. slaframine toxicosis most commonly occurs in the spring or early summer and rarely requires treatment other than removal from the pasture. mowing removes the source in most cases because regrowth in pastures often has less fungal contamination. . the esophagus has no digestive or absorptive functions and serves as a conduit to the stomach for food, water, and salivary secretions. the esophageal mucosa is a keratinized stratified squamous epithelium. the submucosa contains elastic fibers that contribute to the longitudinal folds of the esophagus and confer elasticity to the esophageal wall. a transition occurs in the muscle type composing the tunica muscularis from striated skeletal muscle in the proximal two thirds of the esophagus to smooth muscle in the distal third. in the proximal esophagus the skeletal muscle layers spiral across one another at angles. within the smooth muscle layers of the distal esophagus the outer layer becomes more longitudinal, whereas the inner layer thickens and becomes circular. the wall of the terminal esophagus can be to cm thick. deep cervical fascia, pleura, and peritoneum contribute to the thin fibrous tunica adventitia of the esophagus. motor innervation to the striated skeletal muscle of the esophagus includes the pharyngeal and esophageal branches of the vagus nerve, which originate in the nucleus ambiguus of the medulla oblongata. parasympathetic fibers of the vagus nerve supply the smooth muscle of the distal esophagus. sympathetic innervation of the esophagus is minimal. passage of ingesta through the esophagus can be considered part of the swallowing process, which consists of oral, pharyngeal, and esophageal stages. the oral stage is voluntary and involves transport of the food bolus from the mouth into the oropharynx. during the involuntary pharyngeal stage the food bolus is forced through the momentarily relaxed upper esophageal sphincter by simultaneous contractions of the pharyngeal muscles. in the esophageal phase of swallowing the upper esophageal sphincter closes immediately, the lower esophageal sphincter opens, and esophageal peristalsis propels the bolus into the stomach. unlike a food bolus, liquids do not require peristalsis to reach the lower esophageal sphincter and may precede the food bolus during swallowing. the upper esophageal sphincter prevents esophagopharyngeal reflux during swallowing and air distention of the esophagus during inspiration. upper esophageal pressure increases in response to pressure from a food bolus and to increased intraluminal acidity, as would occur with gastroesophageal reflux. the lower esophageal sphincter is a smooth muscle located at the gastroesophageal junction that is morphologically ill defined but forms an effective functional barrier. normally the lower esophageal sphincter is closed in response to gastric distention to restrict gastroesophageal reflux. relaxation of the lower esophageal sphincter permits passage of ingested material from the esophagus to the stomach. distention of the stomach with ingesta mechanically constricts the lower esophageal sphincter. gastric distention also triggers a the esophagus is a musculomembranous tube that originates from the pharynx dorsal to the larynx and terminates at the cardia of the stomach. in adult thoroughbred horses the esophagus is approximately cm long. the cervical portion is approximately cm long; the thoracic portion, approximately cm long; and the short abdominal portion, only approximately cm long. the cervical esophagus generally lies dorsal and to the left of the trachea in the cervical region. in the thorax the esophagus courses through the mediastinum lying dorsal to the trachea and crosses to the right of the aortic arch dorsal to the heart base. impactions. intramural causes of esophageal obstruction include tumors (squamous cell carcinoma), strictures, diverticula, and cysts. [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] mediastinal or cervical masses (tumors or abscesses) may cause extramural obstructions. congenital anomalies are covered in detail later. the clinician must perform a thorough physical examination, including complete oral and neurologic examination, to help rule out causes of dysphagia and nasal discharge other than esophageal obstruction. the clinical signs associated with esophageal obstructions are related primarily to regurgitation of food, water, and saliva caused by esophageal (postpharyngeal) dysphagia. horses with esophageal obstruction are often anxious and stand with their neck extended. one may note gagging or retching, particularly with acute proximal obstructions. bilateral frothy nasal discharge containing saliva, water, and food material; coughing; odynophagia; and ptyalism are characteristic clinical signs, the severity of which varies with the degree and location of the obstruction. distention in the jugular furrow may be evident at the site of obstruction. one may observe other clinical signs related to regurgitation of saliva, water, and food material, such as dehydration, electrolyte or acid-base imbalances, weight loss, and aspiration pneumonia. in extreme cases, pressure necrosis from the impaction or trauma to the esophagus may cause esophageal rupture. if the rupture is in the cervical esophagus, crepitus or cellulitis may be evident along with signs of systemic inflammation. thoracic auscultation is important to determine whether aspiration pneumonia is present. intrathoracic esophageal rupture may result in pleuritis and its associated clinical signs. passage of a nasogastric tube is an effective way to detect and localize an obstruction but provides little information about the nature of the obstruction or the condition of the esophagus. the most direct method for diagnosis of esophageal obstructions is endoscopic examination. most cases of esophageal obstruction occur at sites of natural narrowing of the esophageal lumen, such as the cervical esophagus, the thoracic inlet, base of the heart, or the terminal esophagus, thus one may need an endoscope longer than m for complete evaluation. endoscopic evaluation is useful before relief of an impaction to localize the obstruction and to investigate the nature of the impaction if one suspects a foreign body. foreign bodies may be retrievable via transendoscopic tethering. one can obtain critical diagnostic and prognostic information following resolution of the impaction. assessing the affected esophagus for mucosal ulceration, rupture, masses, strictures, diverticula, and signs of functional abnormalities is important (figure . - ) . ultrasonography of the cervical region is useful not only to confirm a cervical esophageal impaction but also part ii disorders of specific body systems vagal reflex that increases lower esophageal sphincter tone, a safety mechanism against gastroesophageal reflux. the mechanical and vagal mechanisms that promote lower esophageal sphincter tone prevent spontaneous decompression of the stomach, which along with a lack of a vomiting reflex in the horse, increases the risk of gastric rupture during episodes of severe distention. esophageal obstruction has many causes (table . - ) and most often is manifested clinically by impaction of food material and resulting esophageal dysphagia. esophageal obstruction may be caused by primary impactions (simple choke) of roughage, particularly leafy alfalfa hay, coarse grass hay, bedding, and even grass. prior esophageal trauma or poor mastication caused by dental abnormalities may predispose horses to primary esophageal impaction. wolfing or gulping food may precipitate primary impactions, particularly if the horse is exhausted or mildly dehydrated after a long ride or is weakened from chronic debilitation. impactions also may result from disorders that physically impede the passage of food material and fluid by narrowing the luminal diameter, reduce the compliance of the esophageal wall, or alter the conformation of the esophageal wall such that food material accumulates in a pocket or diverticulum. foreign bodies, intra-or extramural masses, or acquired or congenital anomalies cause these so-called secondary to provide critical information about the location and extent of the impaction and esophageal wall thickness and integrity. ultrasonography may provide information about the cause. radiographic assessment of the esophagus can confirm the presence of esophageal obstruction in cases in which one cannot view the affected area adequately using endoscopy. one can detect impacted food material in the esophagus by a typical granular pattern and often can observe gas accumulation proximal to the obstruction. air or barium contrast radiographic studies are most useful for evaluating the esophagus following relief of the impaction if one suspects a stricture. one often can detect esophageal dilation, diverticula, rupture, functional disorder (megaesophagus), or luminal narrowing caused by extraluminal compression more easily using contrast radiographic studies instead of endoscopy ( figure . - ). - one should take care when interpreting radiographic studies in sedated horses, particularly after passage of a nasogastric tube or other esophageal manipulations that may contribute to esophageal dilation. the primary goal of treatment for esophageal impaction is to relieve the obstruction. parenteral administration of acepromazine ( . mg/kg intravenously), xylazine ( . to . mg/kg intravenously) or detomidine ( . to . mg/kg intravenously), oxytocin ( . to . iu/kg intramuscularly), and/or esophageal instillation of lidocaine ( to ml of % lidocaine) may reduce esophageal spasms caused by pain or may decrease esophageal tone. [ ] [ ] [ ] [ ] some clinicians advocate parasympatholytic drugs such as atropine ( . mg/kg intravenously) to reduce salivary secretions and lessen the risk of aspiration. however, undesirable effects of atropine including excessive drying of the impaction and inhibition of distal gastrointestinal motility may preclude its use. resolution of an impaction may require physical dispersal of the material. one can use a nasogastric tube to displace the impacted material along with external massage if the obstruction is in the cervical region. often, carefully lavaging the esophagus with water via an uncuffed or a cuffed nasogastric tube while the head is lowered is necessary to aid in breaking up the impaction. some clinicians advocate a dual tube method whereby a tube is placed through each nasal passage into the esophagus for ingress and egress of the lavage fluid. because of the risk of aspiration of water and food material, esophageal lavage sometimes is done under general anesthesia with a cuffed nasotracheal tube. in refractory cases, intravenous administration of isotonic fluid containing . % nacl and kcl ( to meq/l) for hours at a rate of to ml/kg/ day along with esophageal relaxants such as oxytocin may promote hydration and softening of the impaction and help prevent or alleviate any electrolyte or acid-base imbalances resulting from salivary losses of chloride, sodium, and potassium. one should note that the effects of oxytocin on esophageal tone occur in the proximal two thirds of the esophagus and may not be effective for mucosa has recovered as assessed by endoscopy, one can feed the horse soft food (moistened pellets and bran mashes). one can return the patient gradually to a highquality roughage diet over to days, depending on the degree of esophageal damage induced by the impaction and the nature of any underlying disease. the prognosis for survival is good ( %), but some horses may require permanent dietary modification if persistent chronic obstruction is a problem. aspiration pneumonia and perforation are potential complications of severe or prolonged esophageal obstructions. if aspiration is suspected, administration of broad-spectrum antibiotics that are effective against gram-positive and gram-negative organisms, including metronidazole ( mg/kg orally every hours) for anaerobes is advisable. a subsequent section describes treatment of esophageal perforation or rupture. esophagitis refers to a clinical syndrome of esophageal inflammation that may or may not be ulcerative. the major protective mechanisms of the esophageal mucosa include salivary and food material buffers, normal peristaltic motility, and the barrier formed by the gastroesophageal sphincter. reflux esophagitis is caused by repeated episodes of gastric fluid regurgitation into the distal esophagus and subsequent chemical injury to the mucosa ( figure distal obstructions. , rarely, esophageal obstruction ultimately may require esophagotomy to relieve the impaction. one must enforce strict restriction of food and water, including access to bedding material, until the obstruction is resolved and the esophagus has regained function. systemic effects of dysphagia associated with esophageal impaction include dehydration, hyponatremia, hypochloremia, and metabolic alkalosis from prolonged loss of salivary free water and electrolytes. if the duration of a complete esophageal obstruction is hours or longer, one should correct dehydration and electrolyte and acid-base imbalances. one can restore fluid and electrolyte balance with oral electrolyte solutions if the patient is less than % to % dehydrated and the esophageal obstruction is resolved. horses that are greater than % to % dehydrated or those that have a refractory obstruction or moderate to severe electrolyte imbalances may require intravenous fluid therapy with solutions containing . % nacl and kcl ( to meq/l). one should perform esophageal endoscopy after relief of the impaction to determine whether any complications of the impaction have developed or if a primary cause of the obstruction is present. endoscopic examination is critical to determine the postobstruction treatment plan and for follow-up evaluation of esophageal healing. one should reevaluate the horse every to weeks following resolution of the impaction if one notes esophageal dilation or mucosal injury. additional evaluation via radiography may be warranted to assess motility and transit times. dilation proximal to the site of obstruction, mucosal injury from trauma, stricture formation, formation of a diverticulum, megaesophagus, and esophagitis are sequelae to esophageal obstruction that predispose patients to reobstruction. the rate of reobstruction may be as high as %. depending on the duration of the obstruction and the degree of trauma or dilation, the risk of reobstruction is high for to hours or longer, thus one should withhold food for at least to hours after resolution of the obstruction. sucralfate ( mg/kg orally every hours) may hasten healing if esophageal ulceration is evident, but the efficacy of sucralfate for this purpose is not established. some clinicians suggest that administration of a nonsteroidal antiinflammatory drug (nsaid) such as flunixin meglumine ( mg/kg orally or intravenously every hours) or phenylbutazone ( to mg/kg orally or intravenously every to hours) for to weeks after resolution of the impaction may reduce the development of strictures. judicious use of nsaids is recommended to prevent nsaid-induced worsening of esophageal mucosal injury. one should avoid orally administered nsaids if esophagitis is present. after to hours or when the esophageal duodenal strictures caused by chronic ulceration commonly have reflux esophagitis. diagnosis requires endoscopic examination of the esophagus. one may note diffuse, patchy, linear, or coalescing erosion or ulcerations (see figures . - and . - ). one also may observe significant edema or hyperemia. determining whether an underlying disease, such as infection, neoplasia, esophageal strictures, or diverticula, is present is important. in addition, one must examine the stomach to determine whether the esophagitis is associated with gastritis, gastric obstruction, or gastric ulcer disease. contrast radiography may be helpful to detect esophageal ulceration and is useful to assess esophageal motility and transit time. the principles of therapy for reflux esophagitis include control of gastric acidity, mucosal protection, and correction of any underlying disorder contributing to gastroesophageal reflux. reduction of gastric acid production with h histamine receptor blockers such as ranitidine or proton pump antagonists such as omeprazole is critical for resolution of the esophagitis. some clinicians advocate using sucralfate to promote healing of ulcerated esophageal mucosa. however, the ability of sucralfate to bind ulcerated esophageal mucosa is not proven, nor is the efficacy of sucralfate for hastening esophageal ulcer healing. horses with reflux esophagitis following delayed gastric outflow caused by gastroduodenal ulcer disease, gastric paresis, or proximal enteritis may benefit from prokinetic drugs that act on the proximal gastrointestinal tract. metoclopramide ( . to . mg/kg subcutaneously every to hours) reduces gastroesophageal reflux by increasing lower esophageal sphincter tone, gastric emptying, and gastroduodenal coordination. one should exercise caution when giving metoclopramide to horses because they are prone to extrapyramidal neurologic side effects of the drug. cholinergic drugs such as bethanechol ( . to . mg/kg subcutaneously every to hours or . to . mg/kg orally every to hours) may improve gastric emptying and are effective for treating reflux esophagitis. for esophagitis from trauma or pressure injury after esophageal impaction, judicious use of nsaids may be warranted to reduce esophageal inflammation and pain. dietary modification may be necessary for patients with esophagitis, depending on the degree of ulceration or if motility is impaired. one should feed horses with mild esophagitis frequent small meals of moistened pellets and fresh grass. severe esophagitis may necessitate withholding food and complete esophageal rest for several days. although the prognosis for esophagitis is good in the absence of underlying disease, the risk of esophagus is delayed, such as in functional disorders of the esophagus. like ulceration of the squamous portion of the stomach in horses, gastric acid and bile salt chemical injury is a major mechanism of esophageal squamous epithelial ulceration. , reflux esophagitis may occur along with gastric ulcer disease, motility disorders, increased gastric volume from gastric outflow obstructions, gastric paresis, intestinal ileus, or impaired lower esophageal sphincter function. , other causes of esophagitis in horses include trauma (foreign bodies, food impactions, nasogastric tubes), infection (mural abscesses), or chemical injury (pharmaceuticals, cantharidin) ( figure . - ). [ ] [ ] [ ] [ ] the clinical signs of esophagitis are nonspecific and similar to esophageal obstruction and gastric ulcer diseases. gagging or discomfort when swallowing may be evident, and hypersalivation and bruxism are signs of esophageal pain. esophageal (postpharyngeal) dysphagia may be evident. one may note partial or complete anorexia such that horses with chronic esophagitis may have significant weight loss. esophageal hypomotility dysfunction caused by the inflammatory process may result in esophageal impaction. clinical signs of underlying diseases that predispose to esophagitis may predominate or mask the signs of esophagitis. horses with gastrointestinal motility disorders such as proximal enteritis or gastric outflow obstruction are at a high risk of developing reflux esophagitis because of the presence of gastric acid and bile salts in the fluid reflux. foals with gastric, pyloric, or stricture formation is high if severe circumferential or coalescing ulcerations are present. esophagitis from severe trauma or infection may be prone to stricture formation. motility dysfunction of the equine esophagus is caused most commonly by hypomotility resulting in esophageal dilation (ectasia) or megaesophagus. although megaesophagus in horses most commonly is acquired, reports indicate idiopathic megaesophagus in young horses may be congenital. [ ] [ ] [ ] [ ] acquired megaesophagus in horses may be a consequence of chronic or recurrent esophageal obstruction. , esophageal impactions of a short duration cause a proximal dilation of the esophagus that is generally reversible. however, if the duration of the obstruction is long enough, the motility of the esophagus proximal to the site of obstruction may be impaired permanently. other causes of acquired megaesophagus include extraesophageal obstruction by tumors or abscesses, pleuropneumonia, and vascular ring anomalies. , acquired megaesophagus also may result from neurologic, neuromuscular, and muscular disorders. neurologic diseases that cause vagal neuropathy-such as equine protozoal myeloencephalitis, equine herpesvirus myeloencephalitis, and idiopathic vagal neuropathy-have been associated with megaesophagus in horses. pleuropneumonia may be associated with a vagal neuropathy resulting in megaesophagus. megaesophagus is an early sign of equine dysautonomia and may be observable in patients with botulism. myasthenia gravis is a well-known cause of megaesophagus in nonequine species but has not been reported in horses. also in other species, electrolyte disorders, cachexia, primary myopathies, myositis, and addison's disease may affect esophageal motility but have not been associated with megaesophagus in horses. one can induce iatrogenic megaesophagus by the α adrenergic agonist detomidine, but this is transient and reversible. , nonetheless, the use of this drug may complicate clinical evaluation of esophageal motility. esophageal inflammation, particularly reflux esophagitis, may affect motility and cause megaesophagus. however, because esophageal hypomotility affects the tone and function of the lower esophageal sphincter, reflux esophagitis also may be a complication of a primary functional disorder. thus assessing esophageal motility in horses with esophagitis that is not responding appropriately to treatment is important. along with a complete physical examination one should include a careful neurologic examination to help rule out primary neurologic causes of megaesophagus. because esophageal hypomotility is a functional obstruction, the clinical signs of esophageal hypomotility or megaesophagus are similar to esophageal obstruction. unlike mechanical obstruction the onset of clinical signs is insidious rather than acute. the clinical signs include those associated with esophageal dysphagia. , , [ ] [ ] [ ] [ ] the cervical esophagus may be dilated enough to be evident externally. weight loss is a common sign. signs attributable to an underlying disease may be evident. diagnosis of esophageal hypomotility requires transit studies. one can measure the transit time of a bolus from the cervical esophagus to the stomach by fluoroscopy or contrast radiography. , other signs of esophageal hypomotility and megaesophagus include pooling of contrast material and an absence of peristaltic constrictions. , , , endoscopy may reveal a dilated esophagus and an absence of peristaltic waves. , one may observe evidence of underlying disease causing obstruction or esophageal dilation. , one should evaluate the esophagus for evidence of esophagitis that is causing esophageal motility dysfunction or is a result of impaired esophageal clearance of gastric fluid. esophageal manometry may be useful to document abnormal postdeglutition contraction pressures, contraction time, and propagation times but is not often available for routine clinical application. , one should perform other diagnostic tests such as a complete blood count and chemistry to help determine a possible underlying cause. cerebral spinal fluid analysis may be indicated to rule out neurologic disorders. specialized testing such as electromyography to detect neuromuscular disorders may also be indicated. treatment of esophageal hypomotility or megaesophagus should aim at treating the underlying cause. dietary modification should aim at improving esophageal transit of food. one should feed the horse slurries of pellets, and feeding from an elevated position to promote transit may be beneficial. metoclopramide or bethanechol may benefit patients with reflux esophagitis associated with megaesophagus by increasing lower esophageal tone, gastric emptying, and reducing gastroesophageal reflux. the prognosis depends on the underlying cause and the degree of dilation. although many cases of megaesophagus associated with reflux esophagitis respond well to treatment, many other forms of megaesophagus including congenital megaesophagus have a poor prognosis. strictures most commonly are caused by pressure necrosis from esophageal impactions that induce circumferential erosion or ulceration of the esophageal mucosa, although esophageal injury caused by oral administration of corrosive medicinal agents and trauma to the neck may also result in stricture formation. congenital strictures also have been reported. strictures caused by mucosal and submucosal trauma are termed esophageal webs or rings. strictures may also originate in the muscular layers and adventitia of the esophagus (mural strictures) or in all of the layers of the esophagus (annular stenosis). , horses with these lesions have a presentation similar to those with simple obstructions, because strictures result in partial obstruction and impaction of food material in the lumen. one can detect esophageal webs or rings with endoscopy (see figure . - ), whereas identification of mural strictures or annular stenosis may require a double-contrast esophogram (see figure . - ). in a retrospective study of horses with esophageal stricture following simple obstruction, maximal reduction in esophageal lumen diameter occurred within days of the esophageal obstruction. although surgery has been used to relieve such strictures, initial medical management is warranted because strictures may resolve with conservative therapy, and the esophagus continues to remodel for up to days following ulceration. in one report, seven horses with esophageal obstruction-induced stricture were treated conservatively by feeding a slurry diet and administering antiinflammatory and antimicrobial medications, and five of seven were clinically normal within days. one of the five successfully treated horses had a -cm area of circumferential ulceration, suggesting that the potential exists for extensive mucosal injury to resolve without permanent stricture formation. if resolution of strictures within days is insufficient, one should investigate other methods to increase esophageal diameter. bougienage has been used successfully in small animal patients and human beings. the technique involves passage of a tubular dilatable instrument down the esophagus and stretching of the stricture. one may perform the technique by passing a nasogastric tube with an inflatable cuff. however, one has to perform the procedure frequently to have any success, and horses do not tolerate it well. alternatively, a number of surgical techniques have been used to resolve strictures, including resection and anastomosis, , temporary esophagostomy with fenestration of the stricture, esophagomyotomy for strictures of the muscularis and adventitia, , or patch grafting with local musculature. however, such surgeries are fraught with complications, largely because of the propensity of the traumatized esophagus to restricture. , the esophagus lacks a serosal layer and does not rapidly form a fibrin seal as does the remainder of the intestinal tract, so anastomoses tend to leak. in addition, tension on the esophagus during swallowing and movement of the neck impairs healing of anastomoses. , in spite of these difficulties, the long-term prognosis for horses with chronic esophageal strictures treated surgically is better than for those treated nonsurgically. two types of diverticula are traction (true) diverticula and pulsion (false) diverticula. traction diverticula result from wounding and subsequent contraction of periesophageal tissues, with resultant tenting of the wall of the esophagus. pulsion diverticula arise from protrusion of esophageal mucosa through defects in the muscular wall of the esophagus and usually result from trauma or acute changes in intraluminal pressure. traction diverticula appear as a dilation with a broad neck on contrast esophagography, whereas pulsion diverticula typically have a flask shape with a small neck on an esophagram (see figure . - ). , although traction diverticula are usually asymptomatic and of little clinical significance, pulsion diverticula may fill with feed material, ultimately leading to esophageal obstruction. [ ] [ ] [ ] a movable mass in the midcervical region may be noticeable before onset of complete obstruction. pulsion diverticula may be corrected surgically by inverting or resecting prolapsed mucosa and closing the defect in the wall of the esophagus. , , inversion of excessive mucosa may reduce the diameter of the esophageal lumen and predispose horses to esophageal obstruction and therefore should be reserved for small diverticula. congenital disorders of the esophagus are rare. reported congenital abnormalities include congenital stenosis, persistent right aortic arch, esophageal duplication cysts, [ ] [ ] [ ] intramural inclusion cysts, , and idiopathic megaesophagus. , , in the one report of congenital stenosis, double-contrast radiography revealed concentric narrowing of the thoracic esophagus in the absence of any vascular abnormalities at the base of the heart. successful treatment included having the foal stand with the forelimbs elevated off the ground following each feeding. persistent right aortic arch is a congenital anomaly in which the right fourth aortic arch becomes the definitive aorta instead of the left aortic arch, which results in constriction of the esophagus by the ligamentum arteriosum as it extends between the anomalous right aorta and the left pulmonary artery. clinical signs may include those associated with esophageal (postpharyngeal) dysphagia, drooling, and distention of the cervical esophagus resulting from partial obstruction of the thoracic esophagus. , endoscopic examination typically reveals dilation of the esophagus cranial to the obstruction section . esophageal diseases with evidence of diffuse esophagitis. successful surgical treatment of persistent right aortic arch has been reported in one foal. esophageal duplication cysts and intramural inclusion cysts cause typical signs of esophageal obstruction, including salivation, esophageal dysphagia, and swelling of the cervical esophagus as the cysts enlarge. , , such signs can make them difficult to differentiate from other forms of esophageal obstruction (choke). endoscopic examination may reveal compression of the esophageal lumen and communication with the esophageal lumen if it exists. ultrasonographic examination may be the most useful method of antemortem diagnosis if the cyst is in the cervical esophagus. examination of an aspirate of the mass may aid in the diagnosis by revealing the presence of keratinized squamous cells. , surgical treatments have included complete surgical resection and surgical marsupialization. , , the latter appears to be more successful and results in fewer complications. , complications of surgical resection have included laryngeal hemiplegia following surgical trauma to the recurrent laryngeal nerve in the region of the esophagus and esophageal fistula formation. perforation typically occurs in the cervical region in response to external trauma, necrosis of the esophageal wall caused by a food impaction, or rupture of an esophageal lesion such as an impacted diverticulum. the esophagus is particularly vulnerable to external trauma in the distal third of the neck because only a thin layer of muscle covers it at this point. iatrogenic perforation may occur in response to excessive force with a stomach tube against an obstruction or a compromised region of the esophagus. esophageal perforations may be open or closed and tend to cause extensive cellulitis and necrosis of tissues surrounding the wound because of drainage of saliva and feed material within fascial planes. systemic inflammation associated with endotoxemia from septic cellulitis may occur. closed perforations of the esophagus are particularly troublesome because food material, water, saliva, and air may migrate to the mediastinum and pleural space via fascial planes. , because of the leakage of air into the tissues surrounding the rupture, extensive subcutaneous and fascial emphysema frequently develops and is usually evident clinically and on cervical radiographs. pneumomediastinum and pneumothorax are potentially fatal complications of esophageal ruptures. treatment should include converting closed perforations to open perforations if possible, extensive debridement and lavage of affected tissues, broadspectrum antibiotics, tetanus prophylaxis, and esophageal rest. the clinician may achieve the latter by placing a feeding tube into the esophagus via the wound. alternatively, one may place a nasogastric tube using a small tube ( -f diameter) . for open perforations, once the wound has granulated and contracted to a small size, one may attempt peroral feeding. extensive loss of saliva via esophageal wounds may lead to hyponatremia and hypochloremia. in addition, transient metabolic acidosis occurs because of salivary bicarbonate loss, followed by progressive metabolic alkalosis. although reports of esophageal wounds healing well by second intention exist, healing takes a prolonged time. in addition, some perforations never completely heal and form permanent esophagocutaneous fistulae that may require surgical correction. the development of esophageal strictures is not common because wounds are usually linear and not circumferential. however, traction diverticula may develop. other complications of esophageal wounds include horner's syndrome and left laryngeal hemiplegia. in a retrospective study on esophageal disorders, only of horses with esophageal perforations survived long-term ; in a report of esophageal trauma following nasogastric intubation, of horses were euthanized. the prognosis is therefore poor in horses with esophageal perforations, largely because of the extent of cellulitis, tissue necrosis, shock, and local wound complications. specialized endoscopic equipment allowing visual inspection of the entire adult equine stomach has become increasingly available to veterinarians in academia and private practice. thus gastric disease in horses recently section . diseases of the stomach has gained increasing awareness among veterinarians, owners, and trainers. peptic ulcer disease is defined as erosions or ulcers of any portion of the gastrointestinal tract normally exposed to acid. mucosal damage can include inflammation, erosion (disruption of the superficial mucosa), or ulceration (penetration of the submucosa). in severe cases, fullthickness ulceration can occur, resulting in perforation. the proximal (orad) portion of the equine stomach is lined by stratified squamous mucosa similar to the esophageal lining. the distal (aborad) portion of the stomach is lined with glandular mucosa, and the distinct junction between the two regions is deemed the margo plicatus. ulceration can occur in either or both gastric regions, although different clinical syndromes and pathophysiologic mechanisms apply. as a result, the broad term equine gastric ulcer syndrome (egus) has been used to encompass the wide array of associated clinical syndromes. egus develops in horses of all ages and continues to be of major clinical and economical importance. the prevalence of gastric ulceration has been reported for a variety of breeds and types of horses; however, most current data involve thoroughbreds in race training. the prevalence of squamous ulceration in horses in race training varies from % to % - and can be as high as % when limited to animals actively racing. in a survey of active show horses, % had gastric ulceration, with only horse having ulceration of the glandular fundus. in one large retrospective study ( adult horses from to ) evaluating incidence of gastric ulceration identified at necropsy, an overall prevalence of . % was found. the highest prevalence was found in thoroughbreds (including arabians) and standardbred trotters, and cold-blooded horses were affected significantly less. lesions were located most commonly in the squamous mucosa along the margo plicatus, followed by the glandular body, proximal squamous mucosa, and antrum. many studies investigating prevalence of gastric ulceration do not differentiate between squamous and glandular lesions or evaluate only squamous disease. in a recent study in which the gastric antrum and pylorus were evaluated in horses in a hospital setting, % had antral or pyloric erosions or ulcerations, % had squamous mucosal lesions, and % had lesions involving the glandular body. a correlation between the presence or severity of squamous disease and antral/pyloric disease was not identified. the reported prevalence of gastric ulceration in foals varies from % to %. [ ] [ ] [ ] an imbalance between inciting and protective factors in the mucosal environment can result in ulcer formation. , the major intrinsic factors promoting ulcer formation include hydrochloric acid, bile acids, and pepsin, with hydrochloric acid being the predominant factor. various intrinsic factors protect against ulcer formation such as the mucus-bicarbonate layer, maintenance of adequate mucosal blood flow, mucosal prostaglandin e and epidermal growth factor production, and gastroduodenal motility. in human beings, extrinsic ulcerogenic factors include nonsteroidal antiinflammatory drugs, helicobacter pylori, stress, changes in diet, or gastrointestinal disorders, especially those resulting in delayed gastric emptying. in human neonates, physiologic stress associated with a major primary illness seems to be associated strongly with gastric ulcers. many of the other factors mentioned previously are believed to be important in horses, but clear evidence of an infectious agent has not yet been identified in horses or foals with egus. , recently, the possibility of helicobacter infection in horses has reemerged with the identification of polymerase chain reaction products from urel, a protongated urea channel unique to gastric-dwelling helicobacter species, in the squamous epithelium of three horses, two of which had squamous erosions. the specific factors involved in injury and the protective mechanisms vary between regions of the proximal gastrointestinal tract. the pathophysiology of squamous mucosal ulceration in the horse appears similar to that in gastroesophageal reflux disease in human beings and ulceration of the nonglandular mucosa in pigs. excess acid exposure is the predominant mechanism responsible for squamous mucosal ulceration, although many details remain unclear. hydrochloric acid is secreted by parietal cells in the gastric glands via a hydrogen-potassium adenosine triphosphatase (h + ,k + -atpase) pump on the luminal side. horses secrete acid continuously, and measured ph of equine gastric contents varies from less than to greater than depending on the dietary state of the horse (fed or fasted). , a protocol of repeated -hour periods of fasting and feeding has been shown to induce squamous erosion and ulceration. because this protocol results in periods of prolonged gastric acidity (ph < . ) and because concurrent administration of the histamine (h ) receptor antagonist ranitidine reduces lesion severity, the protocol supports the role of acid exposure in the pathogenesis of squamous ulcer disease. several peptides can stimulate or inhibit parietal cell secretion of acid. the predominant stimuli for hydrochloric acid secretion are gastrin, histamine, and acetylcholine via the vagus nerve. g cells release gastrin within the antral mucosa, whereas mast cells and enterochromaffin-like cells release histamine in the gastric gland. histamine binds to type receptors on the parietal cell membrane, causing an increase in cyclic adenosine monophosphate and resulting in phosphorylation of enzymes that activate the proton pump. gastrin and acetylcholine can act via calcium-mediated intracellular pathways and also stimulate histamine release directly. isolated equine parietal cells respond maximally to histamine stimulation and only minimally to carbachol and pentagastrin. gastrin release is controlled primarily by gastrin-releasing peptide, which is stimulated by gastric distention and increased luminal ph, but the interaction between gastrin and histamine has not been elucidated fully in the horse. somatostatin, released by fundic and antral d cells, is the primary inhibitor of gastric acid secretion by parietal cells. the inhibitory effect of somatostatin is primarily paracrine, but plasma levels of somatostatin negatively correlate with gastric luminal acidity. epidermal growth factor, a peptide produced in saliva, also inhibits gastric acid secretion. foals can produce significant amounts of gastric acid by the second day of life, with consistent periods of acidity (ph < . ) in clinically normal animals. , in one study, foals tended to have a high gastric ph at day of age, but in a study of critically ill foals, some foals demonstrated periods of gastric acidity on the first day of life. suckling was associated with an immediate rise in gastric ph, whereas periods of rest in which foals did not suck for more than minutes were associated with prolonged periods of acidity. whereas premature human infants are capable of gastric acid production at weeks of gestation, only of premature foals demonstrated an acidic ph recording in a study of gastric ph profiles in critically ill foals. however, multiple factors likely were involved in critically ill foals of this study, and the true ontogeny of gastric acid production in foals is currently unknown. equine squamous mucosa is thin at birth but becomes hyperplastic and parakeratotic within days. the parallel between decreasing ph and proliferation of squamous epithelium correlates with that observed in other species. the combination of a thin gastric epithelium with a high acid output may leave neonatal foals susceptible to ulcer formation at a young age. in addition, one must remember the difference in normal appearance of the squamous mucosa when interpreting gastric endoscopy in a neonatal population. in esophageal squamous mucosa, intercellular tight junctions and bicarbonate secretion are the major factors involved in protection against acid injury in other species, although squamous bicarbonate secretion had not been documented in the horse. [ ] [ ] [ ] the principal barrier is a glycoconjugate substance secreted by cells in the stratum spinosum, with a contribution from the tight junctions in the stratum corneum. this barrier function is considered weak at best, and thus a functioning lower esophageal sphincter, normal salivary flow, and salivary mucins contribute to the prevention of acid injury in human gastroesophageal reflux disease. in horses a mechanical barrier like the lower esophageal sphincter is not available to protect the gastric squamous mucosa from acid exposure. the normal gastric fill line rests just below the cardia, so only the squamous mucosa along the lesser curvature adjacent to the margo plicatus should receive exposure to acidic gastric contents regularly. not surprisingly, this correlates with the most common location of squamous mucosal ulceration. bile salts and pepsin have been implicated as contributing factors to ulcer disease in many species. in rabbit esophageal mucosa, bile salt absorption occurs and is correlated directly with mucosal barrier disruption. the unconjugated bile salts cholate and deoxycholate have a pk a (negative logarithm of the ionization constant of an acid) of and . , respectively, and therefore cannot remain in solution and cause mucosal damage in the presence of acid. alternatively, the conjugated bile salt taurocholate (pk a . ) can cause mucosal injury in the ionized salt form at ph or the un-ionized acid form at ph to . in the pig, bile salts or acid alone cause squamous mucosal damage, whereas a combination of the two result in extensive damage in vitro. in the horse a similar synergistically damaging effect was found with the addition of bile salts and acid (ph . ) to stratified squamous mucosa in vitro in one study. in addition, the investigators were able to document levels of bile salts and acid sufficient to cause mucosal damage in gastric contents within hours of feed deprivation. this is not surprising, given that duodenogastric reflux occurs normally in the horse. in a separate in vitro study of equine squamous mucosa, prolonged exposure to acid alone (ph . ) had a damaging effect, and synergism with exposure to a combination of acid and pepsin or taurocholate was not found. the lack of synergism likely is caused by the lower ph used in this study and stresses the importance of acid exposure in squamous ulcer disease. pepsinogens are secreted primarily by chief cells, although secretion by neck cells, cardiac glands, and antral pyloric glands also occurs. in an acidic environment (ph < . ), pepsinogen is converted to the active pepsin. although the proteolytic activity of pepsin normally is directed toward dietary protein, it also can act on the gastric mucosa. thus acid remains the major contributing factor to squamous mucosal damage, although other factors such as pepsin and bile salts may play an important role as well in the initiation or perpetuation of disease. several mechanisms help protect the glandular mucosa from acid injury. the mucus-bicarbonate layer serves to titrate h + ion from the gastric lumen to co and h o. cellular restitution and prostaglandins of the e series, which enhance mucosal blood flow and secretion of mucus and bicarbonate in the glandular mucosa have not been documented in squamous epithelium. , of these mechanisms, mucosal blood flow is likely the most important contributor to overall gastric mucosal health. nitric oxide is a key regulator of mucosal blood flow and prostaglandin synthesis and thus may play a role in mucosal protection. dietary factors also have been implicated in ulcer disease. horses in race training have a high incidence of gastric ulceration and frequently are fed high-concentrate, low-roughage diets. in one study, higher volatile fatty acid (acetic, propionic, and isovaleric acid) concentrations, higher gastric juice ph, and lower number and severity of nonglandular ulceration were documented after feeding an alfalfa hay-grain diet compared with a bromegrass hay diet. however, many factors differed between the diets, such as digestible energy, bulk, crude protein, and mineral content (especially calcium). thus dietary factors represent an important area of further investigation in the pathophysiology of egus, particularly squamous ulceration. the pathophysiologic correlation between exercise and squamous ulcer disease has not yet been defined despite the high prevalence of ulceration in performance horses. preliminary work suggests that gastric compression occurs during treadmill exercise, presumably because of an increase in intraabdominal pressure. such contracture could result in increased acid exposure to the squamous mucosa by raising the fill line of gastric contents. further studies in this laboratory have provided support for this theory by demonstrating a high ph in the proximal stomach, immediately distal to the lower esophageal sphincter, during resting conditions that decreases during treadmill exercise (m. lorenzo-figueras and a.m. merritt, personal communication, ) . risk factors associated with gastric ulceration include gender and age, and the reported prevalence of gastric ulcers has increased over time. in one study, ulcers were found more commonly in stallions, and the prevalence of gastric ulceration decreased with age, independent of gender, although this trend was only significant in the population of standardbred trotters. interestingly, the frequency of gastric ulceration increased from less than % before to approximately % after . in a study of thoroughbred horses in race training, an increase in squamous ulcer severity was noted in horses years old or older and in those horses that had raced. in the same study, severity of glandular lesions did not change between examinations, and age (> years) was the only factor associated with glandular lesion severity. several studies have failed to document a correlation between nonsteroidal antiinflammatory drug (nsaid) administration and naturally occurring ulcer disease. , , , , however, nsaid administration is a well-known cause of gastric ulceration under experimental conditions. [ ] [ ] [ ] [ ] [ ] nsaid-related ulceration typically is described as predominantly glandular, although nonglandular ulceration also can occur by a mechanism that has not yet been characterized fully. nsaids cause a decrease in prostaglandin e synthesis because of inhibition of the cyclooxygenase pathway. therefore a resultant decrease in glandular mucosal protection, most notably via decreased mucosal blood flow and mucus production, is the most likely mechanism of action. in one study, however, phenylbutazone administration resulted in ulceration of the glandular mucosa at the pyloric antrum but did not alter mucosal prostaglandin e concentration significantly. clinical signs typically associated with gastric ulceration in foals include poor appetite, diarrhea, and colic. many foals probably never exhibit clinical signs, and some do not exhibit clinical signs until ulceration is severe or fatal perforation has occurred. glandular ulceration typically is considered the most clinically significant type of disease in this population. the physiologic stress of a concurrent illness has been associated with gastric ulceration in foals. retrospectively, ( %) of foals up to days of age with a clinical disorder were found to have lesions in the gastric glandular mucosa, and prospectively ( %) of foals up to days of age with a clinical disorder had glandular ulceration. by contrast, only % to % of clinically normal foals examined in endoscopic surveys had lesions observed in the gastric glandular mucosa. , critically ill neonatal foals can have a greatly different ph profile compared with that in clinically normal foals, potentially because of alterations in gastric motility and acid secretion. gastric ulceration was not identified in any animals at necropsy in that study; however, ulceration has been documented in a similar population. thus factors other than acid exposure, most notably mucosal blood flow, may play an important role in the stressrelated ulceration in neonates. subjectively, gastric ulceration and rupture in the hospitalized neonatal population occurs less commonly now than in previous reports. advances in overall neonatal care, especially supportive care, likely have contributed to this decline. in suckling foals less than days old, lesions typically originate in the squamous mucosa adjacent to the margo plicatus along the greater curvature. such lesions can occur in foals as young as days of age and have been observed in % of foals less than days old. histologic examination of these lesions has revealed disruption of the epithelial layers of the mucosa and a neutrophilic infiltration. another phenomenon that occurs in young foals is the shedding, or desquamation, of squamous epithelium, which appears as flakes or sheets of epithelium. desquamation occurs without ulceration in up to % of foals less than days of age, and this process typically is not associated with clinical signs. , , in older foals, lesions become more prevalent in the squamous mucosa, particularly along the lesser curvature. lesions also are found in the squamous mucosa of the fundus and adjacent to the margo plicatus. these lesions can be severe and often are associated with clinical signs such as diarrhea, poor appetite, and poor growth and body condition. diarrhea is the most frequent sign in symptomatic foals with squamous mucosal lesions and is associated with more diffuse erosion or ulceration of the squamous mucosa than that which occurs in asymptomatic foals. in some foals, poor growth, rough hair coat, a potbelly appearance, or all of those occur along with moderate to severe squamous mucosal ulceration. in horses with severe or diffuse squamous ulceration, bruxism or colic may occur. gastroduodenal ulcer disease occurs almost exclusively in suckling and early weanling foals. clinical signs of duodenal ulceration are similar to those described for gastric ulceration (bruxism, colic, salivation, diarrhea), but the consequences are often more severe. lesions occur primarily in the proximal duodenum and range from diffuse inflammation to severe ulceration. foals with duodenal ulceration often have delayed gastric emptying and may have gastroesophageal reflux. complications can include gastric or duodenal rupture, duodenal stricture, and ascending cholangitis. severe squamous and esophageal ulceration and aspiration pneumonia can occur following gastroesophageal reflux. , [ ] [ ] [ ] [ ] the gastroduodenal ulcer disease syndrome can occur in outbreaks and most commonly is identified in intensive breeding operations. the cause of duodenal lesions in foals is not known. one theory is that the problem begins with diffuse duodenal inflammation that can coalesce down to a focal area of ulceration (g.d. lester and a.m. merritt, personal communication, ) . a temporal relationship between gastroduodenal ulcer disease and rotaviral diarrhea has been suggested, but an infectious cause remains unproven. although lesion location and severity associated with rotaviral infection varies among species, duodenal ulceration has not been reported. clinical signs attributable to egus in older horses vary and classically include anorexia and chronic or intermittent colic of varying severity. many horses with endoscopic evidence of disease may appear to be clinically normal or have vague signs that include decreased consumption of concentrates, postprandial episodes of colic, poor performance or failure to train up to expectations, poorquality hair coat, and decreased condition or failure to thrive. diarrhea typically is not associated with gastric ulceration in adult horses, although ulceration can occur concurrently with other causes of diarrhea. horses actively racing are more likely to have squamous ulceration than those solely in training. lesions occur predominantly in the squamous mucosa, particularly adjacent to the margo plicatus ( figure . - ) . in more severe cases, lesions can extend dorsally into the squamous fundus. clinically relevant lesions typically affect a greater portion of the squamous mucosa and can be deep enough to cause bleeding. however, bleeding from ulcers in the gastric squamous mucosa typically is not associated with anemia or hypoproteinemia. according to a recent study, the incidence of glandular lesions, particularly within the pyloric region, may be higher than previously reported, which emphasizes the importance of a thorough endoscopic examination and proper documentation of lesion location when reporting or discussing egus, especially the differentiation between squamous and glandular disease. although one may suspect a diagnosis of egus based on clinical signs and response to treatment, the only current method of confirmation is via gastroendoscopy, which one can perform easily in the standing horse or foal with mild sedation. in adult horses a -m endoscope allows for visual inspection of the entire stomach, pylorus, and proximal duodenum. shorter scopes permit examination of the gastric body and fundus, but not the pyloric antrum in most cases. one should use an endoscope with a maximum external diameter of mm for neonatal foals. numerous scoring systems for lesion severity have been described, but a recent consensus has been published by the equine gastric ulcer council (table . - ). duodenal ulceration can be difficult to confirm. duodenoscopy is the most specific means of diagnosis, although the procedure is more difficult than gastroscopy. additionally, an endoscope at least cm in length is needed for foals up to to months old, and a longer endoscope usually is required for older animals. diffuse reddening or inflammation may be the only recognizable lesion in cases of early duodenal disease. excessive enterogastric reflux of bile through the pylorus suggests duodenal dysfunction. however, the pylorus frequently appears open, and some degree of enterogastric reflux is common under normal conditions. ulceration at the pylorus or pyloric antrum also suggests the presence of a duodenal lesion. if one can perform gastroendoscopy, but not duodenoscopy, the severity of lesions, particularly in the glandular mucosa and in the squamous mucosa of the lesser curvature dorsal to the pyloric antrum, usually will be severe when duodenal ulcers are present. multiple pharmacologic treatments have been suggested for treating egus. because acid has been implicated as the most important pathophysiologic component of squamous ulcer disease, most antiulcer therapy centers on suppression or neutralization of gastric acid. severity and location of gastric lesions and severity and duration of clinical signs, as well as medication cost, can play a role in the therapeutic management of egus (table . - ). if gastroendoscopy is unavailable, some guidelines to therapy can be used, but the efficacy of the treatment is based on clinical signs, which are often vague or nonspecific. signs of colic or diarrhea that result from gastric ulcers often resolve within hours. one can note improvements in appetite, bodily condition, and attitude within to weeks. if one does not observe improvement in clinical signs, treatment has not been effective or gastric ulceration was not the primary problem. the principal therapeutic options for ulcer treatment include h antagonists (cimetidine, ranitidine, famotidine, nizatidine), proton pump blockers (omeprazole, pantoprazole, rabeprazole, esomeprazole), the mucosal adherent sucralfate, and antacids. the h antagonists suppress hydrochloric acid secretion through competitive inhibition of the parietal cell histamine receptor that can be overcome partially with exogenous pentagastrin. use of h antagonists has been successful in raising gastric ph and resolving gastric lesions in foals and adult horses. , , clinical and experimental evidence has demonstrated greater individual variability with lower dosages of h antagonists. thus dosage recommendations are based on levels necessary to increase gastric ph and promote ulcer healing in a majority of horses. commonly recommended dosages are to mg/kg orally every hours or . mg/kg intravenously every hours for cimetidine and . mg/kg orally every hours or . to mg/kg intravenously every hours for ranitidine. famotidine has been used less extensively in the horse, but a dose of to mg/kg/day has been recommended. because gastric perforation caused by glandular ulcer disease has been reported in hospitalized neonates, many clinicians routinely use prophylactic antiulcer therapy in this population. although clinically normal foals respond predictably to ranitidine, sick neonates have shown variability in ph response to intravenously administered ranitidine, with a much shorter duration of action and in some cases no noticeable response. thus currently used dosing schedules for hospitalized foals may be inadequate. because some critically ill foals have a predominantly alkaline gastric ph profile and because gastric acidity may be protective against bacterial translocation in neonates, the need for prophylactic ulcer therapy is controversial. in critically ill human neonates, intravenous administration of ranitidine raises gastric ph and gastric bacterial colonization but does not increase the risk of sepsis. in a retrospective study of hospitalized foals less than days of age, no difference in the frequency of gastric ulceration at necropsy was found between those foals that received prophylactic treatment for gastric ulcers and those that did not. because the study was retrospective, specific details regarding lesion location and severity were not available; however, none of the foals in the study died because of gastric ulcer disease. h antagonist therapy should continue for to days, but complete ulcer healing may take to days. if an animal is kept in race training during therapy, clinical signs may resolve but the lesions may not. currently, cimetidine and ranitidine are available in injectable, tablet, and liquid forms. famotidine and nizatidine are available in tablets. proton pump inhibitors block secretion of h + at the parietal cell membrane by irreversibly binding to the h + ,k + -atpase proton pump of the cell. these agents have a prolonged antisecretory effect, which allows for once-daily dosing. omeprazole, the first proton pump inhibitor to be developed, is the only currently approved agent for the treatment of egus. several studies have documented the safety of orally administered omeprazole in foals and adult horses. , omeprazole has demonstrated efficacy in the healing of nsaid-induced ulcers in horses and in naturally occurring cases of egus. , more importantly, omeprazole has been shown to eliminate or reduce the severity of gastric ulcers in thoroughbreds maintained in race training. the available equine preparation of omeprazole (gastrogard, merial, ltd., duluth, georgia) is recommended at a dose of mg/kg orally every hours. initial reports suggested that to days of omeprazole therapy were necessary to achieve maximum acid suppression; however, an increase in gastric ph and a decrease in acid output are evident to hours after omeprazole paste administration. after initial treatment ( days), treatment with or mg/kg every hours has been shown to decrease or prevent the recurrence of disease in animals maintained in training. the powder form of omeprazole degrades rapidly in an acidic environment, thus one must use an enteric-coated capsule (as used in the human preparation) or a specially formulated paste (such as gastrogard) to allow delivery of the active drug to the small intestine for absorption. many compounding pharmacies prepare omeprazole in liquid or paste formulation for use in horses, but their efficacy has not been evaluated to date. other proton pump inhibitors have been developed recently for use in human beings, including rabeprazole, lansoprazole, esomeprazole, and pantoprazole. in gastroesophageal reflux disease treatment in human beings, esomeprazole has demonstrated a higher rate of healing at and weeks compared with omeprazole, but rabeprazole, lansoprazole, and pantoprazole have similar efficacy. an intravenous formulation of pantoprazole recently became available commercially and may prove beneficial for patients in need of antiulcer therapy that cannot be treated orally. research regarding the pharmacokinetics and efficacy of other proton pump inhibitors in horses is not currently available. sucralfate is effective in treating peptic ulcers and preventing stress-induced ulcers in human beings. the mechanism of action likely involves adherence to ulcerated mucosa, stimulation of mucus secretion, enhanced prostaglandin e synthesis, and increased concentration of growth factor at the site of ulceration, although the prostaglandin effects may not play an important role in ulcer healing. these are factors relevant to glandular mucosa, and the efficacy of sucralfate in treating ulcers in the equine gastric squamous mucosa remains undetermined. sucralfate may be effective in preventing stress-induced ulcers in neonatal foals, because these occur in the glandular mucosa, although no clinical evidence directly supports this concept. in human beings, sucralfate provides protection against stress-induced ulcers with a decreased risk of pathogenic gastric colonization. one should give sucralfate at a dosage of to mg/kg every to hours. the efficacy of sucralfate in an alkaline ph is controversial but appears likely. [ ] [ ] [ ] moreover, at the time of administration of an section . diseases of the stomach h antagonist, the gastric ph likely will have returned to an acidic ph since the last dosage and will remain so for to minutes depending on the route of administration; thus one likely can administer the agents simultaneously if so desired. the use of antacids to treat gastric ulcers has not been examined critically in the horse. research in horses has shown that g aluminum hydroxide per g magnesium hydroxide results in an increase in gastric ph above for approximately hours. thus although antacids may be useful for treating ulcers in horses, a dose of approximately to ml at least every hours is necessary for a standard adult horse. the use of synthetic prostaglandin e analogs, such as misoprostol, has been effective in treating gastric and duodenal ulcers in human beings, and the proposed mechanism of action involves inhibition of gastric acid secretion and mucosal cytoprotection. frequently reported adverse effects of intestinal cramping and diarrhea in human beings have precluded the use of misoprostol in horses. one should consider prokinetic drugs in foals with duodenal disease and gastroesophageal reflux and when one suspects delayed gastric emptying without a physical obstruction. the cholinergic drug bethanechol has been shown to increase the rate of gastric emptying in horses. in cases of acute gastric atony, bethanechol . to . mg/kg administered subcutaneously every to hours has been effective in promoting gastric motility and emptying, followed by oral maintenance dosages of . to . mg/kg to times daily. adverse effects can include diarrhea, inappetance, salivation, and colic, but at the dosages stated, adverse effects have been infrequent and mild. a complete review of ileus and prokinetic therapy is available in chapter . . for foals with severe gastroduodenal ulcer disease that have developed duodenal stricture, surgical therapy is necessary. , these animals require a serious financial commitment because intensive perioperative medical therapy is critical for a successful outcome. even with surgical therapy, these foals often warrant a guarded prognosis. pyloric stenosis is a structural resistance to gastric outflow. congenital pyloric stenosis has been reported in foals and one yearling and results from hypertrophy of the pyloric musculature. [ ] [ ] [ ] acquired pyloric stenosis can result from neoplasia or duodenal ulceration. [ ] [ ] [ ] [ ] clinical signs depend on the degree of obstruction and include abdominal pain, salivation, and teeth grinding. complete or near complete obstruction can result in gastric reflux and reflux esophagitis. in foals with congenital pyloric hypertrophy, clinical signs may begin with the consumption of solid feed. in foals one can make a presumptive diagnosis via gastric endoscopy and radiography (plain and contrast studies). depending on the cause and severity of disease, gastric endoscopy may provide a presumptive diagnosis in the adult horse. measurement of gastric emptying can aid the diagnosis. several methods of measurement are currently available, including nuclear scintigraphy, acetaminophen absorption, and postconsumption [ c] octanoic acid blood or breath testing. , , exploratory laparotomy shows a distended stomach and thickened pylorus accompanied by a relatively empty intestinal tract. if complete obstruction is not present, medical therapy with a prokinetic such as bethanechol can increase the rate of gastric emptying. phenylbutazone and cisapride also have been shown to attenuate the delay in gastric emptying caused by endotoxin administration. , surgical repair is necessary for definitive treatment of complete or near-complete obstruction and consists of gastroenterostomy or pyloroplasty. , gastric dilation can be classified as primary, secondary, or idiopathic. causes of primary gastric dilation include gastric impaction, grain engorgement, excessive water intake after exercise, aerophagia, and parasitism. , secondary gastric dilation occurs more commonly and can result from primary intestinal ileus or small or large intestinal obstruction. time to development of gastric reflux is proportional to the distance to the intestinal segment involved, with duodenal obstruction resulting in reflux within hours. clinical signs of gastric dilation include those associated with acute colic and in severe cases, ingesta appearing at the nares. associated laboratory abnormalities include hemoconcentration, hypokalemia, and hypochloremia. the most common reported cause of gastric rupture in horses varies between reports. in a retrospective study of horses, gastric rupture occurred most commonly as a secondary phenomenon ( %), usually because of small intestinal obstruction, with primary gastric dilation and idiopathic rupture occurring almost equally ( % and %, respectively). in another retrospective study of horses in combination with a search of the veterinary medical database (vmdb), % of the gastric rupture cases were classified as idiopathic. risk factors for gastric rupture include feeding grass hay, not feeding grain, gelding, and a nonautomatic water source. , nasogastric intubation does not preclude the possibility of gastric rupture, and the amount of reflux obtained before rupture varies greatly. because these reports were retrospective, one cannot rule out confounding factors with certainty. regardless of the initiating cause, gastric rupture usually occurs along the greater curvature. in horses with rupture caused by gastric dilation, tears in the seromuscular layer are frequently larger than the corresponding tears in the mucosal layer, indicating that the seromuscularis likely weakens and tears before the mucosa. , in contrast, horses with gastric rupture following gastric ulceration usually demonstrate full-thickness tears of equal size in all layers. gastric rupture is usually fatal because of widespread contamination of the peritoneal cavity, septic peritonitis, and septic shock. initial clinical signs vary with the primary disease; however, when rupture occurs, a previously painful animal can exhibit signs of relief. subsequent signs are consistent with peritonitis and shock, including tachypnea, tachycardia, sweating, and muscle fasciculations. surgical repair is thus limited but has been reported for partial-thickness tears, and in one case of a combined tear of the mucosa and muscularis with only a focal serosal tear, a full-thickness repair was performed with a favorable outcome. gastric impaction can result in acute or chronic signs of colic in the horse. although a specific cause is not always evident, ingestion of coarse roughage (straw bedding, poor-quality forage), foreign objects (rubber fencing material), and feed that may swell after ingestion or improper mastication (persimmon seeds, mesquite beans, wheat, barley, sugar beet pulp) have been implicated. possible predisposing factors include poor dentition, poor mastication and rapid consumption of feedstuffs, and inadequate water consumption. clinical signs can vary from anorexia and weight loss to those consistent with severe abdominal pain. in severe cases, spontaneous reflux may occur, with gastric contents visible at the nares. in cases of acute severe abdominal pain, one often makes a diagnosis during exploratory celiotomy. in animals not exhibiting signs of colic warranting surgical intervention, an endoscopic finding of a full stomach after a normally adequate fast ( to hours) often can confirm the diagnosis. abdominal radiographs are reserved for smaller horses and ponies. in addition to pain management, specific treatment consists of gastric lavage via nasogastric intubation or massage and injection of fluid to soften the impaction during laparotomy. [ ] [ ] [ ] nonulcerative gastritis rarely occurs in the horse; however, a single case of emphysematous gastritis caused by clostridium perfringens has been reported. mimic that of a strangulating or nonstrangulating small intestinal obstruction, so distinguishing between the two syndromes is important because appropriate treatment of small intestinal obstruction usually requires surgical intervention. studies suggest that the survival rate for horses with dpj that endured surgical exploratory laparotomy was poor compared with those treated medically, although differences in disease severity may have accounted for the results in these early reports. , the clinical syndrome of dpj was well described in the s, and although recognized by its classical presentation, varying degrees of focal intestinal and systemic illness may occur. - dpj usually occurs alone but can occur along with gastritis, ileitis, typhlitis, and or colitis. typical pathologic findings in horses with dpj include involvement of the duodenum and usually the proximal jejunum. the ileum and large colon usually are determined to be grossly normal. gastric distention is a common finding and is thought to be caused by hypersecretory mechanisms in the proximal small intestine and a functional ileus of affected enteric segments. the small intestine may be to cm in diameter because of fluid distention with malodorous, red to brown-red intralumenal fluid accumulation. duodenal (and jejunal) serosal surfaces may have varying degrees and distribution of bright-red to dark-red petechial and ecchymotic hemorrhages and yellow to white streaks. the enteric mucosal surfaces are usually hyperemic and have varying degrees of petechiation and ulceration. microscopically, the most severe lesions have been located in the duodenum and proximal jejunum but may extend proximally to the gastric mucosa and aborally to the large intestinal mucosa and submucosa. microscopic lesions consist of varying degrees of mucosal and submucosal hyperemia and edema. more severe lesions include villus degeneration with necrosis and more severely, sloughing of villous epithelium. the lamina propria, mucosa, and submucosa may have varying degrees of granulocyte infiltration (predominantly neutrophils), and the muscular layers and serosal surfaces contain small hemorrhages. proximal small intestinal serosal fibrinopurulent exudate is a common finding in the more severe cases; therefore the term hemorrhagic fibrinonecrotic duodenitis-proximal jejunitis has been suggested as a more descriptive name for this syndrome. horses with dpj often have evidence of multiple organ involvement such as hepatic changes including congestion and varying degrees of biliary duct hyperplasia. additional systemic involvement likely is caused by endotoxin absorption, metabolic imbalances such as acidemia, and circulatory changes. the cause of this syndrome remains an enigma (much like the cause of other inflammatory conditions affecting the intestinal tract). several microorganisms have been implicated as playing a role in triggering dpj, including clostridium spp., salmonella spp., and some mycotoxins, but efforts to reproduce the syndrome experimentally have been futile. a recent dietary change with an abrupt increase in dietary concentrate level has been suggested to predispose a horse to developing dpj because of intraluminal microbial imbalances. two intracellular processes control intestinal secretion, the cyclic nucleotide (cyclic adenosine monophosphate and cyclic guanosine monophosphate) and the calcium systems. agents (inflammatory mediators, microorganisms, toxic agents) can activate adenyl cyclase (vasoactive intestinal peptide, prostaglandin e ) or guanyl cyclase (bacterial enterotoxins) and induce increases in cyclic adenosine monophosphate and cyclic guanosine monophosphate, respectively. this reaction causes phosphorylation of specific protein kinases, which induce the actual mucosal membrane transport events. increases in intracellular free calcium may arise from cyclic nucleotidedependent release of stored calcium within the cell or from increased calcium entry across the cell membrane. calcium may act through calmodulin, which then can activate membrane-phosphorylating protein kinases. the net effect is increased movement of sodium and chloride into the mucosal cell from the interstitium, with secretion of sodium and chloride into the intestinal lumen. water follows the directional flux of sodium and chloride through highly permeable intercellular spaces. several bacterial toxins and endogenous mediators can cause active secretion and contribute to a synergistic mucosal secretory response. passive secretion of protein-rich fluid into the lumen occurs following damage to the mucosal epithelium, capillary endothelium, and submucosal inflammation in the proximal small intestine. the clinically relevant events that result from active and passive fluid secretion are proximal small intestinal distention and nasogastric reflux, dehydration, and circulatory shock. the concentration of protein in the peritoneal fluid from horses with dpj is usually higher than in horses with small intestinal obstruction. a disproportionate increase in total protein concentration relative to nucleated cell count occurs probably by leakage of blood or plasma into the peritoneal cavity without a significant stimulus for leukocyte chemotaxis. suggested mechanisms for increased abdominal fluid protein concentration include serositis associated with inflamed intestine and small intestinal distention causing passive congestion and increased capillary hydrostatic pressure of visceral peritoneal vessels. small intestinal ileus is another hallmark sign of dpj and the pathophysiology is complicated, involving primary and secondary dysfunction of the central, autonomic, and enteric nervous systems and their purported roles in governing intestinal motility. primary role-players in dpj-associated ileus include peritoneal inflammation, inflammatory cell migration/activation within the muscularis, small intestinal mechanical distention, and effects of endotoxin absorption. the use of prokinetic agents for treating ileus and gastric/small intestinal distention in horses with dpj is becoming more common, but veterinarians should realize that a potential restriction on their use is the need for normal intestinal integrity. in spite of that, one may use motility modifiers judiciously. the veterinarian has the challenge of differentiating horses with dpj from horses with small intestinal obstructive lesions so as to avoid surgical intervention (table . - ). horses with dpj typically show signs of acute abdominal pain initially, and then after gastric decompression, volume replacement, and analgesic therapy, the colic signs subside, but signs of lethargy and malaise become more apparent. in contrast, horses with obstructive lesions of the small intestine usually show signs of abdominal pain until the affected viscus is repaired via surgical intervention or the viscus ruptures. another differentiating characteristic is the large volume (> to l with each decompressive effort) of nasogastric reflux that is often malodorous and orange-brown or red-brown. dpj-affected horses have moderate to severe small intestinal distention palpated on rectal examination, temperature of . °to . °c ( . °to . °f), dehydration, brick-red mucous membranes, lethargy and absent borborygmi, prolonged capillary refill time, tachycardia (> beats/min), and tachypnea. although the signs of abdominal pain usually resolve after gastric decompression, most horses remain severely lethargic. without periodic removal of the fluid that accumulates in the proximal intestinal tract, signs of abdominal pain usually recur. horses with dpj often require gastric decompression at -hour intervals, with to l of fluid recovered each time. nasogastric tubes left in place for long periods of time cause varying degrees of pharyngitis, laryngitis, and esophagitis. typical clinical laboratory findings include an increased packed cell volume and total plasma protein reflective of volume depletion, a metabolic acidosis (with elevated anion gap) in longstanding or severe cases, an increased peritoneal fluid protein concentration (often > . g/dl), and a mild to moderate elevation of the peritoneal white blood cell count, although the count usually is less than , cells per microliter. , the peritoneal fluid is usually yellow and turbid, but in severe cases diapedesis occurs resulting in a serosanguinous color. the white blood cell count in the peripheral blood may be normal, decreased, or increased. in addition, hyponatremia, hypochloremia, hypokalemia, and acid-base alterations (elevated anion gap) are often evident. the loss of enteric bicarbonate through evacuation of enterogastric reflux and poor tissue perfusion from hypovolemia can lead to metabolic acidosis. one makes a definitive diagnosis of dpj in most cases by gross examination of the duodenum and proximal jejunum at surgery or at necropsy. some equine practitioners have observed an apparent geographic relationship in the incidence and severity of the syndrome, with more cases occurring in the southeastern united states. horses with dpj appear to share a common characteristic clinical presentation, and the mechanisms leading to electrolyte imbalances, fluid loss, ileus, and endotoxemia and septicemia are similar. treatment regimens are supportive and aim at plasma volume replacement (usually in the form of crystalloid fluid replacement), analgesia and antiinflammatory therapy, gastric decompression, antiendotoxin therapy, antimicrobial therapy if indicated, nutritional support, and nursing care. one should institute aggressive intravenous polyionic fluid therapy immediately in a horse with dpj. one should calculate the total fluid deficit based on clinical assessment of dehydration (e.g., for % or moderate dehydration, . × kg body mass = l) and should administer replacement fluids rapidly (up to to l per hour per -kg adult horse). administering intravenous hypertonic saline ( %) may be useful to treat hypovolemic shock in horses with severe circulatory shock. the use of to l of hypertonic saline ( % nacl) improved systemic blood pressure and cardiac output in horses with hemorrhagic shock and in a model of equine endotoxemia. if one chooses this treatment option, intravenous administration of replacement isotonic fluids must follow immediately to maintain tissue integrity. one should not allow horses with significant volumes of gastric reflux to ingest foodstuffs or liquids orally. once one has administered replacement fluids and the horse is well hydrated, one should administer maintenance fluid amounts, which may be as high as ml/kg/day. unfortunately, the intravenous fluid therapy itself may accelerate the flux of fluid from the vasculature into the intestinal lumen because of a reduction in intravascular oncotic pressure and an increased capillary perfusion pressure, which can result in an increased volume of gastrointestinal reflux. however, the veterinarian should not consider reducing the volume of intravenous fluid therapy because excessive fluid losses continue to occur. one should monitor plasma protein concentration, overall hydration, and the volume of reflux and then determine the rate of intravenous fluid administration. during the initial hours of therapy, even aggressive intravenous fluid administration results in only moderate clinical improvement. the clinical response, as evidenced by improved hydration status, decreased nasogastric reflux, improved attitude, and improvement in values reflecting kidney function (decreased blood urea nitrogen and creatinine), correlates with improvement of intestinal damage. horses with dpj that continue to reflux large volumes of enterogastric fluid frequently for more than to hours most likely will experience protein loss from the inflamed and disrupted intestinal mucosal barrier and from systemic protein catabolism. decreased colloid oncotic pressure leads to decreased effective circulating fluid volume and edema. total plasma protein may decline to below g/dl and the albumin may decrease to below . g/dl. fresh or thawed frozen plasma is ideal for replacement of functional proteins. one should consider treatment with intravenous plasma therapy or a combination of plasma and synthetic colloid (e.g., synthetic amylopectin) as soon as one sees evidence of a consistent decline in total plasma protein or albumin (< . g/dl) or if the horse is developing dependent edema. fresh plasma (preferred) or fresh frozen plasma is the treatment of choice if coagulation disorders accompany protein loss. an average-size horse ( kg) requires to l of plasma (albumin . g/dl) or synthetic colloid to improve plasma oncotic pressure. administration of additional aliquots of to l of a balanced colloidal solution may be necessary if the dpj crisis continues. in addition to albumin (the major colloid component), plasma contains other components that provide overall systemic support (e.g., fibronectins, complement inhibitors, elastase and proteinase inhibitors, antithrombin iii). one may administer a % solution of hydroxyethyl starch (hetastarch ( %), abbott laboratories, north chicago, illinois), a synthetic colloid, at to ml/kg. because of the large size of the starch molecules, this solution is an effective plasma volume expander, resulting in sustained dosedependent decreases in packed cell volume and plasma protein concentration with increased oncotic pressure. the cost of an appropriate amount of commercial plasma or synthetic colloid solution for treatment of adult horses with dpj may be prohibitive but can be life-saving. horses with enteritis frequently absorb large amounts of endotoxin from the disrupted intestinal mucosal barrier, therefore putting these horses at a high risk for laminitis. one should monitor digital pulses every to hours until systemic signs of enteritis have abated (fever, leukopenia, etc.). treatment to combat endotoxemia is critical, and several therapeutic approaches are available. choice of treatment options is based on severity of disease, renal function, hydration status, and economics. the reader is referred to chapter . for a thorough discussion of endotoxemia pathophysiology, treatment, and prevention. nonsteroidal antiinflammatory drugs are the most frequently used group of drugs for treatment of abdominal pain in horses (flunixin meglumine . mg/kg intravenously every hours or phenylbutazone . mg/kg orally or intravenously every hours). the clinician must weigh the benefit of the analgesic effect of nonsteroidal antiinflammatory drugs with the possibility of further damage to the intestine by potentially blocking the protective effects of intestinal mucosal prostaglandins. one should consider other classes of drugs for treating colic associated with dpj. butorphanol (torbugesic; an opioid analgesic) at . to . mg/kg with detomidine (dormosedan; an α-agonist) at . to . mg/kg given intramuscularly every to hours is a useful combination that has minimal effects on gastrointestinal motility. because clostridium spp. are suspected as a causative agent of dpj, penicillin often is administered to affected horses. however, one should consider broad-spectrum antimicrobial coverage for horses with dpj. one can add an aminoglycoside (gentamicin, amikacin) or thirdgeneration cephalosporin (ceftiofur [naxcel], upjohn co., kalamazoo, michigan) to the penicillin therapy, keeping in mind the potential adverse effects of these drugs on renal function. effective antisecretory medications targeting the equine small intestine have not been identified. one should consider the nutritional needs of horses with dpj. most horses have a total body protein loss from cachexia and a protein-losing enteropathy. total parenteral nutrition may be indicated in horses that remain anorectic for more than to days. parenterally administered solutions containing glucose, balanced amino acid solutions, lipid emulsions, balanced electrolyte and trace minerals, and vitamins have been administered to adult horses with small intestinal ileus or enterocolitis. based on a small number of horses, this therapy has proved promising in terms of minimizing protein losses and decreasing the duration of illness. providing for part of the nutritional requirements of the horse ( to , kcal/day) is possible with glucose-amino acid solutions, which are of moderate cost. one may suppose reasonably that providing nutritional support to an anorectic, severely ill horse will facilitate the healing process and even shorten the duration of illness. thus the overall cost of providing parenteral nutritional supplementation to horses with dpj may well be offset by quicker recovery and diminished requirements for other, expensive treatments. normal (healthy) intestine is necessary for optimum performance of prokinetic agents in horses. many motilitymodifying agents likely are ineffective in cases of dpj. however, some benefit may come of the judicious use of prokinetic agents in inflammatory conditions of the equine intestine, particularly if the agent provides additional effects such as analgesia. for example, lidocaine infusion has several actions that may be beneficial in the treatment of ileus, including suppression of primary afferent firing, antiinflammatory properties, an observed analgesic effect, and direct stimulation of smooth muscle. an infusion dose of to mg/min over to hours has been recommended. the reader is referred to chapters . and . for a complete description of motility modifying agents. medical therapy is sufficient in most cases of dpj, but in those cases in which the horse continues to produce copious enterogastric reflux, one may consider surgery as an option. refractory cases have been observed to improve with surgical intervention; however, some horses with refractory dpj have been observed to recover with supportive medical care alone even after days of refluxing large amounts of fluid every to hours (personal observation). the decision of when to intervene surgically often is difficult. one may elect surgery to determine the extent of gross pathologic condition and intestinal distention and to perform intestinal bypass so as to direct enterogastric reflux toward the cecum and colon, where the fluid can be reabsorbed. allen and clark have described two approaches for surgical therapy in such cases. a standing right flank laparotomy with resection of the last rib has been used to approach the duodenum and cecal base. using this approach, one makes a small stoma between the duodenum and cecum using a handsewn . -to . -cm side-to-side anastomosis. the stoma may act as a shunt to decompress the proximal small intestine and deliver the small intestinal fluid to the cecum for reabsorption. following recovery, the stoma likely will close. when a veterinarian is confronted with a horse exhibiting abdominal discomfort, with small intestinal distention palpable per rectum, and greater than l of gastric reflux, the veterinarian should recommend referral of the horse to a facility capable of performing abdominal surgery. the chance that such a horse has an intestinal obstruction is too great to decide to treat it as if it may have dpj. surgery on such horses is not unusual, even though dpj is possible, to rule out an obstruction. at present, the survival of horses with dpj that undergo surgery is much greater than previously described, and certainly greater than that of horses with small intestinal obstruction that do not have surgery. horses with dpj that receive appropriate therapy have a reasonably good chance of making a full recovery. horses that continue to have frequent episodes of voluminous nasogastric reflux and systemic signs of endotoxemia and septicemia have a poorer prognosis for recovery. frequent complications of dpj include laminitis, thrombophlebitis, and weight loss. the clinical signs of chronic wasting and poor body condition, although nonspecific for a diagnosis of malabsorption antemortem, can be attributed to proliferative or inflammatory intestinal disorders, often collectively referred to as chronic inflammatory bowel diseases. clinical signs include alimentary lymphosarcoma, granulomatous enteritis, multisystemic eosinophilic epitheliotropic disease (meed), and lymphocyticplasmacytic enterocolitis-conditions affecting young and adult horses. proliferative enterocolitis, a transmissible disease of foals to months of age characterized by significant small intestinal pathologic changes, will be included in this group. however, several other primarily small intestinal conditions described from a morphologic perspective, such as chronic postinfarctive inflammation and mycobacterial infections, will not be discussed. in addition, a single case of aa amyloid-associated gastroenteropathy in an -year-old morgan stallion that had evidence of severe malabsorption based on poor d-xylose absorption is included. for comparative purposes, table . - lists the clinical and clinicopathologic features of the diseases, and tables . - and . - present the gross morphologic and histopathologic findings, respectively. the extent of small intestinal disease is the key to determine whether one can demonstrate malabsorption based on abnormal carbohydrate absorption. as described in chapter . , this is not an all-or-nothing situation. in the same animal the staging of the pathologic changes differs in different regions of the small and large intestines, thus influencing severity of clinical signs and absorption findings. furthermore, the extent of pathologic changes in different animals with ultimately the same morphologic diagnosis affects absorption studies and progress of the disease. early diagnosis remains a challenge, and even multiple intestinal biopsies taken at exploratory laparotomy may prove unhelpful. by contrast, intestinal infiltration with the predominant cell types can be found in grossly normal appearing intestinal tissue. alimentary lymphosarcoma of the horse may represent a primary neoplasia of the gut associated lymphoid tissue with significant cellular infiltration of the small intestine and associated lymph nodes with minimal large intestinal or systemic involvement. case series and pathology reports indicate that young horses to years of age primarily are affected, although the age range can be broad. [ ] [ ] [ ] no breed or sex predilection exists. prevalence is unknown. despite the progressive nature of lymphomata, onset of clinical signs can be rapid and the animal may become acutely ill. as with all adult cases of chronic inflammatory bowel disease, antemortem diagnosis is by a process of exclusion and usually is confirmed post mortem. frequently, the horse has anemia, thrombocytopenia, neutrophilia or neutropenia, hypoalbuminemia, normal serum protein or hyperproteinemia, and hypergammaglobulinemia. lymphocytosis is rare. one may palpate intraabdominal masses, mainly enlarged mesenteric lymph nodes, rectally. abdominocentesis has been of diagnostic value. carbohydrate absorption tests usually reveal partial to total malabsorption indicative of the severely reduced surface area resulting from significant villous atrophy and the extensive mucosal or transmural infiltration. rectal biopsy has aided diagnosis. early confirmation of a suspected diagnosis necessitates exploratory laparotomy to obtain multiple intestinal and lymph node biopsies. in the future, markers of cancer cells may become available and may be cost-effective to aid diagnosis. prognosis is poor. natural progress of the disease is unknown. most horses are presented in an advanced state of disease. immunosuppressive drugs or chemotherapy may afford temporary improvement. however, outcome is unaffected. the chronic wasting condition granulomatous enteritis was first described in ; of horses were young standardbreds. most affected horses are to years of age. case reports from many countries revealed a predominance of standardbred over thoroughbred horses by three to one. , some of the standardbreds were related, implicating a genetic predisposition. prevalence is low. the condition is sporadic and has an insidious onset, and the course can be protracted. significant diagnostic features include anemia, slight increases or decreases in white blood cell counts, hypoalbuminemia, normal serum protein or hypoproteinemia, occasional increases in serum alkaline phosphatase activity, normal serum γ-glutamyltransferase activity, and enlarged mesenteric lymph nodes on rectal palpation. reduced carbohydrate absorption to the level of partial to total malabsorption is reported frequently, consistent with the severe morphologic changes throughout the small intestine. one can attribute the low proportion of horses exhibiting diarrhea , to the preferential distribution of inflammatory infiltration in the small intestine, with lesser involvement of the large intestine. rectal biopsy can be a useful aid to diagnosis. treatment of horses with granulomatous enteritis with a variety of drugs, particularly corticosteroids, has not affected the outcome except in the short term. one successful response has been reported. prolonged corticosteroid administration produced clinical remission in a -year-old standardbred gelding based on improvement in clinical signs and in d-xylose absorption. five months after cessation of approximately months therapy, d-xylose absorption was normal and the horse was bright, alert, and resumed a level of athletic performance. parenteral administration of dexamethasone sodium phosphate was tapered to achieve a minimal effective dose to reduce intestinal inflammation and abolish clinical signs. adverse effects were not reported. the outcome of this single case is encouraging. surgery may be indicated if the disease is localized. two young horses underwent resection of the thickened terminal small intestine to confirm a diagnosis and provide a means of treatment; one horse died months after surgery, and the other has remained clinically normal for at least years. the cause of granulomatous enteritis is unknown. several infectious agents have been implicated, including mycobacterium avium. the condition may represent a granulomatous hypersensitivity reaction. immunemediated responses to dietary, parasitic, or bacterial antigens may be important initiating factors. recently, six cases purported to represent granulomatous enteritis were linked to environmental contamination with aluminum. although the case definition was flawed and problems existed with the data and interpretation, the report nevertheless raised the possibility that a toxicologic basis may exist for some equine inflammatory bowel disorders. lumen from parasitic, bacterial, or dietary sources. infectious agents have not been identified. , widespread use of the avermectins has tended to reduce parasite loads and composition to favor small strongyles (cyathostomes). eosinophilia is a feature of parasitism in the equine intestinal tract, although nematodes rarely have been identified in any lesions of meed. , however, failure to detect larval structures in these lesions may be attributable to chronicity of the disease and destruction of the parasites in tissue. biopsies of the rectal mucosa or of the skin, liver, intestinal tract, and lymph nodes may assist in diagnosis. treatment has been attempted with a variety of drugs, including antibiotics, corticosteroids, and anthelmintics with larvicidal activity. immediate improvement has not been borne out in the long term. prognosis is poor. the clinical objective is to reach a tentative diagnosis early in the course of the disease for intervention to be more than transient. unlike the other conditions (see table . - ), meed has definitive liver and pancreatic involvement, and thus maldigestion may make a significant contribution to the wasting disease. for example, the lowered albumin and protein could result in part from impaired pancreatic enzyme digestion, and the effects of inflammatory lesions in the liver and ileum may decrease bile salt concentrations. the morphologic findings in lymphocytic-plasmacytic enterocolitis reflect the predominant infiltrative cellular elements of this rarely encountered condition. a retrospective study of horses provided the information presented in the tables. no specific clinical or clinicopathologic features differentiate this condition antemortem from other inflammatory diseases of adult horses. carbohydrate absorption was abnormal or delayed in of horses, consistent with the predominance of small intestinal pathologic changes. rectal biopsies were abnormal in of horses, two of which were reported as having lymphocytic-plasmacytic proctitis. prognosis is poor. treatment has been unsuccessful, probably because of the advanced nature of the condition at the beginning of treatment. proliferative enteropathy has not been associated with abnormal carbohydrate absorption based on three horses subjected to carbohydrate absorption tests. however, the florid mucosal lesions in the jejunum and ileum undoubtedly contribute to impaired digestive function and potential malabsorption of vitamins, minerals, and amino acids in the distal small intestine. the condition meed encompasses disorders characterized by a predominant eosinophilic infiltrate in the gastrointestinal tract, associated lymph nodes, liver, pancreas, skin, and other structures and accompanied by some degree of malabsorption and enteric protein loss. the disorders include chronic eosinophilic gastroenteritis, eosinophilic granulomatosis, chronic eosinophilic dermatitis, and probably basophilic enterocolitis. the condition differs from idiopathic eosinophilic enterocolitis, in which segmental lesions in the small or large intestine induce signs of colic requiring surgical intervention , without evidence of malabsorption or multisystem involvement. although prevalence is low, meed appears to be more common than granulomatous enteritis based on the accumulated published reports and personal experience in australia and the united states. most affected horses are to years of age, and standardbreds and thoroughbred are reported to predominate. the condition is sporadic and has an insidious onset, and the course is protracted with a duration of to months. diarrhea is common in contrast to granulomatous enteritis. severe skin lesions with exudative dermatitis and ulcerative coronitis are prominent and frequently are the principal reason for veterinary attention being sought. despite extensive tissue eosinophilia, systemic eosinophilia is rare. hematologic values are usually unremarkable. notable features include hypoalbuminemia and normal serum protein or hypoproteinemia, and because of liver involvement, serum γ-glutamyltransferase and alkaline phosphatase activities may be increased. most reports of carbohydrate absorption test findings (glucose or d-xylose) indicate retarded absorption and a reduced or normal peak concentration delayed to at least minutes. one can interpret this pattern as the existence of sufficient small intestinal absorptive capacity to enable moderate absorption with possibly delayed gastric emptying or ileocecal ejection. morphologic changes are less pronounced in the small intestine than in the large intestine, and small intestinal lesions predominate segmentally in the proximal duodenum and distal ileum. furthermore, significant hyperkeratosis of the fundic region may contribute to gastric muscle contractile disruption. diarrhea can be a consequence of the severe segmental or multifocal granulomatous lesions in the large intestine with mucosal and transmural thickening and extensive ulceration. abundant fibrosis is a feature of all affected tissues (see table . - ). the cause of meed is unknown and could represent a chronic ongoing immediate hypersensitivity reaction against undefined antigens ingested or excreted into the affects foals to months of age, particularly those that have been weaned recently. the disease is caused by lawsonia intracellulare, an obligate intracellular bacterium found in the cytoplasm of proliferative crypt epithelial cells of the intestine. the condition in a foal was described first as intestinal adenomatosis, because of similarity to the swine disorder of the same name. later, molecular studies showed that intestines from an affected foal contained l. intracellulare sequences as determined by polymerase chain reaction analysis and confirmed by southern blot hybridization. recently, studies of a cluster of affected foals on three breeding farms in canada provided much information on the clinical syndrome, laboratory investigations, and response to treatment. two of the three farms bred arabians, hence a demographic predominance of arabian foals exists. clinical signs included depression, rapid and significant weight loss, edema, diarrhea, and colic. poor body condition, a rough hair coat, and potbelly appearance were common findings. other problems often were concurrent, including respiratory tract infection, dermatitis, intestinal parasitism, and gastric ulceration. significant laboratory findings were anemia, transient leucocytosis, hypoalbuminemia, hypoproteinemia, and elevated serum creatine kinase concentrations. diagnosis was confirmed by identifying characteristic intracellular bacteria within the apical cytoplasm of proliferating crypt epithelial cells using silver stains and by results of polymerase chain reaction analysis and immunohistochemical testing. antemortem diagnosis relied on clinical signs, hypoproteinemia, and exclusion of common enteric infections. one can confirm diagnosis in live animals by fecal polymerase chain reaction analysis (positive in of foals tested) and serologic testing; foals with proliferative enteropathy were evaluated serologically and had antibodies against lawsonia intracellulare. treatment is effective. most foals received erythromycin estolate ( to mg/kg per os every to hours), alone or with rifampin ( to mg/kg per os every hours) for to weeks. foals frequently needed supportive therapy at the outset for stabilization. response to therapy has been excellent. rapid improvement in clinical signs even within hours preceded the rise in plasma protein concentration. the source of the infection was undetermined. no apparent link existed between the three farms and a swine operation or solid and liquid waste disposal on pasture. however, one cannot exclude airborne spread of dried fecal material over distances. comparisons of epidemiologic findings from the swine disease indicated that overcrowding, feed changes, antibiotic usage, and mixing and transportation were potential risk factors at two of the farms. recent weaning appeared to be a key element in the pathogenesis. samuel l. jones acute diarrhea caused by colitis in adult or young horses is a potentially life-threatening disorder of a variety of causes (table . - ) characterized by hypersecretion of fluid, motility disturbances, altered microbial flora in the colon, and an impaired mucosal barrier caused by direct injury or inflammation. many of the clinical and clinicopathologic features are similar regardless of the cause. severe dehydration with profound electrolyte abnormalities is common, as is systemic inflammation from absorption of endotoxin or other bacterial products through the compromised mucosa. gastrointestinal protein loss may result in reduced colloid oncotic pressure from hypoproteinemia, leading to tissue edema. colitis is a highly catabolic disorder, and weight loss may be rapid and severe. some cases of colitis may be complicated by extensive mucosal ulceration, serosal inflammation, or mural ischemia/infarction extending from the inflammation or resulting from coagulopathies. thus diagnostic measures aimed at determining the cause necessarily must be accompanied by clinical and laboratory assessment of hydration, electrolyte and acid-base balance, plasma protein concentration and colloid oncotic pressure, organ function, and evaluation of the degree of systemic inflammation and of the integrity of the intestinal wall. although therapeutic strategies are similar for many causes of colitis, consisting primarily of control of local and systemic inflammation, maintenance of fluid and electrolyte balance, promotion of tissue perfusion, replacement of plasma protein, preservation of colloid oncotic pressure, promotion of mucosal repair, restoration of the microbial ecology of the colon, and nutritional management, some causes of acute colitis have specific therapies aimed at eliminating the cause. a variety of infectious organisms has been identified as causes of acute colitis in adult horses. the clinical syndromes associated with these infections are indistinguishable in most horses. however, appropriate diagnostic tests including fecal bacterial culture, fecal bacterial toxin analysis, pcr, and/or serology may identify specific infectious organisms. salmonella is a genus of gram-negative facultatively anaerobic bacteria that are common gastrointestinal pathogens in horses. many serotypes of salmonella have been reported to infect horses, but those classified in group b appear to be associated more commonly with disease than those in other groups. group b includes s. typhimurium and s. agona, two of the species most frequently isolated from horses. - s. typhimurium is the most pathogenic serotype in horses and is associated with a higher case fatality rate than other species of salmonella. the number of horses that are infected inapparently with and actively shed salmonella in their feces has been reported to be as high as % to %, but actual prevalence of salmonella-shedding in the general horse population is likely to be much lower, less than % to %. horses shedding salmonellae are a potential source of infection to susceptible horses, , as are environmental reservoirs. [ ] [ ] [ ] for these reasons, salmonellosis is one of the most common nosocomial diseases in horses. nosocomial salmonellosis significantly affects morbidity and mortality in hospitalized horses. the emergence of multidrug resistance in equine salmonella isolates has been a cause of concern because of the importance of salmonellosis as a nosocomial disease and because salmonella represents a significant zoonotic pathogen. , [ ] [ ] [ ] the virulence of the bacteria varies tremendously with serotype and even among strains of the same serotype in part because of the important role of host susceptibility in the pathogenicity of particular organisms. the infective dose is generally millions of organisms inoculated orally, but various environmental and host factors can reduce the infective dose to a few thousand or even hundreds of organisms. [ ] [ ] [ ] environmental factors or stresses that increase susceptibility to salmonella infection are not well defined, but high ambient temperature, for example, is known to increase the prevalence of salmonellosis in horses greatly. indeed, the peak incidence of salmonellosis in horses occurs in late summer and fall. , , other environmental and host factors that increase the risk of salmonella infection include transportation, antibiotic administration, gastrointestinal surgery, general anesthesia, preexisting gastrointestinal disease, change in diet, and immunosuppression. , , host factors that restrict gastrointestinal colonization and invasion by pathogens include gastric ph, commensal gastrointestinal flora, gastrointestinal motility, the mucosal barrier and mucosal immunity. , gastric acidity is an important defense mechanism preventing live organisms from reaching the intestine. altering the gastric ph with histamine receptor antagonists, for example, may increase susceptibility to infection. gastrointestinal flora inhibits the proliferation and colonization of salmonella by secreting bacteriocins, short-chain fatty acids, and other substances that are toxic to salmonella. in addition, elements of the normal flora compete for nutrients and space, especially on the mucosa. being predominantly anaerobic, the normal flora maintain a low oxidationreduction potential in the environment of the large intestine, which inhibits the growth of many bacterial pathogens. the importance of normal host gastrointestinal ecology is illustrated by the fact that disturbances of the colonic flora with antibiotics, changes in feed, ileus, or other underlying gastrointestinal disease greatly increase the susceptibility of the host to infection by salmonella, often resulting in serious disease. the immune status of the host may be one of the most important factors determining not only the susceptibility to salmonella infections but also the degree of invasion and subsequent outcome of the infection. local immunity, such as mucosal antibody secretion and enterocyte-derived cationic peptides, prevents colonization of the mucosa. , , opsonizing antibodies and activation of the complement cascade are important in fighting systemic invasion by salmonella by increasing the efficiency of phagocytosis and by direct bactericidal activity. humoral immunity, however, is often ineffective in preventing disease and dissemination once invasion occurs and salmonella has established in its intracellular niche. following invasion, salmonella is capable of surviving and multiplying within macrophages, rendering humoral (noncellular) immune systems ineffective. , specific cellular immunity may be the most effective defense mechanism in the host arsenal against dissemination and systemic infection by salmonella. , oral inoculation with small numbers of virulent organisms may induce protective immunity in horses and calves, but the duration of the immunity is not known. , oral and parenteral vaccines using killed or attenuated organisms and bacterial products have been promising but are effective only against homologous organisms and are usually not cross-protective among different serogroups. [ ] [ ] [ ] in adult horses, salmonella primarily infects the cecum and proximal colon, causing enterocolitis, and the ability to disseminate beyond the intestine and cause enteric fever is limited. in foals, however, salmonellosis often is associated with septicemia. the ability of salmonella to cause enterocolitis depends on the ability of the bacteria to invade the gastrointestinal mucosa. , invasion of the gastrointestinal mucosa occurs preferentially through specialized enterocytes called m cells that overlay intestinal lymphoid tissues such as peyer's patches in nonequine species. a variety of enteric pathogens exploit m cells during infection of intestinal tissue. invasion of the epithelium occurs by self-induced uptake via the apical membrane of the m cell, often killing the cell in the process. salmonellae then invade neighboring cells via the basolateral membrane, eventually spreading the destruction of the epithelium beyond the principle area of attack. virulent salmonellae have a well-developed invasion mechanism involving generation of an apparatus called a type iii secretory system that enables virulence gene products to be injected directly into enterocytes. virulence proteins injected by salmonellae into enterocytes engage the cellular machinery and induce the cell to engulf the bacteria by macropinocytosis. salmonella virulence gene products also induce enterocyte chloride and fluid secretion and upregulate enterocyte transcription of inflammatory cytokines (tumor necrosis factor α and interleukin- β) and chemokines that trigger a mucosal inflammatory response. , , after salmonellae invade the mucosa, they are phagocytosed quickly by macrophages and dendritic cells in the lamina propria and lymphoid tissues. the ability of salmonellae to disseminate systemically and cause enteric fever is associated with the ability to survive and proliferate in macrophages. indeed, phagocytes have an important role in dissemination of the pathogen to blood, lymph nodes, liver, and spleen. most salmonellae in the blood and tissues of infected animals that are competent to cause enteric fever are within phagocytic cells. in adult horses with salmonellosis, dissemination appears to be limited to the intestine and mesenteric lymph nodes, and salmonella rarely is cultured from blood. however, in foals and in some adults, salmonella causes an enteric feverlike disease with dissemination to mesenteric lymph nodes, liver, spleen, and blood. salmonella organisms require specific virulence gene clusters encoded on the chromosome or on plasmids for intracellular survival in macrophages. some of these genes are sensors that signal the bacteria that it has entered an intracellular environment and turn on genes required for intracellular survival. others, like invasion genes, are transported from the bacteria and injected into macrophage cytosol by a type iii secretory system to prevent phagosome/lysosome fusion and subvert other essential macrophage killing mechanisms. salmonellae also possess multiple genes that confer resistance to reactive oxygen and nitrogen metabolites, perhaps the most lethal antimicrobial mechanisms of macrophages. diarrhea associated with salmonellosis has multiple causes. a salmonella cytotoxin inhibits protein synthesis in mucosal cells, causing morphologic damage and altered permeability. virulent salmonellae also produce an enterotoxin similar to the heat-labile toxin (lt) produced by escherichia coli. , the enterotoxin contributes to but is not required in the pathogenesis of diarrhea. , salmonella enterotoxin increases secretion of chloride and water by colonic mucosal cells in many species, including horses, by increasing intracellular cyclic adenosine monophosphate concentrations. the ability of virulent salmonellae to cause diarrhea appears to be associated most closely with the ability to invade enterocytes and to trigger an inflammatory reaction in the intestinal tissue. , gene products injected into enterocyte cytosol by the type iii secretory system of invading salmonellae stimulate chloride and fluid secretion. salmonella invasion of enterocytes is also a potent activator of inflammatory chemokine and cytokine production, resulting in the recruitment of leukocytes, particularly neutrophils, and activation of resident macrophages and mast cells. products of these activated leukocytes, including prostaglandins, leukotrienes, reactive oxygen metabolites, and histamine, are potent stimulators of chloride secretion in the colon of many species. , [ ] [ ] [ ] the enteric nervous system integrates the diverse processes of pathogen recognition, triggering of the inflammatory response, and induction of enterocyte fluid secretion. many of the inflammatory mediators studied stimulate colonic secretion by prostaglandin-dependent mechanisms, resulting in increased intracellular cyclic adenosine monophosphate or calcium concentrations or both in mucosal cells. in addition, these mediators and the enteric nervous system may stimulate secretion by prostaglandin-independent mechanisms, inhibit sodium and water absorption, cause motility disturbances, and potentiate tissue injury, all of which enhance the pathogenicity and dissemination of salmonella and contribute to the pathogenesis of diarrhea. , neutrophils recruited to the mucosa by signals generated by the infected enterocytes physically contribute to mucosal injury by producing a variety of products that are lethal to pathogens but are also toxic to host cells. , moreover, neutrophils attracted to infected epithelial cells accumulate beneath the monolayer, lifting it off the basement membrane in sheets. neutrophils also migrate across the epithelial monolayer in potentially massive numbers. transepithelial migration of neutrophils increases the permeability to macromolecules, bacterial products, and even bacteria. potentially massive losses of electrolytes, water, and protein can occur depending on bacterial and host factors. perhaps most devastatingly, mucosal injury and altered permeability allow systemic absorption of bacterial products and dissemination of bacteria, resulting in systemic inflammatory responses such as occur with endotoxemia and septicemia. four syndromes of salmonella infection have been described clinically and reproduced experimentally in horses: ( ) inapparent infections with latent or active carrier states; ( ) depression, fever, anorexia, and neutropenia without diarrhea or colic; ( ) fulminant or peracute enterocolitis with diarrhea; and ( ) septicemia (enteric fever) with or without diarrhea. inapparent infections can be activated to clinical disease in compromised horses, such as horses with colic or horses being treated with antibiotics, causing mild to severe enterocolitis. in addition, latent infections (nonshedding) can become active infections (shedding) under certain conditions, such as transportation stress and antibiotic treatment. horses with depression, anorexia, fever, and neutropenia without diarrhea generally have a good prognosis and recover in several days without specific treatment. the septicemic form is restricted mostly to neonatal foals and is uncommon in adult horses. this discussion focuses on acute enterocolitis. acute enterocolitis is characterized by severe fibrinonecrotic typhlocolitis, with interstitial edema and variable degrees of intramural vascular thrombosis that may progress to infarction. severe ulceration of the large intestinal mucosa may occur with serosal ecchymoses and congestion. the earliest signs of enterocolitis are usually fever and anorexia. , signs of colic may be apparent early in the course of the disease, especially if ileus is present. clinical signs of endotoxemia are common and range from fever, elevated heart and respiratory rates, poor peripheral perfusion, and ileus to fulminant and rapidly progressive signs of endotoxemic shock. oral mucous membranes are often pale with perigingival hyperemia (a toxic rim) but may be brick red or cyanotic, with prolonged capillary refill time. one may note weakness, muscle fasciculations, cold extremities, and other signs suggestive of hypotensive shock; synchronous diaphragmatic flutter; abdominal pain; and significant metabolic and electrolyte abnormalities in severe cases of enterocolitis. one also may note signs of mild dehydration before diarrhea is apparent. once diarrhea is evident, dehydration may become severe rapidly. occasionally, horses die peracutely, without developing diarrhea. diarrhea may not be apparent for several days but usually occurs by to hours after the fever begins. , the duration of the diarrhea may be days to weeks. the character of the first diarrheal feces is usually watery with particles of roughage but may become fluid rapidly without solid material. finding frank blood and fibrin in the feces is unusual. the volume of feces is often large, with frequent defecation. one may note straining or signs of colic when the patient is defecating, and rectal prolapse may occur occasionally. persistent straining and rectal prolapse may be a sign of colonic infarction. abdominal borborygmi are often absent early in the course of the disease because of ileus but become evident later, usually when diarrhea begins. fluid and gas sounds are commonly audible, but normal progressive motility is less frequently audible than normally. transrectal palpation may reveal edematous rectal mucosa and colon and fluid-filled colon and cecum. one may obtain gastric reflux, especially early in the course when ileus is evident. hematologic abnormalities early in the course of the disease include moderate to severe neutropenia, lymphopenia, and leukopenia, a mild to moderate left shift, and toxic changes in the neutrophils. , thrombocytopenia, moderate to severe hemoconcentration, and hyperfibrinogenemia are also common. neutropenia is an early but nonspecific indicator of salmonellosis, often occurring concurrently with the onset of fever. later in the course of disease, one may see neutrophilic leukocytosis, indicating recovery. a degenerative left shift, with metamyelocytes and myelocytes in the peripheral blood, is a poor prognostic sign. serum biochemical analysis may reveal azotemia, elevations in serum sorbitol dehydrogenase and γglutamine aminotransferase activity, and elevated serum lactic acid concentration. azotemia is often prerenal, but acute hemodynamic renal failure may occur in severely dehydrated, endotoxemic, or septicemic patients. indeed, elevation of creatinine concentration is a poor prognostic indicator in horses with acute colitis. hemodynamic renal disease may be complicated by toxic injury caused by administration of nephrotoxic drugs. hyponatremia may also contribute to prerenal azotemia. elevations in hepatocellular enzymes are usually mild and reflect damage to the hepatocytes from absorbed toxins such as endotoxin and from poor perfusion caused by hypotensive shock or dehydration. lactic acidemia may be present, reflecting poor tissue perfusion. plasma protein rapidly drops as protein is lost in the gastrointestinal tract, causing moderate to severe hypoalbuminemia and hypoglobulinemia. peripheral or organ edema (vascular leak syndrome) may occur if hypoproteinemia is severe, coupled with increases in endothelial permeability induced by systemic inflammation. hypokalemia, hyponatremia, hypochloridemia, and hypocalcemia are common electrolyte abnormalities in patients with enterocolitis. metabolic acidosis also may be present. coagulopathies, such as decreased antithrombin iii activity and disseminated intravascular coagulation, may occur. urinalysis may reveal isosthenuria, proteinuria, hematuria, cylindruria, and glucosuria if hemodynamic or toxic renal injury is present. the number of leukocytes in the feces usually is elevated, and occult blood may be detectable. peritoneal fluid is usually normal except when severe mural inflammation or colonic infarction occurs. routine detection of salmonellae in feces involves five daily cultures of large samples ( to g) of feces using enrichment techniques. , , however, the sensitivity of fecal culture can be as low as % to %, even if one cultures several fecal samples collected daily. concurrent culture of rectal biopsy specimens and feces increases the sensitivity of culture techniques to % to %. currently, the polymerase chain reaction (pcr) is the most sensitive and rapid test for detecting salmonella in feces. a single pcr test applied early in the course of disease is a more sensitive test for the presence of salmonella than repeated fecal cultures. , detection of salmonellae in feces does not prove a diagnosis of salmonellosis, but the positive predictive value of a positive pcr or culture results is high in horses with compatible clinical signs. culture of peripheral blood may allow isolation of the organism if bacteremia or septicemia is present, but blood cultures are not a sensitive test for salmonellosis in adult horses. although foals are more likely than adults to become septicemic, culture of blood is recommended in all cases with signs suggestive of septicemia. increased numbers of fecal leukocytes suggest an invasive process in the colon but are not specific for salmonellosis. early in the course of the disease, dehydration, electrolyte and acid-base imbalances, endotoxemia, and sepsis may be life threatening. aggressive treatment during the acute stages to replace fluids lost in the diarrhea and to control sepsis and endotoxemia is often effective in controlling the primary disease. weight loss and hypoproteinemia are often severe. complications such as multiorgan dysfunction, vascular leak syndrome with peripheral and organ edema, laminitis, acute renal failure, venous thrombosis and septic phlebitis, irreversible protein-losing enteropathy or chronic malabsorption, pulmonary aspergillosis, and gastrointestinal infarction can occur. in many instances, horses recover from acute salmonellosis with aggressive treatment, only to succumb to complications of the disease, partially explaining the high fatality rate of equine salmonellosis compared with human salmonellosis. chronic, mild to moderate diarrhea occasionally occurs in horses after a bout of severe salmonellosis, usually with protein-losing enteropathy. if the chronic diarrhea persists beyond to weeks after the onset of signs, the prognosis for recovery is poor. potomac horse fever (equine monocytic ehrlichiosis) is caused by the obligate intracellular rickettsial organism neorickettsia risticii. [ ] [ ] [ ] [ ] the disease is most common from late summer to early fall, with a peak incidence in july and august. potomac horse fever was described first in the northeast but since has been diagnosed in most areas of the continental united states, with a particularly high prevalence in the northeast and midwest. the geographic distribution is characterized by a significantly higher percentage of cases found along waterways and rivers. , the disease occurs sporadically, temporally and geographically, and can affect any age group of horses. the case fatality rate is % to %. transmission of n. risticii has been reproduced experimentally by oral, intramuscular, intradermal, subcutaneous, and intravenous routes. , however, the natural route of infection has remained a mystery until recently. the epidemiologic data, the fact that many other rickettsial diseases are transmitted by insect vectors, and the finding that the disease can be transmitted via whole blood have implicated insect vectors in the natural transmission of the organism. attempts to transmit the disease experimentally with ticks (dermacentor variablis) or biting flies (stomoxys calcitrans) have been unsuccessful. , recently, n. risticii has been found to infect virgulate cercariae larval stages of trematodes that use operculate freshwater snails (juga spp.) as part of their life cycle in northern california. infected virgulate cercariae have been identified in aquatic snails collected in other parts of the country as well. virgulate cercariae are part of the life cycle of trematodes that are common parasites of many species and use freshwater snails and aquatic insects as intermediate hosts. although the trematode species infected with n. risticii remains to be identified definitively, at least two species are considered potential vectors. during the trematode life cycle, aquatic snails release large numbers of infected cercariae into water, where they seek their next intermediate host. infected metacercaria have been identified in a variety of aquatic insects. preliminary studies suggest that n. risticii in fact may be transmitted via ingestion of mature caddis flies containing infected metacercariae. possibly horses are infected by drinking water containing infected cercaria released from snails or by ingesting infected metacercariae in other aquatic insects. the number of pcr-positive snails in endemic regions corresponds to the seasonal incidence of potomac horse fever and may be as high as %. the pathogenesis of n. risticii is not understood completely. the organism infects and survives in monocytes and monocyte-derived leukocytes and can be found in blood monocytes during natural infections, but the sequence of events resulting in enterocolitis remains speculative. the organism appears first to infect blood monocytes in experimentally infected horses, which may be the vehicle of organ infection. , however, in naturally infected horses, whether leukocytes of monocytic lineage or epithelial cells are infected first is unclear. the target organ is the gastrointestinal mucosa, with the most severe lesions found in the large intestine. , infection of human colonic cells in vitro does not cause major cytopathologic effects for several days. disruption of the microvilli in the region of the plasma membrane where sodium chloride channels are located has been observed in human colonic cell cultures. infection in horses is associated with variable degrees of morphologic damage. , mild morphologic damage and mononuclear cell infiltration of the lamina propria occur early during the infection, but fibrinous, necrotizing typhlocolitis with severe mucosal ulceration and inflammation of the lamina propria may occur later in the disease. vasculitis and intravascular coagulation are consistent features in the large intestine, with perivascular edema. one can observe n. risticii in mucosal cells and macrophages and mast cells of the lamina propria. , n. risticii can survive and multiply in macrophages by inhibiting the production of reactive oxygen intermediates and by avoiding lysosomal digestion by blocking phagosome-lysosome fusion. [ ] [ ] [ ] evidence of impaired sodium chloride absorption in the colon has been suggested to contribute to diarrhea in infected horses and may be related to destruction of the enterocyte membrane structure in the region of sodium chloride channels. , direct injury to the mucosa by n. risticii and colonic inflammation are likely to be prominent features leading to diarrhea, especially later in the disease. fluid, protein, and electrolyte loss likely is caused by mucosal injury and effects on enterocyte fluid secretion caused by the inflammatory response. like other inflammatory conditions of the colon, systemic inflammation caused by absorption of bacteria and bacterial products is a potential complication of n. risticii infections if mucosal injury is severe, which contributes to the clinical signs seen during the disease. n. risticii infection is clinically similar to other forms of enterocolitis and is characterized by anorexia, depression, and fever. , , experimental infections produce a biphasic fever occurring to days apart. decreased gastrointestinal motility, manifested as reduced borborygmi, occurs during the early stages before the onset of diarrhea. diarrhea occurs in % of cases and occurs days after the second fever episode during experimental infections. , the diarrhea can be moderate to severe and dehydrating. ileus can develop at any stage of the disease and can cause signs of moderate to severe colic. systemic signs of endotoxemia, shock, and peripheral edema may occur and are similar to those described for salmonellosis. experimental and natural infection with n. risticii can cause abortion of infected fetuses in pregnant mares. , laminitis is a complication in % to % of naturally occurring cases and is often severe. other complications include protein-losing enteropathy, thrombosis, and renal failure, as described for salmonellosis. hematologic abnormalities reflect endotoxemia, dehydration, and sepsis and are essentially identical to those described for salmonellosis. neutropenia with a left shift is a consistent feature and occurs concurrently with or soon after the onset of diarrhea. thrombocytopenia is common and often severe. neutrophilic leukocytosis occurs later in the course of the disease. hyperfibrinogenemia is usually more pronounced than that with salmonellosis. serum electrolyte, acid-base, and biochemical abnormalities are also similar to those described for salmonellosis. coagulopathies are common during n. risticii infection and reflect activation of coagulation pathways. disseminated intravascular coagulation is common and may be related to the high frequency of laminitis associated with n. risticii infection. one cannot base diagnosis of n. risticii infection solely on clinical signs because the disease is clinically similar to other forms of enterocolitis. however, in endemic areas, acute colitis is likely to be caused by n. risiticii, and thus the clinical signs of acute inflammatory colitis in fact may have a high predictive value. serologic evidence of infection, such as rising antibody titers to n. risticii detected by indirect immunofluorescence or enzymelinked immunosorbent assay in paired serum samples may be helpful in establishing a diagnosis. , one should take care when interpreting the indirect immunofluorescence serologic test for n. risticii because the test appears to have a high false-positive rate. culture of the organism from blood is possible but is difficult and is generally useful only in the research laboratory. recently developed polymerase chain pcr tests for n. risticii dna are rapid, highly sensitive (as sensitive as culture), and specific for n. risticii infection and can be applied to blood or feces. [ ] [ ] [ ] prevention prevention of the disease by reducing exposure to the causative organism is difficult because the mode of transmission is not known. a killed vaccine has been developed that is effective in preventing clinical illness other than fever in % of experimentally challenged horses using the vaccine strain. however, field studies suggest the vaccine has limited benefit for prevention or decreasing the severity of natural infection. vaccine failures have been attributed to strain differences in antigenicity or to poor antibody responses to the vaccine. , clostridiosis is an important cause of acute enterocolitis in foals and adult horses. clostridium perfringens and c. difficile are associated most commonly with intestinal clostridiosis in horses, but other clostridial species, including c. septicum, c. cadaveris, and c. sordellii also have been isolated from horses with enterocolitis. [ ] [ ] [ ] [ ] [ ] [ ] in horses of all ages, clostridial enterocolitis appears to be a common antibiotic-associated and nosocomial cause of enterocolitis. , , however, clostridiosis in neonatal foals is a distinct clinical entity and is discussed in more detail elsewhere. this chapter focuses on adult intestinal clostridiosis. clostridia are obligate anaerobic to aerotolerant spore-forming gram-positive rods that are ubiquitous in the environment in the spore form. clostridia are elements of the normal flora of horses of all ages and are among the first bacteria acquired after birth. however, clostridia inhabiting the gastrointestinal tract normally are found in low numbers and do not produce enterotoxins. clostridiosis is associated with an increase in the number of a particular species of clostridia in the gastrointestinal tract and, perhaps most importantly, exotoxin production. although the conditions resulting in exotoxin production are not understood fully, several factors increase clostridial numbers in the gastrointestinal tract. dietary factors are known to affect the numbers of clostridium species shed in the horse feces. experimental induction of colic increases fecal shedding of clostridium species in the absence of diarrhea. antibiotics, particularly administered orally or recycled via the enterohepatic system, are well documented to increase the recovery of clostridia colony-forming units (cfus) in equine feces and may result in clinical clostridiosis. , , [ ] [ ] [ ] [ ] indeed, clostridiosis associated with c. perfringens or c. difficile is likely to be the most important cause of antibiotic-induced enterocolitis in the horse. clostridium perfringens c. perfringens is made up of many genetically distinct strains of variable virulence that produce one or more of a large group of exotoxins. the pattern of exotoxin production is used to classify c. perfringens into five types, a, b, c, d, and e. c. perfringens type a is the most common clostridium isolate from healthy horses of all ages and adults and foals with diarrhea worldwide. c. perfringens types a, b, c, and d have been associated with hemorrhagic enteritis in foals less than days of age, with type c being the most common cause in north america. the primary toxin produced by c. perfringens type a is α-toxin (phospholipase c), which interferes with glucose uptake and energy production and activates arachidonic acid metabolism and signaling pathways in enterocytes. oral administration of α-toxin does not cause tissue necrosis but causes increased secretion by small intestinal mucosal cells in human beings. , the β-toxin of types b and c is a cytotoxin that causes enterocyte necrosis, ulceration, and ultimately severe intestinal inflammation and hemorrhage. a novel toxin designated β may also have a role in c. perfringens enterocolitis. the biologic activity of the β -toxin is similar to β-toxin, but β -toxin is not related to β-toxin in sequence. the β -toxin was prevalent in two groups of horses with acute enterocolitis but not in healthy horses. the β -toxin appears to be associated predominantly with c. perfringens that would have been classified otherwise as type a but that in fact may represent a previously undescribed type. virulent strains of c. perfringens type a and to a lesser extent type c also may produce enterotoxin. enterotoxin is a cytotoxin that inserts into cell membranes to form pores, which alter permeability to water and macromolecules and ultimately lead to cellular necrosis. massive desquamation of the intestinal mucosa resulting from enterotoxin cytotoxicity triggers an inflammatory response, intestinal edema, mural hemorrhage, and systemic inflammation. enterotoxin also alters tight junction integrity, resulting in increased paracellular permeability by a noncytotoxic mechanism. clostridium difficile c. difficile produces several toxins, but only two, toxin a and toxin b have been studied in detail. toxin b is a potent cytotoxin in vitro, but its role in enterocolitis is less clear than the role of toxin a. toxin b does not induce fluid secretion, inflammation, or characteristic alterations in intestinal morphology. c. difficile induces an inflammatory response with hypersecretory diarrhea that is induced in large part by the enterotoxin toxin a. toxin a induces neutrophil influx into intestinal tissue, mast cell degranulation, and secretion of prostaglandins, histamine, cytokines, and -hydroxytryptamine by these activated leukocytes. , the products of neutrophils and mast cells have a prominent role in the vasodilatory and secretory responses in the intestine during c. difficile infection. the enteric nervous system is central to the induction of intestinal inflammation and mucosal secretion by toxin a. a model for toxin a-induced secretory diarrhea has emerged in which toxin a stimulates substance p-containing afferent sensory nerve fibers, which in turn stimulate mast cell degranulation, recruitment and activation of polymorphonuclear leukocytes, and vasodilation. [ ] [ ] [ ] toxin a-induced stimulation of enterocyte secretion can occur via secretomotor neuronal stimulation by substance p-containing sensory neurons or products of mast cells and polymorphonuclear leukocytes. neural blockade or depletion of substance p abolishes mast cell degranulation, polymorphonuclear leukocyte influx, and enterocyte secretion. how toxin a triggers the sensory component of the enteric nervous system is not known yet, but toxin a-induced necrosis of enterocytes likely exposes afferent neurons to the noxious milieu of the intestinal contents. equine intestinal clostridiosis is clinically similar to other forms of acute enterocolitis in horses. , although the clinical course is usually acute, peracute colitis with rapid death may occur. occasionally, a milder, more prolonged clinical course occurs. one may note fever, anorexia, and depression before the onset of gastrointestinal signs, but more commonly no prodromal signs are apparent. signs of endotoxemia and shock may accompany acute signs of colic and severe, dehydrating diarrhea. diarrhea may not be profuse but is usually dark and foul smelling. like the clinical signs, hematologic and serum biochemical abnormalities are similar to those associated with other forms of enterocolitis and reflect fluid, protein, and electrolyte loss and systemic inflammation from endotoxemia. neutropenia, leukopenia, and hemoconcentration are common. hypoproteinemia may be profound. one often may note hyponatremia, hypokalemia, hypochloremia, hypocalcemia, and a mixed prerenal/renal azotemia, as well as metabolic acidosis and coagulopathies. serum concentrations of hepatocellular enzymes such as sorbitol dehydrogenase may be elevated, and liver function may be reduced. preliminary diagnosis of equine intestinal clostridiosis caused by c. perfringens is based on the isolation of greater than cfus of c. perfringens type a per gram of feces from patients with diarrhea and signs suggestive of toxemia. similar criteria are used to screen human patients for c. perfringens type a infection. normal horses shed fewer than cfus/g of feces, and usually horses with intestinal clostridiosis shed greater than × cfus/g. , however, identification of increased numbers of clostridium organisms in the feces does not prove infection. detection of c. perfringens toxins in feces or intestinal contents in horses with increased numbers of fecal cfus and clinical signs of enterocolitis is more conclusive evidence of an enterotoxigenic infection than culture alone. immunoassays are available that are primarily designed to detect c. perfringens enterotoxin. however, the reliability (specificity) of some immunoassays for diagnosis of c. perfringens infection has come into question. recently, pcr multiplex and gene probe assays have been developed to detect the major lethal toxins in isolates or fecal samples to determine the pattern or toxin production and are currently the preferred methods of detection. [ ] [ ] [ ] as for c. perfringens, diagnosis of c. difficile infection consists of culture of the organism from feces and identification of toxins in the feces. bacterial culture of c. difficile may be difficult and may be an insensitive test in horses. [ ] [ ] detection may require enrichment techniques and culture of multiple fecal samples. , detection of toxins a and b in feces is regarded as the preferred test for diagnosis of c. difficile infection in human beings. commercial enzyme-linked immunosorbent assays are available for toxins a and b that are specific and appear to be more sensitive than a single culture for identifying c. difficile infection in adult horses. , one also may induce toxin production by c. difficile isolates. sensitive pcr methods also have been developed for application to fecal samples and isolates to detect the genes for toxins a and b. proliferative enteropathy is a chronic hyperplastic disorder of the small intestine and has been described in a variety of mammalian and avian species. , the only causative agent identified to date that induces proliferative enteropathy is the obligate intracellular pathogen lawsonia intracellulare. , the pig is the most frequently naturally affected species. however, the reports of equine proliferative enteropathy associated with l. intracellulare have increased in recent years. [ ] [ ] [ ] [ ] the relatedness of the strains of l. intracellulare causing proliferative enteropathy in pigs and horses or even among other affected species is not known. no host restriction is apparent because hamsters and other rodents can be infected with porcine strains of l. intracellulare. before the year , reports of proliferative enteropathy in the literature describing isolated cases were sporadic. [ ] [ ] [ ] however, since , outbreaks on breeding farms have been documented on farms in canada, suggesting that a new strain has emerged. the mode of infection is fecal-oral, and infected animals can shed large numbers of organisms in feces. affected animals shedding the organism in the feces serve as a source of infection for herdmates. possibly nonequine species serve as reservoirs contributing to outbreaks on horse farms. factors that increase the risk of proliferative enteropathy in pigs include overcrowding, ration changes, transport, and weaning. , like pigs, horses are affected as weanlings. factors associated with weaning and other stresses may affect immunity and increase susceptibility to infection. the incubation period is to weeks in nonequine species and is presumed to be similar in horses. experimental l. intracellulare infection produces characteristic pathologic lesions in pigs and hamsters that are identical to lesions in horses with proliferative enteropathy. , a profound hyperplasia of the mucosa predominantly caused by proliferation of crypt epithelium and crypt hyperplasia is induced locally in infected islands of tissue and eventually extends to the entire distal jejunum and ileum. l. intracellulare preferentially infects proliferating cells, thus the tropism for the crypt epithelium. infected cells proliferate far more rapidly than uninfected cells, suggesting that l. intracellulare directly induced the proliferative response. however, the molecular basis for the enhanced proliferation is not known. l. intracellulare penetrates epithelial cells in a membrane-bound vesicle but eventually escapes the vacuole and is found free in the cytoplasm concentrated at the apical pole of the cell. the gross pathologic lesions found in equine proliferative enteropathy are characteristic. [ ] [ ] [ ] [ ] lesions may be segmental and typically are found in the ileum and terminal jejunum in horses. however, lesions also may affect the duodenum. severe mucosal hypertrophy often occurs but may wane during the chronic stages of the disease. the mucosa may become corrugated with focal erosions or ulcers. one often can identify submucosal edema easily on cut sections of affected segments. moderate to severe crypt hyperplasia with atrophy of the intestinal villi is a consistent feature. the hyperplastic crypts are branched and may herniate into the submucosa. necrosis, edema of the submucosal and lamina propria, hemorrhage, mononuclear inflammation and muscular hypertrophy have been reported in affected intestinal segments but are not consistent. special stains such as silver stain are required to detect intracellular organisms. the organisms are curved or comma-shaped rods found clustered in the apical cytoplasm of hyperplastic crypt epithelium. the proliferative response of the intestinal mucosa alters absorption of nutrients and fluid secretion by disrupting the architecture of the villi and by altering the maturation of epithelial cells into absorptive cells, accounting for the secretory diarrhea and often severe weight loss. , the combined effects of the inflammatory part ii disorders of specific body systems response and malabsorption may account for the protein-losing enteropathy. proliferative enteritis most commonly affects weanling foals ages to months. the clinical signs of proliferative enteropathy include ill thrift, weight loss, peripheral edema, diarrhea, and colic. [ ] [ ] [ ] [ ] the diarrhea is usually in the form of soft feces but may be profuse and watery. some foals with mild diarrhea have black, tarry feces. secondary complications such as gastric ulceration, bronchopneumonia, or parasitism may occur concurrently with the proliferative enteropathy. clinicopathologic features include mild to moderate anemia, moderate to severe hypoalbuminemia (often < g/dl), hypoglobulinemia, neutrophilic leukocytosis, and hyperfibrinogenemia. creatine kinase activities may be elevated in affected foals. prerenal azotemia and electrolyte imbalances such as hyponatremia may be associated with diarrhea. peritoneal fluid analysis is usually unremarkable. ultrasonographic examination of the small intestine often reveals significant thickening of the intestinal wall ( figure . - ). intestinal edema may be evident as a hypoechoic appearance to one or more layers of the intestinal wall. methods for antemortem diagnosis include serologic analysis of l. intracellulare antibodies and pcr analysis of feces. serologic analysis using an indirect immunofluorescent antibody test may be the most useful single test available. the pcr test is specific but the sensitivity may be low. by the time clinical signs appear, % of pigs are serologically positive for anti-lawsonia intracellulare immunoglobulin g (igg). in contrast, only % of pigs had positive fecal pcr tests. of the seven foals tested in an outbreak of equine proliferative enteropathy, four foals with confirmed disease and three with suspected proliferative enteropathy had serologic titers against l. intracellulare of : or greater. in contrast, serum samples collected from foals before the outbreak were negative for l. intracellulare antibodies. fecal pcr for l. intracellulare was positive in of foals tested, and half of the serologically positive foals had negative fecal pcr tests. many clinicians combine serologic analysis with fecal pcr testing to increase the sensitivity and specificity of these diagnostic methods. isolation and culture of the organism requires cell culture techniques that are not widely available. thus no practical method exists for culturing the organism from feces or tissues that is available for clinical use. definitive diagnosis requires histopathologic examination of affected tissues. diagnosis is based on typical histopathologic findings of mucosal hypertrophy and submucosal edema and identification of small, curved, rod-shaped intracellular bacteria at the apical pole of epithelial cells in affected segments of intestine. special stains such as warthin-starry silver stain are required to detect the bacteria in histopathologic specimens. pcr analysis of affected intestinal tissue is a specific test for the presence of l. intracellulare and, unlike fecal pcr analysis, appears to be sensitive. strongyle infections in horses are caused by two groups of nematodes: large and small strongyles (see the section cyathostomiasis). large strongyles that are pathogenic in horses include strongylus vulgaris, s. edentatus, and s. equinus. of these species, s. vulgaris is by far the most important cause of disease in the large intestine and in fact is the most pathogenic parasitic infection in horses. s. vulgaris infection in horses is manifested clinically by two forms: acute and chronic disease. the age and resistance of the host, the infective dose, and the size and function of the affected arteries influence the type and degree of disease that occurs. sudden ingestion of large numbers of infective larvae by a naïve host causes acute strongylosis, whereas ingestion of fewer infective larvae over a long period of time by an older, more resistant host causes chronic strongylosis. acute strongylosis is more likely to cause colic than diarrhea and may be rapidly fatal. chronic strongylosis tends to cause debilitation and signs of colic but may also cause diarrhea. diarrhea associated with acute strongylosis occurs within several days of infection and is likely to be caused by migration of the larvae through the intestinal wall. fourth-stage larvae migrate through the mucosa and submucosa into the arterioles of the intestine, causing mural edema, hemorrhage, and infiltration with inflammatory cells into the intestinal wall. , increased secretion and decreased absorption of fluid and electrolytes, stimulated by inflammatory mediators such as prostaglandins and histamine, may play a role in the diarrhea induced by s. vulgaris. interstitial edema and damage to the interstitial matrix and mucosa may result from inflammation and migration of the parasites, causing increased secretion of fluid and albumin loss. abnormal gastrointestinal motility also may play a role in the development of diarrhea. migration of larvae through the intestinal wall early in the course of infection affects myoelectric activity and motility in the large intestine and may affect retention of ingesta and absorption of fluid. , the cause of death in acute strongylosis has not been addressed but may be related to massive migration through the vasculature, causing thrombosis with ischemia and infarction of the intestine. chronic strongylosis causes typical verminous arteritis and is associated more commonly with natural infections in horses than with acute strongylosis. lesions of the large intestinal vasculature caused by migration of larvae through the intima are characterized by thrombus formation, narrowing of the arterial lumen, fibrosis, and thickening of the arterial wall. , embolization may occur, causing acute segmental infarction of the large intestine, but more commonly reduced blood flow without embolization causes ischemia and occasionally infarction. , postmortem examination of horses with colonic infarction failed to reveal embolization as the cause in most cases. reduced blood flow in the tissues of the intestine usually results from narrowing of the arterial lumen by the thrombus and formation of microthrombi at sites independent of the parasites. release of vasoconstrictive inflammatory mediators such as leukotrienes from platelets, neutrophils, and eosinophils and elaboration of parasitic antigens or toxins may cause vasoconstriction and ischemia. horses with experimental strongylosis were found to have a % reduction of blood flow in the colonic vasculature. clearly, reduced blood flow is an important effect of chronic strongylosis, but the relationship between blood flow and diarrhea is unclear. disrupted motility resulting from ischemia may lead to diarrhea by reducing the retention of ingesta and absorption of fluid. acute infarction and mucosal ulceration have been found to cause severe chronic diarrhea in naturally infected horses. release of inflammatory mediators such as prostaglandins, histamine, and kinins from inflammatory cells associated with thrombi and inflamed intestine also may affect secretion, absorption, and motility, leading to diarrhea. the clinical signs of acute strongylosis caused by s. vulgaris infection are characterized by depression, moderate to severe colic, and fever. diarrhea is less often a feature of acute strongylosis than is colic. most cases of acute strongylosis occur in young, naïve horses that are introduced to an infested environment or are inoculated experimentally with infective larvae. this form of strongylosis often is not recognized naturally. chronic strongylosis, however, occurs most commonly as a natural syndrome. weight loss or poor weight gain; chronic, intermittent colic; fever; poor appetite; and diarrhea are frequent signs. , diarrhea may be profuse and watery, or the feces may be soft but of normal volume. transrectal palpation may reveal thickening and fremitus in the cranial mesenteric artery. young horses are most commonly affected, but older horses also may be affected. horses with acute infarction or large intestinal ulceration following chronic strongylosis may have signs of severe abdominal pain, sepsis, and endotoxemia, and profuse, watery diarrhea is common. hematologic abnormalities associated with strongylosis include neutrophilic leukocytosis and eosinophilia. [ ] [ ] [ ] neutrophilia appears to be an early event during the course of the disease, and eosinophilia tends to appear later. , hyperfibrinogenemia also may occur, especially later in the course of the disease. serum αand β-globulin and igg(t) concentrations are characteristically elevated. [ ] [ ] [ ] horses with chronic ulcerative colitis following strongylosis may exhibit severe hypoalbuminemia. peritoneal fluid analysis may reveal an elevated protein concentration and eosinophilia. , tentative diagnosis is based on clinical signs, hematologic abnormalities, and peritoneal fluid analysis. elevated serum αand β-globulin concentrations and igg(t) concentration support the diagnosis. fecal analysis may reveal strongyle eggs, but fecal egg counts are often unreliable because nonpatent larvae cause the disease. appropriate preventive measures are important in controlling this disease, including management procedures such as preventing overcrowding, reducing exposure of susceptible individuals, and instituting proper deworming schedules. ivermectin is the preferred anthelmintic used to control strongylosis in horses. monitoring fecal egg counts as a means of evaluating the efficacy of parasite control measures is recommended. infection with small strongyles (cyathostomiasis) is well recognized as a cause of diarrhea and large intestinal disease in horses of all ages. [ ] [ ] [ ] [ ] [ ] [ ] clinical disease is caused by intramural larval stages. the cyathostome life cycle requires migration by fourth-stage larvae through the mucosa of the large intestine and may include a period of hypobiosis during which the larvae remain encysted within the mucosal layer of the large intestine. after a period of hypobiosis the larvae emerge in response to a largely unknown stimulus. most cases occur when larval emergence takes place, classically in the late winter and spring in the northern temperate zones when larvae are expected to emerge and in the late fall or winter months in the southeastern united states and subtropical regions. sudden emergence of encysted larvae causes the mucosal injury, ulceration, and inflammatory reaction responsible in large part for the clinical disease. , however, migration of the larvae as they penetrate the mucosa affects motility patterns and can cause inflammation that may contribute to diarrhea. chronic, eosinophilic, granulomatous colitis and diarrhea with histopathologic evidence of hypobiotic cyathostome larvae in the large intestine have been reported in two horses during a period in which emergence of larvae would not be expected to occur (early winter). natural emergence of cyathostome larvae causes fibrinous inflammation of the large intestine, focal necrosis, mural hemorrhage, and ulceration of the large intestinal mucosa, which even may result in bleeding into the lumen. mild to moderate eosinophilic and mononuclear inflammation of the lamina propria occurs, and moderate to severe interstitial edema frequently occurs. , colonic inflammation and interstitial edema may contribute to the diarrhea, along with the loss of the mucosal barrier, by causing increased active and passive secretion of fluid, electrolytes, and protein. protein loss is often significant, resulting in profound hypoalbuminemia and interstitial edema of skin and other organs. chronic, granulomatous colitis has been reported to occur in response to encysted larvae and may cause diarrhea by increased secretion following granulomatous inflammation or disruption of the interstitium by granulomatous infiltration. administration of an anthelmintic to horses with a heavy load of encysted larvae also may cause rapid larval death and acute and often severe inflammation similar to natural emergence. cyathostomiasis may be the most commonly identified cause of chronic diarrhea in the horse. [ ] [ ] [ ] however, an acute syndrome also has been associated with cyathostomiasis. clinical signs of cyathostomiasis are characterized by moderate to severe weight loss or poor weight gain, ill thrift, ventral edema, intermittent fever, and intermittent, mild colic. acute onset of diarrhea is typically profuse and progresses to chronic diarrhea that is often mild, is the consistency of bovine feces, and may be intermittent. [ ] [ ] [ ] [ ] [ ] [ ] appetite is usually normal, but affected horses occasionally have a ravenous appetite. transrectal palpation usually does not reveal any abnormalities. cyathostomiasis may affect any age of horse, and clinical signs are more common during periods of emergence of larvae, corresponding to late winter and spring in northern temperate zones. the deworming history may appear to be adequate. neutrophilic leukocytosis is typically evident, but the white blood cell count may be normal. [ ] [ ] [ ] [ ] [ ] [ ] profound hypoalbuminemia is a characteristic feature of cyathostomiasis, manifested clinically by ventral edema. plasma αand β-globulin concentrations may be elevated, which can result in a normal total plasma protein concentration in spite of hypoalbuminemia. [ ] [ ] [ ] the serum igg(t) concentration, however, has been reported to be normal, which may help distinguish cyathostomiasis from s. vulgaris infection. , , in addition, peritoneal fluid analysis does not usually reveal any abnormalities, in contrast to horses with s. vulgaris infection. fecal analysis may be unrewarding because the infection is often not patent when clinical signs are apparent. measurement of plasma fructosamine may provide a measure of protein catabolism or protein loss in the absence of hypoalbuminemia. plasma fructosamine concentrations are significantly lower in horses with experimental cyathostomiasis than in normal controls, , suggesting that this test may be a useful diagnostic tool. however, the test has not yet been validated in naturally occurring cases, and neither the specificity nor the sensitivity is known. rectal scrapings or rectal mucosal biopsies may reveal evidence of cyathostome larvae. , definitive diagnosis usually requires microscopic examination of biopsy specimens of the cecum and ascending colon, collected by laparotomy. examination of biopsy specimens collected from the small intestine is recommended to rule out other causes of weight loss and diarrhea. one should include appropriate diagnostic tests, such as culture of feces for pathogenic bacteria, in the workup to rule out other causes. preventive measures are appropriate for other horses on premises known to have a problem with cyathostomiasis, particularly frequent deworming (every weeks) during times of high infectivity (spring and summer in the north and fall, winter, and early spring in the south) to eliminate parasites before they become patent. because of high levels of resistance to benzimidazoles, avermectins (ivermectin or moxidectin) are the drugs of choice. [ ] [ ] [ ] resistance to ivermectin has been demonstrated, but the prevalence of ivermectin resistance appears to remain low. although daily pyrantel pamoate administration also has been reported to reduce worm burdens and pasture infectivity in young and mature horses effectively, cyathostome resistance has been reported and is a concern for the use of this drug as a routine preventive anthelmintic. , diarrhea in adult horses may also occur secondary to administration of antimicrobial or antiinflammatory medications or after ingestion of toxic compounds. affected horses exhibit clinical signs that may be indistinguishable from signs exhibited by horses with diarrhea of infectious etiology. antibiotic-associated diarrhea has been reported in many species, including horses. certain antibiotics-such as trimethoprim-sulfonamide combinations, erythromycin, penicillins, tetracyclines, clindamycin, and lincomycinare associated with naturally occurring and experimental enterocolitis syndromes in horses. , [ ] [ ] [ ] [ ] in some cases, such as with trimethoprim-sulfonamide combinations, the geographic incidence of antibiotic-associated diarrhea appears to differ considerably. clostridium perfringens, c. difficile, and salmonella spp. are apparently the most common causes of antibioticassociated diarrhea in horses. outbreaks of c. difficile have been reported in hospitalized horses being treated with antibiotics. , in sweden, accidental erythromycin ingestion has been associated with c. difficile enterocolitis in mares in which their foals were being treated for rhodococcus equi. , , interestingly, this phenomenon has not been reported in other areas of the world. foals being treated with erythromycin are at a higher risk for diarrhea than foals being treated with other antibiotics. tetracycline administration has been shown to be associated with an increase in the numbers of gram-negative enteric bacteria and c. perfringens in the feces of horses and reactivation of salmonellosis and prolongation of fecal shedding of salmonella. , the most common mechanism by which antibiotics cause diarrhea is by disrupting the gastrointestinal flora. the normal large intestinal flora, comprised of mainly obligate anaerobes and streptococci, protects the host from pathogenic bacteria by colonization resistance. ecologic factors play an important role in colonization resistance. for example, surface bacteria in the large intestine interact with receptors on the mucosal cells, facilitating adherence to the mucosa. , in doing so, the normal organisms compete more successfully for this important niche. competition for space and nutrients is an important means of preventing colonization and proliferation of pathogenic bacteria. in addition, anaerobic bacteria produce short-chain fatty acids (scfas) and other metabolites that are toxic to facultative anaerobic bacteria, especially in the conditions of the large intestine. , , organisms of the normal flora also produce bacteriocins that inhibit growth of potential pathogens. antibiotics that deplete the population of obligate anaerobes and streptococci efficiently decrease colonization resistance. production of fatty acids diminishes, thus reducing competition for space and nutrients. as a result, gram-negative enteric bacteria such as salmonella are able to proliferate. in addition, pathogenic anaerobes normally found in low numbers can proliferate. antibiotic-resistant strains of bacteria, especially gram-negative enteric bacteria and possibly clostridia, may be selected by antibiotic administration, allowing proliferation of pathogenic bacteria resistant to many antibiotics. obligate anaerobic commensal organisms, perhaps the most critical group of microbes for maintaining colonization resistance, are usually susceptible to macrolides, tetracyclines, β-lactams, and lincosamides, perhaps explaining the high incidence of diarrhea associated with the administration of these antibiotics. in addition to reduction of colonization resistance, depletion of the normal anaerobic microbial population in the intestine decreases carbohydrate fermentation and production of scfas, which contributes to the pathogenesis of antibiotic-associated diarrhea by decreasing absorption of sodium and water by the colonic mucosa. ampicillin decreases colonic fermentation of carbohydrates in human beings. human patients with antibioticassociated diarrhea have greatly impaired colonic fermentation and low production of scfas. erythromycin, ampicillin, or metronidazole treatment is associated with decreased production of scfas in patients with and without diarrhea. absorption of sodium and water is stimulated by absorption of scfas in the equine colon, suggesting that reduction of colonic scfa content by antibiotic-induced depletion of anaerobic flora has similar effects in horses as in human beings. broad-spectrum antibiotics exert a more profound effect on the gastrointestinal flora than narrow-spectrum antibiotics. antibiotics administered orally, especially those that are poorly absorbed, are more likely to cause diarrhea than are parenterally administered antibiotics. for instance, clindamycin is less likely to cause diarrhea in human beings when administered intravenously than when administered orally. antibiotics with extensive enterohepatic circulation, such as tetracyclines and erythromycin, are excreted in high concentrations in the bile and are associated more commonly with diarrhea than antibiotics that do not undergo enterohepatic circulation. antibiotics may cause diarrhea by means other than by disrupting the normal flora. direct toxic effects may play a role in producing irritation, increasing secretion, and disrupting motility patterns. tetracyclines are irritating to the gastrointestinal mucosa and may cause inflammation and increase secretion. erythromycin has been shown to interact with smooth muscle cells, stimulating gastrointestinal motility. , normal peristalsis plays an important role in suppressing the population size of potentially pathogenic bacteria. normally, bacteria that are prevented from adhering to the mucosa by colonization resistance are swept aborally by peristalsis and are excreted in the feces. disruption of normal motility patterns may prevent clearance of pathogenic bacteria, contributing to the colonization of mucosal surfaces. diarrhea induced by antibiotics usually occurs within days of antibiotic administration or can occur several days after cessation of antibiotic treatment. the clinical syndrome of antibiotic-associated diarrhea can vary from mild diarrhea to fulminant enterocolitis with severe diarrhea. mild cases of diarrhea are common, especially in foals receiving erythromycin, trimethoprim-sulfonamide combinations, or rifampin. , mild cases of diarrhea are usually not clinically significant. however, acute, severe enterocolitis can occur in all ages of horses receiving antibiotics and can be life threatening. clinical signs are identical to other causes of acute enterocolitis. severe, dehydrating diarrhea, endotoxemia, sepsis, and shock may occur. hemoconcentration, neutropenia, hypoproteinemia, and electrolyte and acid-base imbalances are common. severe hyponatremia may occur in foals with antibiotic-associated diarrhea, especially if trimethoprimsulfonamide and rifampin combinations are the cause. more detailed descriptions of the clinical and laboratory findings were given previously. diagnosis is presumptive, because definitive diagnosis of antibiotic-associated diarrhea is impossible. fecal culture and pcr testing may reveal salmonella or clostridium infection. toxicity resulting from nonsteroidal antiinflammatory drug (nsaid) administration has been well documented in several species, including horses. [ ] [ ] [ ] [ ] [ ] [ ] [ ] in horses and human beings, nsaid toxicity is manifested by renal and gastrointestinal disease. elderly human patients are more susceptible to nsaid toxicity, but the effects of age on nsaid toxicity in horses are less well defined. foals are considered to be more susceptible than adult horses to gastrointestinal disease following nsaid administration, and ponies may be more susceptible than horses. all nsaids are capable of inducing gastrointestinal and renal damage at toxic concentrations, and the toxicity is not significantly different among products. aspirin is potentially more toxic than other nsaids because it irreversibly inactivates cyclooxygenase by acetylation, whereas other nsaids reversibly inhibit cyclooxygenase. however, phenylbutazone is the drug most commonly reported to cause toxicity in horses, perhaps because of its widespread use by veterinarians and horse owners. phenylbutazone toxicity in horses is characterized by mucosal ulceration throughout the gastrointestinal tract, oral ulceration, renal papillary necrosis, vasculopathy, thrombosis, and protein-losing enteropathy with hypoalbuminemia. [ ] [ ] [ ] this discussion focuses on the toxic effects of nsaids on the large intestine but necessarily includes elements of upper gastrointestinal and renal disease. horses with large intestinal disease resulting from nsaid toxicity generally are receiving inappropriately large doses. the dosage regimen recommended for phenylbutazone ( . mg/kg twice in day and then . mg/kg twice daily) is considered to be safe. experimental studies in horses, however, have shown toxicity to occur when greater than the recommended dosage ( . mg/kg/day) is administered for several days. , in most reported cases of phenylbutazone toxicosis horses were receiving higher than recommended dosages. , , regardless, administration of phenylbutazone at the recommended dosage has been reported to cause a significant decrease in plasma protein concentration and gastrointestinal disease. , moreover, signs of nsaid toxicity have been reported in normovolemic horses treated with appropriate doses of phenylbutazone. , dehydration, sepsis, and endotoxemia exacerbate the renal and gastrointestinal toxicity of nsaids. clearly, the margin of safety is narrow for phenylbutazone and probably for other nsaids used in horses as well. gastrointestinal disease induced by nsaids is manifested by mucosal ulceration, inflammation, bleeding, and protein-losing enteropathy. , , , in addition to direct effects on the mucosal barrier, nsaid administration has been shown to cause an acute relapse of preexisting colonic inflammatory disease and worsen colonic inflammation in human beings. , , whether this occurs in horses is not clear. the mechanism by which nsaids induce mucosal damage is probably multifactorial. direct irritation may play a role in oral and gastric irritation and ulceration; however, parenteral administration of nsaids produces oral and gastric ulceration as well. inhibition of prostaglandin synthesis by inhibition of cyclooxygenase (the constitutive cox) and cyclooxygenase (the inducible cox) appears to be the most important mechanism of mucosal injury. prostaglandins, particularly pge and pgi , are critical for mucosal health. , pge has been shown to increase mucosal blood flow; increase secretion of mucus, water, and bicarbonate; increase mucosal cell turnover rate and migration; stimulate adenyl cyclase activity; and exert other protective effects in the gastric mucosa of several species. , , perhaps most importantly, pge and pgi have a role in maintaining epithelial tight junction integrity, which is indispensable for mucosal barrier function and repair after mucosal injury. in spite of the overwhelming amount of information about the role of prostaglandins in maintaining the mucosal barrier in other species and clear clinical and experimental evidence that nsaids injure the equine colonic mucosa, the role of prostaglandins in mucosal protection in the equine colon is not yet well defined. inhibition of cox- and cox- in equine colonic mucosa with flunixin meglumine resulted in reduced electric resistance of the mucosa and increased permeability to macromolecules in vitro (a.t. blikslager and s.l. jones, ) , suggesting that flunixin treatment disrupts the epithelial tight junctions in the equine colon. mucosal changes were correlated with a profound inhibition of pge and pgi concentrations in the treated tissues. in other studies, administration of a pge analog prevented the gastrointestinal manifestations of phenylbutazone toxicosis in ponies. recent development of nsaids specific for cox- have greatly reduced the frequency and severity of gastrointestinal side effects in human beings taking nsaids for chronic musculoskeletal conditions. thus cox- -specific nsaids hold promise for use in horses to treat arthritis and reduce the incidence of toxicity. for example, the cox- -specific inhibitor etodolac was less harmful to equine colonic mucosa than flunixin meglumine in vitro (a.t. blikslager and s.l. jones, ) . moreover, etodolac was significantly more permissive than flunixin for recovery of the mucosa in equine ischemic-injured intestinal tissues, and in fact, recovery was no different than control tissues. however, their use is at present limited because the specificity of the so-called cox- selective inhibitors and their efficacy as analgesics have not been demonstrated in the horse. nsaid-induced mucosal injury is associated with a significant inflammatory response to microbial products exposed to the lamina propria. this inflammation exacerbates mucosal dysfunction and injury associated with nsaid toxicity. for example, depletion of neutrophils or blockade of neutrophil influx into gastrointestinal tissues or inhibition of neutrophil activation and release of toxic products prevents many of the pathophysiologic effects of nsaid toxicity in the gastrointestinal tract. [ ] [ ] [ ] [ ] the inflammatory response alone may result in moderate to severe gastrointestinal ulceration, mural vascular thrombosis and edema, fluid secretion, protein-losing enteropathy, and mucosal hemorrhage. nsaid colitis manifests as two clinical syndromes: right dorsal colitis (rdc) and generalized nsaid toxicity. rdc is an ulcerative disorder isolated to the right dorsal segment of the large intestine. , , the most prominent clinical signs of rdc are anorexia, lethargy, and colic. anorexia, depression, diarrhea, fever, and signs of section . inflammatory diseases of the gastrointestinal tract causing diarrhea endotoxemia also may be features. if the rdc is chronic, weight loss, intermittent colic, lethargy, anorexia, and ventral edema are common clinical signs, along with soft and unformed feces. severe ulceration of the right dorsal colonic mucosa results in proteinlosing enteropathy and significant hypoproteinemia attributable mainly to hypoalbuminemia. hypoproteinemia may be severe enough to cause peripheral (usually ventral) edema.in some cases, one may note dehydration, electrolyte abnormalities, neutropenia or anemia, azotemia, and biochemical abnormalities if the ulceration and diarrhea are severe or if systemic inflammation is present. clinical signs of generalized nsaid toxicity may vary from mild diarrhea with no systemic signs to severe dehydrating diarrhea with anorexia, fever, depression, peripheral edema, oral ulceration, and colic. , , clinical signs of systemic inflammation caused by endotoxemia may occur, manifested as poor peripheral perfusion, tachycardia, tachypnea, weakness, trembling, and cyanotic or hyperemic oral mucous membranes. hematuria or oliguria may be present if renal involvement is present. complications associated with other forms of severe enterocolitis, such as laminitis, thrombophlebitis, and severe weight loss, may occur. hematologic abnormalities of generalized nsaid toxicity are nonspecific and include neutropenia with a left shift or leukocytosis and hemoconcentration. serum biochemical analysis is characterized by profound hypoproteinemia, hyponatremia, and metabolic acidosis. , hypocalcemia, hypokalemia, hypochloremia, and elevated hepatocellular enzyme activities also may occur. hypoproteinemia may occur without signs of diarrhea. azotemia may be prerenal from dehydration but frequently is caused by renal failure resulting from a combination of hemodynamic and toxic renal injury. urinalysis frequently reveals hematuria, proteinuria, cylindruria, and isosthenuria. fecal occult blood is frequently detectable. diagnosis of either form of nsaid colitis is often presumptive, with a history of overdose of nsaids being strong evidence of nsaid toxicity. but as discussed earlier, toxicity may occur with dosage regimens that are not considered inappropriate, particularly if the horse experiences a concurrent period of dehydration. one can use ultrasonographic examination of the right dorsal colon to confirm a diagnosis of rdc, but the sensitivity of this method is questionable. ultrasonography ( . -to -mhz transducer at the right twelfth to fifteenth intercostal spaces below the margin of the lung axial to the liver) may reveal a thickened right dorsal colon (> . cm) and evidence of colonic edema in horses with rdc. however, the sensitivity of this method of diagnosis is questionable. one can use nuclear scintigraphy of horses after infusion with technetium -labeled white blood cells to document inflammation of the right dorsal colon. diagnosis of rdc may require one to perform laparotomy or laparoscopic examination of the right dorsal colon. one must rule out other causes of enterocolitis, such as salmonellosis, potomac horse fever, clostridiosis, and antibiotic-associated diarrhea. cantharidin is the toxic principle found in beetles of the genus epicauta, commonly known as blister beetles. [ ] [ ] [ ] ingestion of the beetles in contaminated alfalfa hay causes release of the toxin from the tissues of the beetle and absorption through the gastrointestinal tract. transcutaneous absorption may occur but appears to be rare in horses. blister beetles feed on the flowers of alfalfa and may be incorporated into processed alfalfa hay if the hay is cut and processed simultaneously, as by crimping. [ ] [ ] [ ] the beetles often swarm, and one may find large numbers of beetles in small portions of hay. the lethal dose of cantharidin is less than mg/kg, but the concentration of cantharidin varies from species to species of blister beetles and between sexes. , as many as to as few as to beetles may be lethal. usually, only one or a few horses fed contaminated hay ingest beetles because the beetles are concentrated in a small portion of the hay. however, outbreaks involving many horses on a farm have occurred. most cases occur in texas and oklahoma, but horses in other states may be affected as well, especially if hay is imported from states where blister beetles are common. peak incidence is in late summer and fall. the fatality rate may be % or greater, , but if the patient survives several days, recovery is probable. cantharidin is absorbed from the gastrointestinal tract and excreted via the kidney. cantharidin is a potent irritant, causing acantholysis and vesicle formation when applied topically. , , the chemical is thought to disrupt oxidative metabolism in the mitochondria, causing mitochondrial swelling, plasma membrane damage, and changes in membrane permeability. the mucosa of the gastrointestinal tract is affected most commonly in horses because they ingest the toxin. cell swelling and necrosis occur, resulting in mucosal ulceration. oral, esophageal, gastric, and small and large intestinal ulceration have been observed in natural and experimental canthariasis. , , severe fibrinous to pseudomembranous inflammation and submucosal edema of the intestine also have been reported. diarrhea probably results from the severe ulceration and inflammation of the large intestine, causing increased secretion of water, electrolytes, and protein and decreased absorption of fluid. large volumes of fluid and protein are lost in the gastrointestinal tract, causing hemoconcentration and profound hypoalbuminemia in some cases. , , cystitis and myocarditis occur in natural and experimentally produced cases of cantharidin toxicity. , , cystitis occurs because renal excretion of cantharidin results in high concentrations in urine. occasionally, hemorrhagic cystitis may occur, with hematuria or frank hemorrhage into the bladder. the cause of the myocarditis and myocardial necrosis is unknown but also may be a direct effect of the toxin on the myocardium. elevated plasma creatine kinase activity often occurs and has been postulated to arise from the damaged myocardium. , horses have a characteristically stiff gait, but histopathologic evidence of skeletal muscle injury that explains the elevated plasma creatine kinase activity has not been observed. the kidneys are often pale, swollen, and moist, with occasional infarcts. hypocalcemia and hypomagnesemia are biochemical features of cantharidin toxicity in horses that have not been explained. , , hypocalcemia may occur from hypoalbuminemia, but the ionized calcium concentration often is decreased along with the total calcium concentration, indicating that hypoalbuminemia is not responsible for the hypocalcemia. in addition, clinical signs of hypocalcemia, such as synchronous diaphragmatic flutter, are often associated with hypocalcemia from cantharidin toxicity. hypocalcemia associated with hypoalbuminemia alone does not produce clinical signs. cantharidin toxicity can cause a range of clinical signs from mild depression and abdominal discomfort to fulminant signs of toxemia and rapid death, depending on the ingested dose of toxin. most commonly, clinical signs include depression, sweating, irritability, abdominal pain, elevated heart and respiratory rates, fever, polyuria, polydypsia, and profuse diarrhea. , , blood is rarely visible in the feces. affected horses frequently posture to urinate; indeed, stranguria and pollakiuria are characteristic of cantharidin toxicity. signs of hypocalcemia include synchronous diaphragmatic flutter and tremors. a stiff and stilted gait may be evident. one may note neurologic signs such as head pressing, swaying, and disorientation. signs of systemic inflammation from endotoxemia may be apparent in severe cases. some horses develop severe depression and toxemia and may die within hours after ingestion of cantharidin without developing diarrhea. , hematologic abnormalities include hemoconcentration and neutrophilic leukocytosis. occasionally, neutropenia and leukopenia may accompany endotoxemia. serum biochemical analysis usually reveals elevated creatine kinase activity, hypocalcemia, and hypoalbuminemia. , biochemical abnormalities include hypocalcemia (ionized and total calcium concentrations), hypomagnesemia, and azotemia. , , urine specific gravity is characteristically in the hyposthenuric range. , microscopic hematuria and mild proteinuria may be evident. fecal occult blood is often present, but hematochezia is unusual. one can make a tentative diagnosis based on clinical signs and the finding of blister beetles in the hay. determining the species of the insects may be necessary to estimate the amount of cantharidin ingested. all species of epicauta contain cantharidin, but some have small amounts. definitive diagnosis requires the measurement of the cantharidin concentration in gastric or intestinal contents and urine. , measurement of cantharidin concentration in the beetles is often done but is not necessary. arsenic toxicosis is an unusual cause of diarrhea in horses, resulting from ingestion of arsenic-containing herbicides, insecticides, and other pest control products contaminating water or roughage used as a food source. the toxicity of arsenic depends on the valence of the element. , arsenate may be reduced to arsenite in mammalian systems, and arsenite is thought to be more toxic than arsenate and less rapidly excreted in urine. arsenate and arsenite uncouple oxidative phosphorylation, leading to breakdown of energy metabolism in the cells of many tissues. widespread cellular injury and death occur rapidly during acute arsenic toxicosis. multiorgan failure usually results. in fact, cardiomyopathy and pulmonary disease are common causes of death in human beings. damage to the large intestine is probably caused in part by direct cellular toxicity and corrosion by the compound. however, vasculitis is a hallmark of the disease in human beings and horses and is thought to be the most important mechanism of large intestinal disease in human beings. , acute hemorrhagic colitis is a feature of arsenic toxicosis, with severe mural edema and mucosal ulceration. profuse, hemorrhagic diarrhea and abdominal pain result. chronic arsenic toxicity can occur but appears to be rare in horses. acute depression, weakness, abdominal pain, hemorrhagic diarrhea, and shock are characteristic of acute arsenic toxicosis in horses. death may occur before diarrhea is evident. initial clinical signs may be difficult to distinguish from other peracute forms of colitis and are related to endotoxic shock, metabolic disturbances, and dehydration. later, cardiac arrhythmias, pulmonary edema, acute renal failure, and neurologic deficits (ataxia and stupor) may develop. one may observe anuria or polyuria. hemolytic anemia caused by preferential binding of arsenic compounds to red blood cells is a feature of arsenic poisoning in human beings. hematologic abnormalities may be apparent after the peracute stages from injury to bone marrow cells and ongoing hemolysis. leukopenia and thrombocytopenia have been described in human patients. serum biochemical analysis may reveal azotemia, hepatocellular enzyme activities higher than generally attributed to endotoxemia, and elevated creatine kinase activity. urine specific gravity may be in the isosthenuric range, with hematuria, cylindruria, and proteinuria evident by urinalysis. diagnosis may be possible by measuring blood and urine arsenic concentration, but these tests may not be diagnostic. postmortem diagnosis is by measuring the arsenic concentration in liver and kidney samples. history of exposure and clinical signs remain the primary means of diagnosis. other disorders associated with diarrhea in adult horses include anaphylaxis, carbohydrate overload, and sand enteropathy. careful evaluation of history, environment, and management will assist the clinician in arriving at an accurate diagnosis. severe intestinal anaphylaxis is a syndrome in horses characterized by peracute, rapidly fatal colitis. the severe syndrome is clinically and pathologically similar to other known causes of peracute colitis, such as salmonellosis, clostridiosis, and antibiotic-associated diarrhea. some cases are less severe and manifest as mild to moderate diarrhea or colic. an ige-mediated type i hypersensitivity or an ige-independent anaphylactoid reaction can produce the syndrome of intestinal anaphylaxis. , local gastrointestinal exposure to a food, environmental contaminant, drug, or other allergen usually induces intestinal anaphylaxis, , but anaphylaxis also may occur with systemic exposure to an allergen. [ ] [ ] [ ] [ ] massive mast cell degranulation, secretion of inflammatory mediators, and activation of enteric neural reflexes in the intestine causes profound alterations in blood flow, increased vascular permeability and interstitial edema, recruitment of neutrophils, altered motility, mucosal injury, absorption of microbial products, and mucosal hypersecretion. [ ] [ ] [ ] [ ] [ ] systemic signs may be caused by the anaphylactic reaction or may be associated with systemic inflammation triggered by microbial products (endotoxin) absorbed through the injured and hyperpermeable mucosa. intestinal anaphylaxis in horses may be a peracute, fulminant enterocolitis with endotoxemia that may be fatal. , this form is characterized by severe intramural edema and hemorrhagic inflammation of the large intestine, often producing submucosal thickening on the order of many centimeters. vascular thrombosis may be widespread with mucosal and serosal petechia and ecchymoses. less severe forms of intestinal anaphylaxis may manifest as patchy areas of intestinal edema and congestion. diarrhea results from intestinal inflammation initiated by the type i hypersensitivity response. many of the mediators of type i hypersensitivity, such as histamine and -hydroxytryptamine, have well-documented stimulatory effects on mucosal secretory activity, vascular and epithelial permeability, and motility [ ] [ ] [ ] in the intestine. systemic inflammation from endotoxemia may be overwhelming once the mucosal barrier breaks down. infarction of intestinal segments and other organs may occur from intravascular coagulation. ileus, abdominal distention, and moderate to severe abdominal pain may result from motility disturbances and infarction of the large intestine. the clinical signs are similar to those described for other forms of peracute colitis. however, the severity may vary, manifesting as colic or moderate diarrhea. characteristically, severe shock, signs of systemic inflammation from endotoxemia, and severe metabolic disturbances are observable. , heart and respiratory rates may be elevated greatly, with other signs of cardiovascular collapse such as weak and thready peripheral pulses and peripheral vasoconstriction. however, peripheral vasodilation may occur later in the course of disease. dark red, muddy, or cyanotic mucous membranes with a prolonged capillary refill time signify sepsis. borborygmi are usually absent, and abdominal tympany may be heard on percussion, following ileus. moderate to severe colic may accompany ileus. severe diarrhea may occur, but death may occur before diarrhea is evident. multiorgan failure from disseminated intravascular coagulation is not unusual. rapid onset of weakness, staggering, and trembling commonly precedes death. the syndrome may cause death in to hours. hematologic abnormalities include severe neutropenia and leukopenia, thrombocytopenia, and hemoconcentration. serum biochemical alterations include hyponatremia, hypokalemia, hypocalcemia, and severe metabolic acidosis. blood urea nitrogen and creatinine may be elevated from prerenal or renal azotemia. if acute renal failure accompanies the colitis, hyperkalemia may result. hepatocellular enzyme activity may be elevated in the serum from endotoxemia. severe coagulopathies are common, resulting in prolonged coagulation times, elevated fibrinogen, decreased antithrombin iii activity, and elevated plasma concentration of fibrin degradation products. analysis of peritoneal fluid may be valuable because infarction of the large intestine is not unusual. protein concentration and the white blood cell count may be elevated. red blood cell counts are less likely to be elevated, because infarction and not strangulation of the intestine occurs. diagnosis is based on clinical signs, postmortem findings, and exclusion of other causes. cultures and toxicologic analysis of fecal samples and gastrointestinal tissues fail to demonstrate a clear cause. other diagnostic tests are also inconclusive. if an antigen is suspected as the trigger of the anaphylaxis, a prausnitz-küstner passive cutaneous anaphylaxis sensitization test can confirm the presence of antigen-specific ige in the patient serum. overeating of soluble carbohydrates, especially so-called hot grains such as corn, overwhelms the digestive capability of the small intestine, resulting in a high percentage of the soluble carbohydrates entering the large intestine. the amount of soluble carbohydrates that produce diarrhea varies according to the previous dietary history of the individual. horses fed diets higher in soluble carbohydrates are more resistant to the deleterious effects of carbohydrate overload. gradual accommodation to a diet high in carbohydrates can be accomplished over several weeks. however, horses fed an unusually large amount of grains or other form of soluble carbohydrates often develop diarrhea and may, depending on the amount ingested, develop severe colitis, systemic inflammation from endotoxemia, metabolic acidosis, and laminitis. [ ] [ ] [ ] [ ] the pathogenesis of colitis from carbohydrate overload is caused primarily by the toxic effects on the microbial flora in the large intestine. a sudden delivery of soluble carbohydrates to the large intestine causes rapid fermentation by gram-positive lactic acid-producing bacteria and a sudden increase in organic acid production. the cecal ph rapidly decreases, and the lactic acid concentration rapidly increases. rapid organic acid production overwhelms the buffering capacity of the large intestine not only by directly depleting the buffers found in the contents but also by reducing the efficiency of buffer secretion. bicarbonate secretion is linked to absorption of volatile fatty acids, which are produced in low amounts by fermentation of soluble carbohydrates. the contents of the large intestine become profoundly acidic, resulting in unfavorable conditions for the microbial flora. lactic acid-producing bacteria flourish, while the gram-negative bacteria, especially the enterobacteriaceae, are killed in large numbers by the acids. large quantities of endotoxin are released from the dying bacteria. the osmotic load from the lactic acid produced in the large intestine is an important factor in the development of diarrhea because organic acids such as lactic acid are absorbed poorly. mild cases of carbohydrate overload may result purely from osmotic diarrhea. in more severe cases, the acidic contents of the large intestine are toxic to the mucosa, causing necrosis of the mucosal tissues, similar to that occurring in ruminal acidosis. mucosal ulceration allows absorption of large quantities of endotoxin and lactic acid produced by the massive die-off of acid-intolerant microbes and fermentation of soluble carbohydrates, normally poorly absorbed by intact mucosa. systemic inflammation from endotoxemia may be overwhelming, and profound metabolic acidosis may occur. secretory diarrhea caused by the direct effects of acid luminal contents on the mucosa, as well as the effects of inflammatory mediators on enterocyte secretion, worsens the acidosis and dehydration. systemic inflammation from endotoxemia, along with intestinal inflammation, adversely affects intestinal motility, and ileus develops. ileus and gas production from fermentation of the carbohydrates may cause severe distention of the large intestine and signs of abdominal pain. laminitis is a frequent complication of endotoxemia and lactic acidosis. in fact, carbohydrate overload is used to induce laminitis as an experimental model because of the consistency of the laminitis produced. [ ] [ ] [ ] clinical signs of colitis from carbohydrate overload can vary according to the amount of carbohydrates ingested and accommodation of the flora to a high-carbohydrate diet. mild cases may result in a transient osmotic diarrhea with no systemic effects. more severe cases are characterized by signs similar to those described for other forms of colitis, including abdominal pain, moderate to severe diarrhea, and dehydration. signs of endotoxemia and sepsis are frequently present in severe cases. elevated heart and respiratory rates are common, with peripheral vasoconstriction early in the disease, followed by peripheral vasodilation as the disease progresses. depression may be profound from metabolic acidosis and endotoxemia. abdominal auscultation and percussion may reveal ileus and intestinal tympany. nasogastric intubation may yield significant gastric acidic reflux. one may note particles of grain in the gastric reflux and the feces, if grain overload is the source of the carbohydrate overload. laminitis may complicate mild and severe cases of carbohydrate overload, especially if the animal has had previous bouts of laminitis. hematologic abnormalities include neutropenia and leukopenia. severe dehydration may result in profound hemoconcentration. protein loss later in the course of disease may result in hypoproteinemia. serum biochemical abnormalities include azotemia, elevated hepatocellular enzyme activity, hyponatremia, and hypokalemia. severe hypocalcemia and metabolic acidosis are characteristic of the disease. serum lactate concentrations are elevated in the absence of evidence of intestinal strangulation or infarction. peritoneal fluid analysis often reveals no abnormalities. sand enteropathy is described in more detail under the heading of obstructive diseases, because acute obstruction is often associated with abnormally large amounts of sand in the large intestine. however, chronic sand-induced diarrhea is a distinct syndrome that can occur at any age from abnormal accumulation of sand in the large intestine. , chronic diarrhea and signs of colic may occur without obstruction. the pathogenesis of sand accumulation in individual horses, other than simple ingestion of large quantities, is unclear. presumably the sand causes irritation and may disrupt motility, leading to diarrhea. the diarrhea is usually not severe and dehydrating and may be intermittent. weight loss is characteristic and can be severe in some cases. complications may occur such as peritonitis and acute obstruction. diagnosis usually is based on finding abnormal amounts of sand in the feces. because sand-induced chronic diarrhea is associated primarily with sand accumulation in the ventral colon, auscultation of the ventral abdomen immediately behind the xiphoid process may reveal characteristic sand sounds. this technique is only sensitive if peristalsis is present. ultrasonography also may be useful to identify sand in the ventral colon but is not useful to quantitate the amount of sand. occasionally, radiography may be required to detect sand in the colon. the principles of therapy of acute diarrhea from colitis are similar regardless of the cause and include replacement of fluid and electrolyte losses, control of colonic inflammation and reduction of fluid secretion, promotion of mucosal repair, control of endotoxemia and sepsis, and reestablishment of normal flora. this section focuses on a review the principles of therapy with references to specific therapies for particular causes as they arise. replacement of fluid and electrolyte losses is of primary concern in treating horses with salmonellosis. depending on the severity of the disease, fluid losses may be minimal or massive. one can administer fluid and electrolytes orally or intravenously. some horses with mild to moderate diarrhea may maintain hydration and electrolyte balance by consuming water and electrolytes voluntarily. freshwater and water containing electrolytes should be available in all cases. in many instances, periodic nasogastric intubation and administration of water and electrolytes via the tube may be sufficient to maintain hydration. in more severe cases, one can maintain indwelling nasogastric tubes and can administer up to to l of fluid by the tube every to minutes, if ileus is not evident. however, intravenous administration of fluids is preferred in most cases, requiring significant quantities of fluid to replace and maintain hydration and electrolyte balance. for patients with severe diarrhea to require large volumes ( to l/day) of intravenous fluids to maintain hydration is not unusual. frequent monitoring of packed cell volume, serum electrolyte concentration, venous blood gases or total serum carbon dioxide, blood urea nitrogen and creatinine, urine protein and cytologic findings, and body weight is important to monitor hydration, electrolyte and acid-base balance, and renal function. isotonic sodium chloride or lactated ringer's solution frequently is used to restore and maintain fluid and electrolyte balance. one can add potassium chloride to the fluids and administer it at a rate up to . to . meq/ kg/hr. generally, a rate of less than . meq/kg/hr is used. hypertonic nacl solutions ( to l of % to % nacl) have been used in horses that are severely hyponatremic (< meq/dl). one should not administer hypertonic solutions to severely dehydrated horses, but such solutions have been used clinically without complication and with considerable beneficial effect in patients with endotoxemia. the beneficial effects of hypertonic nacl are short-lived ( to minutes). one should administer isotonic solutions concurrently or immediately following administration of hypertonic nacl solutions. isotonic ( . %) or hypertonic ( . %) sodium bicarbonate solutions are used to correct metabolic acidosis. prolonged administration of sodium-containing fluids may promote diuresis and renal water loss or accumulation of peripheral edema and should be used conservatively when one notes a free water loss. administration of isotonic dextrose ( %) or . % dextrose/ . % nacl solutions may be beneficial when free water loss (sodium excess) is evident. many horses with acute colitis are concurrently hypoproteinemic because of gastrointestinal losses and are absorbing bacterial products that induce a systemic inflammatory response. thus plasma oncotic pressures are abnormally low in the face of increased vascular permeability. interstitial edema formation is a clinical problem in these patients and contributes to organ dysfunction. crystalloid fluids, although critical for replacing water and electrolyte losses from diarrhea, actually may contribute to a drop in plasma oncotic pressure because of hemodilution. , administration of colloid solutions are important for volume expansion and to maintain plasma oncotic pressures, which improve tissue perfusion and oxygenation and organ function in hypovolemic, hypotensive, and hypoproteinemic patients with or without systemic inflammatory response syndrome. colloids are more effective than crystalloid fluids at expanding plasma volume and thus require smaller volumes. moreover, the effect of colloid volume expansion is longer lasting than crystalloid fluid volume expansion, because colloids are retained in the vasculature better. , natural colloids, such as plasma and purified albumin are used commonly. in addition to its beneficial colloidal properties, plasma harvested from donor horses immunized with rough mutants of escherichia coli (j ) or salmonella typhimurium may have other benefits for treatment of endotoxemia from gastrointestinal disease. , the horse may require large volumes ( to l/day) to increase and maintain plasma protein concentration significantly. synthetic colloids such as dextrans, starches, or polymerized hemoglobin are also available for use in the horse. hetastarch ( to ml/kg of a % solution) increases colloidal oncotic pressures for up to hours in hypoproteinemic horses and has beneficial effects on cardiac output and other cardiorespiratory parameters, vascular permeability, interstitial fluid content, and tissue perfusion in models of hypoproteinemia and systemic inflammatory response syndrome. when one administers synthetic or even natural colloids, monitoring plasma oncotic pressure may be more relevant than monitoring plasma protein concentrations as a means of assessing the need for plasma or other colloid administration. hetastarch may prolong bleeding times by altering von willebrand's factor function; thus one should use this synthetic colloid cautiously in horses with suspected coagulopathies, active hemorrhage, or other bleeding problems. control of colonic inflammation and secretion is a difficult and poorly studied aspect of equine acute colitis. the role of inflammation and mediators such as prostaglandins as causes of fluid loss is well known for salmonella and clostridium infections. cox inhibitors (nsaids) have antisecretory effects in the equine colon and in models of salmonellosis that appear to extend to clinical management of salmonellosis. , , [ ] [ ] [ ] indeed, nsaids commonly are administered to horses with salmonellosis. however, prostaglandins such as pge and pgi are also cytoprotective to gastrointestinal mucosa and critical for mucosal repair. the doses of nsaids used pharmacologically to inhibit colonic inflammation and secretion in fact may be detrimental to the mucosa if not used judiciously. nsaids have been shown to exacerbate colonic inflammation in human beings with inflammatory colitis, impede mucosal healing in several models of mucosal injury, and have well-documented detrimental effects on colonic mucosa in horses. , , in addition to toxicity to the colonic mucosa, gastric ulceration is not unusual in horses with enterocolitis and may be related to treatment with nsaids. in addition to nsaids, other drugs occasionally are used as antiinflammatory or antisecretory therapy. metronidazole has beneficial effects in experimental models of gastrointestinal inflammation, including nsaid toxicity and may be useful for treating horses with colitis, but evidence supporting its use is lacking. bismuth subsalicylate solutions administered orally often are used to decrease inflammation and secretion in the colon. in adult horses the volume of solution necessary to be beneficial is large ( to l every to hours). often the solution is administered twice daily instead of to times daily. if one does not achieve a beneficial effect within to days of treatment, one should discontinue administration of bismuth subsalicylate solution. one can administer the treatment more frequently in foals, and clinical improvement occurs more often in foals than in adult horses. in light of the role of reactive oxygen metabolites in colonic inflammation, free radical scavengers have been advocated to reduce the effects of these molecules. sulfasalazine metabolites have been shown to reduce reactive oxygen metabolite-induced colonic inflammation in other species, and sulfasalazine has been used to treat chronic inflammatory disease in horses but has not been used to treat acute colitis. the only free radical scavenger used commonly in horses with colitis is dimethyl sulfoxide, which at a dosage of . to . g/kg intravenously every to hours in a % solution has been used in clinical cases of colitis, but evidence of efficacy has not been established. systemic inflammatory response syndrome associated with endotoxemia frequently occurs in patients with salmonellosis. the principles of therapy for endotoxemia are covered in detail elsewhere in this chapter. oral administration of activated charcoal and mineral oil is used commonly to reduce absorption of endotoxin in horses with colitis. low doses of nsaids (such as flunixin meglumine at . to . mg/kg intravenously every to hours) inhibit eicosanoid synthesis induced by endotoxin. in addition, administration of nsaids prevents laminitis from endotoxemia, a devastating complication of salmonellosis. one must remember that prostaglandins are important for mucosal healing and may worsen mucosal injury in colitis. although the benefits of low doses of nsaids administered to horses with systemic inflammatory response syndrome are believed to outweigh the risks of worsening gastrointestinal damage, judicious use is recommended. sucralfate ( mg/kg orally every hours) has been advocated to aid in healing the colonic mucosa, but the efficacy in the large intestine is questionable. misoprostol ( µg/kg orally to times daily) and other synthetic pge analogs have been shown in several species including horses to enhance mucosal healing in the intestine and promote recovery in experimental models of colitis. misoprostol may be particularly useful for treating nsaid toxicity, the generalized form or rdc. however, the efficacy of misoprostil for hastening mucosal healing is clinically unproven in equine colitis. the primary drawbacks of prostaglandin analogs such as misoprostol are the side effects of the drug, including abdominal cramping, diarrhea, sweating, and abortion in pregnant mares. one can add psyllium mucilloid to the diet ( tablespoons once or twice daily) to increase the production of scfas in the colon. amylase-resistant fermentable fiber such as psyllium is hydrolyzed by colonic bacteria to scfas such as butyrate, which represent a major energy source for colonocytes. butyrate and other scfas hasten epithelial maturation and stimulate salt (and thus fluid) absorption in the colon, improve the clinical course of ulcerative colitis, and hasten colon healing. psyllium is itself a source of butyrate in the colon and also promotes the movement of amylase sensitive carbohydrates into the distal colon, which then are fermented to scfas. thus psyllium is thought to be clinically useful for promoting mucosal healing in colitis. many horses with salmonellosis or other forms of colitis have mild to severe signs of abdominal pain from gas and fluid distention of the colon, colonic ischemia, or infarction. one can accomplish analgesia with nsaids such as flunixin, but the potential for worsening mucosal injury or nephrotoxicity may prevent the use of analgesic doses, especially in horses with suspected nsaid toxicity. newer nsaids that specifically target cox- (the inducible cox) but have little activity against cox- (the constitutive cox) may be useful analgesics that spare the gastrointestinal mucosa. for example, etodolac ( to mg/kg intravenously or orally once daily) has analgesic properties in horses and may spare the intestinal mucosa from the detrimental effects associated with nonselective cox inhibitors (a.t. blikslager, personal communication, ) . however, the specificity for cox- in horses is unproven. thus avoiding the use of any nsaids in horses with rdc or other forms of nsaid toxicity is advisable. xylazine or detomidine may provide temporary relief of pain. butorphanol is a useful analgesic that one can administer intramuscularly ( . mg/kg every hours) or as a continuous infusion. an infusion of . µg/kg/hr in isotonic crystalloid fluid such as lactated ringer's solution has been suggested. continuous lidocaine infusions ( . mg/kg intravenous loading dose administered slowly over minutes and followed by mg/kg/hr infusion in isotonic crystalloid fluids) can provide profound visceral analgesia and may have added prokinetic benefits if ileus is present. broad-spectrum antibiotic treatment often is recommended in neutropenic horses or horses with signs of septicemia. neutropenia is associated with an increased risk of septicemia and septic complications such as septic phlebitis and infection of surgical site. septicemia is a potentially life-threatening complication of enterocolitis and may be caused directly by salmonella, clostridium, other invasive enteric bacteria, or indirectly by toxic injury to the colonic mucosa that breaks down the barrier to luminal microbes. neutropenia possibly may weaken host defenses enough to render horses susceptible to organisms that breach the mucosal barrier. although most attempts to culture bacteria from the blood of adult horses with colitis fail to isolate organisms, no detailed studies have been undertaken to determine the prevalence of bacteremia or septicemia in these patients. disseminated aspergillosis has been reported in horses as a complication of acute colitis, demonstrating the potential for systemic infections with rarely pathogenic organisms stemming from colonic mucosal injury in the face of potential immunosuppression from neutropenia. , broad-spectrum antibiotics lessen septic complications in human patients. however, evidence supporting this principle in horses with colitis is lacking. treatment with antibiotics is controversial in horses with salmonellosis and is not thought to alter the course of the enterocolitis. antibiotics directly targeted at the salmonella are reserved for patients with the enteric fever (septicemia) form of salmonellosis, documented with positive blood cultures. lipid-soluble antibiotics are suited ideally for salmonella infections, because the bacteria persist intracellularly. trimethoprim-sulfadiazine or other potentiated sulfa drugs, enrofloxacin, and chloramphenicol are preferred antibiotics for the enteric fever form of salmonellosis for this reason. as with other causes of enterocolitis, the use of antibiotics for equine monocytic ehrlichiosis is controversial. fear of inducing salmonellosis or other forms of antibiotic-induced diarrhea and the difficulty of diagnosing the disease early have caused most authors to recommend judicious use of antibiotics. however, in patients with a high suspicion of neorickettsia risticii infection, treatment with antibiotics often is indicated before definitive diagnosis. lipid-soluble drugs are desirable because the organism can live within cells. oxytetracycline ( . mg/kg intravenously every hours), doxycycline ( mg/kg orally every hours), trimethoprim-sulfadiazine ( mg/kg trimethoprim orally or intravenously every to hours and mg/kg sulfadiazine every to hours), or erythromycinrifampin ( mg/kg and mg/kg, respectively, orally every hours) have been used effectively to treat clinical cases. , [ ] [ ] [ ] the tetracyclines appear to be the most effective antibiotics for treatment of potomac horse fever. treatment is most successful if initiated before the onset of diarrhea. , clostridiosis if one has administered antibiotics since the onset of enterocolitis, one should discontinue administration as soon as possible. specific treatment with metronidazole ( to mg/kg orally every hours) is effective for treating clostridiosis in human beings and appears to be effective in horses. , metronidazole resistance in clinical isolates of clostridium difficile has been reported in one outbreak but appears to be rare in most human and equine cases. metronidazole-resistant isolates were sensitive to vancomycin, which may be effective for treating clinical cases if one suspects metronidazole resistance. however, metronidazole remains the treatment of choice. some authors describe the off-label use of c. perfringens type c antitoxin in cases of neonatal clostridiosis, described in more detail elsewhere. antitoxin preparations generally are not advocated for use in adult horses with clostridiosis. lawsonia intracellulare is susceptible to a variety of antibiotics in vitro, including chlortetracycline, erythromycin, penicillin, difloxacin, and ampicillin. lipid-soluble antibiotics with a large volume of distribution usually are chosen to treat proliferative enteropathy because l. intracellulare is an intracellular organism. erythromycin estolate ( to mg/kg orally every to hours) alone or with rifampin ( mg/kg orally every hours) is the most commonly reported efficacious treatment for proliferative enteropathy. chloramphenicol ( mg/kg orally every hours) has also been reported to be effective if erythromycin worsens the diarrhea. anecdotal reports suggest that oxytetracycline and doxycycline also may be effective. supportive care including maintenance of hydration and electrolyte balance and plasma or colloid administration to increase colloid oncotic pressure in hypoalbuminemic patients is also indicated. one should treat affected foals until clinical signs, hypoproteinemia, and ultrasonographic evidence of intestinal thickening resolve. the prognosis depends on the duration of the disease and the degree of fibrosis and destruction of the intestinal architecture. hypercoagulability is a common complication of enterocolitis, associated with systemic inflammation from endotoxemia. administration of heparin ( to iu/kg subcutaneously or intravenously every to hours) may prevent thrombosis in these patients, provided antithrombin iii concentrations are adequate in the plasma. concentrated sources of antithrombin iii are not available for use in horses, but whole plasma may provide an important source. treatment with heparin is thought to decrease thrombosis, especially of the jugular vein, a serious complication of salmonellosis. low-dose aspirin treatment ( mg/kg orally every to hours) along with heparin treatment may provide added benefit by irreversibly inhibiting platelet function. heparin and aspirin may have protective effects on the digital lamina. , heparin also may enhance the phagocytic activity of the reticuloendothelial system by enhancing the efficiency of opsonins such as fibronectin and immunoglobulin, thereby stimulating phagocytosis of products of coagulation and possibly other particles, including bacteria. , maintenance of the bacterial flora and antagonism of pathogenic bacteria such as salmonella in the gastrointestinal tract are important defense mechanisms preventing colonization by pathogenic bacteria. the use of probiotic preparations containing beneficial bacteria has been shown to prevent colonization of pathogenic bacteria, including salmonella, in poultry. little work has been done to investigate the efficacy of these products in preventing salmonellosis in horses, but ongoing studies may provide important information. probiotic and other preparations designed to restore normal flora to the gastrointestinal tract, such as fecal suspensions, sour milk, and yogurt, have been used clinically to shorten the course of salmonellosis, with variable results. therefore prevention of infection by using probiotic agents and other means is important. exposure of susceptible horses to salmonella should be avoided, but the task is difficult, especially because asymptomatic infections are common and the bacteria are ubiquitous in the environment. prophylactic use of probiotic preparations, judicious use of antibiotics in susceptible horses, control of environmental conditions such as temperature, and restricted exposure to pathogenic bacteria are important for control of salmonellosis. because altered large intestinal flora appears to play an important role in the pathogenesis of equine intestinal clostridiosis or any antibiotic-associated diarrhea, probiotic preparations have been advocated to treat affected horses. sour milk, a product containing lactose-producing streptococcus species, appears to improve the clinical course greatly in horses suspected of having clostridium perfringens type a infection. sour milk may benefit the patient by altering the flora and antagonizing enterotoxigenic c. perfringens type a but also is reported to be bactericidal against c. perfringens type a. preparations of saccharomyces boulardi are effective for reducing diarrhea and the frequency of c. difficile recurrence in human beings. however, whether relapse is a problem in horses with c. difficile colitis is not clear. lactobacillus preparations have a protective effect in human beings and decrease the severity and duration of antibioticassociated diarrhea. , however, evidence of their clinical usefulness in horses is lacking. good nursing care and adequate nutrition are vital to the treatment of horses with salmonellosis. salmonellosis is a severely catabolic disease, increasing caloric requirements greatly. normal intake of roughage to provide energy may be inadequate; however, one should avoid feeding of grains to prevent carbohydrate overload. dietary management usually consists of restricting or eliminating long-stem roughage (hay) from the diet and feeding exclusively a complete pelleted diet (at least % dietary fiber). the rationale behind this recommendation is to reduce the mechanical load on the colon. frequent meals ( to times a day) are recommended. one can add corn oil ( cup every to hours) to the pellets to increase the caloric intake without adding roughage or grain. one should note that if a horse with colitis refuses to eat pelleted feed, then one should feed good-quality grass hay. in anorectic or severely catabolic patients, enteral and parenteral nutrition (total and partial) has been used successfully to provide calories and nutritional support. strongylus vulgaris infection requires treatment of the migrating parasite larvae and the lesions produced by the parasite. fenbendazole ( mg/kg orally every hours for days or mg/kg orally every hours for days) and ivermectin ( mg/kg orally) are effective in killing fourth-stage larvae. other anthelmintics also may be part ii disorders of specific body systems effective when given at higher doses than those required to kill adult worms. the efficacy of these anthelmintics against larvae within thrombi is not known. thrombolytic and antithrombotic therapy has been advocated in horses with suspected strongylosis. , heparin ( to iu intravenously or subcutaneously every to hours) is often administered as an anticoagulant. aspirin ( to mg/kg orally every to hours) is usually combined with heparin to inhibit platelet adhesion. aspirin also may inhibit release of platelet products such as thromboxane that affect the motility of the large intestine. low-molecular-weight dextrans have been advocated as antithrombotics that act by inhibiting platelet function and coagulation. , the clinical efficacy of dextran administration appears to be good, but no controlled studies have been performed. anthelmintic administration is usually the only treatment necessary for mild to moderate cases of cyathostomiasis treated early in the course of the disease (within to weeks of onset). fenbendazole is effective against many larval stages, but resistance is increasing. although the reported efficacy of ivermectin varies against certain stages, one study reported an overall efficacy of %. currently, fenbendazole ( . to mg/kg orally every hours for days) followed on day by ivermectin ( mg/kg orally) is the most commonly advocated treatment regimen. , moxidectin ( µg/kg orally once daily) also may be effective against adults and l and l larval stages and may be useful for treating cyathostomiasis. antiinflammatory therapy also may be beneficial, especially in severe or refractory cases. nsaid administration may have limited value, but dexamethasone appears to be efficacious in refractory cases when used with larvicidal anthelmintics. , pretreatment with dexamethasone or prednisolone is indicated before anthelmintic administration if heavy larval loads are suspected to prevent an acute exacerbation of the disease by rapid death of encysted larvae. bismuth subsalicylate often is administered orally as an antisecretory agent in young animals. supportive care may be necessary in severe cases, particularly if hypoproteinemia is severe. horses occasionally require administration of intravenous crystalloid fluids and plasma or other colloids. proper nutritional support is also important. supportive care is the most important principle of therapy for cantharidin toxicity. intravenous fluid administration; maintenance of electrolyte balance, especially calcium; and prevention of further renal and urinary tract damage is important. , diuresis by intravenous fluid administration is often sufficient to prevent renal failure. furosemide often is administered after rehydration of the patient to further promote diuresis and to decrease the concentration of the toxin in the urine, which may ameliorate some of the effects on the urinary tract mucosa. diuresis also has been suggested to increase clearance of the toxin, but no evidence for this has been found. judicious use of nsaids may be necessary to control abdominal pain but should be reserved until the patient is rehydrated and renal failure has been ruled out. cantharidin is lipid-soluble; therefore oral administration of mineral oil may prevent further absorption of the toxin. activated charcoal often is administered with the mineral oil. to reduce arsenic absorption, one should initiate administration of cathartics such as mineral oil and magnesium sulfate slurries and activated charcoal by nasogastric tube immediately. chelation therapy with sodium thiosulfate to g in ml of water orally and dimercaprol (bal) mg/kg intramuscularly every hours is indicated. dimercaprol is a specific antidote for trivalent arsenicals, but its efficacy in horses is questionable. intravenous fluid administration may help treat shock, replace fluid lost in feces, and promote diuresis but should be monitored carefully because pulmonary edema is a frequent complication. the horse may require more specific treatment of renal, cardiac, pulmonary, or neurologic disease. treatment of intestinal anaphylaxis is in principle similar to treatment of other forms of colitis but is often unsuccessful because of the rapidly progressive nature of the syndrome. inclusion of heparin in intravenous fluids ( to iu/kg intravenously every to hours) may help prevent vascular thrombosis. administration of hypertonic saline solutions or colloids may prove to be useful during initial periods of shock. early treatment with prednisolone succinate ( to mg/kg intravenously) or dexamethasone ( . to . mg/kg intravenously) may be essential for successful treatment. mild cases of carbohydrate overload may not require treatment other than exclusion of grains from the diet for several days to weeks and gradual reintroduction of grain into the diet later if the horse needs the extra energy. patients showing signs of colic or diarrhea without other systemic signs may benefit from administration of mineral oil, charcoal, and fluids via nasogastric tube. one also may lavage residual carbohydrates from the stomach with the nasogastric tube. nsaids such as phenylbutazone ( . to . mg/kg/day intravenously) or flunixin meglumine ( mg/kg intravenously every hours) often are administered to prevent laminitis. phenoxybenzamine and heparin given before the onset of laminitis may prevent or decrease the severity of laminitis. , more severe cases with dehydrating diarrhea, systemic signs of endotoxemia, or metabolic acidosis require intravenous fluid support to maintain water, electrolyte, and acid-base balance in addition to the previously mentioned treatments. large amounts of bicarbonatecontaining solutions may be required. one should take care when administering hypertonic bicarbonate solutions, because many patients already may be hyperosmotic from lactic acidemia. isotonic sodium bicarbonate . % may be useful in the hyperosmotic patient. careful attention to calcium balance is also important, because severe hypocalcemia may occur. one should institute aggressive therapy for systemic inflammation from endotoxemia. one should administer broad-spectrum antibiotics intravenously to combat bacteremia and septicemia, which frequently complicate colitis induced by carbohydrate overload. in extreme cases, especially if the patient has ingested a large quantity of grain, surgical removal of the grain from the large intestine may be indicated, especially if one can accomplish surgery before the onset of severe clinical signs. however, administration of oral cathartics, such as magnesium sulfate slurries or mineral oil, or a combination of these, is often sufficient to clear the carbohydrates from the large intestine before fermentation, mucosal damage, and absorption of endotoxin and lactic acid occur. oral administration of activated charcoal may prevent absorption of endotoxin by binding the molecules in the lumen of the bowel. in any case, one should discontinue feeding of the source of the soluble carbohydrates, such as grains. one should feed the horse low-carbohydrate and low-protein roughage such as grass or oat hays until the microbial flora recovers. oral administration of probiotic preparations containing lactobacillus is contraindicated; however, other sources of normal equine large intestinal microbial flora, such as fecal extracts from normal feces, may be useful to reintroduce appropriate microorganisms. complications from laminitis and sepsis are common and often cause death. treatment of sand enteropathy requires removal of the sand from the gastrointestinal tract using psyllium products and magnesium sulfate slurries administered orally. analgesics may be required initially to relieve pain and stimulate appetite. a diet high in roughage often stimulates further passage of sand. treatment may require several weeks to remove as much sand as possible. prevention of the disease is important, and recurrence is not unusual. lumen results in clinical signs similar to those of simple obstruction, occlusion of the blood supply results in a more rapid deterioration of the intestinal mucosa and subsequent onset of endotoxemic shock. although a great deal of interest in the relevance and treatment of intestinal reperfusion injury has arisen recently, - the lesion that develops during strangulation is often severe, leaving little viable bowel for further injury during reperfusion. although extensive lengths of strangulated small intestine may be resected, strangulation of the large colon presents a much greater treatment dilemma because strangulated intestine usually extends beyond the limits of surgical resection. therefore horses with large intestinal strangulation often recover with extensive intestinal injury left in place. thus subtle degrees of reperfusion injury may be important in horses with large colon disease, warranting further work in this area in an attempt to reduce mortality. strangulating obstruction may be divided into hemorrhagic and ischemic forms. , hemorrhagic strangulating obstruction, which is most common, involves initial occlusion of veins before occlusion of arteries because of the greater stiffness of arterial walls. this lesion is characterized by a darkened appearance to affected bowel and increased thickness as blood is pumped into the lesion. ischemic strangulating obstruction occurs if the intestine is twisted tightly enough to occlude arteries and veins simultaneously. in the case of the colon, such strangulation has been suggested to be determined by how much ingesta is in the colon, because intestinal contents may prevent the intestine from twisting tightly. tissue involved in ischemic strangulating obstruction appears pale and of normal or reduced thickness because of a complete lack of blood flow ( figure . - ). bowel peripheral to strangulating lesions also may become injured because of distention, which reduces mural blood flow once it reaches critical levels. furthermore, as this intestine is decompressed, it also may undergo reperfusion injury. [ ] [ ] [ ] small intestinal strangulation horses with small intestinal strangulating obstruction typically have moderate to severe signs of abdominal pain that are only intermittently responsive to analgesic medications. during the latter stages of the disease process, horses may become profoundly depressed rather than painful as affected intestine necroses. horses have progressive signs of endotoxemia, including congested mucous membranes, delayed capillary refill time, and an elevated heart rate (> beats/min in most cases). in addition, one typically obtains reflux following passage of a stomach tube, and one usually can detect loops of distended small intestine on rectal palpation of the abdomen. however, these latter findings vary depending on the duration and location of the obstruction. for example, horses with ileal obstructions tend to reflux later in the course of the disease process than horses with a jejunal obstruction. furthermore, a horse that has an entrapment of small intestine in the epiploic foramen may not have palpable loops of small intestine because of the cranial location of these structures. abdominocentesis can provide critical information on the integrity of the intestine and is indicated in horses in which one suspects strangulation of the small intestine. a horse that has signs compatible with a small intestinal obstruction and additionally has serosanguinous abdominal fluid with an elevated protein level (> . mg/dl) is likely to require surgery, although one must differentiate these a b figure . - ischemic strangulating obstruction of the small colon by a mesenteric lipoma. a, the lipoma (arrow) has encircled a segment of small colon tightly. b, following resection of the lipoma, a pale area of strangulated small colon clearly is demarcated (arrows), the appearance of which is consistent with ischemic strangulating obstruction. cases from proximal enteritis. in general, horses with small intestinal strangulation show continued signs of abdominal pain, whereas horses with proximal enteritis tend to be depressed after initial episodes of mild abdominal pain. in addition, horses with small intestinal strangulation continue to deteriorate clinically despite appropriate medical therapy and will likely begin to show an increased white blood cell count (> , cells/µl) in the abdominal fluid as the duration of strangulation increases. however, cases occur in which the differentiation between small intestinal strangulation and proximal enteritis is not clear, at which point one may elect surgery rather than risking delay of abdominal exploration of a horse with a potential strangulating lesion. the prognosis for survival in horses with small intestinal strangulating lesions is generally lower than for most forms of colic. however, recent studies indicate that in excess of % of horses with small intestinal strangulating lesions are discharged from the hospital. nonetheless, veterinarians should warn owners that the long-term survival rates are reduced substantially to below %, in part because of long-term complications such as adhesions. , in addition, the prognosis is particularly low for some forms of strangulation, including entrapment of small intestine within a mesenteric rent. the epiploic foramen is a potential opening (because the walls of the foramen are usually in contact) to the omental bursa located within the right cranial quadrant of the abdomen. the foramen thus is bounded dorsally by the caudate process of the liver and caudal vena cava and ventrally by the pancreas, hepatoduodenal ligament, and portal vein. intestine may enter the foramen from the visceral surface of the liver toward the right body wall or the opposite direction. studies differ as to which is the most common form. , in the case of entrapments that enter the foramen in a left-to-right direction, the omental bursa ruptures as the intestine migrates through the epiploic foramen, which may contribute to intraabdominal hemorrhage often seen with this condition. clinical signs include acute onset of severe colic with examination findings compatible with small intestinal obstruction. the condition tends to be more prevalent in older horses, possibly because of enlargement of the epiploic foramen as the right lobe of the liver undergoes ageassociated atrophy. however, the disease also has been recognized in foals as young as months of age. one makes a definitive diagnosis at surgery, although ultrasonographic findings of distended loops of edematous small intestine adjacent to the right middle body wall suggest epiploic foramen entrapment. in general, thickened, amotile intestine on ultrasonographic examination is highly predictive for small intestinal strangulating obstruction. small intestine entrapped in the epiploic foramen may be limited to a portion of the intestinal wall (parietal hernia), and the large colon may become entrapped within the epiploic foramen. in treating epiploic foramen entrapment, one must not enlarge the epiploic foramen by blunt force or with a sharp instrument, because rupture of the vena cava or portal vein and fatal hemorrhage may occur. prognosis has improved substantially over the last decade, with current short-term survival rates (discharge from the hospital) ranging from % to %. preoperative abdominocentesis has been found consistently to be the most predictive test of postoperative survival. , lipomata form between the leaves of the mesentery as horses age and develop mesenteric stalks as the weight of the lipoma tugs on the mesentery. the stalk of the lipoma subsequently may wrap around a loop of small intestine or small colon causing strangulation. one should suspect strangulating lipomata in aged (> years old) geldings with acute colic referable to the small intestinal tract. , ponies also appear to be at risk of developing disease, suggesting alterations in fat metabolism may predispose certain horses to development of mesenteric lipomata. one usually makes the diagnosis at surgery, although on rare occasions one can palpate a lipoma per rectum. treatment involves surgical resection of the lipoma and strangulated bowel, although strangulated intestine is not always nonviable. studies indicate that approximately % to % of horses are discharged from the hospital following surgical treatment. a volvulus is a twist along the axis of the mesentery, whereas torsion is a twist along the longitudinal axis of the intestine. small intestinal volvulus theoretically is initiated by a change in local peristalsis or the occurrence of a lesion around which the intestine and its mesentery may twist (such as an ascarid impaction). volvulus is reportedly one of the most commonly diagnosed causes of small intestinal obstruction in foals. , the theory is that young foals may be at risk of small intestinal volvulus because of changing feed habits and adaptation to a bulkier adult diet. onset of acute, severe colic, a distended abdomen, and radiographic evidence of multiple loops of distended small intestine in a young foal suggest small intestinal volvulus. however, one cannot differentiate volvulus from other causes of small intestinal obstruction preoperatively. in adult horses, volvulus frequently occurs in association with another disease process, during which small intestinal obstruction results in distention and subsequent rotation of the small intestine around the root of the mesentery. although any segment of the small intestine may be involved, the distal jejunum and ileum are affected most frequently because of their longer mesenteries. one makes the diagnosis at surgery by palpating a twist at the origin of the cranial mesenteric artery. treatment includes resection of devitalized bowel, which may not be an option because of the extent of small intestinal involvement (similar to large colon volvulus). prognosis is based on the extent of small intestine involved and its appearance following surgical correction of the lesion. in general, horses with greater than % of the small intestine devitalized are considered to have a grave prognosis. a number of structures, when torn, may incarcerate a segment of intestine (typically the small intestine), including intestinal mesentery, the gastrosplenic ligament, the broad ligament, and the cecocolic ligament. horses with such incarcerations have signs typical of a horse with strangulating small intestine, including moderate to severe signs of abdominal pain, endotoxemia, absent gastrointestinal sounds, distended small intestine on per rectal palpation, nasogastric reflux, and serosanguinous abdominal fluid. however, the prognosis for many of these horses appears to be lower than for horses with other types of small intestinal strangulations. for example, in horses with small intestine entrapped in a mesenteric rent, only of horses were discharged from the hospital, and only of horses for which follow-up information was available survived long term (> months). poor outcome may result from the difficulty in unentrapping incarcerated intestine, the degree of hemorrhage, and the length of intestine affected. inguinal herniae are more common in standardbred and tennessee walking horses that tend to have congenitally large inguinal canals. inguinal herniae also may occur in neonatal foals but differ from herniae in mature horses in that they are typically nonstrangulating. the nature of the hernia (direct versus indirect) is based on the integrity of the parietal vaginal tunic. in horses in which the bowel remains within the parietal vaginal tunic, the hernia is referred to as indirect, because strictly speaking the bowel remains within the peritoneal cavity. direct herniae are those in which strangulated bowel ruptures through the parietal vaginal tunic and occupies a subcutaneous location. these direct herniae most commonly occur in foals and should be suspected when a congenital inguinal hernia is associated with colic, swelling that extends from the inguinal region or the prepuce, and intestine that may be palpated subcutaneously. , although most congenital indirect inguinal herniae resolve with repeated manual reduction or application of a diaper, surgical intervention is recommended for congenial direct herniae. historical findings in horses with strangulating inguinal herniae include acute onset of colic in a stallion that recently had been used for breeding. a cardinal sign of inguinal herniation is a cool, enlarged testicle on one side of the scrotum (figure . - ). , however, inguinal herniae also have been reported in geldings. one also can detect inguinal herniae on rectal palpation, and one can use manipulation of herniated bowel per rectum to reduce a hernia, but this is generally not recommended because of the risk of rectal tears. in many cases, the short segment of herniated intestine greatly improves in appearance after reduction and in some cases can be left unresected. the affected testicle will be congested because of vascular compromise within the spermatic cord, and although the testicle may remain viable, resection generally is recommended. the prognosis in adult horses is good, with up to % of horses surviving to months. horses that have been treated for inguinal herniae may be used for breeding. in these horses, the remaining testicle will have increased sperm production, although an increased number of sperm abnormalities will be noticeable following surgery because of edema and increased temperature of the scrotum. although umbilical herniae are common in foals, strangulation of herniated bowel is rare. in one study, of ( %) horses with umbilical herniae had incarcerated intestine. clinical signs include a warm, swollen, firm, and painful hernia sac associated with signs of colic. the affected segment of bowel is usually small intestine, but herniation of cecum or large colon also has been reported. in rare cases, one may find a hernia that involves only part of the intestinal wall, called a richter's hernia. in foals that have a richter's hernia, an enterocutaneous fistula may develop. in one study, of foals with strangulating umbilical herniae survived to discharge, although at least died of long-term complications. an intussusception involves a segment of bowel (intussusceptum) that invaginates into an adjacent aboral segment of bowel (intussuscipiens). the reason for such invagination is not always clear but may involve a lesion at the leading edge of the intussusception, including small masses, foreign bodies, or parasites. in particular, tapeworms (anoplocephala perfoliata) have been implicated. ileocecal intussusceptions are the most common intestinal intussusceptions in the horse and typically affect young animals. in one study evaluating cases of ileocecal intussusception, the median age of the horses was year old. acute ileocecal intussusceptions are those in which the horses has a duration of colic of less than hours and involve variable lengths of intestine that ranged in one study from to cm long. in acute cases the involved segment of ileum typically has a compromised blood supply. chronic ileocecal intussusceptions typically involve short segments of ileum (up to cm long), and the ileal blood supply is frequently intact. abdominocentesis results vary because strangulated bowel is contained within the adjacent bowel. obstruction of the small intestine often is evident, including nasogastric reflux and multiple distended loops of small intestine on rectal palpation. horses with chronic ileocecal intussusceptions have mild, intermittent colic, often without evidence of small intestinal obstruction. in one study, a mass was palpated in the region of the cecal base in approximately % of cases. transabdominal ultrasound may be helpful in discerning the nature of the mass. the intussusception has a characteristic target appearance on cross section. other segments of the small intestine also may be intussuscepted, including the jejunum (figure . - ) . in one study of jejunojejunal intussusceptions, the length of bowel involved ranged from . to . m. attempts to reduce intussusceptions at surgery are usually futile because of intramural swelling of affected bowel. one should resect jejunojejunal intussusceptions. for acute ileocecal intussusceptions, one should transect the small intestine as far distally as possible and perform a jejunocecal anastomosis. in horses with particularly long intussusceptions (up to m has been reported), one may attempt an intracecal resection. for horses with chronic ileocecal intussusceptions, one should perform a jejunocecal bypass without small intestinal transection. the prognosis is good for horses with chronic ileocecal intussusceptions and guarded to poor for horses with acute ileocecal intussusceptions, depending on the length of bowel involved. herniation of intestine through a rent in the diaphragm is rare in the horse and may involve any segment of bowel, although small intestine is herniated most frequently. diaphragmatic rents may be congenital or acquired, but acquired herniae are more common. congenital rents may result from incomplete fusion of any of the four embryonic components of the diaphragm: pleuroperitoneal membranes, transverse septum, and esophageal mesentery. in addition, abdominal compression of the foal at parturition may result in a congenital hernia. acquired herniae are presumed to result from trauma to the chest or a sudden increase in intraabdominal pressure, such as might occur during parturition, distention of the abdomen, a sudden fall, or strenuous exercise. herniae have been found in a number of different locations, although large congenital herniae are typically present at the ventral most aspect of the diaphragm, and most acquired herniae are located at the junction of the muscular and tendinous portions of the diaphragm. a peritoneopericardial hernia has been documented in at least one horse. figure . - jejunojejunal intussusception in a horse presented for colic. the intussusceptum has become ischemic because of invagination of intestine and its mesenteric blood supply into the intussuscipiens. clinical signs usually are associated with intestinal obstruction rather than respiratory embarrassment. however, careful auscultation may reveal an area of decreased lung sounds associated with obstructed intestine and increased fluid within the chest cavity. such signs may prompt thoracic radiography or ultrasound, both of which one can use to make a diagnosis. auscultation also may reveal thoracic intestinal sounds, but differentiating these from sounds referred from the abdomen typically is not possible. in one report, two of three horses diagnosed with small intestinal strangulation by diaphragmatic hernia had respiratory acidemia attributable to decreased ventilation. treatment of horses with diaphragmatic hernia is fraught with complications because of the need to reduce and resect strangulated bowel and the need to repair the defect in the diaphragm. , because dorsal defects in the diaphragm are among the common forms of diaphragmatic defect, closing the diaphragmatic hernia via the approach used for abdominal exploration may not be possible. however, because herniation is likely to recur, scheduling a second surgery using an appropriate approach to resolve the diaphragmatic defect is appropriate. horses with large colon volvulus have rapid onset of severe, unrelenting abdominal pain, most often in postpartum broodmares. once the large colon strangulates (≥ -degree volvulus), gas distention is significant, leading to gross distention of the abdomen, compromised respiration as the distended bowel presses up against the diaphragm, and visceral pooling of blood as the caudal vena cava is compressed. horses with this condition are frequently refractory even to the most potent of analgesics. these horses may prefer to lie in dorsal recumbency, presumably to take weight off the strangulated colon. an abbreviated physical examination is warranted in these cases, because the time elapsed from the onset of strangulation to surgical correction is critical. under experimental conditions, the colon is irreversibly damaged within to hours of a -degree volvulus of the entire colon. despite severe pain and hypovolemia, horses may have a paradoxically low heart rate, possibly related to increased vagal tone. in addition, results of abdominocentesis often do not indicate the degree of colonic compromise , and in many cases are not worth attempting because of extreme colonic distention. palpation per rectum reveals severe gas distention of the large colon, often restricting access to the abdomen beyond the pelvic brim. one may make the diagnosis tentatively based on signalment, severity of pain, and degree of distention. at surgery, the volvulus typically is located at the mesenteric attachment of the colon to the dorsal body wall and the most common direction of the twist is dorsomedial using the right ventral colon as a reference point. however, the colon may twist in the opposite direction, twist greater than degrees (up to degrees has been reported) or twist at the level of the diaphragmatic and sternal flexures. in all cases, one should decompress the colon as much as possible, and in many cases a colonic evacuation via a pelvic flexure enterotomy greatly aids correction of the volvulus. one must determine after correction of the volvulus whether the colon has been injured irreversibly and should base the determination on mucosal color and bleeding (if an enterotomy has been performed), palpation of a pulse in the colonic arteries, serosal color, and appearance of muscular motility. if one judges the colon to be damaged irreversibly, one can consider the feasibility of a large colon resection. although % of the colon can be resected (that part of the colon distal to the level of the cecocolic fold), damage from the volvulus usually exceeds that which can be resected. in these cases, surgeons may elect to resect as much damaged bowel as possible or may advise euthanasia. the prognosis is guarded to poor because of the rapid onset of this disease. in one study the survival rate was %. in a more recent report the survival rate was % for horses with -degree volvulus of the large colon compared with % for horses with -degree volvulus. however, one study in central kentucky documented a high success rate, possibly because of early recognition of the disease and the proximity of the hospital to the surgical caseload. postoperative complications include hypovolemic and endotoxic shock, extensive loss of circulating protein, disseminated intravascular coagulation, and laminitis. in addition, large colon volvulus has a propensity to recur. although one study documented a recurrence rate of less than %, some authors believe recurrence may be as high as %. therefore one should consider methods to prevent recurrence in patients at risk of recurrence, particularly broodmares that tend to suffer from the disease recurrently during the foaling season. , the most common intussusceptions of the large intestine are cecocecal and cecocolic intussusceptions. , both are likely attributable to the same disease process, with variable inversion of the cecum. these conditions doughnut-shaped prolapse of rectal mucosa and submucosa. type ii prolapses involve full-thickness rectal tissue, whereas type iii prolapses additionally have invagination of small colon into the rectum. type iv prolapses involve intussusception of proximal rectum or small colon through the anus in the absence of prolapse of tissue at the mucocutaneous junction at the anus. one can differentiate type iv from other forms of prolapse by their appearance and a palpable trench between prolapsed tissue and the anus. type i prolapses occur most frequently in horses with diarrhea, in which the rectal mucosa becomes irritated and protrudes intermittently during episodes of tenesmus. if tenesmus persists, rectal mucosa can remain prolapsed. rectal mucosa rapidly becomes congested and edematous under these conditions, which one should treat with osmotic agents such as glycerin or magnesium sulfate and by massaging and reducing the prolapse. a purse-string suture may be required to keep the mucosa inside the rectum. topical application of lidocaine solution or jelly, epidural anesthesia, and sedation may help reduce tenesmus that incites and exacerbates rectal prolapse. one can apply similar treatments to type ii rectal prolapses. however, these more severe prolapses may not be reducible without surgical resection of mucosa and submucosa from the prolapsed bowel. , type iii and iv rectal prolapses are more serious injuries because of involvement of small colon. in horses with type iii prolapses, one should perform an abdominocentesis to determine if injured small colon has resulted in peritonitis. one should reduce the small colon component manually if possible, although prolapsed rectal tissue typically requires mucosal/ submucosal resection. one should perform surgical exploration of the abdomen to determine the status of the small colon, although one can use serial abdominocenteses in lieu of surgery to detect progressive necrosis of bowel. type iv prolapses occur most commonly in horses with dystocia. these prolapses are almost always fatal because of stretching and tearing of mesenteric vasculature, with subsequent infarction of affected bowel. therefore euthanasia usually is warranted tend to occur in young horses ( % were less than years old in one study) and may be associated with intestinal tapeworms. horses show highly variable clinical signs, including acute severe colic, intermittent pain over a number of days, or chronic weight loss. these variable presentations likely relate to the degree to which the cecum has intussuscepted. initially, the cecal tip inverts, creating a cecocecal intussusception, which does not obstruct flow of ingesta. as the intussusception progresses, the cecum inverts into the right ventral colon (cecocolic intussusception), obstructs flow of ingesta, and often causes severe colic. the cause of abdominal pain is often difficult to differentiate in these cases, although detecting a mass on the right side of the abdomen by per rectal palpation or ultrasound examination sometimes is possible. , treatment involves manual surgical reduction by retracting the intussusceptum directly or via an enterotomy in the right ventral colon. however, a number of cases occur in which one cannot reduce the cecum readily because of severe thickening or in which surgical procedures result in fatal contamination. for example, one report stated that of horses were euthanized in the perioperative period because of complications, and another report stated that of horses were euthanized before or during surgery. the latter included all of the horses with chronic disease because of irreversible changes to the cecum. however, one recent report on cecocolic intussusceptions indicated that seven of eight horses that underwent right ventral colon enterotomy and cecal resection survived long-term, suggesting that continued improvements in surgical techniques may improve the prognosis. colocolic intussusceptions are rare but have been reported to affect the pelvic flexure and the left colons. [ ] [ ] [ ] [ ] although the condition is reported to be more common in young horses, - the condition may affect older horses. clinical findings may include a palpable mass on the left side of the abdomen. ultrasonography also may be useful. treatment requires manual reduction of the intussusception at surgery, , or resection of affected bowel. because the left colons may be exteriorized extensively and manipulated at surgery, - the prognosis is fair. rectal prolapse may occur following any disease that causes tenesmus, including diarrhea, rectal neoplasia, and parasitism, or prolapse can occur following elevations in intraabdominal pressure during parturition or episodes of coughing. , rectal prolapses are classified into four categories (table . - ) based on the extent of tissue prolapsed and the severity. type i rectal prolapse is most common and is characterized by a intussusception of rectum and poor small colon through the anus based on physical examination findings. however, confirmation of severe small colonic injury requires abdominal exploration via a midline approach or laparoscopy. a horse with compromised small colon conceivably could undergo a colostomy of the proximal small colon, but the compromised small colon typically necroses beyond that which can be resected via a midline abdominal approach. nonstrangulating infarction occurs following cranial mesenteric arteritis caused by migration of strongylus vulgaris and has become a rare disorder since the advent of broad-spectrum anthelmintics. although thromboemboli have been implicated in the pathogenesis of this disease, careful dissection of naturally occurring lesions has not revealed the presence of thrombi at the site of intestinal infarctions in most cases. these findings suggest that vasospasm plays an important role in this disease. clinical signs vary greatly depending on the extent to which arterial flow is reduced and the segment of intestine affected. any segment of intestine supplied by the cranial mesenteric artery or one of its major branches may be affected, but the distal small intestine and large colon are more commonly involved. no clinical variables exist that one can use to differentiate this disease from strangulating obstruction reliably. in some cases, massive infarction results in acute, severe colic. occasionally, one may detect an abnormal mass and fremitus on palpation of the root of the cranial mesenteric artery per rectum. one should consider this disease a differential diagnosis in horses with a history of inadequate anthelmintic treatment and the presence of intermittent colic that is difficult to localize. although one should perform fecal parasite egg counts, they are not indicative of the degree of parasitic infestation. in addition to routine treatment of colic, dehydration, and endotoxemia, medical treatment may include aspirin ( mg/kg every hours) to decrease thrombosis. definitive diagnosis requires surgical exploration. however, these cases are difficult to treat because of the patchy distribution of the lesions and the possibility of lesions extending beyond the limits of surgical resection. in addition, further infarction may occur following surgery. the prognosis is fair for horses with intermittent mild episodes of colic that may be amenable to medical therapy but is poor in horses that require surgical intervention. , surgical exploration of a horse with on-going intestinal injury exacerbates shock induced largely by endotoxin traversing damaged mucosa, and this in turn correlates with mortality. the initial clinical step in the workup of horses with colic is taking a thorough history. however, one may have to delay taking a complete history until after the physical examination and initial treatment, because management of abdominal pain may take precedence. if possible, one should obtain the vital components of the history before examination and treatment: the duration and severity of colic symptoms, analgesics already administered, and a history of any adverse drug reactions. the two most critical factors from a history that would support a decision to explore a horse with colic surgically are the duration of signs and the extent of pain. one deduces the latter from asking the owner about the presence and frequency of pawing, looking at the flanks, rolling, repeatedly going down and getting back up, posturing as if to lie down or urinate, among other clinical evidence of pain. table . - lists other important components of the history one should obtain to try to ascertain why colic has occurred. just as the history necessarily may need to be brief to allow rapid treatment of colic, so the clinician must be able to alter the extent of the physical examination to treat the horse in a timely fashion. the most critical examination finding is the heart rate of the horse, because it provides an excellent assessment of the cardiovascular status of the horse. the heart rate is likely the single most reliable predictor of the need for surgery and survival. , because analgesics can alter the heart rate dramatically, if possible, one should obtain the heart rate before administering analgesics. other components of the examination are designed specifically to gather information about the cardiopulmonary status of the horse (quality of the pulse, mucous membrane color, capillary refill time, respiratory rate, and full auscultation of the chest), and the nature of the intestinal obstruction (auscultation of gastrointestinal sounds, per rectal palpation of the abdomen, and presence of nasogastric reflux). although classic presentations exist for horses with obstructions of the small or large intestine (table . clinical management of colic is distinctly different from management of many other clinical syndromes because the initial focus is often not on defining the definitive diagnosis but rather on deciding whether a horse requires surgical exploration. therefore the clinician must collect historical, physical examination, and clinicopathologic information and make a decision whether these findings warrant medical management or whether to perform surgical exploration of the abdomen because of a suspected obstructive or ischemic lesion. for example, one may examine a horse with signs of severe abdominal pain, poor cardiovascular status, and abdominal distention that may be compatible with an extensive list of differential diagnoses but that more importantly indicate the need for abdominal exploration to minimize the extent of intestinal injury. the speed with which one can make this clinical decision has a tremendous effect on the well-being of the patient, , because delaying gastric fluid accumulation because of direct compression of the small intestine by distended colon or via tension on the duodenocolic ligament. the most useful diagnostic test for determining the type of intestinal obstruction is rectal palpation of the abdomen. however, one can reach only approximately one third of the abdomen via the rectum, and this percentage may be substantially less in large horses or heavily pregnant horses. nonetheless, attempting to determine the type of obstruction present (small intestine versus large intestine, and simple obstruction versus strangulating obstruction) is worthwhile; this information directly affects prognosis. in one study, interns and residents at a veterinary teaching hospital were able to predict the type of lesion with a specificity exceeding %. findings from palpation are helpful in educating the client about the potential findings in surgery and the likelihood of survival for the horse. before considering how to manage signs of colic, one should remember that such signs are poorly localized. therefore although colic is most frequently associated with intestinal disease, one should consider dysfunction of other organ systems, including urinary obstruction, , biliary obstruction, uterine torsion or tears, , ovarian artery hemorrhage, and neurologic disease as differential diagnoses. however, the duration and severity of colic the most immediately useful clinicopathologic information in horses with colic are the packed cell volume and total protein, because one can use them to substantiate clinical estimates of dehydration and they correlate strongly with prognosis. , a serum biochemical profile is useful for assessing electrolyte imbalances, tissue perfusion (anion gap or lactate), and kidney and liver function. one can use serum biochemical or blood gas analysis to assess acid-base status. horses with colic most frequently show evidence of metabolic acidosis associated with poor tissue perfusion caused by hypovolemia or endotoxemia, but one may note other abnormalities such as metabolic alkalosis in association with extensive loss or sequestration of stomach chloride. metabolic acidosis has been investigated further in horses with colic by measuring blood lactate, although this test is not offered routinely in many laboratories. lactate levels also have been inferred from measurement of the anion gap, although one study noted that lactate in horses with colic did not account for the entire anion gap. lactate levels and anion gap closely correlate with prognosis for survival. , , other key components of assessment of the horse with colic are abdominocentesis and complete blood count. the total white blood cell count and differential can provide crucial evidence of systemic inflammation associated with endotoxemia stemming from colic attributable to colitis (leukopenia, neutropenia, and a left shift) rather than an obstruction (highly variable complete blood count findings). peritoneal fluid may be helpful in determining the integrity of the intestine. specifically, as the intestine becomes progressively devitalized, the peritoneal fluid becomes serosanguinous as red blood cells leak into the abdomen, followed by an elevation in the total protein (> . g/dl) and progressive increases in total nucleated cell count (> , cells/µl). however, these findings do not always correlate well with the condition of the intestine, particularly in horses with large colon volvulus. for example, in a study of horses with large colon volvulus, the average total protein ( . g/dl) and total nucleated cell count ( cells/µl) were normal despite the fact that only % with a -degree volvulus survived. , these measures may appear normal because the development of severe mucosal injury following large colon volvulus is rapid and may not allow enough time for protein and leukocytes to equilibrate with the abdominal fluid. investigators have taken all the variables routinely assessed during evaluation of horses for colic and have attempted to develop models to predict accurately the need for surgery and the prognosis for life. [ ] [ ] [ ] [ ] none of these predictor models has taken the place of clinical decision making, although these studies have added part ii disorders of specific body systems signs are excellent predictors of whether a horse requires surgical exploration of the abdomen. in fact, refractory pain supersedes all other predictors of the need for surgery in the colic patient. once signs of colic have been recognized and categorized as to their severity, rapidly and effectively relieving the pain is critical for the well-being of the horse and to reduce the owner's anxiety. in addition, pain is best managed before it becomes severe. several classes of analgesics are readily available to treat horses with colic (table . - ), including α -agonists (xylazine, detomidine), opiates (butorphanol), and nonsteroidal antiinflammatory drugs (nsaids, such as flunixin meglumine). although much of this information is familiar to most practitioners, several principles deserve emphasis. the short-duration drugs xylazine and butorphanol, which provide analgesia for to minutes, allow the veterinarian to determine if pain is recurrent within the time period of the typical examination. in contrast, flunixin meglumine is not as potent as an analgesic but has a much longer duration of action. to avoid deleterious effects on gastrointestinal mucosa and the kidneys, one should not administer flunixin meglumine more frequently than recommended. , the recent discovery of two isoforms of cyclooxygenase (cox), the enzyme inhibited by nsaids, has resulted in discovery of drugs that can more selectively inhibit proinflammatory cox- while permitting continued constitutive production of prostanoids. such specificity may be advantageous in horses with colic, particularly when one considers recent evidence of reduced intestinal recovery from an ischemic event with flunixin compared with a drug that is more selective for cox- . one should reserve the α agonist detomidine for horses with severe, unrelenting pain because of its tremendous potency. in addition, one should remember that α -agonists reduce the heart rate associated with a transient increase in blood pressure, , thereby reducing the predictive value of the heart rate and pulse pressure. tremendously to understanding of the importance of some prognostic factors, particularly those reflecting cardiovascular function. simple obstruction involves intestinal obstruction of the lumen without obstruction of vascular flow. however, because a tremendous volume of fluid enters the small intestinal lumen daily, , the obstructed intestine tends to become distended, which in turn may reduce mural blood flow. ultimately, such distention may result in necrosis of tissues, particularly in the immediate vicinity of the obstruction. few are the causes of simple obstruction in the small intestine, and the incidence of these obstructions is low (approximately % of all referred horses in one large hospital-based study). however, in some geographic regions, this type of obstruction is prevalent. for example, in the southeastern united states, ileal impactions are common. ileal impactions most commonly occur in adult horses in the southeastern united states. although feeding of coastal bermuda hay has been implicated in the regional distribution of the disease, separating geographic location from regional hay sources as risk factors has been difficult. nonetheless, feeding coastal bermuda hay likely places horses at risk of ileal impaction, particularly if the coarse fiber content of the hay is high. furthermore, sudden changes in feed from an alternate type of hay to coastal bermuda hay likely places a horse at risk of ileal impaction. studies in england have revealed tapeworm infection as another important risk factor for ileal impaction. based on risk analysis, the data suggested that in excess of % of the ileal impaction cases studied were associated with serologic or fecal evidence of tapeworm infection. because of the poor sensitivity of fecal analysis for tapeworms, proudman and trees have developed a serologic test (enzyme-linked immunosorbent assay) with a sensitivity of approximately % and a specificity of %. clinical signs of horses with ileal impaction are typical for a horse with small intestinal obstruction, including onset of moderate to severe colic and loops of distended small intestine palpable per rectum as the condition progresses. because the ileum is the distal most aspect of the small intestinal tract, nasogastric reflux may take a considerable time to develop and is found in approximately % of horses requiring surgical correction of impacted ileum. , one usually makes the diagnosis at surgery, although on occasion one may palpate an impacted ileum per rectum. multiple loops of distended small intestine frequently make the impaction difficult to palpate. ileal impactions may resolve with medical treatment but frequently require surgical intervention ( figure . - ) . at surgery, one can infuse fluids directly figure . - appearance of roundworms that have been retrieved within the nasogastric reflux from a foal with an ascarid impaction. the large size of these ascarids (bar = cm) contributes to the risk of impaction following sudden kills of these parasites by broad-spectrum anthelmintics. into the mass, allowing the surgeon to breakdown the impaction. the surgeon may include dioctyl sodium sulfosuccinate in the infused fluid to aid in disruption of the mass. extensive small intestinal distention and intraoperative manipulation of the ileum may lead to postoperative ileus, but recent studies indicate that this complication is less frequent as the duration of disease before admission decreases. recent studies indicate that the prognosis for survival is good. , ileal hypertrophy is a disorder in which the muscular layers (circular and longitudinal) of the ileum hypertrophy for unknown reasons (idiopathic) or following an incomplete or functional obstruction. for idiopathic cases, proposed mechanisms include parasympathetic neural dysfunction resulting in chronically increased muscle tone and subsequent hypertrophy of the muscular layers of the ileal wall. such neural dysfunction possibly could result from parasite migration. alternative hypotheses include chronic increases in the muscular tone of the ileocecal valve, leading to muscular hypertrophy of the ileum as it contracts against a partially occluded ileocecal valve. the jejunum also may be hypertrophied, alone or with the ileum. clinical signs include chronic intermittent colic as the ileum hypertrophies and gradually narrows the lumen diameter. in one study, partial anorexia and chronic weight loss ( to months) were documented in % of the horses, most likely because of intermittent colic and reduced appetite. because hypertrophy does not affect the ileal mucosa, no reason exists to believe that these horses experience malabsorption of nutrients. one usually makes the diagnosis at surgery, although one may palpate the hypertophied ileum per rectum in some cases. for treatment, one performs an ileocecal or jejunocecal anastomosis to bypass the hypertrophied ileum. without surgical bypass, intermittent colic persists and the thickened ileum ultimately may rupture. the prognosis is fair with surgical treatment. secondary ileal hypertrophy is most commonly notable in horses that previously have had colic surgery and that may have a partial or functional obstruction at an anastomotic site. for example, in one case report, a horse developed ileal hypertrophy after surgical correction of an ileocecal intussusception. ileal hypertrophy also was noted in a horse with cecal impaction in which an ileocolic anastomosis was oriented incorrectly. horses are typically re-presented for recurrence of colic in these cases. surgical therapy is directed at addressing the cause of small intestinal obstruction and resecting hypertrophied intestine. meckel's diverticulum is an embryonic remnant of the vitelloumbilical duct, which fails to atrophy completely and becomes a blind pouch projecting from the antimesenteric border of the ileum. , however, similar diverticula also have been noted in the jejunum. these diverticula may become impacted, resulting in partial luminal obstruction, or may wrap around an adjacent segment of intestine, causing strangulation. occasionally, an associated mesodiverticular band may course from the diverticulum to the umbilical remnant and serve as a point around which small intestine may become strangulated. mesodiverticular bands also may originate from the embryonic ventral mesentery and attach to the antimesenteric surface of the bowel, thereby forming a potential space within which intestine may become entrapped. clinical signs range from chronic colic for an impacted meckel's diverticulum to acute severe colic for intestine strangulated by a mesodiverticular band. one makes the diagnosis at surgery, and treatment requires resection of the diverticulum and any associated bands. the prognosis is good for horses with simple impaction of a meckel's diverticulum and is guarded for horses with an associated small intestinal strangulation. adhesions of one segment of bowel to another or of a segment of intestine to other organs and the body wall most typically occur following abdominal surgery and may be clinically silent, cause chronic colic attributable to partial obstruction, or result in acute obstruction. these differing clinical syndromes are attributable to the type of adhesions that develop. for example, a fibrous adhesion that does not by itself obstruct the intestinal lumen might serve as the pivot point for a volvulus, whereas an adhesion between adjacent segments of the intestinal tract may create a hairpin turn that causes chronic partial obstruction. the number of adhesions that develop also may vary greatly from horse to horse. some horses may develop a single adhesion adjacent to an anastomotic site or a discrete segment of injured intestine, whereas other horses may develop diffuse adhesions involving multiple segments of intestine, likely because of widespread inflammation of the peritoneum at the time of the original surgery. the mechanism whereby adhesions develop is complex but likely involves initial injury to the serosa initiated by intestinal ischemia, reperfusion injury, and luminal distention. importantly, such injury involves infiltration of neutrophils into the serosa accompanied by loss of mesothelial cells. in one study assessing the margins of resected small intestine, extensive neutrophil infiltration was documented in the serosa, particularly in the proximal resection margin that had been distended before correction of a variety of strangulating lesions. regions of serosal injury and inflammation subsequently undergo reparative events similar to any wound, including local production of fibrin, de novo synthesis of collagen by infiltrating fibroblasts, and ultimately maturation and remodeling of fibrous tissue. unfortunately, during this process, fibrin may result in injured intestinal surfaces adhering to adjacent injured bowel or an adjacent organ. once a fibrinous adhesion has developed, new collagen synthesis may result in a permanent fibrous adhesion. alternatively, proteases released by local phagocytes may lyse fibrinous exudate, thereby reversing the adhesive process. thus one can view formation of adhesions as an imbalance of fibrin deposition and fibrinolysis. prevention of adhesions depends on inhibition of the mechanisms involved in adhesion formation, including reduction of serosal injury with early intervention and good surgical technique, reduction of inflammation by administration of antiinflammatory medications, physical separation of inflamed serosal surfaces (e.g., carboxymethylcellulose and hyaluronan), [ ] [ ] [ ] and pharmacologic modulation of fibrinous adhesion formation (e.g., heparin). in addition, early return of motility in the small intestine after surgery may reduce contact time between inflamed surfaces of intestine, thereby reducing the chances of adhesion formation. horses at greatest risk of developing adhesions after colic surgery appear to be those that have small intestinal disease. , in one study of horses undergoing surgical correction of small intestinal obstruction, % developed a surgical lesion associated with adhesions. foals appear to have an increased incidence of adhesions compared with mature horses regardless of the nature of the abdominal surgery. one study indicated that % of foals developed lesions attributable to adhesions regardless of the type of initial surgery. studies conflict as to whether the degree of surgical intervention influences adhesion formation, but in one study, horses that require enterotomy or resection and anastomosis were at greatest risk of developing adhesions. as an indication of the importance of postoperative adhesion formation, adhesions were among the most common reasons for repeat laparotomy in postoperative colic patients. , clinical signs of horses with adhesions vary greatly depending on whether the adhesion is causing partial obstruction or complete luminal obstruction or involves intestinal vasculature. adhesions would be an important differential diagnosis for intermittent colic in the postoperative period, particularly if such colic was not relieved by nasogastric decompression of the stomach. continued intermittent colic should prompt abdominocentesis to determine if septic peritonitis is present, which may contribute to adhesion formation. placement of a large bore drain and peritoneal lavage ( figure surgery should prompt immediate nasogastric intubation to decompress the stomach. treatment should include attempts at obtaining reflux from the horse at frequent intervals rather than relying on passive flow of reflux. in addition, administration of intravenous fluids should account for the maintenance requirement ( ml/kg/day, about l/hr in the average horse) and fluid losses via reflux. in practice, this requires frequent monitoring of packed cell volume and total protein to ensure that the horse remains well hydrated. although concerns have arisen that overhydrating horses may contribute to increased nasogastric reflux, keeping horses well-hydrated to avoid hypovolemic shock is critical. additionally, one should monitor electrolytes frequently, particularly considering their potential role in smooth muscle contraction and nerve excitability. because of the important role of inflammation in postoperative ileus, including elaboration of cox- -produced prostanoids, administration of nsaids is indicated. nsaid administration is particularly necessary if postoperative ileus is associated with endotoxemia, because lipopolysaccharide-induced prostanoid production disrupts propulsive motility in horses. , interestingly, phenylbutazone is more effective than flunixin meglumine at reducing the deleterious actions of lipopolysaccharide on intestinal motility. however, one should use caution when administering nsaids to patients with postoperative ileus in light of research suggesting that complete inhibition of prostanoid production can alter motility patterns in normal equine intestine. the advent of selective cox- inhibitors may provide optimal antiinflammatory treatment in the future. other treatments aimed at specifically modulating intestinal motility include lidocaine (bolus of . mg/kg followed by . mg/kg/min for hours), erythromycin ( . to . mg/kg slow intravenous infusion in l saline every hours), and metoclopramide ( . mg/kg/hr). , , the mechanism of lidocaine is presumed to be inhibition of sensory nerve activity within the wall of the intestine, thereby reducing reflex sympathetic inhibitory activity. in addition, intravenously administered lidocaine appears to be an effective analgesic. thus an important feature of intravenous lidocaine therapy may be to control postoperative pain-induced reduction of gastrointestinal motility and mucosal secretory activity. metoclopramide may stimulate intestinal motility by several mechanisms, including dopamine receptor blockade, cholinergic stimulation, and adrenergic blockade. although metoclopramide has been shown to be beneficial for reversing postoperative ileus in clinical patients and research animals, it has central nervous system excitatory side effects in the horse that make its use difficult. nonetheless, administration of metoclopramide to horses with postoperative ileus resulted in elect repeat laparotomy or laparoscopy. in one study of adhesions, % of repeat laparotomies were performed within days, suggesting that surgical colic attributable to adhesions typically occurs within months of an initial surgical procedure. unfortunately, the prognosis for horses with colic attributable to adhesions is low, with only % of horses in one study surviving from adhesion-induced colic. the definition of ileus is intestinal obstruction, including physical and functional obstructions. however, in veterinary medicine, the term typically is used to designate a lack of progressive aboral propulsion of ingesta resulting in functional obstruction. one typically bases the diagnosis of postoperative ileus on the presence of excessive gastric fluid accumulation (reflected as excessive nasogastric reflux). postoperative ileus may occur following any abdominal exploratory procedure. however, horses undergoing surgery for strangulating small intestinal lesions or small intestinal obstructive lesions such as an ileal impaction are at greatest risk. recently, the syndrome of postoperative ileus in horses has been broadened to include those horses that may have delayed transit of ingesta through the large intestine following surgery. this large intestinal ileus may follow any type of surgery, particularly horses that have had orthopedic surgery, and is characterized by reduced fecal output (fewer than three piles of manure per day) rather than excessive nasogastric reflux. however, horses with excessive nasogastric reflux are unlikely to have normal fecal output, so the distinction between these two manifestations of ileus is not absolute. mechanisms involved in precipitating postoperative ileus characterized by small intestinal dysfunction likely involve local inflammation, reduced coordination of progressive motility, and increased sympathetic tone. a recent series of studies in the rat has shown that surgical manipulation of intestine results in delayed transit time associated with infiltration of neutrophils into intestinal longitudinal muscle [ ] [ ] [ ] and upregulation of inducible nitric oxide synthase and cox- . the mechanisms in the horse may be similar in that extensive manipulation of the intestine resulted in abnormal intestinal motility in ponies, and prostanoids and nitric oxide alter or reduce intestinal motility in horses. [ ] [ ] [ ] clinical signs of postoperative ileus following colic surgery include evidence of abdominal pain, increased heart rate, reduced gastrointestinal sounds, and reflux of gastric fluid via a nasogastric tube. of these signs, heart rate is critical because it appears to be a more sensitive indicator of pain in the postoperative period than overt evidence of colic. therefore a sudden increase in the heart rate of a postoperative patient following colic a significantly reduced duration of reflux and shorter postoperative hospital stays compared with horses not receiving this drug. in the same study, constant infusion of metoclopramide was superior to intermittent infusion. recent in vitro studies indicate that metoclopramide effectively increases smooth muscle contractile activity throughout the small intestine. similarly, the motilin agonist erythromycin had stimulatory effects on equine small intestine, although the results were not uniform throughout the small intestine. erythromycin stimulates contractile activity in the longitudinal muscle of the pyloric antrum but inhibits contractile activity in circular smooth muscle in this segment of the gastrointestinal tract. the latter may be attributable to activation of motilin receptors on inhibitory nerves and may result in enhanced gastric emptying. in vivo studies on erythromycin confirmed the stimulatory action of this drug on the distal small intestine and indicated this drug also stimulates contractile activity in the cecum and pelvic flexure. however, the stimulation depends on the temporal association with surgery. erythromycin stimulated contractile activity in the postoperative period in the ileum and pelvic flexure but not the cecum, suggesting this drug may be useful for treating select cases of postoperative ileus. for horses with presumed ileus of the large colon, signs included reduced fecal output (fewer than three piles of manure per day), reduced gastrointestinal sounds, variable presence of colic, and on occasion a palpable impaction of the cecum or large colon. risk factors for this syndrome include orthopedic surgery, length of the operative period, and most importantly inadequate treatment with phenylbutazone, presumably resulting from insufficient control of postoperative pain. although treatment of large colon impaction in the postoperative period typically is uncomplicated, onset of cecal impaction is fatal in many cases because of the difficulty in recognizing horses that have cecal dysfunction. therefore one should pay close attention to fecal production and optimal analgesic treatment in any horse following an orthopedic procedure. other painful procedures, including ophthalmologic procedures, also likely place horses at risk of developing ileus of the large intestine. simple obstructions of the large intestine such as impaction tend to have a more gradual onset than those of the small intestine, although horses may become acutely and severely painful with some forms of colon displacement. in fact, some of these cases mimic and may progress toward large colon volvulus. medical therapy is frequently successful in correcting large colon impactions. however, cecal impactions present much more of a dilemma because of the greater propensity of this organ to rupture, the relative difficulty of surgically manipulating the cecum, and the onset of cecal dysfunction that may prevent the cecum from emptying following surgical resolution of impaction. cecal impaction may be divided into two syndromes: primary cecal impactions that result from excessive accumulation of ingesta in the cecum and secondary cecal impactions that develop while a horse is being treated for a separate problem. , although primary impactions typically consist of impacted, relatively dry fecal material and secondary cecal impactions tend to have fluid contents, considerable overlap exists between the two syndromes, and one must approach each case carefully. in horses with primary cecal impactions, onset of abdominal pain occurs over a number of days, reminiscent of the development of a large colon impaction. one should differentiate cecal impactions from large colon impactions on the basis of rectal palpation findings. cecal impactions have a propensity to rupture before the development of severe abdominal pain or systemic deterioration and therefore must be monitored closely. secondary cecal impactions typically develop following unrelated surgical procedures that result in postoperative pain (particularly orthopedic surgeries). secondary cecal impactions may be even more difficult to detect because one may attribute postoperative depression and decreased fecal output to the operative procedure rather than to colic. by the time horses with secondary cecal impactions show noticeable signs of colic, the cecum may be close to rupture. in many cases, no signs of impending rupture are evident. therefore all horses that undergo surgeries in which considerable postoperative pain may develop should have feed intake and manure production closely monitored. a recent study indicated that horses that produce fewer than three piles of manure daily in the postoperative period are at risk of developing a large intestinal impaction. furthermore, horses that underwent prolonged (> hour) orthopedic surgery that received inadequate treatment with phenylbutazone were at considerable risk of reduced postoperative fecal output. these results are in contrast to statements indicating that nsaids may place horses at risk of impaction, statements that appear to be based largely on clinical impressions rather than on risk analysis. the diagnosis of primary cecal impaction is based on palpation of a firm, impacted cecum per rectum. in some cases, cecal impactions may be difficult to differentiate from large colon impactions. however, careful palpation reveals the inability to move the hand completely dorsal to the impacted viscus because of the attachment of the cecum to the dorsal body wall. treatment for horses with primary cecal impactions may include initial medical therapy, including aggressive administration of intravenous fluids and judicious use of analgesics. however, if the cecum is distended grossly or if medical therapy hasno effect within a reasonable period of time, surgical evacuation of the cecum via a typhlotomy is indicated. in addition, performing an ileocolostomy to bypass the cecum is advisable, because postoperative cecal motility dysfunction with recurrence of the impaction is common. , in horses that develop secondary cecal impactions, diagnosis is based on palpation of a greatly distended cecum filled with semifluid intestinal contents. the nature of the contents likely is related to the more rapid progression of this disease compared with primary cecal impaction. one should not delay surgery because of the risk of cecal rupture. however, if the cecum appears healthy following typhlotomy and evacuation, bypass of the cecum is not as critical as it is for primary impactions as long as one can control the inciting cause of the impaction (such as orthopedic pain). the prognosis is guarded for surgical treatment of all cecal impactions because of the potential for the cecum to rupture during prolonged medical treatment or during surgical manipulation, the possibility of abdominal contamination during surgery, and the extensive surgical procedures required. in a recent report, seven of nine horses for which cecal impaction was treated by typhlotomy and ileocolostomy or jejunocolostomy lived long term. however, a separate report indicated that all horses with cecal impaction following another disease process had cecal rupture without any signs of impending rupture. ingesta impactions of the large colon occur at sites of anatomic reductions in luminal diameter, particularly the pelvic flexure and the right dorsal colon. although a number of risk factors have been reported, most have not been proved. however, a sudden restriction in exercise associated with musculoskeletal injury appears frequently to be associated with onset of impaction. another consideration is equine feeding regimens, which usually entail twice daily feeding of concentrate. such regimens are associated with large fluxes of fluid into and out of the colon, associated with readily fermentable carbohydrate in the colon and subsequent increases in serum aldosterone, respectively. one may prevent these fluid fluxes, which may cause dehydration of ingesta during aldosterone-stimulated net fluid flux out of the colon, with frequent small feedings. amitraz, an acaricide associated with clinical cases of colon impaction, can induce impaction of the ascending colon. , this effect may provide some clues as to the pathogenesis of large colon impaction. in particular, amitraz appears to alter pelvic flexure pacemaker activity, resulting in uncoordinated motility patterns between the left ventral and left dorsal colon and excessive retention of ingesta. absorption of water from the ingesta increases with retention time, dehydrates the contents of the colon, and results in impaction. conceivably, parasite migration in the region of a pacemaker may have a similar action. other factors implicated in large colon impaction include limited exercise, poor dentition, coarse roughage, or dehydration. clinical signs of large colon impaction include slow onset of mild to moderate colic. fecal production decreases, and the feces are often hard, dry, and mucuscovered because of delayed transit time. the heart rate may be elevated mildly during episodes of pain but is often normal. signs of abdominal pain are typically well controlled with administration of analgesics but become increasingly more severe and refractory if the impaction does not resolve. the diagnosis is based on palpation of a firm mass in the large colon per rectum. however, one may underestimate the extent of the impaction by rectal palpation alone because much of the colon is out of reach. adjacent colon may be distended if the impaction has resulted in complete obstruction. one should attempt initial medical treatment. administration of analgesics (e.g., flunixin meglumine at . to . mg/kg intravenously every to hours; butorphanol at . to . mg/kg intramuscularly every to hours; or xylazine at . to . mg/kg intravenously as needed) controls intermittent abdominal pain. administration of oral laxatives such as mineral oil ( to l by nasogastric tube every to hours) and the anionic surfactant dioctyl sodium sulfosuccinate ( to g/ kg diluted in to l of water by nasogastric tube every to hours) are used commonly to soften the impaction. saline cathartics such as magnesium sulfate ( . mg/kg in to l by nasogastric tube) also may be useful. one should not permit access to feed. for impactions that persist, one should institute aggressive oral and intravenous fluid therapy ( to times the maintenance fluid requirement). if the impaction remains unresolved, the horse becomes uncontrollably painful, or extensive gas distention of the colon occurs, surgery is indicated. in addition, one can monitor abdominal fluid serially to determine the onset of intestinal compromise. at surgery, one evacuates the contents of the colon via a pelvic flexure enterotomy. the prognosis is good for those horses in which impactions resolve medically ( % long-term survival in one study) and fair in horses that require surgical intervention ( % long-term survival in the same study). enteroliths are mineralized masses typically composed of magnesium ammonium phosphate (struvite). however, magnesium vivianite also has been identified in enteroliths, along with variable quantities of sodium, sulfur, potassium, and calcium. the formation of magnesiumbased minerals is puzzling because of the relative abundance of calcium in colonic fluids, which would favor the formation of calcium phosphates (apatite) rather than struvite. however, elevated dietary intake of magnesium and protein may play a role. many horses that develop enteroliths are located in california and are fed a diet consisting mainly of alfalfa hay. analysis of this hay has revealed a concentration of magnesium approximately times the daily requirements of the horse. furthermore, the high protein concentration in alfalfa hay may contribute to calculi formation by increasing the ammonia nitrogen load in the large intestine. enteroliths most commonly form around a nucleus of silicon dioxide (a flintlike stone), but nidi have included ingested nails, rope, and hair. enteroliths usually are found in the right dorsal and transverse colons. although enterolithiasis has a wide geographic distribution, horses in california have the highest incidence. in one california study, horses with enterolithiasis represented % of the surgical colic population, and arabians, morgans, american saddlebreds, and donkeys were at greatest risk of this disease. in a study of enterolithiasis in texas, risk factors also included feeding of alfalfa hay and arabian breed. however, in that study, miniature horses were also at risk. horses with enteroliths are rarely under years old, although an enterolith in an -month-old miniature horse has been reported recently. enterolithiasis is characterized by episodic, mild to moderate, intermittent abdominal pain. progressive anorexia and depression may develop. the amount of pain depends on the degree of obstruction and amount of distention. partial luminal obstruction allows the passage of scant, pasty feces. heart rate varies and depends on the degree of pain. in some cases, an enterolith is forced into the small colon, where it causes acute small colon obstruction. one may diagnose enteroliths by abdominal radiography or at surgery. on rare occasions, one may palpate an enterolith per rectum, particularly if it is present in the distal small colon. in general, these cases require surgery, although enteroliths being retrieved per rectum have been reported. in fact in one study, % of horses presented for treatment of enterolithiasis had a history of passing an enterolith in the feces. however, enteroliths typically are located in the right dorsal colon, transverse colon, or small colon. at surgery, one gently pushes the enterolith toward a pelvic flexure enterotomy, but removal frequently requires a separate right dorsal colon enterotomy to prevent rupture of the colon. following removal of an enterolith, one must conduct further exploration to determine if other enteroliths are present. solitary enteroliths are usually round, whereas multiple enteroliths have flat sides. the prognosis is good ( % -year survival in one study of cases), unless the colon ruptures during removal of an enterolith. in one recent study, rupture occurred in % of cases. sand impactions are common in horses with access to sandy soils, particularly horses eating feed placed on the ground. some horses, especially foals, deliberately eat sand. fine sand tends to accumulate in the ventral colon, whereas coarse sand may accumulate in the dorsal colon. , however, individual differences in colonic function may contribute to accumulation of sand, because some horses can clear consumed sand, whereas others cannot. distention from the impaction itself, or gas proximal to the impaction, causes abdominal pain. in addition, sand may trigger diarrhea, presumably because of irritation of the colonic mucosa. in horses with sand impactions, clinical signs are similar to those of horses with large colon impactions. one may find sand in the feces, and auscultation of the ventral abdomen may reveal sounds of sand moving within the large colon. however, unlike sand-induced diarrhea, one may not hear sand impactions easily because of the lack of colonic motility. to determine the presence of fecal sand, one places several fecal balls in a rectal palpation sleeve or other container, which subsequently is filled with water. if sand is present, it accumulates at the bottom of the container. in addition, one may detect mineral opacity within the colon on abdominal radiographs, particularly in foals, ponies, and small horses. abdominal paracentesis typically yields normal fluid and poses some risk because large quantities of sand in the ventral colon make inadvertent perforation of the colon more likely. peritoneal fluid is often normal but may have an elevated protein concentration. initially, medical therapy is warranted. administration of psyllium hydrophilic mucilloid ( . to . kg/ kg in to l of water by stomach tube) may facilitate passage of sand. one should administer the solution rapidly because it will form a viscous gel. an alternative method of administration is to mix psyllium with l of mineral oil, which will not form a gel and can be pumped through a nasogastric tube easily. one then pumps to l of water through the tube. the psyllium separates from the oil phase and mixes with the water, forming a gel within the gastrointestinal tract. psyllium is thought to act by stimulating motility or by agglutinating the sand. however, a recent experimental study failed to show a benefit of this treatment for clearing sand from the colons of otherwise normal horses. if a severe impaction is present, one should not give the psyllium until softening the impaction by administrating intravenous or oral fluids and other laxatives. perforation is a potential complication in horses with sand impactions because the sand stretches and irritates the intestinal wall and causes inflammation. therefore if colic becomes intractable, one should perform surgical evacuation of the large colon. the prognosis is generally good. , displacement of the ascending colon is a common cause of large intestinal obstruction. the ascending colon is freely movable except for the right dorsal and ventral colons. contact with adjacent viscera and the abdominal wall tends to inhibit movement of the ascending colon from a normal position; however, accumulation of gas and fluid or ingesta may cause the colon to migrate. feeding practices, including feeding of large concentrate meals, likely plays a role in initiating displacement of the large colon. large concentrate meals increase the rate of passage of ingesta, allowing a greater percentage of soluble carbohydrates to reach the large intestine, which in turn increases the rate of fermentation and the amount of gas and volatile fatty acids produced. the production of large amounts of volatile fatty acids stimulates the secretion of large volumes of fluid into the colon. the association between feeding concentrate and development of displacements of the large colon is illustrated by studies indicating that ascending colon displacement is more prevalent in horses fed a highconcentrate, low-roughage diet. abnormal motility patterns of the ascending colon also have been suggested to contribute to the development of colonic displacement. feeding stimulates colonic motility via the gastrocolic reflex, but large meals may alter normal motility patterns and concurrently allow rapid accumulation of gas and fluid from fermentation. , migration of parasite larvae (strongyles) through the intestinal wall also has been shown to alter colonic motility patterns. other experimental studies also have shown that strongylus vulgaris infection results in reduced blood flow to segments of the large intestine without necessarily causing infarction. electric activity of the colon and cecocolic junction increases after infection with s. vulgaris and cyathostome larvae, probably reflecting a direct effect of migration through the intestine and an early response to reduced blood flow. displacements of the ascending colon generally are divided into three types: left dorsal displacement, right dorsal displacement, and retroflexion. left dorsal displacement is characterized by entrapment of the ascending colon in the renosplenic space. the colon often is twisted degrees such that the left ventral colon is situated in a dorsal position relative to the left dorsal colon. the entrapped portion may be only the pelvic flexure or may involve a large portion of the ascending colon, with the pelvic flexure situated near the diaphragm. the colon may become entrapped by migrating dorsally between the left abdominal wall and the spleen or may migrate in a caudodorsal direction over the nephrosplenic ligament. occasionally, one can palpate the ascending colon between the spleen and abdominal wall, lending support to the first mechanism of displacement. gastric distention is thought to predispose horses to left dorsal displacement of the ascending colon by displacing the spleen medially, allowing the colon room to migrate along the abdominal wall. right dorsal displacement begins by movement of the colon cranially, medial (medial flexion) or lateral (lateral flexion) to the cecum. according to one author, the proportion of right dorsal displacements with medial versus lateral flexion is approximately : . in either case the pelvic flexure ends up adjacent to the diaphragm. retroflexion of the ascending colon occurs by movement of the pelvic flexure cranially without movement of the sternal or diaphragmatic flexures. displacement of the ascending colon partially obstructs the lumen, resulting in accumulation of gas or ingesta and causing distention. secretion of fluid in response may exacerbate the distention. tension and stretch of the visceral wall is an important source of the pain associated with colonic displacement. tension on mesenteric attachments and the root of the mesentery by the enlarged colon also may cause pain. ischemia rarely is associated with nonstrangulating displacement of the colon. however, vascular congestion and edema often occur in the displaced segments of colon, resulting from increased hydrostatic pressure from reduced venous outflow. morphologic damage to the tissues is usually minor. clinically, displacement of the ascending colon is characterized by intermittent signs of mild to moderate abdominal pain of acute onset. however, one also may note an insidious onset of colic. one may note dehydration if the duration of the displacement is prolonged. the heart rate may be elevated during periods of abdominal pain but is often normal. abdominal distention may be present if the colon is enlarged by gas, fluid, or ingesta. fecal production is reduced because progressive motility of the large intestine is absent. one often diagnoses left dorsal displacements by palpation per rectum. one can feel the left ventral colon in a dorsal position; it often is filled with gas. one can trace the ascending colon to the nephrosplenic space, and the spleen may be displaced medially. alternatively, one can reach a tentative diagnosis using abdominal ultrasonography. the spleen is visible on the left side of the abdomen, but the gasdistended bowel obscures the left kidney. evaluation of this technique indicates that false positives occur in few instances, although false negatives occasionally may occur. a definitive diagnosis therefore may require surgery. right dorsal displacements are characterized by the presence of the distended ventral colon running across the pelvic inlet and may be felt between the cecum and the body wall if a lateral flexion is present. the pelvic flexure is usually not palpable. retroflexion of the ascending colon may produce a palpable kink in the colon. if the displaced colons are not distended by gas in the instance of right dorsal displacement and retroflexion, the ascending colon may not be palpable and is conspicuous by its absence from a normal position. peritoneal fluid may increase in amount, but the color, protein concentration, and white blood cell count are usually normal. however, as the displaced segment becomes edematous, fluid leaking through the serosa into the peritoneal fluid increases the protein concentration. surgical correction of colon displacement is the most effective means of resolving this disorder. however, nonsurgical intervention has been successful in select cases of nephrosplenic entrapment of the large colon. [ ] [ ] [ ] before attempting such manipulations, the clinician must be certain of a diagnosis. one anesthetizes the horse and places it in right lateral recumbency, rotates the horse up to dorsal recumbency, rocking it back and forth for to minutes, and then rolls the horse down into left lateral recumbency. one should palpate the nephrosplenic space per rectum to determine whether the entrapment has been relieved before recovering the horse from anesthesia. one may administer phenylephrine ( - µg/kg/min over minutes) to decrease the size of the spleen. more recently, phenylephrine has been used successfully with to minutes of exercise to reduce nephrosplenic entrapments in four of six horses. the authors suggested that the technique be used on horses with mild to moderate colonic distention, particularly when financial constraints are severe. a number of cases occur in which nonsurgical interventions do not correct the problem and others in which nonsurgical manipulations correct the entrapment but result in large colon volvulus or displacement. one should take horses in such condition to surgery promptly. the prognosis for horses with large colon displacement is good. in one study on horses with nephrosplenic entrapment of the large colon, survival exceeded %. the horse, particularly young horses, may ingest foreign material that can cause obstruction, such as bedding, rope, plastic, fence material, and feedbags. these foreign bodies may result in impaction with ingesta and distention of the intestine, typically in the transverse or descending colon. young horses usually are affected. in one study the obstructing mass could be palpated per rectum in three of six horses. fecaliths are common in ponies, miniature horses, and foals. older horses with poor dentition also may be predisposed to fecaliths because of the inability to masticate fibrous feed material fully. fecaliths commonly cause obstruction in the descending colon and may cause tenesmus. other clinical signs are similar to those of enterolithiasis. abdominal radiography may be useful in smaller patients to identify the obstruction, especially if gas distention around the foreign body or fecalith provides contrast. the horse usually requires surgical treatment. mural masses such as abscesses, tumors (adenocarcinoma, lymphosarcoma), granulomata, and hematomas ( figure . - ) can cause luminal obstruction and impaction, typically in older horses. impaction may result from obstruction of the lumen or impaired motility in the segment of intestine with the mass. abscesses may originate from the lumen of the intestine or may extend from the mesentery or mesenteric lymph nodes. intramural hematomas form most commonly in the descending colon and cause acute abdominal pain. once the acute pain from the hematoma subsides, impaction proximal to the hematoma develops because of impaired motility through the affected portion of the colon. trauma, ulceration of the mucosa, and parasitic damage are speculated causes of intramural hematomas. , stricture of the large intestine occurs when fibrous tissue forms in a circular pattern around or within the intestine, reducing the luminal diameter and the ability of the wall to stretch. strictures may be congenital or may follow peritonitis, previous abdominal surgery, or inflammatory bowel disease. in a report of horses with inflammatory bowel disease, horses had strictures, four of which were in the small intestine and two of which were in the large colon. clinical signs vary according to the degree of luminal obstruction. partial obstruction and impaction tend to produce mild to moderate abdominal pain of insidious onset. mural hematomas tend to produce signs of acute abdominal pain. , per rectal palpation of the abdomen may reveal the presence of a mass or simply the impacted segment but not the mass itself. one may note fever, weight loss, and anorexia if an abscess or tumor is the cause. an elevated white blood cell count; hyperfibrinogenemia; hyperglobulinemia; or normocytic, normochromic anemia may occur with abscesses or tumors. peritoneal fluid may reflect the cause of the mass. tumor cells may occur infrequently. one may note evidence of inflammation with bacteria if the cause of colic is an abscess or granuloma, in which case one should culture the fluid. hematomas may cause hemorrhage into the peritoneal fluid. treatment usually requires surgical resection of the mass. one may treat abscesses with appropriate antibiotics if the impaction can be resolved medically with oral or intravenous analgesics and laxatives. streptococcus spp, actinomyces pyogenes, corynebacterium pseudotuberculosis, rhodococcus equi, anaerobic bacteria, and gram-negative enteric organisms commonly are involved in abscesses. atresia of a segment of the colon is a rare congenital abnormality in horses. the heritability and causes of the condition are unknown. one potential mechanism for development of the lesion is intestinal ischemia during fetal life, which results in necrosis of a segment of intestine. clinical signs include a failure to pass meconium and colic within the first to hours of life. secondary abdominal distention results from complete intestinal obstruction, and abdominal radiographs may reveal gas-distended colon. one makes the diagnosis at surgery. any portion of the colon may be absent, but the distal segment of the large colon or the proximal small colon usually is affected most severely. if sufficient tissue is present, one may attempt anastomosis to the proximal blind end of the colon. the prognosis depends on which segment of the colon is absent but is usually poor because of an absence of distal colon. neoplasia in the alimentary tract of the horse is uncommon. primary and metastatic neoplasia can affect multiple locations within the oral cavity and gastrointestinal tract (boxes . - and . - ). neoplasia is not limited to older horses. the average age of horses with squamous cell carcinoma is . to . years. , the alimentary form of lymphoma occurs most commonly in horses less than years of age. identification of benign versus malignant tumors is imperative to justify treatment and predict survival. clinical signs associated with alimentary neoplasia are related to the tumor location. clinical signs of oral neoplasia can include enlargement or ulceration of the mandible or maxilla. neoplasia of the tongue results in weight loss, quidding, prepharyngeal dysphagia, halitosis, and nasal discharge containing feed material if the oropharynx is involved. [ ] [ ] [ ] tumors of the esophagus cause signs typical of esophageal dysphagia, ptyalism, choke, intermittent colic, fever, weight loss, and halitosis. , , gastric neoplasia can be associated with abnormal chewing and swallowing behavior, anorexia, weight loss, chronic intermittent colic, abdominal distention, and intermittent fever. abdominal neoplasia has been implicated in % of horses with intermittent or chronic colic. , altered stool character, weight loss, ventral edema, and recurrent fever have been associated with intestinal neoplasia. acute signs of abdominal discomfort can occur in intestinal obstructions from malignant and benign neoplastic disease. paraneoplastic syndromes may occur in the horse. the most common syndromes are cancer cachexia, ectopic hormone production, anemia, leukocytosis, thrombocytopenia, hypergammaglobulinemia, fever, and neurologic abnormalities. horses with cancer cachexia have profound weight loss despite adequate consumption of calories. diagnosis of alimentary neoplasia can be challenging. data collected from a complete blood count, biochemistry panel, and urinalysis may support the diagnosis of neoplasia but rarely confirms it. normocytic normochromic anemia indicates chronic disease and is the most likely cause of anemia associated with neoplasia. blood loss anemia (via gastrointestinal tract) and immune-mediated hemolytic anemia (lymphoma) are less frequent causes of anemia associated with abdominal neoplasia. peripheral eosinophilia has been reported in association with multisystemic eosinophilic, epitheliotropic disease with lymphoma. leukocytosis and hyperfibrinogenemia are common findings. serum chemistry can confirm hypoalbuminemia caused by inflammation of the bowel wall. hyperglobulinemia can be characterized with serum electrophoresis, which is nonspecific and can reveal chronic inflammation. a few cases of lymphoma have been identified with monoclonal hypergammaglobulinemia. ectopic hormone production may result in hypercalcemia (calcium > mg/dl), which is associated with alimentary neoplasia such as lymphoma, multiple myeloma, carcinomata, and ameloblastoma. , hypoglycemia (blood glucose < mg/dl) can occur with neoplasia of the pancreas or liver. rectal examination may detect an abdominal mass, thickening of the intestinal wall, lymph node enlargement, or a gritty texture in horses with carcinomatosis. rectal biopsy can reveal lymphoma in some cases. fecal occult blood test is nonspecific for neoplastic disease but can reveal blood loss through the gastrointestinal tract. occasionally, abdominocentesis can identify neoplasia if the tumor exfoliates cells into the abdomen. one can diagnose squamous cell carcinoma, adenocarcinoma, and mesothelioma from peritoneal fluid. , , , characterization of peritoneal fluid as an inflammatory exudate or modified transudate without any neoplastic cells present is common. cytologic analysis of peritoneal fluid samples collected by abdominocentesis accurately predicted the presence of neoplasia in of cases in one study. cytologic examination of two or more peritoneal fluid samples increases the sensitivity of this test for detecting abdominal neoplasia. measurement of peritoneal fluid glucose concentration and ph is valuable to differentiate inflammation in the peritoneum caused by neoplasia from bacterial peritonitis. abdominal neoplasia typically is associated with peritoneal glucose concentrations similar to blood and ph higher than . . d-xylose absorption tests can reveal malabsorptive diseases that include lymphoma. , immunoglobulin m deficiency is associated with lymphoma in some young adult horses, but the prevalence of immunoglobulin m deficiency in horses with lymphoma and the value of measuring serum immunoglobulin m concentrations for the diagnosis of lymphoma have not been evaluated. dna cell cycle analysis of suspect neoplastic cells has been used to detect lymphoma in equine patients confirmed with the disease. this method of evaluating fluid or tissues aspirates may increase the accuracy for diagnosing neoplasia in the future. a complete evaluation of the oral cavity may include using a full-mouth speculum, radiographs, and endoscopy of the pharynx. evaluation of the esophagus and stomach with a -m endoscope can reveal intralumenal masses. pleuroscopy has been used to obtain biopsy samples of extralumenal masses surrounding the esophagus. contrast radiography can assist in the diagnosis of neoplasia within the wall or outside of the esophagus. , ultrasonography of the stomach, small intestine, cecum, and large colon is useful in detecting intestinal wall thickness, abdominal masses, and excessive peritoneal fluid. identification of neoplasia in the liver, kidney or spleen may support metastasis to other parts of the gastrointestinal tract or lymph nodes. laparoscopy and exploratory laparotomy often are required to obtain a final diagnosis. lymphoma is the most common neoplasia in the horse and has been divided into four categories. this section covers only the intestinal/alimentary form. in the past, lymphoma has been called lymphosarcoma, but the preferred term by oncologists is lymphoma because no benign form of this disease exists. lymphoma originates from lymphoid tissue and predominantly affects the small intestine and intestinal lymph nodes. chronic weight loss from malabsorption, intermittent colic, and fever are the most common clinical findings. , chronic diarrhea has been reported in some cases. paraneoplastic pruritus and alopecia have been identified in one case of diffuse lymphoma. one generally does not note peripheral lymphadenopathy, but one may palpate enlargement of the intestinal lymph nodes on rectal examination. large colon resection for treatment of lymphoma in horses has increased short-term survival in two horses. chemotherapy in two mares that were pregnant extended their lives long enough to foal normally. long-term prognosis for intestinal lymphoma is poor. squamous cell carcinoma (scc) is a malignant tumor of the gastrointestinal epithelium. scc is the second most common neoplasia in the horse and is the most common oral neoplasia. however, the incidence of scc is rare. , , in the oral cavity scc may affect the lips, tongue, hard palate, pharynx, and mucosa. , treatment for scc in the oral cavity may involve surgical resection, iridium- brachytherapy, -fluorouracil, or intralesional cisplatin. , [ ] [ ] [ ] the prognosis for survival is good if complete removal of the tumor is possible. metastasis beyond the regional lymph nodes is rare for oral scc. squamous cell carcinoma is the most common tumor of the stomach and esophagus , and can invade these areas and metastasize to the lymph nodes and lungs. abnormal masses were palpated on rectal examination in four of five cases of gastric scc. treatment by surgical resection is not possible in most cases and the horses die or are euthanized. adenocarcinoma is a malignant tumor that can occur in the small intestine, cecum, and large colon. the tumor arises from the glandular crypts of the gastrointestinal tract and has been reported in middle-aged and older horses. metastasis to the lymph nodes, liver, and lungs can occur. intestinal adenocarcinoma has been reported to metastasize to the bone and was diagnosed using nuclear scintigraphy following injection of technetium- m hydroxymethylene diphosphate. , no reports of successful surgical resection have been published. leiomyosarcoma is a malignant tumor of the smooth muscle lining the gastrointestinal tract and has been reported in the stomach, small intestine, and rectum. , , , , in one case report, gastroscopy could not identify the mural mass in the stomach that was found during exploratory surgery. another report describes a favorable outcome for surgical resection of a leiomyosarcoma that was protruding from the anal sphincter in a -year-old quarter horse. prognosis for survival is favorable if surgical resection is possible. leiomyoma is a benign tumor of the smooth muscle of the gastrointestinal tract that can occur in the stomach, small intestine, and small colon. , clinical signs are consistent with intestinal obstruction. surgical resection and anastomosis of the affected portion of the intestine have been performed without complications. lipoma is a benign tumor that occurs in older horses ( to years) and arises from mesenteric adipocytes. the tumor grows on a stalk that wraps around the intestine, causing a strangulating lesion manifested clinically by acute obstructive colic. intestinal injury caused by pedunculated lipomata may occur in the small intestine, small colon, and rectum. long-term survival with surgical resection and anastomosis of the affected segment has been reported to be % to %. , oral neoplasia oral cavity neoplasia may involve the dental tissue (odontogenic tumors), bone (osteogenic tumors), or soft tissues (see box . - ). ameloblastoma occurs in horses greater than years old and mainly affects the mandible. ameloblastic odontoma affects younger horses and usually involves the maxilla. both are benign but locally invasive. radiographs may distinguish the difference between an ameloblastoma (radiolucent lesion) and ameloblastic odontoma (radiolucent lesion with partially mineralized density). the best treatment option is surgical resection and radiation therapy regardless of the type. juvenile mandibular ossifying fibroma occurs in the rostral mandible of young horses between the ages of months and years. the fibroma may cause significant distortion of the bone. with early diagnosis and surgical excision of the mass, the horse has a good prognosis. melanomas, sarcoids, and oral papilloma occur on the mouth and lips. melanomas rarely metastasize, but they commonly are found in the parotid salivary glands and lymph nodes. sarcoids are the most common skin tumor that can involve the mouth. ulcerations of the buccal mucosa are difficult to treat. intralesional cisplatin, cryosurgery, radiation, and laser excision have been tried with limited success. equine papilloma virus is responsible for the common skin wart found on the lips and muzzle of young horses. these lesions are usually selflimiting but may be removed successfully by cryosurgery or excision. a number of detailed and informative reviews are available describing the anatomy, physiology, and pathophysiology of the equine peritoneum. [ ] [ ] [ ] [ ] [ ] the peritoneum consists of a single layer of mesothelial cells lining the peritoneal cavity and serosal surfaces of the intraabdominal viscera. the mesothelial lining of the diaphragm, abdominal walls, and pelvic cavity is termed parietal peritoneum. the visceral peritoneum includes the serosal surfaces of the intraabdominal organs. the parietal and visceral portions of the peritoneum are contiguous with each other through the omentum, mesenteries, and ligaments. caudally, the peritoneum reflects over the surfaces of the pelvic organs (portions of the urogenital tract and rectum), excluding them from the peritoneal space, and thus much of the pelvic cavity and contents are described as retroperitoneal. the peritoneal space communicates with the lumen of the uterus (and thus the external environment) via the fallopian tubes in females. in males the peritoneum forms a true blind sac. the vascular supply and nervous innervation of the visceral peritoneum are supplied by the splanchnic vessels and visceral autonomic nerves, respectively. branches of the intercostal, lumbar, and iliac arteries supply the parietal peritoneum, and the phrenic and intercostal nerves provide nervous innervation. the clinical relevance is that inflammation of the parietal peritoneum is perceived as somatic pain, resulting in a splinted abdominal wall, pain on external palpation, and reluctance to move. visceral pain is mediated by small type c sensory fibers, which are believed to be stimulated by bowel distention, smooth muscle spasms, tension on the mesentery, and ischemia. the peritoneal lining functions as a semipermeable barrier to the diffusion of water and low-molecular weight solutes between the blood and the abdominal cavity. the peritoneum secretes a serous fluid that lubricates the abdominal cavity, inhibits adhesion formation, and has minor antibacterial properties. , macrophages, mast cells, mesothelial cells, and lymphocytes provide immune function within the peritoneum. , peritoneal macrophages impart antibacterial activity via complement receptors, phagocytic activity, interaction with t lymphocytes, neutrophil chemotaxis, and fibroblast activation. the peritoneal surface maintains a high level of fibrinolytic activity through the production of plasminogen activators by mesothelial cells. this function, together with the lubricant properties of the peritoneal fluid, helps to maintain gliding surfaces within the peritoneum and prevent adhesion formation. in quadrupeds, peritoneal fluid produced by the mesothelium tends to move ventrally and cranially, aided largely by diaphragmatic movement. peritoneal fluid, waste products, and foreign material (including bacteria) exit the peritoneal cavity to enter the lymphatic system through diffusely distributed subendothelial pores or via the large diaphragmatic stomata, depending on particle size. large molecules and particles greater than approximately , mw (such as bacteria) exit through the diaphragmatic stomata and ultimately enter the thoracic duct. peritonitis is inflammation of the mesothelial lining of the peritoneal cavity and is characterized by desquamation and transformation of mesothelial cells; chemotaxis of neutrophils; release of several soluble mediators of inflammation; exudation of serum, fibrin, and protein into the peritoneal cavity; and depression of fibrinolytic activity. peritonitis occurs in association with a variety of disorders that result in mechanical, chemical, or infectious insult to the peritoneal lining. [ ] [ ] [ ] [ ] any process resulting in disruption or irritation of the peritoneal lining, inflammation or infection of abdominal organs, or compromise of the intestinal wall can result in peritonitis (box . - ). common mechanical injuries include blunt or perforating trauma to the abdominal wall, breeding and foaling accidents, and abdominal surgery. a variety of traumatic insults of iatrogenic origin can cause peritonitis, such as the pathogenesis of peritonitis as a series of stages, as reviewed and described by trent, is useful. the contamination stage, lasting to hours, involves introduction of bacteria into the peritoneum and initiation of the acute inflammatory response previously described. if the organisms are not eliminated and infection is established, the process evolves to the stage of acute diffuse peritonitis. although the overall movement of contaminants is toward the diaphragmatic stomata and into the thoracic duct, the nature of the peritoneal circulation is such that regardless of the location of the initial contamination, bacteria spread throughout the peritoneum within several hours. the stage of acute diffuse peritonitis lasts up to days. the inflammatory response persists and escalates with continued exudation of proteinaceous fluid and influx of inflammatory cells. offending organisms are delivered to the lymphatic system and may be eliminated by the immune system. organisms, however, may gain access to the systemic circulation in sufficient numbers to result in clinically relevant septicemia. in human beings and laboratory animals having undergone polymicrobial contamination of the peritoneum, the organisms causing septicemia at this stage are usually coliforms, e. coli in particular. this stage of the disease process has the highest mortality because of the effects of severe peritoneal inflammation, endotoxemia, and septicemia. if the animal survives this stage but fails to eliminate the infection in the peritoneal cavity, the disease enters a transitional phase referred to as the acute adhesive (or localizing) stage. this stage occupies a time frame of perhaps to days after the initial insult. neutrophils are still active, macrophages are increasing in numbers, and fibrin aggregates are being organized or lysed. in human beings and laboratory animals, selective reduction and synergism continue such that anaerobes and gram-negative aerobes predominate. if infection persists beyond this point, organization of fibrin proceeds and organisms become isolated from host defenses. at this point, the disease process enters the stage of chronic abscessation. this stage can begin as early as days after inoculation and persists indefinitely. clinical signs of peritonitis depend on the primary disease process, the duration of the problem, and the extent of peritoneal inflammation. localized peritonitis may have few or no systemic manifestations, whereas severe localized or generalized peritonitis often is accompanied by severe toxemia or septicemia or both. septic peritonitis usually causes more severe clinical signs because of the inflammatory mediators released in response to bacterial toxins and because of the presence of endotoxin when gram-negative organisms are involved. most clinical signs are nonspecific and include fever, depression, inappetance, decreased borborygmi, and dehydration. additional signs, reported in horses (ages months to years) with peritonitis, were colic, ileus, weight loss, and diarrhea. horses with peracute peritonitis, as occurs with rupture of the bowel or rectal tear, have severe toxemia, weakness, depression or severe colic, tachycardia, tachypnea, and circulatory failure. fever may not be present depending on the degree of shock. typical clinical findings include sweating, pawing, muscle fasciculations, weak peripheral pulses, red to purple mucous membranes, prolonged capillary refill time, and decreased skin elasticity. parietal pain, characterized by reluctance to move, splinting of the abdominal wall, and sensitivity to external abdominal pressure occur in some acute cases. urination or defecation may be painful for the horse, and urine and fecal retention may be evident on rectal examination. palpation of the abdomen externally may elicit flinching, aversion movements, or groaning. with extensive abdominal fecal contamination, rectal examination may reveal a gritty feeling of the serosal and parietal surface of the peritoneum because of fibrin deposition and a dry texture of the peritoneum. in horses with more chronic peritonitis, rectal examination findings can include pain on palpation of fibrinous or fibrous adhesions, intestinal impaction or distention following ileus and dehydration, an abdominal mass (abscess or neoplasia), or an impression of bowel floating in fluid. in many cases, one can detect no abnormalities on rectal examination. horses with localized, subacute, or chronic peritonitis may have signs of chronic or intermittent colic, depression, anorexia, weight loss, intermittent fever, ventral edema, exercise intolerance, decreased or absent intestinal sounds, and mild dehydration. heart rate and respiratory rate may be normal. fecal output may be normal; however, horses with chronic diarrhea and weight loss have been reported. foals with peritonitis usually exhibit signs of colic (acute or chronic) and are febrile, depressed, and inappetant. in some foals with primary peritonitis, pleural effusion occurs. in young foals, peritonitis can cause rapid metabolic deterioration, and determination and correction of the primary problem requires immediate attention. in older foals, peritonitis may occur insidiously, as occurs following s. equi or r. equi infections. clinicopathologic abnormalities vary depending on the time of onset and severity of peritonitis. horses with acute, septic peritonitis can have leukopenia, hemoconcentration, metabolic acidemia, azotemia, and electrolyte imbalances reflective of systemic inflammation from endotoxemia and hypovolemia. horses with peritonitis of a few days' duration may have leukocytosis and hyperfibrinogenemia. plasma protein levels vary depending on the hydration status, degree of exudation into the peritoneum, and type of underlying problem. in chronic peritonitis, hyperproteinemia with hyperglobulinemia may be present. neonates with uroperitoneum caused by urinary bladder rupture or urachal disease tend to develop azotemia, hyponatremia, hypochloremia, hyperkalemia, and acidosis. foals with peritonitis following septicemia, severe enterocolitis, severe meconium impaction, intussusception, small intestinal volvulus, gastric or duodenal rupture, or ascarid impactions usually have clinicopathologic findings reflective of systemic inflammation, such as inflammatory leukogram or leukopenia, hemoconcentration, and acidosis. chronic abscessation, as occurs in foals with r. equi and streptococcal infections, results in clinicopathologic findings reflecting chronic inflammation (anemia, hyperfibrinogenemia, hyperglobulinemia). abnormalities in the composition of peritoneal fluid occur with peritoneal inflammation, and peritoneal fluid analysis is principal to the diagnosis of peritonitis. one collects peritoneal fluid through puncture of the abdomen on the ventral midline. one should clip and prepare an area aseptically. usually, the lowest point of the abdomen, to cm caudal to the xiphoid cartilage, is prepared for puncture; although in some cases one may perform paracentesis more caudally, particularly when one suspects a specific area of sequestered fluid or abscessation. in addition, one may choose a site to the right of midline in an attempt to avoid the spleen. one can perform peritoneal puncture using a / -inch, -gauge needle or, following local anesthesia and a stab incision with a no. scalpel blade, using a sterile cannula. one collects fluid by gravity flow and should collect fluid in a tube containing anticoagulant, preferably edta for cytologic examination, and in a sterile tube without anticoagulant for visual inspection and, if desired, for culture. one should fill the edta tube to half its volume, because the edta will alter the refractive index of the fluid, resulting in a falsely elevated value for total solids when one collects only a small volume and tests it with a refractometer. one should evaluate peritoneal fluid routinely as to color, turbidity, total protein, white blood cell (wbc) count and differential, and the presence of bacteria as determined by gram stain. normal peritoneal fluid is clear and straw-colored and does not coagulate spontaneously. peritoneal fluid becomes turbid when increased numbers of white blood cells and concentration of protein are present. pink or red fluid indicates free hemoglobin or hemorrhage. blood introduced into the peritoneal fluid iatrogenically in some cases may be differentiated from blood from internal hemorrhage based on the presence of platelets and hematocrit. fluid with iatrogenic blood contamination contains platelets, whereas fluid with blood following internal hemorrhage or diapedesis often does not have platelets. blood contamination resulting from splenic puncture often results in the packed cell volume of the sample being greater than that of the blood. large volumes of dark brown or green fluid with a fetid odor obtained from several sites strongly suggest bowel rupture, but one should perform cytologic examination for confirmation. the distribution of polymorphonuclear and mononuclear cells varies widely, and one should interpret the results of cell counts and differentials as supporting a number of disorders rather than a specific diagnosis. normal equine peritoneal fluid contains fewer than nucleated cells per microliter. , wbc counts in acute peritonitis (> , /µl) are reported to be higher than those in chronic peritonitis ( , to , /µl) [ ] [ ] [ ] ; however, this is not always the case, and the wbc count depends most on the cause of the peritonitis. the wbc level does not always correlate with severity of peritonitis or the prognosis. the peritoneal fluid wbc count can be greater than , /µl following enterocentesis, with no clinical signs or problem. conversely, peritoneal wbc counts of fewer than , /µl may be found in foals or horses with intraabdominal abscesses. the peritoneal wbc count can increase to greater than , /µl following celiotomy and can be higher if an enterotomy is done. postoperatively, the wbc count normally continues to decline and returns to near normal by to days. failure of the wbc count to decrease suggests peritonitis resulting from a postoperative complication. finally, peritoneal fluid wbc counts greater than , /µl indicate severe focal or generalized peritoneal sepsis. the distribution of polymorphonuclear and mononuclear cells varies in normal peritoneal fluid, , but polymorphonuclear cells usually predominate. with acute peritonitis, polymorphonuclear cells typically increase to a greater degree than mononuclear cells, but this depends on the cause. in horses that have bowel disease accompanied by endotoxemia, the number of peritoneal mononuclear cells increases, as does transformation of mesothelial cells to macrophages. in chronic cases, one easily may mistake transforming mesothelial cells for neoplastic cells, which can make diagnosis difficult, particularly when the presenting problem is compatible with a neoplastic process. in such cases, consultation with a clinical pathologist regarding cytologic findings is prudent. factor, and interleukin- have been measured in the peritoneal fluid of horses with abdominal disorders, but the diagnostic and prognostic implications of the presence or absence of these enzymes and analytes is limited. [ ] [ ] [ ] one should submit peritoneal fluid samples in appropriate media (port-a-cul vial, bbl microbiology system) for aerobic and anaerobic cultures in an attempt to identify the pathogenic organism(s). obligate anaerobic bacteria such as bacteroides are difficult to culture, because one must collect, transport, and culture the sample under strict anaerobic conditions. frequently, bacterial cultures are negative when bacteria are present in peritoneal fluid. to enhance recovery of bacteria, one can inoculate peritoneal fluid into blood culture medium (septi-chek columbia, hoffmann-laroche inc., nutley, new jersey), and if the horse has received antimicrobial treatment, one first should pass fluid through an antimicrobial removal device (a.r.d., becton dickinson & co., cockeysville, maryland). early and aggressive therapy is required if treatment of peritonitis is to be successful. the goals of treatment are to resolve the primary problem, minimize the inflammatory response, and prevent long-term complications. in the acute phase, one gives primary consideration to the arrest of endotoxic, septic, or hypovolemic shock; correction of metabolic and electrolyte abnormalities and dehydration; and management of pain. in the absence of blood gas and electrolyte determinations, adequate volumes of a balanced electrolyte solution are required to correct dehydration and support the cardiovascular system. if the plasma protein concentration of the horse is less than . g/dl, one should consider administration of plasma or synthetic colloids. one should administer flunixin meglumine (banamine) for its local and systemic antiinflammatory effects. dosages vary depending on the severity of peritonitis, degree of toxemia, severity of pain, and hydration status of the horse and range from . mg/kg intramuscularly or intravenously every to hours to . mg/kg intramuscularly or intravenously every hours. the higher dosage provides greater visceral analgesia, whereas the lower dosage is effective in modifying the effects of experimental endotoxemia. in addition to analgesic and general antiinflammatory effect, flunixin meglumine may be effective in retarding adhesion formation when administered early in the acute, diffuse stage of septic peritonitis. heparin therapy has been recommended to prevent adhesion formation and to render bacteria more susceptible to cellular and noncellular clearing mechanisms. in experimental models using laboratory animals, normal peritoneal fluid protein concentration is less than . g/dl. protein levels between . g/dl and . g/dl can be difficult to interpret, but one should consider levels greater than . g/dl to be elevated abnormally. fibrinogen concentration increases with inflammation, and levels greater than mg/dl in the peritoneal fluid suggest that an acute inflammatory process is present. fibrinogen content will also increase from blood contamination. the presence of free and phagocytosed bacteria in peritoneal fluid indicates generalized suppuration, abscessation, or compromised bowel. if one observes numerous microorganisms of mixed types free in the peritoneal fluid or if one observes plant material, massive bacterial contamination of the abdomen following bowel rupture likely has occurred. the presence of toxic or degenerate neutrophils and bacteria within polymorphonuclear cells helps to distinguish peritoneal fluid from intestinal contents in such cases. enterocentesis yields discolored fluid containing mixed microorganisms and plant material and that is largely devoid of white blood cells. bacterial contamination of a sample can occur during collection of the sample, and iatrogenic contamination of a sample can result in free and intracellular bacteria in peritoneal fluid, particularly if processing is delayed. in such cases the bacterial numbers are few and the neutrophils appear healthy. in some cases of gastrointestinal perforation the luminal material, inflammatory cells, and protein may be sequestered by the omentum and further contained by fibrinous adhesions. abdominal fluid obtained via standard ventral paracentesis may have low cellularity and protein content but large numbers of mixed bacteria indicating bowel rupture. examples include gastric rupture along the greater curvature of the stomach between the omental layers (omental bursa) and perforated gastric or duodenal ulcers in foals. correlating all cytologic findings with clinical and clinicopathologic findings is important for interpreting the results of peritoneal fluid cytologic examination. biochemical analysis of peritoneal fluid may be useful in detecting sepsis when cytologic examination and culture are negative or otherwise unavailable. in a prospective study by van hoogmoed, rodger, spier, et al., peritoneal fluid ph and glucose concentrations from horses with septic peritonitis were significantly lower than horses with nonseptic peritonitis and healthy horses. peritoneal fluid ph less than . , glucose less than mg/dl, and fibrinogen concentration greater than mg/dl were considered highly predictive of septic peritonitis. serum to peritoneal glucose concentration differences of greater than mg/dl was considered the most diagnostically useful test for septic peritonitis in the study. increased activities of alkaline phosphatase, lactic dehydrogenase, creatine kinase, aspartate aminotransferase, tumor necrosis heparin therapy was associated with decreased adhesions in septic peritonitis. heparin has not yet been demonstrated clearly to have similar efficacy in horses, although it may. suggested dosages range from to iu subcutaneously every hours for hours to to iu/kg subcutaneously every hours. one should note that heparin induces red blood cell aggregation in horses, which may adversely affect capillary blood flow. one should initiate antimicrobial therapy after making a diagnosis of peritonitis and before the results of peritoneal culture are available, because isolating an organism may take several days and often culture fails to isolate the organism(s). intravenous administration of antimicrobials is preferred over oral or intramuscular routes in acute, diffuse, septic peritonitis because more reliable levels of drug are achieved in the tissues and peritoneal fluid than otherwise would be obtained in horses with hypovolemia or decreased intestinal motility. the combination of a β-lactam antibiotic with an aminoglycoside is appropriate in most circumstances and certainly in the acute diffuse stage of septic peritonitis. these drugs act synergistically to provide a broad spectrum of activity against a variety of gram-positive and gramnegative aerobic and anaerobic bacteria. potassium penicillin ( , to , iu/kg intravenously every hours) combined with gentamicin ( . mg/kg every hours) is an appropriate regimen. in most cases, peritonitis will have resulted from bowel contamination, and thus one should presume a mixed infection with gram-negative aerobic bacteria and gram-positive and gram-negative anaerobic bacteria. one also should presume the same in many cases of traumatic peritonitis, as occurs with foreign body puncture, breeding trauma, or foaling trauma. therefore a strong possibility exists of infection involving penicillin resistant bacteroides fragilis, so that adding metronidazole ( mg/kg orally every to hours) to the regimen is prudent. combination therapy with β-lactam and aminoglycoside antibiotics (and metronidazole when indicated) is a standard and generally effective protocol. one can modify this antimicrobial regimen when culture and antimicrobial sensitivity results become available. aminoglycosides and nonsteroidal antiinflammatory drugs have the potential to induce acute renal tubular damage, particularly when dehydration and decreased renal perfusion are present. therefore adequately rehydrating the patient and ensuring that renal function is intact before initiating treatment with these drugs are important. furthermore, maintaining hydration and monitoring renal function during the course of treatment are important. monitoring serum creatinine concentration; performing serial uninalysis observing for pigment, red blood cells; and casts; determining the ratio of γ-glutamyltransferase to creatinine in the urine; and therapeutic drug monitoring of aminoglycoside levels are useful in this regard. sodium ampicillin and ceftiofur sodium are β-lactam antibiotics that may be useful in combination therapy. these drugs have an extended gram-negative spectrum compared with penicillin. however, as a third-generation cephalosporin, ceftiofur is less effective against anaerobes than penicillin. one also may consider ceftiofur, trimethoprim-potentiated sulfonamides, amikacin, and enrofloxacin for treatment of gram-negative infection based on culture and sensitivity results. enrofloxacin is a quinolone drug with excellent activity against gramnegative pathogens, including pseudomonas, and also can be effective against resistant staphylococci (personal observation). such staphylococci may be involved in infections caused by traumatic puncture of the abdominal wall. enrofloxacin has a variety of potential toxic effects, including cartilage damage in young growing animals. however, a recent study concluded the drug was safe when administered to adult horses intravenously at mg/kg every hours for weeks. one probably should avoid using the drug in young, growing animals until the issue of cartilage damage is resolved. administration of enrofloxacin to horses constitutes off-label usage. one should treat horses with acute, diffuse, septic peritonitis with antibiotics until the white blood cell count, plasma fibrinogen, and abdominal fluid analysis are normal. in horses that respond to therapy, this process takes a variable amount of time depending on the offending organisms and stage of disease at the time treatment is initiated. horses with abdominal abscessation resulting from polymicrobial infection may require many months of antibiotic treatment. abdominal abscesses caused by streptococci and corynebacterium pseudotuberculosis also may require long-term treatment (weeks to months). long-term antibiotic treatment generally necessitates the use of oral antibiotics, and the options are limited. trimethoprim-potentiated sulfonamides are administered orally and are effective against a variety of gram-positive and gram-negative organisms, although some streptococci are resistant. metronidazole is an orally administered drug effective against anaerobic bacteria, as previously discussed. other orally administered antimicrobials one may consider for long-term use include doxycycline (broad spectrum), erythromycin (gram-positive spectrum), and enrofloxacin (mostly gram-negative spectrum). importantly, rifampin, when used with other drugs, can be effective in penetrating and resolving abscesses. combination therapy with erythromycin and rifampin is the standard treatment for rhodococcus equi infection in foals. peritonitis caused by actinobacillus equuli usually is manifested as a diffuse, supporative peritoneal exudate. the same is true for some cases involving streptococci (personal observation). these infections generally respond well to combination therapy with penicillin and gentamicin. if streptococci are involved as the sole pathogen, then penicillin alone should be effective. streptococci potentially can be involved in mixed, synergistic peritoneal infections in horses. drainage or lavage of the peritoneal cavity may be of benefit in removing toxic bacterial by-products and products of inflammatory cells. high numbers of inflammatory cells and release of their mediators can persist even after the primary stimulus of the inflammatory response has resolved. infusing large volumes of isotonic, warmed fluid into the peritoneal cavity also dilutes the inflammatory mediators, possibly reducing their deleterious effects. when successful, peritoneal lavage decreases the peritoneal fluid wbc count and total protein, potentially reflecting a decrease in diffuse inflammation. the benefits of peritoneal lavage are controversial, and a positive effect may be more likely during the acute, diffuse stage of disease. , some studies suggest peritoneal lavage, along with heparin therapy, may reduce the incidence of adhesions. one should perform peritoneal drainage and lavage using a drain of no less than f diameter. foley-type catheters can be used, but "mushroom" drains provide a larger area for fluid to enter the drain. two approaches to peritoneal lavage are ( ) retrograde irrigation through a ventrally placed ingress-egress drain and ( ) placement of ingress catheter(s) in the paralumbar fossa(e) for infusion of fluids, with a drain placed ventrally for removal of infused fluid. one must recognize that thorough peritoneal lavage can be achieved only via ventral midline laparotomy. complications associated with the use of abdominal drains or repeated peritoneal penetration to drain fluid include retrograde infection, local irritation, pneumoperitoneum, and subcutaneous seepage around the drain and resultant cellulitis. if the patient is hypovolemic or hypoproteinemic, one should consider volume replacement and administration of plasma before removing large quantities of fluid from the abdomen. in horses with suspected parasite involvement, such as verminous arteritis, one should give larvicidal doses of an anthelmintic once the condition of the horse is stabilized. ivermectin, fenbendazole, and thiabendazole have been recommended as larvacidal therapies. visualization of auscultation sounds of the large intestine small intestinal betagalactosidase activity in the horse rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases equine lymphocyticplasmacytic enterocolitis: a retrospective study of cases eosinophilic enterocolitis and dermatitis in two horses breath hydrogen measurements in ponies: a preliminary study the oral glucose tolerance test in the horse small intestinal malabsorption in the horse: an assessment of the specificity of the oral glucose tolerance test transient glucose malabsorption in two horses: fact or artifact? chronic diarrhoea in adult horses: a review of referred cases effect of food deprivation on d-xylose absorption test results in mares malabsorption syndromes in the horse the diagnostic value of the d-xylose absorption test in horses with unexplained chronic weight loss small intestinal malabsorption in horses effects of extensive small intestinal resection in the pony effects of extensive resection of the small intestine in the pony clinical remission of granulomatous enteritis in a standardbred gelding following long term dexamethasone administration clinical and pathophysiological features of granulomatous enteritis and eosinophilic granulomatosis in the horse inflammatory bowel disease in horses: cases segmental eosinophilic colitis: a review of cases chronic idiopathic inflammatory bowel diseases of the horse section . malabsorption syndromes and maldigestion: pathophysiology, assessment, management, and outcome references of critical care medicine consensus conference: definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis sisson and grossman's the anatomy of domestic animals observational study of "urine testing" in the horse and donkey stallion oral examination and diagnosis: management of oral disease fluoroscopic investigations of swallowing in the horse esophageal dysfunction in a weanling thoroughbred cervical esophagostomy to permit extraoral feeding of the horse use of a liquid diet as the sole source of nutrition in six dysphagic horses and as a dietary supplement in seven hypophagic horses acid-base and electrolyte alterations associated with salivary loss in the pony dysphagia caused by squamous cell carcinoma in horses diseases of the teeth comparative dental pathology (with particular reference to caries and paradental disease in the horse and the dog) some aspects of equine dental radiology some aspects of equine dental decay the median cleft of the lower lip and mandible and its surgical correction in a donkey sisson and grossman's the anatomy of domestic animals esophageal disorders of the horse esophageal disorders in horses: results of nonsurgical and surgical management recurrent esophageal obstruction due to squamous cell carcinoma in a horse squamous cell carcinoma of the lower cervical oesophagus in a pony squamous cell carcinoma: an unusual cause of choke in a horse megaesophagus and aspiration pneumonia secondary to gastric ulceration in a foal persistent right aortic arch in a yearling horse intramural esophageal cyst in a horse surgical correction of esophageal diverticulum in a horse on the true definition of dysphagia retrieval of an esophageal foreign body in a horse dysphagia caused by squamous cell carcinoma in two horses peptic ulcer pathophysiology recommendations for the diagnosis and treatment of equine gastric ulcer syndrome (egus) gastric ulcers in horses: a comparison of endoscopic findings in horses with and without clinical signs pipers fs et al: factors associated with gastric lesions in thoroughbred racehorses cross-sectional study of gastric ulcers of the squamous mucosa in thoroughbred racehorses gastric ulceration in mature thoroughbred horses epidemiological study of gastric ulceration in the thoroughbred racehorse: horses - endoscopic evaluation of the relationship between training, racing, and gastric ulcers prevalence of gastric ulcers in show horses postmortem findings of gastric ulcers in swedish horses older than age one year: a retrospective study of horses ( - ) endoscopic findings of the gastric antrum and pylorus in horses: cases gastric and duodenal ulcers in foals: a retrospective study endoscopic appearance of gastric lesions in foals: cases prevalence of gastric lesions in foals without signs of gastric disease: an endoscopic survey gastroduodenal ulceration in foals regional gastric ph measurement in horses and foals peptic ulcer disease in the pediatric population is helicobacter (campylobacter) pylori associated with gastritis/ulcer disease in asymptomatic foals? aetiopathogenesis and treatment of peptic ulcer in the horse: a comparative review evidence of helicobacter infection in the horse. proceedings of the society for microbiologists comparative pathophysiology of nonglandular ulcer disease: a review of experimental studies section . diseases of the stomach references . blackwell rb, white na: duodenitis-proximal jejunitis in the horse hemorrhagic fibrinonecrotic duodenitis-proximal jejunitis in horses: cases comparison of duodenitis-proximal jejunitis and small intestinal obstruction in horses: cases ( - ) prognostic indicators for horses with duodenitis-proximal jejunitis: horses ( - ) duodenitis-proximal jejunitis integrative immunophysiology in the intestinal mucosa immunophysiology of intestinal electrolyte transport peritoneal fluid constituents in horse with colic due to small intestinal disease modification of gastrointestinal motility in horses. proceedings of the eighteenth annual forum of the american college of veterinary internal medicine haemodynamic effects of small volume hypertonic saline in experimentally induced haemorrhagic shock equine proliferative enteropathy: a cause of weight loss, colic, diarrhea and hypoproteinemia in foals on three breeding farms in canada chronic inflammatory and lymphoproliferative lesions of the equine small intestine aa amyloid-associated gastroenteropathy in a horse malabsorption in the horse associated with alimentary lymphosarcoma clinical aspects of lymphosarcoma in the horse: a clinical report of cases alimentary lymphosarcoma in the horse equine granulomatous enteritis clinical and pathophysiological features of granulomatous enteritis and eosinophilic granulomatosis in the horse chronic idiopathic inflammatory bowel diseases of the horse pathology of equine granulomatous enteritis rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases chronic enteritis associated with malabsorption and protein-losing enteropathy in the horse clinical remission of granulomatous enteritis in a standardbred gelding following long term dexamethasone administration granulomatous enteritis in nine horses a cluster of granulomatous enteritis cases: the link with aluminum equine granulomatous enteritis linked with aluminum chronic eosinophilic gastroenteritis in the horse chronic eosinophilic dermatitis: a manifestion of a multisystemic, eosinophilic, epitheliotropic disease in five horses basophilic enterocolitis in a horse segmental eosinophilic colitis: a review of cases inflammatory bowel disease in horses: cases equine lymphocyticplasmacytic enterocolitis: a retrospective study of cases proliferative and inflammatory intestinal diseases associated with malabsorption and maldigestion references . smith bp: salmonella infection in horses prevalence and epizootiology of equine salmonellosis emergence of antibiotic-resistant salmonella agona in horses in kentucky fecal shedding of salmonella spp by horses in the united states during and and detection of salmonella spp in grain and concentrate sources on equine operations epidemiologic study of salmonellae shedding in the feces of horses and potential risk factors for development of the infection in hospitalized horses risk factors for nosocomial salmonella infection among hospitalized horses an outbreak of salmonellosis among horses at a veterinary teaching hospital outbreak of salmonella infantis infection in a large animal veterinary teaching hospital influence of fecal shedding of salmonella organisms on mortality in hospitalized horses control of an outbreak of salmonellosis caused by drug-resistant salmonella anatum in horses at a veterinary hospital and measures to prevent future infections antimicrobial resistance in salmonella and escherichia coli isolated from horses utilization of both phenotypic and molecular analyses to investigate an outbreak of multidrugresistant salmonella anatum in horses understanding the role of endotoxins in gramnegative sepsis salmonellosis in hospitalized horses: seasonality and case fatality rates salmonellosis in equidae: a study of cases pathogenesis of acute bacterial diarrheal disorders the alimentary canal as a microbial habitat enteric defensins: antibiotic peptide components of intestinal host defense regional specialization in the mucosal immune system: what happens in the microcompartments? salmonella: a model for bacterial pathogenesis pathogenesis of bacterial infections in animals review of veterinary microbiology a preliminary evaluation of some preparations of salmonella typhimurium vaccines in horses aromatic-dependent salmonella typhimurium as modified live vaccines for calves intranasal immunogenicity of a delta cya delta crp-paba mutant of salmonella enterica serotype typhimurium for the horse m cells as ports of entry for enteroinvasive pathogens: mechanisms of interaction, consequences for the disease process type iii secretion machines: bacterial devices for protein delivery into host cells epithelial cells as sensors for microbial infection extraintestinal dissemination of salmonella by cd -expressing phagocytes oxygen-dependent anti-salmonella activity of macrophages pathogenesis of experimental salmonellosis: inhibition of protein synthesis by cytotoxin pathogenesis of salmonella-mediated intestinal fluid secretion: activation of adenylate cyclase and inhibition by indomethacin elevated camp in intestinal epithelial cells during experimental cholera and salmonellosis role of salmonella enterotoxin in overall virulence of the organism mutation of invh, but not stn, reduces salmonella-induced enteritis in cattle importance of the intestinal inflammatory reaction in salmonella-mediated intestinal secretion pathophysiology of infectious diarrhea: changes in intestinal structure and function digestive physiology of the large intestine in adult horses. . pathophysiology of colitis neuroimmunophysiology of the gastrointestinal mucosa: implications for inflammatory diseases transepithelial signaling to neutrophils by salmonellae: a novel virulence mechanism for gastroenteritis review article: pathobiology of neutrophil interactions with intestinal epithelia equine salmonellosis: experimental production of four syndromes characteristics and risk factors for failure to survive of horses with acute diarrhea: cases ( - ) diagnosing salmonellosis in horses: culturing of multiple versus single faecal samples comparison of rectal mucosal cultures and fecal cultures in detecting salmonella infection in horses and cattle comparison of polymerase chain reaction and microbiological culture for detection of salmonellae in equine feces and environmental samples rapid pcr detection of salmonella in horse faecal samples potomac horse fever equine monocytic ehrlichiosis (potomac horse fever): a review experimental reproduction of potomac horse fever in horses with a newly isolated ehrlichia organism causative ehrlichial organisms in potomac horse fever ehrlichial diseases attempted ehrlichia risticii transmission with dermacentor variabilis (acari: ixodidae) attempted transmission of ehrlichia risticii (rickettsiaceae) with stomoxys calcitrans (diptera: muscidae) production and characterization of ehrlichia risticii, the agent of potomac horse fever, from snails (pleuroceridae: juga spp.) in aquarium culture and genetic comparison to equine strains infection of aquatic insects with trematode metacercariae carrying ehrlichia risticii, the cause of potomac horse fever transmission of ehrlichia risticii, the agent of potomac horse fever, using naturally infected aquatic insects and helminth vectors: preliminary report infection rate of ehrlichia risticii, the agent of potomac horse fever, in freshwater stream snails (juga yrekaensis) from northern california ultrastructural study of ehrlichial organisms in the large colons of ponies infected with potomac horse fever enterocolitis caused by ehrlichia sp. in the horse (potomac horse fever) growth of ehrlichia risticii in human colonic epithelial cells respiratory burst activity associated with phagocytosis of ehrlichia risticii by mouse peritoneal macrophages in vitro killing of ehrlichia risticii by activated and immune mouse peritoneal macrophages lack of lysosomal fusion with phagosomes containing ehrlichia risticii in p d cells: abrogation of inhibition with oxytetracycline pathophysiological changes in the large colon of horses infected with ehrlichia risticii disease features in horses with induced equine monocytic ehrlichiosis (potomac horse fever) clinical and hematologic variables in ponies with experimentally induced equine ehrlichial colitis (potomac horse fever) evaluation of fetal infection and abortion in pregnant ponies experimentally infected with ehrlichia risticii identification of ehrlichia risticii as the causative agent of two equine abortions following natural maternal infection effect of equine ehrlichial colitis on the hemostatic system in ponies detection of serum antibodies against ehrlichia risticii in potomac horse fever by enzyme-linked immunosorbent assay evidence for a high rate of false-positive results with the indirect fluorescent antibody test for ehrlichia risticii antibody in horses detection and quantitation of ehrlichia risticii genomic dna in infected horses and snails by real-time pcr comparison of pcr and culture to the indirect fluorescent-antibody test for diagnosis of potomac horse fever diagnostic application of polymerase chain reaction for detection of ehrlichia risticii in equine monocytic ehrlichiosis (potomac horse fever) evaluation of vaccination of horses as a strategy to control equine monocytic ehrlichiosis association of deficiency in antibody response to vaccine and heterogeneity of ehrlichia risticii strains with potomac horse fever vaccine failure in horses equine intestinal clostridiosis: an acute disease in horses associated with high intestinal counts of clostridium perfringens type a a method for reproducing fatal idiopathic colitis (colitis x) in ponies and isolation of a clostridium as a possible agent hemorrhagic necrotizing enterocolitis associated with clostridium difficile infection in four foals apparent outbreaks of clostridium difficile-associated diarrhea in horses in a veterinary medical teaching hospital a prospective study of the roles of clostridium difficile and enterotoxigenic clostridium perfringens in equine diarrhoea clostridial enterocolitis prevalence of clostridium perfringens enterotoxin and clostridium difficile toxin a in feces of horses with diarrhea and colic clostridium difficile associated with acute colitis in mature horses treated with antibiotics the effect of colic on the microbial activity of the equine intestine comparative effects of oral administration of trimethoprim/sulphadiazine or oxytetracycline on the faecal flora of horses colonic fermentation to short-chain fatty acids is decreased in antibiotic-associated diarrhea clostridium difficile associated with acute colitis in mares when their foals are treated with erythromycin and rifampicin for rhodococcus equi pneumonia lincomycin-induced severe colitis in ponies: association with clostridium cadaveris an investigation into clostridium perfringens enterotoxin-associated diarrhoea enterotoxigenic clostridium perfringens esophageal duplication cyst as a cause of choke in the horse beta toxin, a novel toxin produced by clostridium perfringens prevalence of beta -toxigenic clostridium perfringens in horses with intestinal disorders an overview of clostridium perfringens enterotoxin the effects of clostridium perfringens type a enterotoxin in shetland ponies: clinical, morphologic and clinicopathologic changes an update on clostridium perfringens enterotoxin clostridium difficile infection direct evidence of mast cell involvement in clostridium difficile toxin a-induced enteritis in mice neutrophil recruitment in clostridium difficile toxin a enteritis in the rabbit neuronal involvement in the intestinal effects of clostridium difficile toxin a and vibrio cholerae enterotoxin in rat ileum increased substance p responses in dorsal root ganglia and intestinal macrophages during clostridium difficile toxin a enteritis in rats cp- , , a substance p antagonist, inhibits rat intestinal responses to intestinal infarction associated with mesenteric vascular thrombotic disease in the horse the role of parasites in colic retropulsion-propulsion in equine large colon diarrhoea in horses associated with ulceration of the colon and caecum resulting from s. vulgaris larval migration clinical response of pony foals experimentally infected with strongylus vulgaris host response to experimentally induced infections of strongylus vulgaris in parasite-free and naturally infected ponies morphologic and clinicopathologic changes following strongylus vulgaris infections of immune and nonimmune ponies increase of immunoglobulin t concentration in ponies as a response to experimental infection with the nematode strongylus vulgaris larval cyathostomiasis immature stages of trichonema spp as a cause of diarrhoea in adult horses in spring larval cyathostomiasis (immature trichonema-induced enteropathy): a report of clinical cases diagnosis and successful treatment of diarrhoea in horses caused by immature small strongyles apparently insusceptible to anthelmintics recurrent diarrhoea in aged ponies associated with larval cyathostomiasis outbreak of larval cyathostomiasis among a group of yearling and two-year-old horses pathogenicity of cyathostome infection chronic diarrhoea in adult horses: a review of referred cases diarrhoea in adult horses: a survey of clinical cases and an assessment of some prognostic indices the pathogenic effects of experimental cyathostome infections in ponies fructosamine measurement in ponies: validation and response following experimental cyathostome infection re-evaluation of ivermectin efficacy against equine gastrointestinal parasites prevalence and clinical implications of anthelmintic resistance in cyathostomes of horses equine cyathostome infection: suppression of faecal egg output with moxidectin experimental cyathostome challenge of ponies maintained with or without benefit of daily pyrantel tartrate feed additive: comparison of parasite burdens, immunity and colonic pathology identification and characterization of a pyrantel pamoate resistant cyathostome population lethal complications following administration of oxytetracycline in the horse lincomycin-associated colitis in horses risk of adverse effects in pneumonic foals treated with erythromycin versus other antibiotics: cases ( - ) case control and historical cohort study of diarrhea associated with administration of trimethoprim-potentiated sulphonamides to horses and ponies the association of erythromycin ethylsuccinate with acute colitis in horses in sweden effects of transportation, surgery, and antibiotic therapy in ponies infected with salmonella the influence of the normal flora on clostridium difficile colonisation of the gut studies on experimental enteric salmonellosis in ponies physiology of diarrhea: large intestine impaired colonic fermentation of carbohydrate after ampicillin the relationship of absorption characteristics and gastrointestinal side effects of oral antimicrobial agents prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period hypovolemic hyponatremia and signs of neurologic disease associated with diarrhea in a foal toxicology of nonsteroidal antiinflammatory drugs colitis induced by nonsteroidal anti-inflammatory drugs: report of four cases and review of the literature the effects of phenylbutazone on the intestinal mucosa of the horse: a morphological, ultrastructural and biochemical study experimentally induced phenylbutazone toxicosis in ponies: description of the syndrome and its prevention with synthetic prostaglandin e phenylbutazone toxicosis in the horse: a clinical study ulceration and stricture of the right dorsal colon after phenylbutazone administration in four horses phenylbutazone toxicity in a horse biochemical and haematological effects of phenylbutazone in horses medical management of right dorsal colitis in horses: a retrospective study mechanisms of intestinal mucosal repair prostaglandins in the gut and their relationship to non-steroidal anti-inflammatory drugs the future of antiinflammatory therapy the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse mechanisms of acute and chronic intestinal inflammation induced by indomethacin mechanisms of nsaid-induced gastrointestinal injury defined using mutant mice pathogenesis of nsaid gastropathy: are neutrophils the culprits? icam- and p-selectin expression in a model of nsaid-induced gastropathy gastric ulceration induced by nonsteroidal anti-inflammatory drugs is a neutrophil-dependent process the recognition and medical management of right dorsal colitis in horses the application of technetium- m hexamethylpropyleneamine oxime ( mtc-hmpao) labeled white blood cells for the diagnosis of right dorsal ulcerative colitis in two horses cantharidin toxicosis in horses experimental cantharidiasis in the horse pathology of blister beetle (epicauta) poisoning in horses etiologic agents, incidence, and improved diagnostic methods of cantharidin toxicosis in horses clinical features of blister beetle poisoning in equids: cases clinical and diagnostic veterinary toxicology environmental biochemistry of arsenic trace metal poisoning gee, honey, why does the iced tea have a garlic taste? arsenic intoxication acute diarrheal disease in the horse the mucosal barrier, ige-mediated gastrointestinal events, and eosinophilic gastroenteritis ige-mediated (and food-induced) intestinal disease induction of anaphylaxis in mouse intestine by orally administered antigen and its prevention with soluble high affinity receptor for ige vaccine-associated anaphylactic-like reaction in a horse equine anaphylaxis equine anaphylaxis pathologic changes in experimental equine anaphylaxis role of intestinal mast cells in modulating gastrointestinal pathophysiology role of -hydroxytryptamine in intestinal water and electrolyte movement during gut anaphylaxis intestinal anaphylaxis in the rat: mediators responsible for the ion transport abnormalities motility effects of intestinal anaphylaxis in the rat intestinal anaphylaxis: rapid changes in mucosal ion transport and morphology exhaustion shock in the horse lactic acidosis: a factor associated with equine laminitis changes in the caecal flora associated with the onset of laminitis intracecal endotoxin and lactate during the onset of equine laminitis: a preliminary report plasma endotoxin levels in horses subjected to carbohydrate induced laminitis surgical treatment of sand colic: results in horses diarrhea associated with sand in the gastrointestinal tract of horses sand-induced diarrhea in a foal abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation nasogastric electrolyte replacement in horses acute colitis in horses. . initial management effect of hydroxyethyl starch infusion on colloid oncotic pressure in hypoproteinemic horses oncotic, hemodilutional, and hemostatic effects of isotonic saline and hydroxyethyl starch solutions in clinically normal ponies colloid volume expanders: problems, pitfalls and possibilities immunity targeting common core antigens of gram-negative bacteria protection against clinical endotoxemia in horses by using plasma containing antibody to an rc mutant e. coli (j ) enterotoxin activity of a salmonella typhimurium of equine origin in vivo in rabbits and the effect of salmonella culture lysates and cholera toxin on equine colonic mucosa in vitro enterotoxin-induced fluid accumulation during experimental salmonellosis and cholera: involvement of prostaglandin synthesis by intestinal cells nacl transport across equine proximal colon and the effect of endogenous prostanoids nonsteroidal anti-inflammatory drugs activate quiescent inflammatory bowel disease misoprostol provides a colonic mucosal protective effect during acetic acidinduced colitis in rats rationale for the luminal provision of butyrate in intestinal diseases pharmacokinetics and adverse effects of butorphanol administered by single intravenous injection or continuous intravenous infusion in horses necrotizing mycotic vasculitis with cerebral infarction caused by aspergillus niger in a horse with acute typholocolitis pulmonary aspergillosis in horses: cases ( - ) effect of antibiotics on clinical, pathologic and immunologic responses in murine potomac horse fever: protective effects of doxycycline effect of treatment with oxytetracycline during the acute stages of experimentally induced equine ehrlichial colitis in ponie effect of treatment with erythromycin and rifampin during the acute stages of experimentally induced equine ehrlichial colitis in ponies use of metronidazole in equine acute idiopathic toxaemic colitis antimicrobial susceptibilities of equine isolates of clostridium difficile and molecular characterization of metronidazole-resistant strains equine neonatal clostridiosis: treatment and prevention antimicrobial susceptibility of ileal symbiont intracellularis isolated from pigs with proliferative enteropathy antithrombotic actions of aspirin in the horse evaluation of heparin for prophylaxis of equine laminitis: cases ( - ) phagocytosis of gelatin-latex particles by a murine macrophage line is dependent on fibronectin and heparin cold insoluble globulin and heparin interactions in phagocytosis by macrophage monolayers: mechanism of heparin enhancement probiotics in man and animals double-blind report on the efficacy of lactic acid-producing enterococcus sf in the prevention of antibiotic-associated diarrhoea and in the treatment of acute diarrhoea effect of lactobacillus gg yoghurt in prevention of antibiotic associated diarrhoea comparative efficacy of moxidectin and ivermectin against hypobiotic and encysted cyathostomes and other equine parasites efficacy of oral ivermectin paste against mucosal stages of cyathostomes elimination of mucosal cyathostome larvae by five daily treatments with fenbendazole moxidectin: spectrum of activity and uses in an equine anthelmintic program references . laws eg, freeman de: significance of reperfusion injury after venous strangulation obstruction of equine jejunum how important is intestinal reperfusion injury in horses? clinical relevance of intestinal reperfusion injury in horses strangulating volvulus of the ascending colon in horses mucosal alterations in experimentally induced small intestinal strangulation obstruction in ponies histologic findings in the gastrointestinal tract of horses with colic large colon resection evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal diseases of the small intestine small intestinal herniation through the epiploic foramen: cases ( - ) determining the diagnosis and prognosis of the acute abdomen duodenitis-proximal jejunitis risk factors and clinical signs associated with cases of equine colic short-term survival and prevalence of postoperative ileus after small intestinal surgery in horses short-and longterm survival and prevalence of postoperative ileus after small intestinal surgery in the horse survival after small intestine resection and anastomosis in horses abdominal adhesions after small intestinal surgery in the horse mesenteric rents as a source of small intestinal strangulation in horses: cases ( - ) small intestine incarceration through the epiploic foramen of the horse right hepatic lobe atrophy in horses: cases incarceration of the jejunum in the epiploic foramen of a four month old foal use of diagnostic ultrasonography in horses with signs of acute abdominal pain parietal hernia of the small intestine into the epiploic foramen of a horse transection of the pelvic flexure to reduce incarceration of the large colon through the epiploic foramen in a horse incarceration of the small intestine in the epiploic foramen: report of cases pedunculated lipomas as a cause of intestinal obstruction in horses: cases ( - ) an analysis of cases of intestinal obstruction caused by pedunculated lipomas strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma abdominal surgery in foals intestinal surgery in the foal effects of extensive resection of the small intestine in the pony incarceration of small intestine through rents in the gastrosplenic ligament in the horse jejunal displacement through the mesometrium in a pregnant mare strangulating obstruction caused by intestinal herniation through the proximal aspect of the cecocolic fold in horses congenital inguinal hernias associated with a rent in the common vaginal tunic in five foals ruptured inguinal hernia in new-born colt foals: a review of cases acquired inguinal hernia in the horse: a review of cases surgical treatment of acquired inguinal hernia in the horse: a review of cases different types of inguinal herniation in two stallions and a gelding surgery of the small intestine complications of umbilical hernias in horses: cases strangulated umbilical hernias in horses: cases ( - ) surgical management of intussusception in the horse ileocecal intussusception in horses: cases ultrasonographic diagnosis of small-intestinal intussusception in three foals jejunal intussusception in adult horses: cases ileocecal intussusception corrected by resection within the cecum in two horses diaphragmatic hernias in horses and cattle diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases peritoneopericardial hernia in a horse diaphragmatic herniation as a cause of lethargy and exercise intolerance in a mare diaphragmatic hernia repair in three young horses surgical repair of a diaphragmatic hernia in a racehorse diaphragmatic hernias in the horse: a review of the literature and an analysis of six additional cases morphologic alterations observed during experimental ischemia of the equine large colon equine large intestinal volvulus: a review of cases diseases and surgery of the large colon large colon volvulus: surgical treatment of horses colopexy in broodmares: cases cecocolic intussusception in horses: cases ( - ) cecocolic and cecocecal intussusception in horses: cases ( - ) cecal amputation via a right ventral colon enterotomy for correction of nonreducible cecocolic intussusception in horses resection of intussuscepted large colon in a horse intussusception of the large colon in a horse intussusception of the left dorsal colon in a horse intussusception of the colon in a filly rectal prolapse in the horse rectal prolapse and cystic calculus in a burro rectal prolapse in a foaling mare management of rectal injuries rectum and anus disruption to the blood supply to the small colon following rectal prolapse and small colon intussusception in a mare laparoscopic diagnosis of ischemic necrosis of the descending colon after rectal prolapse and rupture of the mesocolon in two postpartum mares intestinal infarction associated with mesenteric vascular thrombotic disease in the horse diseases of the large colon diagnostic and prognostic procedures for equine colic surgery examination of the horse with colic detection of endotoxin in cases of equine colic determining the diagnosis and prognosis of the acute abdomen risk factors and clinical signs associated with cases of equine colic accuracy of clinicians in predicting site and type of lesion as well as outcome in horses with colic struvite urethral calculus in a three-month-old thoroughbred colt urolithiasis in horses cholelithiasis in horses: ten cases review of cases of colic in the pregnant mare surgical management of uterine torsion in the mare: a review of cases rabies in horses: cases mechanisms of pain and their therapeutic implications gastrointestinal pharmacology nonsteroidal anti-inflammatory drugs the role of cyclooxygenase inhibitors in repair of ischaemic-injured jejunal mucosa in the horse alpha adrenoceptor agonists in the horse: a review cardiovascular effects of medetomidine, detomidine and xylazine in horses cardiovascular effects of xylazine and detomidine in horses prognosis in equine colic: a study of individual variables used in case assessment a: study of variables commonly used in examination of equine colic cases to assess prognostic value clinical evaluation of blood lactate levels in equine colic use of clinical pathology in evaluation of horses with colic the anion gap as a prognostic indicator in horses with abdominal pain strangulating volvulus of the ascending colon in horses diseases and surgery of the large colon multivariable prediction model for the need for surgery in horses with colic prognosis in equine colic patients using multivariable analysis prognosis in equine colic: a comparative study of variables used to assess individual cases prognostic index for acute abdominal crisis (colic) in horses feeding and digestive problems in horses: physiologic responses to a concentrated meal effect of meal feeding on plasma volume and urinary electrolyte clearance in ponies evaluation of the microcirculation of the equine small intestine after intraluminal distention and subsequent decompression morphologic effects of experimental distention of equine small intestine surgical reduction of ileal impactions in the horse: cases medical treatment of horses with ileal impactions: cases ascarids: recent advances the purported role of coastal bermuda hay in the etiology of ileal impactions: results of a questionnaire (abstract) tapeworm infection is a significant risk factor for spasmodic colic and ileal impaction colic in the horse use of excretory/secretory antigens for the serodiagnosis of anoplocephala perfoliata cestodosis ileal impaction in the horse: cases evaluation of factors associated with postoperative ileus in horses: cases idiopathic muscular hypertrophy of the equine small intestine: cases ( - ) obstruction of the ileum in the horse: a report of clinical cases ileal muscular hypertrophy and rupture in a pony three years after surgery for ileocaecal intussusception jejunocolostomy or ileocolostomy for treatment of cecal impaction in horses: nine cases ( - ) small intestinal strangulation caused by meckel's diverticulum in a horse volvulus associated with meckel's diverticulum in the horse congenital jejunal diverticulum in a foal mesodiverticular bands as a cause of small intestinal strangulation and volvulus in the horse abdominal adhesions after small intestinal surgery in the horse induction of peritoneal adhesions with small intestinal ischaemia and distention in the foal the characteristics of intestinal injury peripheral to strangulating obstruction lesions in the equine small intestine current concepts in management of abdominal adhesions one percent sodium carboxymethylcellulose prevents experimentally induced abdominal adhesions in horses effect of carboxymethylcellulose and a hyaluronate-carboxymethylcellulose membrane on healing of intestinal anastomoses in horses intraperitoneal use of sodium carboxymethylcellulose in horses undergoing exploratory celiotomy prevention of intraabdominal adhesions in ponies by low-dose heparin therapy retrospective analysis of the results of exploratory laparotomies in horses with gastrointestinal disease surgical treatment for colic in the foal retrospective evaluation of repeat celiotomy in horses with acute gastrointestinal disease risk factors for reduced postoperative fecal output in horses: cases ( - ) role of inducible nitric oxide synthase in postoperative intestinal smooth muscle dysfunction in rodents surgically induced leukocytic infiltrates within the rat intestinal muscularis mediate postoperative ileus surgical manipulation of the gut elicits an intestinal muscularis inflammatory response resulting in postsurgical ileus pathophysiology of equine postoperative ileus: effect of adrenergic blockade, parasympathetic stimulation and metoclopramide in an experimental model the effect of prostaglandin e on motility of the equine gut in vitro investigation of the effect of prostaglandins and nonsteroidal antiinflammatory drugs on contractile activity of the equine smooth muscle of the dorsal colon, ventral colon, and pelvic flexure evaluation of nitric oxide as an inhibitory neurotransmitter in the equine ventral colon prostanoid production via cox- as a causative mechanism of rodent postoperative ileus the action of low dose endotoxin on equine bowel motility sir frederick hobday memorial lecture: all wind and water-some progress in the study of equine gut motility antagonism of endotoxin-induced disruption of equine bowel motility by flunixin and phenylbutazone cyclooxygenase inhibitors in equine practice in vitro effects of erythromycin, lidocaine, and metoclopramide on smooth muscle from the pyloric antrum, proximal portion of the duodenum, and middle portion of the jejunum of horses efficacy of metoclopramide for treatment of ileus in horses following small intestinal surgery: cases ( - ) role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea prokinetic effects of erythromycin on the ileum, cecum, and pelvic flexure of horses during the postoperative period cecal impaction in the horse caecal disease in equids ileocolostomy: a technique for surgical management of equine cecal impaction diseases and surgery of the cecum treatment of impaction colics large colon impaction in horses: cases ( - ) effects of amitraz, several opiate derivatives and anticholinergic agents on intestinal transit in ponies experimental studies of druginduced impaction colic in the horse retropulsion-propulsion in equine large colon clinical and structural features of equine enteroliths petrographic and geochemic evaluation of equine enteroliths enteroliths in horses evaluation of enterolithiasis in equids: cases risk factors for enterolithiasis among horses in texas obstructive enterolith in an -month-old miniature horse surgical treatment of sand colic in equids: cases ( - ) surgical treatment of sand colic: results in horses diarrhea associated with sand in the gastrointestinal tract of horses abdominal auscultation in the detection of experimentally induced gastrointestinal sand accumulation failure of psyllium mucilloid to hasten evaluation of sand from the equine large intestine nonstrangulated colonic displacement in horses functions of the equine large intestine and their interrelationship in disease comparisons of age, breed, history and management in horses with colic motor functions of the intestine the effect of strongylus vulgaris larvae on equine intestinal myoelectrical activity displacement of the large colon reversal of colonic net absorption to net secretion with increased intraluminal pressure use of ultrasound in horses for diagnosis of left dorsal displacement of the large colon and monitoring its nonsurgical correction renosplenic entrapment of the large colon in horses: cases renosplenic entrapment of the large colon in horses: cases further experiences with non-surgical correction of nephrosplenic entrapment of the left colon in the horse effect of phenylephrine on hemodynamics and splenic dimensions in horses displacement of the large colon associated with nonsurgical correction of large-colon entrapment in the renosplenic space in a mare foreign body obstruction of the small colon in six horses fecalith impaction in four miniature foals obstruction of the small colon by intramural haematoma in three horses submucosal haematoma as a cause of obstruction of the small colon in the horse: a review of four cases inflammatory bowel disease in horses: cases intestinal atresia in horses clinical survey of tumours and tumourlike lesions in horses in south east queensland abdominal neoplasia (excluding urogenital tract) clinical manifestation of squamous cell carcinoma in horses lymphoma (lymphosarcoma) in horses oral and dental tumors in equine denistry t cell-rich b cell lymphosarcoma in the tongue of a horse multiple myeloma in a horse rhabdomyosarcoma of the tongue in a horse paraneoplastic bullous stomatitis in a horse gastric squamous cell carcinoma in three horses use of esophagoscopy in the diagnosis of esophageal squamous cell carcinoma in a horse gastric hyperplastic polyp in a horse gastric leiomyosarcoma in a horse multisystemic, eosinophilic, epitheliotropic disease with intestinal lymphosarcoma in a horse an immunohistochemical study of equine b-cell lymphoma squamous cell carcinoma of the equine stomach: a report of five cases six cases of squamous cell carcinoma of the stomach of the horse small intestinal adenocarcinoma in a horse ganglioneuroma as a cause of small intestinal obstruction in the horse: a case report intestinal carcinoid in a mare: an etiologic consideration for chronic colic in horses leiomyoma of the small intestine in a horse jejunal intussusception associated with leiomyoma in an aged horse duodenal leiomyoma associated with colic in a two-year-old horse leiomyosarcoma of the duodenum in two horses pedunculated lipomas as a cause of intestinal obstruction in horses: cases ( - ) clinical aspects of lymphosarcoma in the horse: a clinical report of cases multiple peripheral nerve sheath tumors in the small intestine of a horse equine adenocarcinomas of the large intestine with osseous metaplasia intestinal myxosarcoma in a thoroughbred mare gastrointestinal stromal tumors of the equine cecum colonic adenocarcinoma with osseous metaplasia in a horse intestinal adenocarcinoma causing recurrent colic in the horse equine colonic lipomatosis large colon resection for treatment of lymphosarcoma in two horses colic in a mare caused by a colonic neurofibroma small colon intussusception associated with an intralumenal leiomyoma in a pony leiomyoma of the small colon in a horse an analysis of cases of intestinal obstruction caused by pedunculated lipomas rectal leiomyosarcoma in a horse strangulation of the rectum of a horse by the pedicle of a mesenteric lipoma rectal biopsy diagnosis in horses with clinical signs of intestinal disorders: a retrospective study of cases standing rectal and tail surgery disseminated peritoneal leiomyomatosis in a horse ascites as a result of peritoneal mesotheliomas in a horse a case of peritoneal mesothelioma in a thoroughbred mare omental fibrosarcoma in a horse neoplasia of the mouth and surrounding structure pleuroscopic diagnosis of gastroesophageal squamous cell carcinoma in a horse recurrent esophageal obstruction due to squamous cell carcinoma in a horse chronic colic in the mature horse: a retrospective review of cases recurrent colic in the mature horse: a retrospective review of cases paraneoplastic syndromes lymphosarcoma and associated immune-mediated hemolytic anemia and thrombocytopenia in horses differentiation of chronic lymphocytic leukemia in the horse: a report of two cases hypercalcemia associated with malignancy in a horse adenocarcinoma of intestinal origin in a horse: diagnosis by abdominocentesis and laparoscopy differentiation between intra-abdominal neoplasms and abscesses in horses, using clinical and laboratory data: cases ( - ) malabsorption in the horse associated with alimentary lymphosarcoma immunodeficiency associated with lymphosarcoma in a horse flow cytometric methods to diagnose selected equine immune-mediated disorders pleural effusion associated with squamous cell carcinoma of the stomach of a horse use of diagnostic ultrasonography in horses with signs of acute abdominal pain the indications for equine laparotomy: an analysis of cases equine lymphosarcoma diarrhoea in the horse as a result of alimentary lymphosarcoma paraneoplastic pruritus and alopecia in a horse with diffuse lymphoma lymphoma in the horse. proceedings of the twelfth annual meeting of the american college of veterinary internal medicine squamous cell carcinoma of the pharyngeal wall in a horse squamous cell carcinoma of the oral, pharyngeal and nasal mucosa in the horse treatment of superficial ulcerative squamous cell carcinoma in three horses with topical -fluorouracil intratumoral chemotherapy with cisplatin in oily emulsion in horses excision of oral squamous cell carcinoma in a horse extensive resection and anastomosis of the descending (small) colon in a mare following strangulation by a mesenteric lipoma section . peritonitis references . hosgood g: peritonitis. . a review of the pathophysiology and diagnosis the peritoneum and peritoneal cavity large animal internal medicine diseases of the peritoneum and mesentery equine internal medicine peritonitis associated with actinobacillus equuli in horses: cases veterinary gastroenterology peritonitis and other intra-abdominal infection antibiotic susceptibility of bacterial pathogens from horses peritonitis in horses: cases biologic reactions to endotoxin effect of dietary alpha-linoleic acid on equine monocyte procoagulant activity and eicosanoid synthesis review of cases of peritonitis in the horse reference values for equine peritoneal fluid diagnostic cytology in the equine species: overview of effusions (peritoneal, pleural, and synovial joint) and transtracheal wash effects of enterocentesis on peritoneal fluid constituents in the horse internal abdominal abscesses in the horse: a study of cases equine peritoneal fluid analysis following celiotomy analysis of equine peritoneal fluid evaluation of peritoneal fluid ph, glucose concentration, and lactate dehydrogenase activity for detection of septic peritonitis in horses tumor necrosis factor and interleukin- activity and endotoxin concentration in peritoneal fluid and blood of horses with acute abdominal disease low dose flunixin meglumine: effects on eicosanoid production and clinical signs induced by experimental endotoxemia in horses heparin in the treatment of experimental peritonitis erythrocyte agglutination associated with heparin treatment in three horses therapeutic strategies involving antimicrobial treatment of large animals with peritonitis effect of long-term administration of injectable enrofloxacin solution on physical and musculoskeletal variables in adult horses peritoneal lavage in the horse pharmacologic principles therapeutic drug monitoring: a tool for rational drug therapy. proceedings of the seventh american college of veterinary internal medicine forum toxicity of quinolones although a number of potential causes of peritonitis exist, sepsis is a common and serious complication, and the identification and control of bacterial sepsis is critical for a successful outcome. bowel leakage (as well as external trauma) results in contamination of the peritoneum with large numbers of many types of bacteria. the intestinal tract contains a mixed population of bacteria, and the quantity of bacteria and prevalence of anaerobic species increase in the distal segments. [ ] [ ] [ ] [ ] [ ] [ ] [ ] there are approximately × anaerobic and × aerobic bacteria per milliliter of cecal and colonic fluid, thus the potential for bacterial contamination of the peritoneum is great. high mortality is associated with contamination from the lower bowel because of the large numbers of bacteria present. hirsch and jang reported isolation of an infective agent from equine peritoneal fluid in approximately % of attempts. obligate anaerobic bacteria were cultured most frequently, followed by members of the enterobacteriaceae family (escherichia coli). penicillin-resistant bacteroides fragilis was isolated from % to % of cases. in another study in which bacteria were identified in equine abdominal fluid by cytologic examination or culture, e. coli was the organism most commonly isolated. in human beings and laboratory animals the well-established fact is that despite the variety of organisms initially introduced subsequent to these events, established infections are characterized by only a few types of bacteria, which are often gram-negative aerobes and anaerobic bacteria. this selectivity occurs through the processes of selective reduction of bacterial populations and bacterial synergism. a well-known example of synergism in human beings and laboratory animals is peritonitis involving e. coli and b. fragilus. the presence of each organism is beneficial to the survival of the other, and each is important in the overall pathogenesis of the disease. e. coli is associated with septicemia and early mortality, whereas b. fragilis infection tends to result in chronic abscessation with delayed morbidity and mortality. some evidence suggests that in horses, in addition to coliforms and anaerobes, streptococci and perhaps c. psuedotuberculosis may survive selective reduction and participate in synergistic infection following polymicrobial contamination.biologic events resulting from contamination of the abdomen or injury to the mesothelial cells have been described [ ] [ ] [ ] [ ] and include release of catecholamines, histamine, and serotonin from peritoneal mast cells; vasodilation and hyperemia; increase in peritoneal vascular permeability; secretion of protein-rich fluid into the peritoneum; transformation of mesothelial cells into macrophages; and influx of polymorphonuclear cells, humoral opsonins, natural antibodies, and serum complement into the peritoneal cavity. additionally, depression of the peritoneal fibrinolytic activity, fibrin deposits on the peritoneal surface, and sympathetic-mediated ileus of the gastrointestinal tract can occur.these processes benefit the animal by confining contamination and infection, and indeed, with clean, minimally invasive procedures such as enterocentesis or trocharization, this is effective. however, with greater severity of peritoneal contamination or irritation, these processes are magnified and become deleterious, resulting in problems such as hypovolemia, hypoproteinemia, ileus with resultant bowel distention, ischemia of the bowel wall with subsequent absorption of bacteria and toxins, and ultimately adhesion and abscess formation. additionally, systemic responses to bacterial toxins, particularly lipopolysaccharide, , can compromise the metabolic condition of the patient further. equine peritoneal macrophages release several mediators when exposed to bacterial lipopolysaccharide, undoubtedly an important component of septic peritonitis.pathologic description of peritonitis includes origin (primary or secondary), onset (peracute, acute, chronic), distribution (localized versus diffuse), and presence of bacteria (septic versus nonseptic). , clinically, viewing key: cord- -hnf vayd authors: ford, richard b.; mazzaferro, elisa m. title: emergency care date: - - journal: kirk and bistner's handbook of veterinary procedures and emergency treatment doi: . /b - - - / - sha: doc_id: cord_uid: hnf vayd nan in the event that you suspect peritonitis and have a negative tap with abdominal paracentesis, a diagnostic peritoneal lavage can be performed. to perform abdominal paracentesis, follow this procedure: . place the patient in left lateral recumbency and clip a -to -inch square with the umbilicus in the center. . aseptically scrub the clipped area with antimicrobial scrub solution. . wearing gloves, insert a -or -gauge needle or over-the-needle catheter in four quadrants: cranial and to the right, cranial and to the left, caudal and to the right, and caudal and to the left of the umbilicus. as you insert the needle or catheter, gently twist the needle to push any abdominal organs away from the tip of the needle. local anesthesia typically is not required for this procedure, although a light sedative or analgesic may be necessary if severe abdominal pain is present. in some cases, fluid will flow freely from one or more of the needles. if not, gently aspirate with a -to -ml syringe or aspirate with the patient in a standing position. avoid changing positions with needles in place because iatrogenic puncture of intraabdominal organs may occur. . save any fluid collected in sterile red-and lavender-topped tubes for cytologic and biochemical analyses and bacterial culture. monitor hemorrhagic fluid carefully for the presence of clots. normally, hemorrhagic effusions rapidly become defibrinated and do not clot. clot formation can occur in the presence of ongoing active hemorrhage or may be due to the iatrogenic puncture of organs such as the spleen or liver. if abdominal paracentesis is negative, a diagnostic peritoneal lavage can be performed. peritoneal dialysis kits are commercially available but are fairly expensive and often impractical. to perform a diagnostic peritoneal lavage, follow this procedure: . clip and aseptically scrub the ventral abdomen as described previously. . wearing sterile gloves, cut multiple side ports in a -or -gauge over-the needle catheter. use care to not cut more than % of the circumference of the catheter, or else the catheter will become weakened and potentially can break off in the patient's abdomen. . insert the catheter into the peritoneal cavity caudal and to the right of the umbilicus, directing the catheter dorsally and caudally. . infuse to ml of sterile lactated ringer's solution or . % saline solution that has been warmed to the patient's body temperature. during the instillation of fluid into the peritoneal cavity, watch closely for signs of respiratory distress because an increase in intraabdominal pressure can impair diaphragmatic excursions and respiratory function. . remove the catheter. . in ambulatory patients, walk the patient around while massaging the abdomen to distribute the fluid throughout the abdominal cavity. in nonambulatory patients, gently roll the patient from side to side. . next, aseptically scrub the patient's ventral abdomen again, and perform an abdominal paracentesis as described previously. save collected fluid for culture and cytologic analyses; however, biochemical analyses may be artifactually decreased because of dilution. remember that you likely will retrieve only a small portion of the fluid that you instilled. during the early stage of repair, granulation tissue, some exudate, and minor epithelialization is observed. place a nonadherent bandage with some antibacterial properties (petroleum or nitrofurazone-impregnated gauze) or absorbent material (foam sponge, hydrogel, or hydrocolloid dressing) in direct contact with the wound to minimize disruption of the granulation tissue bed. next, place an absorbent intermediate layer, followed by a porous outer layer, as previously described. granulation tissue can grow through gauze mesh or adhere to foam sponges and can be ripped away at the time of bandage removal. hemorrhage and disruption of the granulation tissue bed can occur. later in the repair process, granulation tissue can exude sanguineous drainage and have some epithelialization. a late nonadherent bandage is required. the contact layer should be some form of nonadherent dressing, foam sponge, hydrogel, or hydrocolloid substance. the intermediate layer and outer layers should be absorbent material and porous tape, respectively. with nonadherent dressings, wounds with viscous exudates may not be absorbed well. this may be advantageous and enhance epithelialization, provided that complications do not occur. infection, exuberant granulation tissue, or adherence of absorbent materials to the wound may occur and delay the healing process. moist healing is a newer concept of wound management in which wound exudates are allowed to stay in contact with the wound. in the absence of infection a moist wound heals faster and has enzymatic activity as a result of macrophage and polymorphonuclear cell breakdown. enzymatic degradation or "autolytic debridement" of the wound occurs. moist wounds tend to promote neutrophil and macrophage chemotaxis and bacterial phagocytosis better than use of wet-to-dry bandages. a potential complication and disadvantage of moist healing, however, is the development of bacterial colonization, folliculitis, and trauma to wound edges that can occur because of the continuously moist environment. use surfactant-type solutions (constant clens; kendall, mansfield, massachusetts) for initial wound cleansing and debridement. use occlusive dressings for rapid enzymatic debridement with bactericidal properties to aid in wound healing. bandage wet necrotic wounds with a dressing premoistened with hypertonic saline (curasalt [kendall] , % saline) to clean and debride the wounds. hypertonic saline functions to desiccate necrotic tissue and bacteria to debride the infected wound. remove and replace the hypertonic saline bandage every to hours. next, place gauze impregnated with antibacterial agents (kerlix amd [kendall] ) over the wound in the bandage layer to act as a barrier to bacterial colonization. if the wound is initially dry or has minimal exudate and is not obviously contaminated or infected, place amorphous gels of water, glycerin, and a polymer (curafil [kendall] ) over the wound to promote moisture and proteolytic healing. discontinue moisture gels such as curafil once the dry wound has become moist. finally, the final stage of moist healing helps to promote the development of a healthy granulation tissue bed. use calcium alginate dressings (curasorb or curasorb zn with zinc [kendall] ) in noninfected wounds with a moderate amount of drainage. alginate gels promote rapid development of a granulation tissue bed and epithelialization. foam dressings also can be applied to exudative wounds after a healthy granulation bed has formed. change foam dressings at least once every to days. for closed wounds without any drainage, such as a laceration that has been repaired surgically, a simple bandage with a nonadherent contact layer (telfa pad [kendall] , for example), intermediate layer of absorbent material, and an outer porous layer (elastikon, vetrap) can be placed to prevent wound contamination during healing. the nonadherent pad will not stick to the wound and cause patient discomfort. because there usually is minimal drainage from the wound, the function of the intermediate layer is more protective than absorptive. any small amount will be absorbed into the intermediate layer of the bandage. it is important in any bandage to place the tape strips or "stirrups" on the patient's limb and then overlap in the bandage, to prevent the bandage from slipping. place the intermediate and tertiary layers loosely around the limb, starting distally and working proximally, with some overlap with each consecutive layer. this method prevents excessive pressure and potential to impair venous drainage. leave the toenails of the third and fourth digits exposed, whenever possible, to allow daily examination of the bandage to determine whether the bandage is impairing venous drainage. if the bandage is too tight and constricting or impeding vascular flow, the toes will become swollen and spread apart. when placed and maintained properly (e.g., the bandage does not get wet), there usually are relatively few complications observed with this type of bandage. in some cases, it is necessary to cover a wound in which a penrose drain has been placed to allow drainage. in many cases, there is a considerable amount of drainage from the drain and underlying soft tissues. the function of the bandage is to help obliterate dead space created by the wound itself, absorb the fluid that drains from the wound and that will contaminate the environment, and prevent external wicking of material from the external environment into the wound. when the bandage is removed, the clinician can examine the amount and type of material that has drained from the wound in order to determine when the drain should be removed. when placing a bandage over a draining wound, the contact layer should be a commercially available nonadherent dressing and several layers of absorbent wide-mesh gauze placed directly over the drain at the distal end of the incision. overlay the layers of gauze with a thick layer of absorbent intermediate dressing to absorb fluid that drains from the wound. if the gauze and intermediate layers are not thick or absorbent enough, there is a potential for the drainage fluid to reach the outer layer of the bandage and provide a source of wicking of bacteria from the external environment into the wound, leading to infection. some wounds such as lacerations have minor bleeding or hemorrhage that require an immediate bandage until definitive care can be provided. to create a pressure bandage, place a nonadherent dressing immediately in contact with the wound, followed by a thick layer of absorbent material, topped by a layer of elastic bandage material such as elastikon or vetrap. unlike the bandage for a closed wound, the top tertiary outer layer should be wrapped with some tension and even pressure around the limb, starting from the distal extremity (toes) and working proximally. the pressure bandage serves to control hemorrhage but should not be left on for long periods. pressure bandages that have been left on for too long can impair nerve function and lead to tissue necrosis and slough. therefore, pressure bandages should be used in the hospital only, so that the patient can be observed closely. if hemorrhage through the bandage occurs, place another bandage over the first until the wound can be repaired definitively. removal of the first bandage will only disrupt any clot that has formed and cause additional hemorrhage to occur. fractures require immediate immobilization to prevent additional patient discomfort and further trauma to the soft tissues of the affected limb. as with all bandages, a contact layer, intermediate layer, and outer layer should be used. place the contact layer in accordance with any type of wound present. the intermediate layer should be thick absorbent material, followed by a top layer of elastic bandage material. an example is to place a telfa pad over a wound in an open distal radius-ulna fracture, followed by a thick layer of cotton gauze cast padding, followed by an elastic layer of kling (johnson & johnson medical, arlington, texas) , pulling each layer tightly over the previous layer with some overlap until the resultant bandage can be "thumped" with the clinician's thumb and forefinger and sound like a ripe watermelon. the bandage should be smooth with consecutive layers of even pressure on the limb, starting distally and working proximally. leave the toenails of the third and fourth digits exposed to monitor for impaired venous drainage that would suggest that the bandage is too tight and needs to be replaced. finally, place a top layer of vetrap or elastikon over the intermediary layer to protect it from becoming contaminated. if the bandage is used with a compound or open fracture, drainage may be impaired and actually lead to enhanced risk of wound infection. bandages placed for initial fracture immobilization are temporary until definitive fracture repair can be performed once the patient's cardiovascular and respiratory status are stable. wounds with exuberant granulation tissue must be handled carefully so as to not disrupt the healing process but to keep an overabundance of tissue from forming that will impair epithelialization. to bandage a wound with exuberant granulation tissue, place a corticosteroid-containing ointment on the wound, followed by a nonadherent contact layer. the corticosteroid will help control the exuberant growth of granulation tissue. next, carefully wrap an absorbent material over the contact layer, followed by careful placement of and overlay of elastic bandage material to place some pressure on the wound. leave the toenails of the third and fourth digits exposed so that circulation can be monitored several times daily. bandages that are too tight must be removed immediately to prevent damage to neuronal tissue and impaired vascularization, tissue necrosis, and slough. because wound drainage may be impaired, there is a risk of infection. gaping wounds or those that have undermined in between layers of subcutaneous tissue and fascia should be bandaged with a pressure bandage to help obliterate dead space and prevent seroma formation. an example of a wound that may require this type of bandage is removal of an infiltrative lipoma on the lateral or ventral thorax. use caution when placing pressure bandages around the thorax or cervical region because bandages placed too tightly may impair adequate ventilation. to place a pressure bandage and obliterate dead space, place a nonadherent contact layer over the wound. usually, a drain is placed in the wound, so place a large amount of wide-mesh gauze at the distal end of the drain to absorb any wound exudate or drainage. place several layers of absorbent material over the site to further absorb any drainage. place a layer of elastic cotton such as kling carefully but firmly over the dead space to cause enough pressure to control drainage. place at least two fingers in between the animal's thorax and the bandage to ensure that the bandage is not too tight. in many cases, the bandage should be placed once the animal has recovered from surgery and is able to stand. if the bandage is placed while the animal is still anesthetized and recumbent, there is a tendency for the bandage to be too tight. finally, the tertiary layer should be an elastic material such as elastikon or vetrap. many wounds require a pressure relief bandage to prevent contact with the external environment. wounds that may require pressure relief for healing include decubitus ulcers, pressure bandage or cast ulcers, impending ulcer areas (such as the ileum or ischium of recumbent or cachexic patients), and surgical repair sites of ulcerated areas. pressure relief bandages can be of two basic varieties: modified doughnut bandage and doughnut-shaped bandage. to create a cup or clamshell splint, follow this procedure (figures - to - ): . place a nonadherent contact layer directly over the wound. . place stirrups of tape in contact with the skin of the dog, to be placed over the intermediate layer and prevent the bandage from slipping. . place a fairly thick layer of absorbent intermediate bandage material over the contact layer such that the bandage is well-padded. pull the tape stirrups and secure them to the intermediate layer. . place a length of cast material that has been rolled to the appropriate length, such that the cast material is cupped around the patient's paw, and lies adjacent to the caudal aspect of the limb to the level of the carpus or tarsus. in the case of a clamshell splint, place a layer of cast material on the cranial and caudal aspect of the paw and conform it in place. . take the length of cast padding and soak it in warm water after it has been rolled to the appropriate length. wring out the pad, and secure/conform it to the caudal (or cranial and caudal, in the case of a clamshell splint) aspect of the distal limb and paw. . secure the cast material in place with a layer of elastic cotton gauze (kling). . secure the bandage in place with a snug layer of elastikon or vetrap. short or long splints made of cast material can be incorporated into a soft padded bandage to provide extra support of a limb above and below a fracture site. for a caudal or lateral splint to be effective, it must be incorporated for at least one joint above any fracture site to prevent a fulcrum effect and further disruption or damage to underlying soft tissue structures. a short lateral or caudal splint is used for fractures and luxations of the distal metacarpus, metatarsus, carpus, and tarsus. to place a short lateral or caudal splint, follow this procedure: . secure a contact layer as determined by the presence or absence of any wound in the area. . place tape stirrups on the distal extremity to be secured later to the intermediate bandage layer and to prevent slipping of the bandage distally. . place layers of roll cotton from the toes to the level of the mid tibia/fibula or mid radius/ulna. place the layers with even tension, with some overlap of each consecutive layer, moving distally to proximally on the limb. . secure the short caudal or lateral splint and conform it to the distal extremity to the level of the toes and proximally to the level of the mid tibia/fibula or mid radius/ulna. . secure the lateral or caudal splint to the limb with another outer layer of elastic cotton (kling). . cover the entire bandage and splint with an outer tertiary layer of vetrap or elastikon. make sure that the toenails of the third and fourth digits remain visible to allow daily evaluation of circulation. long lateral or caudal splints are used to immobilize fractures of the tibia/fibula and radius/ulna. the splints are fashioned as directed for short splints but extend proximally to the level of the axilla and inguinal regions to immobilize above the fracture site. • packed cell volume drops rapidly to less than % in the dog and less than % to % in the cat • acute loss of more than % of blood volume ( ml/kg in dog, ml/kg in cat) • clinical signs of lethargy, collapse, hypotension, tachycardia, tachypnea (acute or chronic blood loss) • ongoing hemorrhage is present • poor response to crystalloid and colloid infusion • life-threatening hemorrhage caused by thrombocytopenia or thrombocytopathia • surgical intervention is necessary in a patient with severe thrombocytopenia or thrombocytopathia plasma support • life-threatening hemorrhage with decreased coagulation factor activity • severe inflammation (pancreatitis, systemic inflammatory response syndrome) • replenish antithrombin (disseminated intravascular coagulation, protein-losing enteropathy or nephropathy) • surgery is necessary in a patient with decreased coagulation factor activity • severe hypoproteinemia is present; to partially replenish albumin, globulin, and clotting factors type a cats typically possess weak anti-b antibodies of igg and igm subtypes. transfusion of type b blood into a type a cat will result in milder clinical signs of reaction and a markedly decreased survival half-life of the infused rbcs to just days. because type ab cats possess both moieties on their cell surface, they lack naturally occurring alloantibodies; transfusion of type a blood into a type ab cat can be performed safely if a type ab donor is not available. the life span of an rbc from a type-specific transfusion into a cat is approximately days. . indications for fresh whole blood transfusion include disorders of hemostasis and coagulopathies including disseminated intravascular coagulation, von willebrand's disease, and hemophilia. fresh whole blood and platelet-rich plasma also can be administered in cases of severe thrombocytopenia and thrombocytopathia. stored whole blood and packed rbcs can be administered in patients with anemia. if pcv drops to below % or if rapid hemorrhage causes the pcv to drop below % in the dog or less than % to *indicates that this must be done for each donor being tested. minor crossmatch* . obtain a crossmatch segment from blood bank refrigerator for each donor to be crossmatched, or use an edta tube of donor's blood. make sure tubes are labeled prop-erly. . collect ml of blood from recipient and place in an edta tube. centrifuge blood for minutes. . extract blood from donor tubing. centrifuge blood for minutes. use a separate pipette for each transfer because cross-contamination can occur. . pipette plasma off of donor and recipient cells and place in tubes labeled dp and rp, respectively. . place µl of donor and recipient cells in tubes labeled dr and rr, respectively. . add . ml . % sodium chloride solution from wash bottle to each red blood cell (rbc) tube, using some force to cause cells to mix. . centrifuge rbc suspension for minutes. . discard supernatant and resuspend rbcs with . % sodium chloride from wash bottle. . repeat steps and for a total of three washes. . place drops of donor rbc suspension and drops of recipient plasma in tube labeled ma (this is the major crossmatch). . place drops of donor plasma and drops recipient rbc suspension in tube labeled mi (this is the minor crossmatch). . prepare control tubes by placing drops donor plasma with drops donor rbc suspension (this is the donor control); and place drops recipient plasma with drops recipient rbc suspension (this is the recipient control). . incubate major and minor crossmatches and control tubes at room temperature for minutes. . centrifuge all tubes for minute. . read tubes using an agglutination viewer. . check for agglutination and/or hemolysis. . score agglutination with the following scoring scale: + one solid clump of cells + several large clumps of cells + medium-sized clumps of cells with a clear background + hemolysis, no clumping of cells neg = negative for hemolysis; negative for clumping of red blood cells fresh whole blood coagulopathy with active hemorrhage (disseminated intravascular coagulation, thrombocytopenia; massive acute hemorrhage; no stored blood available) stored whole blood massive acute or ongoing hemorrhage; hypovolemic shock caused by hemorrhage that is unresponsive to conventional crystalloid and colloid fluid therapy; unavailability of equipment required to prepare blood components packed red blood cells nonregenerative anemia, immune-mediated hemolytic anemia, correction of anemia before surgery, acute or chronic blood loss fresh frozen plasma factor depletion associated with active hemorrhage (congenital: von willebrand's factor, hemophilia a, hemophilia b; acquired: vitamin k antagonist, rodenticide intoxication, dic); acute or chronic hypoproteinemia (burns, wound exudates, body cavity effusion; hepatic, renal, or gastrointestinal loss); colostrum replacement in neonates frozen plasma acute plasma or protein loss; chronic hypoproteinemia; (contains stable colostrum replacement in neonates; hemophilia b and clotting factors) selected clotting factor deficiencies platelet-rich plasma* thrombocytopenia with active hemorrhage (immune-mediated thrombocytopenia, dic); platelet function abnormality (congenital: thrombasthenia in bassett hounds; acquired: nsaids, other drugs) cryoprecipitate congenital factor deficiencies (routine or before surgery): (concentration of factor hemophilia a, hemophilia b, von willebrand's disease, viii, von willebrand's hypofibrinogenemia; acquired factor deficiencies factor, and fibrinogen) *must be purchased because logistically one cannot obtain enough blood simultaneously to provide a significant amount of platelets; platelets infused have a very short (< hours) half-life. dic, disseminated intravascular coagulation; nsaids, nonsteroidal antiinflammatory drugs. universal donor (e.g., should be administered whenever possible. because there is no universal donor in the cat and because cats possess naturally occurring alloantibodies, all cat blood should be typed and crossmatched before any transfusion. if fresh whole blood is not available, a hemoglobin-based oxygen carrier (oxyglobin, to ml/kg iv) can be administered until blood products become available. table - indicates blood component dose and administration rates. blood products should be warmed slowly to °c before administering them to the patient. blood warmer units are available for use in veterinary medicine to facilitate rapid transfusion without decreasing patient body temperature (thermal angel; enstill medical technologies, inc., dallas, texas). red blood cell and plasma products should be administered in a blood administration set containing a -µm in-line filter. smaller in-line filters ( µm) also can be used in cases in which extremely small volumes are to be administered. blood products should be administered over a period of hours, whenever possible, according to guidelines set by the american association of blood banks. the volume of blood components required to achieve a specific increment in the patient's pcv depends largely on whether whole blood or packed rbcs are transfused and whole blood ml/kg will increase max rate: ml/kg/ max: ml/kg/ volume by % hours hour packed red ml/kg will increase critically ill blood cells volume by % patients (e.g., cardiac failure or renal failure): - ml/kg/hour fresh frozen ml/kg body mass (repeat - ml/minute or use rates as for plasma in - days or in - days whole blood (infuse within - hours) or until bleeding stops); monitor act, aptt, and pt before and hour after transfusion cryoprecipitate general: unit/ kg/ hours - ml/minute or use rates as for whole or until bleeding stops blood (infuse within - hours) hemophilia a: - units factor viii/kg; unit of cryoprecipitate contains approximately units of factor viii platelet-rich unit/ kg ( unit of ml/minute plasma platelet-rich plasma will check platelet count before and hour increase platelet count after transfusion hour after transfusion by , /µl) whether there is ongoing hemorrhage or rbc destruction. because the pcv of packed rbcs is unusually high ( % for greyhound blood), a smaller total volume is required than whole blood to achieve a comparable increase in the patient's pcv. in general, ml/kg of packed rbcs or ml/kg whole blood will raise the recipient's pcv by %. the "rule of ones" states that ml per lb of whole blood will raise the pcv by %. if the patient's pcv does not raise by the amount anticipated by the foregoing calculation(s), causes of ongoing hemorrhage or destruction should be considered. the goal of red blood component therapy is to raise the pcv to % to % in dogs and % to % in cats. if an animal is hypovolemic and whole blood is administered, the fluid is redistributed into the extravascular compartment within hours of transfusion. this will result in a secondary rise in the pcv hours after the transfusion in addition to the initial rise to hours after the rbc transfusion is complete. the volume of plasma transfused depends largely on the patient's need. in general, plasma transfusion should not exceed more than ml/kg during a -hour period for normovolemic animals. thaw plasma at room temperature, or place it in a ziplock freezer bag and run under cool (not warm) water until thawed. then administer the plasma through a blood administration set that contains an in-line blood filter or through a standard driptype administration set with a detachable in-line blood administration filter. the average rate of plasma infusion in a normovolemic patient should not exceed ml/kg/hour. in acute need situations, plasma can be delivered at rates up to to ml/kg/minute. for patients with cardiac insufficiency or other circulatory problems, plasma infusion rates should not exceed ml/kg/hour. plasma or other blood products should not be mixed with or used in the same infusion line as calcium-containing fluids, including lactated ringer's solution, calcium chloride, or calcium gluconate. the safest fluid to mix with any blood product is . % sodium chloride. administer fresh frozen plasma, frozen plasma, and cryoprecipitate at a volume of ml/kg until bleeding is controlled or source of ongoing albumin loss ceases. the goal of plasma transfusion therapy is to raise the albumin to a minimum of . g/dl or until bleeding stops as in the case of coagulopathies. monitor the patient to ensure that bleeding has stopped, coagulation profiles (act, aptt, and pt) have normalized, hypovolemia has stabilized, and/or total protein is normalizing, which are indications for discontinuing ongoing transfusion therapy. plasma cryoprecipitate can be purchased or manufactured through the partial thawing and then centrifugation of fresh frozen plasma. cryoprecipitate contains concentrated quantities of vwf, factor viii, and fibrinogen and is indicated in severe forms of von willebrand's disease and hemophilia a (factor viii deficiency). platelet-rich plasma must be purchased from a commercial source. one unit of fresh whole blood contains to platelets. the viability of the platelets contained in the fresh whole blood is short-lived, just to hours after transfusion into the recipient. because platelet-rich plasma is difficult to obtain, animals with severe thrombocytopenia or thrombocytopathia should be treated with immunomodulating therapies and the administration of fresh frozen plasma. in dogs, blood and plasma transfusions can be administered intravenously or intraosseously. the cephalic, lateral saphenous, medial saphenous, and jugular veins are used most commonly. fill the recipient set so that the blood in the drip chamber covers the filter (normal -µm filter). with small amounts of blood ( ml) or critically ill patients, use a -µm filter. avoid latex filters for plasma and cryoprecipitate administration. blood can emergency care be administered at variable rates, but the routine figure of to ml/minute often is used. normovolemic animals can receive blood at ml/kg/day. dogs in heart failure should receive infusions at no more than ml/kg/hour. volume is given as needed. to calculate the approximate volume of blood needed to raise hematocrit levels, use the following formula for the dog: anticoagulated blood volume (ml) = body mass (kg) × × pcv desired − pcv of recipient pcv of donor in anticoagulant an alternative formula is the following: . × recipient body mass (kg) × (dog) × pcv desired − pcv of recipient pcv of donor in anticoagulant surgical emergencies and shock may require several times this volume within a short period. if greater than % of the patient's blood volume is lost, supplementation with colloids, crystalloids, and blood products is indicated for fluid replacement. one volume of whole blood achieves the same increase in plasma as two to three volumes of plasma. if the patient's blood type is unknown and type a-negative whole blood is not available, any dog blood can be administered to a dog in acute need if the dog has never had a transfusion before. if mismatched blood is given, the patient will become sensitized, and after days, destruction of the donor rbcs will begin. in addition, any subsequent mismatched transfusions may cause an immediate reaction (usually mild) and rapid destruction of the transfused rbcs. the clinical signs of a transfusion reaction typically only are seen when type a blood is administered to a type a-negative recipient that has been sensitized previously. incompatible blood transfusions to breeding females can result in isoimmunization and in hemolytic disease in the puppies. the a-negative bitch that receives a transfusion with a-positive and that produces a litter from an a-positive stud can have puppies with neonatal isoerythrolysis. cats with severe anemia in need of a blood transfusion are typically extremely depressed, lethargic, and anorexic. the stress of restraint and handling can push these critically ill patients over the edge and cause them to die. extreme gentleness and care are mandatory in restraint and handling. the critically ill cat should be cradled in a towel or blanket. supplemental flow-by or mask oxygen should be administered, whenever possible, although it may not be clinically helpful until oxygen-carrying capacity is replenished with infusion of rbcs or hemoglobin. blood can be administered by way of cephalic, medial saphenous, or the jugular vein. intramedullary infusion is also possible, if vascular access cannot be accomplished. the average -to -kg cat can accept to ml of whole blood injected intravenously over a period of to minutes. administer filtered blood at a rate of to ml/kg/hour. the following formula can be used to estimate the volume of blood required for transfusion in a cat: anticoagulated blood volume (ml) = body mass (kg) × × pcv desired − pcv of recipient pcv of donor in anticoagulant the exact overall incidence and clinical significance of transfusion reactions in veterinary medicine are unknown. several studies have been performed that document the incidence of transfusion reactions in dogs and cats. overall, the incidence of transfusion reactions in dogs and cats is . % and %, respectively. transfusion reactions can be immune-mediated and non-immune-mediated and can happen immediately or can be delayed until after a transfusion. acute reactions usually occur within minutes to hours of the onset of transfusion but may occur up to hours after the transfusion has been stopped. acute immunologic reactions include hemolysis and acute hypersensitivity including rbcs, platelets, and leukocytes. signs of a delayed immunologic reaction include hemolysis, purpura, immunosuppression, and neonatal isoerythrolysis. acute nonimmunologic reactions include donor cell hemolysis before onset of transfusion, circulatory volume overload, bacterial contamination, citrate toxicity with clinical signs of hypocalcemia, coagulopathies, hyperammonemia, hypothermia, air embolism, acidosis, and pulmonary microembolism. delayed nonimmunologic reactions include the transmission and development of infectious diseases and hemosiderosis. clinical signs of a transfusion reaction typically depend on the amount of blood transfused, the type and amount of antibody involved in the reaction, and whether the recipient has had previous sensitization. monitoring the patient carefully during the transfusion period is essential in recognizing early signs of a transfusion reaction, including those that may become life threatening. a general guideline for patient monitoring is first to start the transfusion slowly during the first minutes. monitor temperature, pulse, and respiration every minutes for the first hour, hour after the end of the transfusion, and every hours minimally thereafter. also obtain a pcv immediately before the transfusion, hour after the transfusion has been stopped, and every hours thereafter. monitor coagulation parameters such as an act and platelet count at least daily in patients requiring transfusion therapy. the most common documented clinical signs of a transfusion reaction include pyrexia, urticaria, salivation/ptyalism, nausea, chills, and vomiting. other clinical signs of a transfusion reaction may include tachycardia, tremors, collapse, dyspnea, weakness, hypotension, collapse, and seizures. severe intravascular hemolytic reactions may occur within minutes of the start of the transfusion, causing hemoglobinemia, hemoglobinuria, disseminated intravascular coagulation, and clinical signs of shock. extravascular hemolytic reactions typically occur later and will result in hyperbilirubinemia and bilirubinuria. pretreatment of patients to help decrease the risk of a transfusion reaction remains controversial, and in most cases, pretreatment with glucocorticoids and antihistamines is ineffective at preventing intravascular hemolysis and other reactions should they occur. the most important component of preventing a transfusion reaction is to screen each recipient carefully and process the donor component therapy carefully before the administration of any blood products. treatment of a transfusion reaction depends on its severity. in all cases, stop the transfusion immediately when clinical signs of a reaction occur. in most cases, discontinuation of the transfusion and administration of drugs to stop the hypersensitivity reaction will be sufficient. once the medications have taken effect, restart the transfusion slowly and monitor the patient carefully for further signs of reaction. in more severe cases in which a patient's cardiovascular or respiratory system become compromised and hypotension, tachycardia, or tachypnea occurs, immediately discontinue the transfusion and administer diphenhydramine ( mg/kg im), dexamethasone-sodium phosphate ( . to . mg/kg iv), and epinephrine to the patient. the patient should have a urinary catheter and central venous catheter placed for measurement of urine output and central venous pressures. aggressive fluid therapy may be necessary to avoid renal insufficiency or renal damage associated with severe intravascular hemolysis. overhydration with subsequent pulmonary edema generally can be managed with supplemental oxygen administration and intravenous or intramuscular administration of furosemide ( to mg/kg). plasma products with or without heparin can be administered for disseminated intravascular coagulation. the hbocs can be stored at room temperature and have a relatively long shelf life compared with red blood component products. the hbocs function to carry oxygen through the blood and can diffuse oxygen past areas of poor tissue perfusion. an additional characteristic of hbocs is as a potent colloid, serving to maintain fluid within the vascular space. for this reason, hbocs must be used with caution in euvolemic patients and patients with cardiovascular insufficiency. central venous pressure (cvp) measures the hydrostatic pressure in the anterior vena cava and is influenced by vascular fluid volume, vascular tone, function of the right side of the heart, and changes in intrathoracic pressure during the respiratory cycle. the cvp is not a true measure of blood volume but is used to gauge fluid therapy as a method of determining how effectively the heart can pump the fluid that is being delivered to it. thus the cvp reflects the interaction of the vascular fluid volume, vascular tone, and cardiac function. measure cvp in any patient with acute circulatory failure, large volume fluid diuresis (i.e., toxin or oliguric or anuric renal failure), fluid in-and-out monitoring, and cardiac dysfunction. the placement of central venous catheters and thus cvp measurements is contraindicated in patients with known coagulopathies including hypercoagulable states. to perform cvp monitoring, place a central venous catheter in the right or left jugular vein. in cats and small dogs, however, a long catheter placed in the lateral or medial saphenous vein can be used for trends in cvp monitoring. first, assemble the equipment necessary for jugular catheter (see vascular access techniques for how to place a jugular or saphenous long catheter) and cvp monitoring (box - ). after placing the jugular catheter, take a lateral thoracic radiograph to ensure that the tip of the catheter sits just outside of the right atrium for proper cvp measurements (see to establish an intravenous catheter for cvp, follow this procedure: . assemble the cvp setup such that the male end of a length of sterile intravenous catheter extension tubing is inserted into the t port of the jugular or medial/lateral saphenous catheter. make sure to flush the length of tubing with sterile saline before connecting it to the patient to avoid iatrogenic air embolism. . next, insert the male end of a three-way stopcock into the female end of the extension tubing. . attach a -ml syringe filled with heparinized sterile . % saline to one of the female ports of the three-way stopcock and either a manometer or a second length of intravenous extension tubing attached to a metric ruler. . lay the patient in lateral or sternal recumbancy. . turn the stopcock off to the manometer/ruler and on to the patient. infuse a small amount of heparinized saline through the catheter to flush the catheter. . next, turn the stopcock off to the patient and on to the manometer. gently flush the manometer or length of extension tubing with heparinized saline from the syringe. use care not to agitate the fluid and create air bubbles within the line or manometer that will artifactually change the cvp measured. . next, lower the cm point on the manometer or ruler to the level of the patient's manubrium (if the patient is in lateral recumbancy) or the point of the elbow (if the patient is in sternal recumbancy). . turn the stopcock off to the syringe, and allow the fluid column to equilibrate with the patient's intravascular volume. once the fluid column stops falling and the level rises and falls with the patient's heartbeat, measure the number adjacent to the bottom of the meniscus of the fluid column. this is the cvp in centimeters of water (see figure - ). . repeat the measurement several times with the patient in the same position to make sure that none of the values has been increased or decreased artifactually in error. alternately, attach the central catheter to a pressure transducer and perform electronic monitoring of cvp. there is no absolute value for normal cvp. the normal cvp for small animal patients is to cm h o. values less than zero are associated with absolute or relative hypovolemia. values of to cm h o are borderline hypervolemia, and values greater than cm h o suggest intravascular volume overload. values greater than cm h o may be correlated with congestive heart failure and the development of pulmonary edema. in individual patients, the trend in change in cvp is more important than absolute values. as a rule of thumb, when using cvp measurements to gauge fluid therapy and avoid vascular and pulmonary overload, the cvp should not increase by more than cm h o in any -hour period. if an abrupt increase in cvp is found, repeat the measurement to make sure that the elevated value was not obtained in error. if the value truly has increased dramatically, temporarily discontinue fluid therapy and consider administration of a diuretic. delaforcade am, rozanski ea: central venous pressure and arterial blood pressure measurements, vet clin north am small anim pract ( ) the diagnosis of intracellular fluid deficit is difficult and is based more on the presence of hypernatremia or hyperosmolality than on clinical signs. an intracellular fluid deficit is expected when free water loss by insensible losses and vomiting, diarrhea, or urine is not matched by free water intake. consideration of the location of the patient's fluid deficit, history of vomiting and diarrhea, no visible clinical signs of deficit % dry mucous membranes, mild skin tenting % increased skin tenting, dry mucous membranes, mild tachycardia, normal pulse* % increased skin tenting, dry mucous membranes, tachycardia, weak pulse pressure % increased skin tenting, dry corneas, dry mucous membranes, % elevated or decreased heart rate, poor pulse quality, altered level of consciousness* the respiratory system further contributes to acid-base status by changes in the elimination of carbon dioxide. hyperventilation decreases the blood pco and causes a respiratory alkalosis. hypoventilation increases the blood pco and causes a respiratory acidosis. depending on the altitude, the pco in dogs can range from to mm hg. in cats, normal is to mm hg. venous pco values are to mm hg in dogs and to mm hg in cats. use a systematic approach whenever attempting to interpret a patient's acid-base status. ideally, obtain an arterial blood sample so that you can monitor the patient's oxygenation and ventilation. once an arterial blood sample has been obtained, follow these steps: . determine whether the blood sample is arterial or venous by looking at the oxygen saturation (sao ). the sao should be greater than % if the sample is truly arterial, although it can be as low as % if a patient has severe hypoxemia. . consider the patient's ph. if the ph is outside of the normal range, an acid-base disturbance is present. if the ph is within the normal range, an acid-base disturbance may or may not be present. if the ph is low, the patient is acidotic. if the ph is high, the patient is alkalotic. . next, look at the base excess or deficit. if the base excess is increased, the patient has higher than normal bicarbonate. if there is a base deficit, the patient may have a low bicarbonate or increase in unmeasured anions (e.g., lactic acid or ketoacids). . next, look at the bicarbonate. if the ph is low and the bicarbonate is low, the patient has a metabolic acidosis. if the ph is high and the bicarbonate is elevated, the patient has a metabolic alkalosis. . next, look at the paco . if the patient's ph is low and the paco is elevated, the patient has a respiratory acidosis. if the patient's ph is high and the paco is low, the patient has a respiratory alkalosis. . finally, if you are interested in the patient's oxygenation, look at the pao . normal pao is greater than mm hg. the metabolic acidosis early in renal failure may be hyperchloremic and later may convert to typical increased anion gap acidosis. . next, you must determine whether the disorders present are primary disorders or an expected compensation for disorders in the opposing system. for example, is the patient retaining bicarbonate (metabolic alkalosis) because of carbon dioxide retention (respiratory acidosis)? use the chart in table - to evaluate whether the appropriate degree of compensation is occurring. if the adaptive response falls within the expected range, a simple acid-base disorder is present. if the response falls outside of the expected range, a mixed acid-base disorder is likely present. . finally, you must determine whether the patient's acid-base disturbance is compatible with the history and physical examination findings. if the acid-base disturbance does not fit with the patient's history and physical examination abnormalities, question the results of the blood gas analyses and possibly repeat them. the most desirable method of assessing the acid-base status of an animal is with a blood gas analyzer. arterial samples are preferred over venous samples, with heparin used as an anticoagulant (table - ) . potassium primarily is located in the intracellular fluid compartment. serum potassium is regulated by the actions of the sodium-potassium-adenosinetriphosphatase pump on cellular membranes, including those of the renal tubular epithelium. inorganic metabolic acidosis artifactually can raise serum potassium levels because of redistribution of extracellular potassium in exchange for intracellular hydrogen ion movement in an attempt to correct serum ph. metabolic acidosis potassium is one of the major players in the maintenance of resting membrane potentials of excitable tissue, including neurons and cardiac myocytes. changes in serum potassium can affect cardiac conduction adversely. hyperkalemia lowers the resting membrane potential and makes cardiac cells, particularly those of the atria, more susceptible to depolarization. characteristic signs of severe hyperkalemia that can be observed on an ecg rhythm strip include an absence of p waves, widened qrs complexes, and tall tented or spiked t waves. further increases in serum potassium can be associated with bradycardia, ventricular fibrillation, and cardiac asystole (death). treatment of hyperkalemia consists of administration of insulin ( . to . units/kg, iv regular insulin) and dextrose ( g dextrose per unit of insulin administered, followed by . % dextrose iv cri to prevent hypoglycemia), calcium ( to ml of % calcium gluconate administered iv slowly to effect), or sodium bicarbonate ( meq/kg, iv slowly). insulin plus dextrose and bicarbonate therapy help drive the potassium intracellularly, whereas calcium antagonizes the effect of hyperkalemia on the myocardial cells. all of the treatments work within minutes, although the effects are relatively short-lived ( minutes to hour) unless the cause of the hyperkalemia is identified and treated appropriately (box - ). dilution of serum potassium also results from restoring intravascular fluid volume and correcting metabolic acidosis, in most cases. treatment with a fluid that does not contain potassium (preferably . % sodium chloride) is recommended. hypokalemia elevates the resting membrane potential and results in cellular hyperpolarization. hypokalemia may be associated with ventricular dysrhythmias, but the ecg changes are not as characteristic as those observed with hyperkalemia. causes of hypokalemia include renal losses, anorexia, gastrointestinal loss (vomiting, diarrhea), intravenous fluid diuresis, loop diuretics, and postobstructive diuresis (box - ). if the serum potassium concentration is known, potassium supplementation in the form of potassium chloride or potassium phosphate can be added to the patient's intravenous fluids. correct serum potassium levels less than . meq/l or greater than . meq/l. potassium rates should not exceed . meq/kg/hour (table - ) . metabolic acidosis from bicarbonate depletion often corrects itself with volume restoration in most small animal patients. patients with moderate to severe metabolic acidosis may benefit from bicarbonate supplementation therapy. the metabolic contribution to acid-base balance is identified by measuring the total carbon dioxide concentration or calculating the bicarbonate concentration. if these measurements are not available, the degree of expected metabolic acidosis can be estimated subjectively by the severity of underlying disease that often contributes to metabolic acidosis: hypovolemic or traumatic shock, septic shock, diabetic ketoacidosis, or oliguric/anuric renal failure. if the metabolic acidosis is estimated to be mild, moderate, or severe, add sodium bicarbonate at , , and meq/kg body mass, respectively. patients with diabetic ketoacidosis may not require bicarbonate administration once volume replacement and perfusion is restored, and the ketoacids are metabolized to bicarbonate. if the bicarbonate measurement of base deficit is known, the following formula can be used as a gauge for bicarbonate supplementation: base deficit × . = body mass (kg) = meq bicarbonate to administer osmolality osmolality is measured by freezing point depression or a vapor pressure osmometer, or it may be calculated by the following formula: mosm/kg = [(na + ) + (k + )] + bun/ . + glucose/ where sodium and potassium are measured in milliequivalents, and bun and glucose are measured in milligrams per deciliter. osmolalities less than mosm/kg or greater emergency care than mosm/kg are serious enough to warrant therapy. the difference between the measured osmolality and the calculated osmolality (the osmolal gap) should be less than mosm/kg. if the osmolal gap is greater than mosm/kg, consider the presence of unmeasured anions such as ethylene glycol metabolites. the volume of extracellular fluid is determined by the total body sodium content, whereas the osmolality and sodium concentration are determined by water balance. serum sodium concentration is an indication of the amount of sodium relative to water in the extracellular fluid and provides no direct information about the total body sodium content. unlikely to cause hyperkalemia in presence of normal renal function unless iatrogenic (e.g., continuous infusion of potassium-containing fluids at an excessively rapid rate) acute mineral acidosis (e.g., hydrochloric acid or ammonium chloride) insulin deficiency (e.g., diabetic ketoacidosis) acute tumor lysis syndrome reperfusion of extremities after aortic thromboembolism in cats with cardiomyopathy hyperkalemic periodic paralysis (one case report in a pit bull) mild hyperkalemia after exercise in dogs with induced hypothyroidism infusion of lysine or arginine in total parenteral nutrition solutions nonspecific β-blockers (e.g., propranolol)* cardiac glycosides (e.g., digoxin)* urethral obstruction ruptured bladder anuric or oliguric renal failure hypoadrenocorticism selected gastrointestinal disease (e.g., trichuriasis, salmonellosis, or perforated duodenal ulcer) late pregnancy in greyhound dogs (mechanism unknown but affected dogs had gastrointestinal fluid loss) chylothorax with repeated pleural fluid drainage hyporeninemic hypoaldosteronism † angiotensin-converting enzyme inhibitors (e.g., enalapril)* angiotensin receptor blockers (e.g., losartan)* cyclosporine and tacrolimus* potassium-sparing diuretics (e.g., spironolactone, amiloride, and triamterene)* nonsteroidal antiinflammatory drugs* heparin* trimethoprim* from dibartola sp: fluid, electrolyte and acid-base disorders in small animal practice, st louis, , saunders. *likely to cause hyperkalemia only in conjunction with other contributing factors (e.g., other drugs, decreased renal function, or concurrent administration of potassium supplements). † not well documented in veterinary medicine. if refractory hypokalemia is present, supplement magnesium at . meq/kg/day for hours. alone unlikely to cause hypokalemia unless diet is aberrant administration of potassium-free (e.g., . % sodium chloride or % dextrose in water) or potassium-deficient fluids (e.g., lactated ringer's solution over several days) bentonite clay ingestion (e.g., cat litter) alkalemia insulin/glucose-containing fluids catecholamines hypothermia hypokalemic periodic paralysis (burmese cats) albuterol overdosage patients with hyponatremia or hypernatremia may have decreased, normal, or increased total body sodium content (boxes - and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ). an increased serum sodium concentration implies hyperosmolality, whereas a decrease in serum sodium concentration usually, but not always, implies hypoosmolality. the severity of clinical signs of hypernatremia and hyponatremia is related primarily to the rapidity of the onset of the change rather than to the magnitude of the associated plasma hyperosmolality or hypoosmolality. clinical signs of neurologic disturbances include disorientation, ataxia, and seizures, and coma may occur at serum sodium concentrations less than meq/l or greater than meq/l in dogs. therapy of hypernatremia or hyponatremia with fluid containing low or higher concentrations of sodium should proceed with caution, for rapid changes (decreases or increases) of serum sodium and osmolality can cause rapid changes in the intracellular and extracellular fluid flux, leading to intracellular dehydration or edema, even though the serum sodium has not been returned to normal. a rule of thumb is to not raise or lower the serum sodium by more than meq/l during any one -hour period. restoration of the serum sodium concentration over a period of to hours is better. in almost all circumstances, an animal will correct its sodium balance with simple fluid restoration. if severe hypernatremia exists that suggests a free water deficit, however, the free water deficit should be calculated from the following formula: hypernatremia can be corrected slowly with . % sodium chloride plus . % dextrose, % dextrose in water, or lactated ringer's solution (sodium content: meq/l). correct hyponatremia initially with . % sodium chloride. sodium is balanced predominantly by chloride and bicarbonate. the difference between these concentrations, (na , has been called the anion gap. the normal anion gap is between and meq/l. when the anion gap exceeds , consider the possibility of an accumulation of unmeasured anions (e.g., lactate, ketoacids, phosphate, sulfate, ethylene glycol metabolites, and salicylate). abnormalities in the anion gap may be helpful in determining the cause of metabolic acidosis (boxes - and - ). the colloid oncotic pressure of blood is associated primarily with large-molecular-weight colloidal substances in circulation. the major player in maintaining intravascular and interstitial oncotic pressure, the water-retaining property of each fluid compartment, is albumin. albumin contributes roughly % to the colloidal oncotic pressure of blood. the majority of albumin is located within the interstitial space. hypoalbuminemia can result from increased loss in the form of protein-losing enteropathy or nephropathy and wound exudates, or it may be due to lack of hepatic albumin synthesis. serum albumin pools are in a constant flux with interstitial albumin. once interstitial albumin pools become depleted from replenishing serum albumin, serum albumin levels can continue to decrease, which can lead to a decrease in colloidal oncotic pressure. serum albumin less than . g/dl has been associated with inadequate intravascular fluid retention and the development of peripheral edema and third spacing of fluid. oncotic pressure can be restored with the use of artificial or synthetic colloids or natural colloids (see colloids). maintenance fluid requirements have been extrapolated from the formulas used to calculate a patient's daily metabolic energy requirements because it takes ml of water to metabolize kcal of energy (table - ) . the patient's daily metabolic water (fluid) requirements can be calculated by the following formula: administration of an isotonic crystalloid fluid for maintenance requirements often can produce iatrogenic hypokalemia. in most cases, supplemental potassium must be added to prevent hypokalemia resulting from inappetance, kalliuresis, and supplementation with isotonic crystalloid fluids. the most reliable method of determining the degree of fluid deficit is by weighing the animal and calculating acute weight loss. acute weight loss in a patient with volume loss in the form of vomiting, feces, wound exudates, and urine is due to fluid loss and not loss of muscle or fat. lean body mass normally is not gained or lost rapidly enough to cause major changes in body weight. one milliliter of water weighs approximately g. this fact allows calculation of the patient's fluid deficit, if ongoing losses can be measured. when a patient first presents, however, the body weight before a fluid deficit has occurred rarely is known. instead, one must rely on subjective measures of dehydration to estimate the patient's percent dehydration and to calculate the volume of fluid required to rehydrate the patient over the next hours. to calculate the volume deficit, use the following formula: body mass (kg) × (% dehydration) × = fluid deficit (ml) the patient's fluid deficit must be added to the daily maintenance fluid requirements and administered over a -hour period. ongoing losses can be determined by measuring urine output, weighing the patient at least to times a day, and measuring the volume or weight of vomitus or diarrhea. a crystalloid fluid contains crystals of salts with a composition similar to that of the extracellular fluid space and can be used to maintain daily fluid requirements and replace fluid deficits or ongoing fluid losses (table - ) . metabolic, acid-base, and electrolyte imbalances also can be treated with isotonic fluids with or without supplemental electrolytes and buffers. depending on the patient's clinical condition, choose the specific isotonic crystalloid fluid to replace and maintain the patient's acid-base and electrolyte status ( table - ) . crystalloid fluids are readily available, are relatively inexpensive, and can be administered safely in large volumes to patients with no preexisting cardiac or renal disease or cerebral edema. following infusion, approximately % of the volume of a crystalloid fluid infused will redistribute to the interstitial fluid compartment. as such, crystalloid fluids alone are ineffective for ongoing intravascular volume depletion when given as a bolus. the crystalloid fluid bolus must be followed by a constant rate infusion, taking into consideration the patient's daily maintenance fluid requirements and ongoing fluid losses. administration of a large volume of crystalloid fluids can cause dilutional anemia and coagulopathies. * × bw kg + = kcal/day = ml/day. note: this formula will slightly underestimate the requirements for patients that are less than kg and will slightly overestimate the requirements for patients greater than kg. retain fluid in the vascular space, the volume of crystalloid fluid infused (maintenance + deficit + ongoing losses) should be decreased by % to % to avoid vascular volume overload. two major classes of colloids exist: natural and synthetic. natural colloids (whole blood, packed rbcs, plasma) are discussed elsewhere in this text. concentrated human albumin is a natural purified colloid that recently has become more popular in the treatment of advanced hypoalbuminemia and hypoproteinemia and will be discussed here. synthetic colloids are starch polymers and include dextrans and hetastarch. concentrated human albumin is available as a % or % solution. the % solution has an osmolality similar to that of serum ( mosm/l), whereas the % solution is hyperoncotic ( mosm/l). a % albumin solution draws fluid from the interstitial space into the intravascular space. concentrated albumin solutions often are used to restore circulating volume when synthetic colloids are not available. albumin not only is important at maintaining the colloidal oncotic pressure of blood but also serves as a valuable free-radical scavenger and carrier of drugs and hormones necessary for normal tissue function and healing. albumin levels less than . g/dl have been associated with increased morbidity and mortality. concentrated human albumin solutions can be administered as an effective method of restoring interstitial and serum albumin concentrations in situations of acute and chronic hypoalbuminemia. albumin ( %) is available in -and -ml vials and is more cost-efficient as an albumin replacement than procurement and administration of fresh frozen plasma. recommended albumin infusion rates are to ml/kg over hours, after pretreatment with diphenhydramine. although concentrated human albumin is structurally similar to canine albumin, closely monitor the patient for signs of allergic reaction during and after the infusion. dextran- is a synthetic high-molecular-weight polysaccharide (sucrose polymer) with a molecular weight of , d. particles less than , d, are cleared rapidly by the kidneys, whereas larger particles are cleared more slowly by the hepatic reticuloendothelial system. dextran- can coat platelets and inhibit platelet function and so must be used with caution in patients with known coagulopathies. the total daily dosage should not exceed ml/kg/day. hetastarch (hydroxyethyl starch) is a large-molecular-weight amylopectin polymer, has molecules with a molecular weight that exceeds , d, and has an average half-life of to hours in circulation. hetastarch can bind with vwf and cause prolongation of the act and aptt; however, it does not cause a coagulopathy. recommended rates of hetastarch infusion are -to -ml incremental boluses for the treatment of hypotension and to ml/kg/day as a constant rate infusion for maintenance of colloidal oncotic pressure. many are the acceptable ways to administer the fluids prescribed for each patient based on the degree of dehydration, estimation of ongoing losses, ability to tolerate oral fluid, and metabolic, acid-base, and electrolyte derangements. administer the fluids in a manner that is best for the patient and most appropriate for the practice. to determine the rate of intravenous fluid infusion, take the total volume of fluids that have been prescribed and divide the total volume by the total number of hours in a day that intravenous fluids can be delivered safely and monitored. the safest and most accurate way to deliver intravenous fluids, particularly in extremely small animals or those with congestive heart failure, is through an intravenous fluid pump. fluid should not be administered intravenously if the patient cannot be monitored to make sure that the fluids are being delivered at a safe rate and that the fluid line has not become disconnected. supplement fluids over as many hours as possible to allow the patient as much time as possible to redistribute and fully utilize the fluids administered. fluids administered too quickly can cause a diuresis to occur, such that the majority of the fluids administered will be excreted in the urine. if time is limited or if extra time is needed for safe administration of fluids, consider using a combination of intravenously and subcutaneously emergency care administered fluids. intravenous is the preferred route of administration of fluids in any patient with dehydration and hypovolemia. as intravascular volume depletion occurs, reflex peripheral vasoconstriction occurs to restore core perfusion. the subcutaneous tissue are not perfused well and therefore fluids administered subcutaneously will not be absorbed well into the interstitial and intravascular spaces. subcutaneously administered fluids can be absorbed slowly and delivered effectively in the management of mild interstitial dehydration and in the treatment of renal insufficiency. subcutaneously administered fluids should never take the place of intravenously administered fluids in a hypovolemic patient or one with severe interstitial dehydration. intramedullary (intraosseous) infusion works well in small patients in which vascular access cannot be established. shock doses of fluids and other substances, including blood products, can be administered under pressure through an intraosseous cannula. because of the inherent discomfort and risk of osteomyelitis with intraosseous infusion, establish vascular access as soon as possible. the safest and most efficient method of intravenous fluid infusion is through a fluid pump. in cases in which a fluid pump is unavailable, infusion by gravity feed is the next option. infusion sets from various manufacturers have calibrated drip chambers such that a specific number of drops will equal ml of fluid. fluid rates can be calculated based on the number of drops that fall into the drip chamber per minute: fluid volume to be infused (ml) = ml/hour number of hours available many pediatric drip sets deliver drops/ml, such that milliliters/hour equals drops/ minute. carefully record fluid orders so that the volume to be administered is recorded as milliliters/hour, milliliters/day, and drops/minute. this will allow personnel to detect major discrepancies and calculation errors more readily. the volume actually delivered should be recorded in the record by nursing personnel. all additives should be listed clearly on the bottle on a piece of adhesive tape or a special label manufactured for this purpose. a strip of adhesive tape also can be attached to the bottle and marked appropriately to provide a quick visualization of the estimate of volume delivered. includes a large-bore flexible orogastric lavage tube, permanent marker or white tape, lubricating jelly, warm water, two large buckets, a roll of -inch white tape, and a manual lavage pump. to perform the orogastric lavage, follow this procedure: . place all animals under general anesthesia with a cuffed endotracheal tube in place to protect the airway and prevent aspiration of gastric contents into the lungs. . place a roll of -inch white tape into the animal's mouth, and secure the tape around the muzzle. you will insert the tube through the hole in the center of the roll of tape. . next, place the distal end of the tube at the level of the last rib, directly adjacent to the animal's thorax and abdomen. measure the length of the tube from the most distal end to the point where it comes out of the mouth, and label this location on the tube with a permanent marker or piece of white tape. . lubricate the distal portion of the tube, and gently insert it through the roll of tape in the animal's mouth. . gently push the tube down the esophagus. palpate the tube within the esophagus. two tubes should be palpable, the orogastric tube, and the patient's trachea. push the tube down into the stomach. you can verify location by blowing into the proximal end of the tube and simultaneously auscultating the stomach for borborygmi. . insert the manual pump to the proximal end of the tube, and instill the warm water. alternate instilling water with removal of fluid and gastric debris by gravity. repeat the process until the efflux fluid is clear of any debris. . save fluid from the gastric efflux fluid for toxicologic analyses. hackett tb: emergency approach to intoxications, clin tech small anim pract ( ): - , . hypoxia, or inadequate tissue oxygenation, is the primary reason for supplemental oxygen therapy. major causes of hypoxia include hypoventilation, ventilation-perfusion mismatch, physiologic or right-to-left cardiac shunt, diffusion impairment, and decreased fraction of inspired oxygen (table - ) . inadequate tissue perfusion caused by low cardiac output or vascular obstruction also can result in circulatory hypoxia. finally, histiocytic hypoxia results from inability of cells to use oxygen that is delivered to them. this form of hypoxia can be observed with various toxin ingestions (bromethalin, cyanide) and in septic shock. a patient's oxygenation status can be monitored invasively by drawing of arterial blood gas samples or noninvasively through pulse oximetry, in most cases (see acid-base physiology and pulse oximetry). inspired air at sea level has a po of mm hg. as the air travels through the upper respiratory system to the level of the alveolus, the po drops to mm hg. tissue oxygen saturation in a normal healthy animal is mm hg. after oxygen has been delivered to the tissues, the oxygen left in the venous system (pvo ) is approximately mm hg. normally, oxygen diffuses across the alveolar capillary membrane and binds reversibly with hemoglobin in rbcs. a small amount of oxygen is carried in an unbound diffusible form in the plasma. when an animal has an adequate amount of hemoglobin and hemoglobin becomes fully saturated while breathing room air, supplemental oxygen administration will only increase the sao a small amount. the unbound form of oxygen dissolved in plasma will increase. if, however, inadequate hemoglobin saturation is obtained by breathing room air, as in a case of pneumonia or pulmonary edema, for example, breathing a higher fraction of inspired oxygen (fio ) will improve bound and unbound hemoglobin levels. the formula for calculating oxygen content of arterial blood is as follows: where cao is the arterial oxygen content, . is the amount of oxygen that can be carried by hemoglobin (hb), sao is the hemoglobin saturation, and . × pao is the amount of oxygen dissolved (unbound) in plasma. dissolved oxygen actually contributes little to the total amount of oxygen carried in the arterial blood, and the majority depends on the amount or availability of hemoglobin and the ability of the body (ph and respiratory status) to saturate the hemoglobin at the level of the alveoli. oxygen therapy is indicated whenever hypoxia is present. the underlying cause of the hypoxia also must be identified and treated, for chronic, lifelong oxygen therapy is rarely feasible in veterinary patients. if hemoglobin levels are low due to anemia, oxygen supplementation must occur along with rbc transfusions to increase hemoglobin mass. whenever possible, use arterial blood gas analyses or pulse oximetry to gauge a patient's response to oxygen therapy and to determine when an animal can be weaned from supplemental oxygen. the goal of oxygen therapy is to increase the amount of oxygen bound to hemoglobin in arterial blood. oxygen supplementation can be by hood, oxygen cage or tent, nasal or nasopharyngeal catheter, or tracheal tube. in rare cases, administration of oxygen with mechanical ventilation may be indicated. administration of supplemental oxygen to patients with chronic hypoxia is sometimes necessary but also dangerous. with chronic hypoxia the patient develops a chronic respiratory acidosis (elevated paco ) and depends almost entirely on the hypoxic ventilatory drive to breathe. administration of supplemental oxygen increases pao and may inhibit the central respiratory drive, leading to hypoventilation and possibly respiratory arrest. therefore, closely monitor animals with chronic hypoxia that are treated with supplemental oxygen. oxygen hoods can be purchased from commercial sources or can be manufactured in the hospital using a rigid elizabethan collar, tape, and plastic wrap. to make an oxygen hood, place several lengths of plastic wrap over the front of the elizabethan collar and tape them in place. leave the ventral third of the collar open to allow moisture and heat to dissipate and carbon dioxide to be eliminated. place a length of flexible oxygen tubing under the patient's collar into the front of the hood, and run humidified oxygen at a rate of to ml/kg/minute. animals may become overheated with an oxygen hood in place. carefully monitor the patient's temperature so that iatrogenic hyperthermia does not occur. commercially available plexiglass oxygen cages can be purchased from a variety of manufacturers. the best units include a mechanical thermostatically controlled compressor cooling unit, a circulatory fan, nebulizers or humidifiers to moisten the air, and a carbon dioxide absorber. alternately, a pediatric (infant) incubator can be purchased from hospital supply sources, and humidified oxygen can be run into the cage at to l/minute (depending on the size of the cage). high flow rates may be required to eliminate nitrogen and carbon dioxide from the cage. in most cases, the fio inside the cage reaches % to % using this technique. disadvantages of using an oxygen cage are high consumption/ use of oxygen, rapid decrease in the fio within the cage whenever the cage must be opened for patient treatments, lack of immediate access to the patient, and potential for iatrogenic hyperthermia. one of the most common methods for oxygen supplementation in dogs is nasal or nasopharyngeal oxygen catheters: . to place a nasal or nasopharyngeal catheter, obtain a red rubber catheter ( f to f, depending on the size of the patient). a. for nasal oxygen supplementation, measure the distal tip of the catheter from the medial canthus of the eye to the tip of the nose. b. for nasopharyngeal oxygen supplementation, measure the catheter from the ramus of the mandible to the tip of the nose. . mark the tube length at the tip of the nose with a permanent marker. . instill topical anesthetic such as proparacaine ( . %) or lidocaine ( %) into the nostril before placement. . place a stay suture adjacent to (lateral aspect) the nostril while the topical anesthetic is taking effect. . lubricate the tip of the tube with sterile lubricant. . gently insert the tube into the ventral medial aspect of the nostril to the level made with the permanent marker. if you are inserting the tube into the nasopharynx, push the nasal meatus dorsally while simultaneously pushing the lateral aspect of the nostril medially to direct the tube into the ventral nasal meatus and avoid the cribriform plate. . once the tube has been inserted to the appropriate length, hold the tube in place with your fingers adjacent to the nostril, and suture the tube to the stay suture. if the tube is removed, you can cut the suture around the tube and leave the stay suture in place for later use, if necessary. . suture or staple the rest of the tube dorsally over the nose and in between the eyes to the top of the head, or laterally along the zygomatic arch. . attach the tube to a length of flexible oxygen tubing, and provide humidified oxygen at to ml/kg/minute. . secure an elizabethan collar around the patient's head to prevent the patient from scratching at the tube and removing it. the rule of s states that if a patient's pao is less than mm hg, or if the paco is mm hg, mechanical ventilation should be considered. for mechanical ventilation, anesthetize the patient and intubate the patient with an endotracheal tube. alternately, a temporary tracheostomy can be performed and the patient can be maintained on a plane of light to heavy sedation and ventilated through the tracheostomy site. this method, a noninvasive means of determining oxygenation is through the use of pulse oximetry. a pulse oximeter uses different wavelengths of light to distinguish characteristic differences in the properties of the different molecules in a fluid or gas mixture, in this case, oxygenated (oxyhemoglobin) and deoxygenated hemoglobin (deoxyhemoglobin) in pulsatile blood. the process is termed pulse oximetry. oxyhemoglobin and deoxyhemoglobin are different molecules that absorb and reflect different wavelengths of light. oxyhemoglobin absorbs light in the infrared spectrum, allowing wavelengths of light in the red spectrum to transmit through it. conversely, deoxyhemoglobin absorbs wavelengths of the red spectrum and allows wavelengths in the infrared spectrum to transmit through the molecule. the spectrophotometer in the pulse oximeter transmits light in the red ( nanometers) and infrared ( nanometers) spectra. the different wavelengths of light are transmitted across a pulsatile vascular bed and are detected by a photodetector on the other side. the photodetector processes the amount of light of varying wavelengths that reaches it, then transmits an electrical current to a processor that calculates the difference in the amount of light originally transmitted and the amount of light of similar wavelength that actually reaches the photodetector. the difference in each reflects the amount of light absorbed in the pulsatile blood and can be used to calculate the amount or ratio of oxyhemoglobin to deoxyhemoglobin in circulation, or the functional hemoglobin saturation by the formula: where hbo is oxygenated hemoglobin, and hb is deoxygenated hemoglobin. four molecules of oxygen reversibly bind to hemoglobin for transport to the tissues. carbon monoxide similarly binds to hemoglobin and forms carboxyhemoglobin, a molecule that is detected similarly as oxygenated hemoglobin. thus sao as detected by a pulse oximeter is not reliable if carboxyhemoglobin is present. in most cases, pulse oximetry or sao corresponds reliably to the oxyhemoglobin dissociation curve. oxygen saturation greater than % corresponds to a pao greater than mm hg. above this value, large changes in pao are reflected in relatively small changes in sao , making pulse oximetry a relatively insensitive method of determining oxygenation status when pao is normal. because pulse oximetry measures oxygenated versus nonoxygenated hemoglobin in pulsatile blood flow, it is fairly unreliable when severe vasoconstriction, hypothermia, shivering or trembling, or excessive patient movement are present. additionally, increased ambient lighting and the presence of methemoglobin or carboxyhemoglobin also can cause artifactual changes in the sao , and thus the measurement is not reliable or accurate. most pulse oximeters also display a waveform and the patient's heart rate. if the photodetector does not detect a good quality signal, the waveform will not be normal, and the heart rate displayed on the monitor will not correlate with the patient's actual heart rate. the efficiency of ventilation is evaluated using the paco value on an arterial blood gas sample. alternatively, a noninvasive method to determine end-tidal carbon dioxide is through use of a capnograph. the science of capnometry uses a spectrophotometer to measure carbon dioxide levels in exhaled gas. the capnometer is placed in the expiratory limb of an anesthetic circuit. a sample of exhaled gas is aliquoted from the breath, and an infrared light source is passed across the sample. a photodetector on the other side of the sample flow measures the amount or concentration of carbon dioxide in the sample of expired gas. the calculated value is displayed as end-tidal carbon dioxide. this value also can be displayed as a waveform. when placed in graphic form, a waveform known as a capnograph is displayed throughout the ventilatory cycle. normally, at the onset of exhalation, the gas exhaled into the expiratory limb of the tubing comes from the upper airway or physiologic dead space and contains relatively little carbon dioxide. as exhalation continues, a steep uphill slope occurs as more carbon dioxide is exhaled from the bronchial tree. near the end of exhalation, the capnogram reaches a plateau, which most accurately reflects the carbon dioxide level at the level of the alveolus. because carbon dioxide diffuses across the alveolar basement membrane so rapidly, this reflects arterial carbon dioxide levels. if a plateau is not reached and notching of the waveform occurs, check the system for leaks. if the baseline waveform does not reach zero, the patient may be rebreathing carbon dioxide or may be tachypneic, causing physiologic positive end-expiratory pressure. the soda-sorb in the system should be replaced if it has expired. conversely, low end-tidal carbon dioxide may be associated with a decrease in perfusion or blood flow. decreased perfusion can be associated with low end-tidal carbon dioxide values, particularly during cardiopulmonary cerebral resuscitation. end-tidal carbon dioxide levels are one of the most accurate predictors of the efficacy of cardiopulmonary cerebral resuscitation and patient outcome. additionally, the difference between arterial carbon dioxide levels (paco ) and end-tidal carbon dioxide can be used to calculate dead-space ventilation. increases in the difference also occur with poor lung perfusion and pulmonary diffusion impairment. thoracocentesis refers to the aspiration of fluid or air from within the pleural space. thoracocentesis may be diagnostic to determine whether air or fluid is present and to characterize the nature of the fluid obtained. thoracocentesis also can be therapeutic when removing large volumes of air or fluid to allow pulmonary reexpansion and correction of hypoxemia and orthopnea. to perform thoracocentesis, follow this procedure: . first, assemble the equipment necessary (box - ). . next, clip a -cm square in the center of the patient's thorax on both sides. . aseptically scrub the clipped area. . ideally, thoracocentesis should be performed within the seventh to ninth intercostal space. rather than count rib spaces in an emergent situation, visualize the thoracic cage as a box, and the clipped area as a box within the box. you will insert your needle or catheter in the center of the box and then direct the bevel of the needle dorsally or ventrally to penetrate pockets of fluid or air present. . attach the needle or catheter hub to the length of intravenous extension tubing. attach the female port of the intravenous extension tubing to the male port of the three-way stopcock. attach the male port of the -ml syringe to one of the female ports of the three-way stopcock. the apparatus is now assembled for use. . insert the needle through the intercostal space such that the bevel of the needle initially is directed downward. . next, push down on the hub of the needle such that the needle becomes parallel with the thoracic wall. by moving the hub of the needle in a clockwise or counterclockwise manner, the bevel of the needle will move within the thoracic cavity to penetrate pockets of air or fluid. in general, air is located dorsally and fluid is located more ventrally, although this does not always occur. . aspirate air or fluid. save any fluid obtained for cytologic and biochemical analyses and bacterial culture and susceptibility testing. in cases of pneumothorax, if the thoracocentesis needs to be repeated more than times, consider using a thoracostomy tube. place a thoracostomy tube in cases of pneumothorax whenever negative suction cannot be obtained or repeated accumulation of air requires multiple thoracocentesis procedures. thoracostomy tubes also can be placed to drain rapidly accumulating pleural effusion and for the medical management of pyothorax. before attempting thoracostomy tube placement, make sure that all necessary supplies are assembled (box - ; table - ) . to place a thoracostomy tube, follow this procedure: . lay the patient in lateral recumbency. . clip the patient's entire lateral thorax. . aseptically scrub the lateral thorax. . palpate the tenth intercostal space. . have an assistant pull the patient's skin cranially and ventrally toward the point of the elbow. this will facilitate creating a subcutaneous tunnel around the thoracostomy tube. . draw up mg/kg % lidocaine ( mg/kg for cats) along with a small amount of sodium bicarbonate to take away some of the sting. . insert the needle at the dorsal aspect of the tenth intercostal space and to the seventh intercostal space. inject the lidocaine into the seventh intercostal space at the point where the trocarized thoracic drainage catheter will penetrate into the thoracic cavity. slowly infuse the lidocaine as you withdraw the needle to create an anesthetized tunnel through which to insert the catheter. . while the local anesthetic is taking effect, remove the trocar from the catheter and cut the proximal end of the catheter with a mayo scissors to facilitate adaptation with the christmas tree adapter. . attach the christmas tree adapter to the three-way stopcock and the three-way stopcock to a length of intravenous extension tubing and the -ml syringe so that the apparatus can be attached immediately to the thoracostomy tube after placement. . aseptically scrub the lateral thorax a second time and then drape it with sterile huck towels secured with towel clamps. . wearing sterile gloves, make a small stab incision at the dorsal aspect of the tenth intercostal space. . insert the trocar back into the thoracostomy drainage tube. insert the trocar and tube into the incision. tunnel the tube cranially for approximately intercostal spaces while an assistant simultaneously pulls the skin cranially and ventrally toward the point of the elbow. . at the seventh intercostal space, direct the trocar and catheter perpendicular to the thorax. grasp the catheter apparatus at the base adjacent to the thorax to prevent the trocar from going too far into the thorax. . place the palm of your dominant hand over the end of the trocar, and push the trocar and catheter into the thoracic cavity, throwing your weight into the placement in a swift motion, not by banging the butt of your hand on the end of the stylette. for small individuals, standing on a stool, or kneeling over the patient on the triage table can create leverage and make this process easier. the tube will enter the thorax with a pop. . gently push the catheter off of the stylette, and remove the stylette. . immediately attach the christmas tree adapter and have an assistant start to withdraw air or fluid while you secure the tube in place. . first, place a horizontal mattress suture around the tube to cinch the skin securely to the tube. use care to not penetrate the tube with your needle and suture. . next, place a purse-string suture around the tube at the tube entrance site. leave the ends of the suture long, so that you can create a finger-trap suture to the tube, holding the tube in place. . place a large square of antimicrobial-impregnated adhesive tape over the tube for further security and sterility. . if antimicrobial adhesive is not available, place a gauze pad × inches square over the tube, and then wrap the tube to the thorax with cotton roll gauze and elastikon adhesive tape. . draw the location of the tube on the bandage to prevent cutting it with subsequent bandage changes. an alternate technique to use if a trocar thoracic drainage catheter is not available is the following: . prepare the lateral thorax and infuse local lidocaine anesthetic as listed before. . make a small stab incision with a no. scalpel blade, as listed before. . obtain the appropriately sized red rubber catheter and cut multiple side ports in the distal end of the catheter, taking care to not cut more than % of the circumference of the diameter of the tube. . insert a rigid, long urinary catheter into the red rubber catheter to make the catheter more rigid during insertion into the pleural space. . grasp the distal end of the catheter(s) in the teeth of a large carmalt. tunnel a metzenbaum scissors under the skin to the seventh intercostal space and make a puncture through the intercostal space. . remove the metzenbaum scissors, and then tunnel the carmalt and red rubber tube under the skin to the hole created in the seventh intercostal space with the metzenbaum scissors. . insert the tips of the carmalt and the red rubber catheter through the hole, and then open the teeth of the carmalt. . push the red rubber catheter cranially into the pleural cavity. . remove the carmalt and the rigid urinary catheter, and immediately attach the suction apparatus. secure the red rubber catheter in place as listed before. placement of a temporary tracheostomy can be lifesaving to relieve upper respiratory tract obstruction, to facilitate removal of airway secretions, to decrease dead space ventilation, to provide a route of inhalant anesthesia during maxillofacial surgery, and to facilitate mechanical ventilation. in an emergent situation in which asphyxiation is imminent and endotracheal intubation is not possible, any cutting instrument placed into the trachea distal to the point of obstruction can be used. to perform a slash tracheostomy, quickly clip the fur and scrub the skin over the third tracheal ring. make a small cut in the trachea with a no. scalpel blade, and insert a firm tube, such as a syringe casing. alternately, insertion of a -gauge needle attached to intravenous extension tubing and adapted with a -ml syringe case to attach to a humidified oxygen source also temporarily can relieve obstruction until a temporary tracheostomy can be performed. in less emergent situations, place the patient under general anesthesia and intubate the patient. assemble all the equipment necessary before starting the temporary tracheostomy procedure (box - ). to perform a tracheostomy, follow this procedure: . place the patient in dorsal recumbency. . clip the ventral cervical region from the level of the ramus of the mandible caudally to the thoracic inlet and dorsally to midline. . aseptically scrub the clipped area, and then drape with sterile huck towels secured with towel clamps. . make a -cm ventral midline skin incision over the third to sixth tracheal rings, perpendicular to the trachea. . bluntly dissect through the sternohyoid muscles to the level of the trachea. . carefully pick up the fascia overlying the trachea and cut it away with a metzenbaum scissors. . place two stay sutures through/around adjacent tracheal rings. . incise in between trachea rings with a no. scalpel blade. take care to not cut more than % of the circumference of the trachea. . using the stay sutures, pull the edges of the tracheal incision apart, and insert the tracheostomy tube. the shiley tube contains an internal obturator to facilitate placement into the tracheal lumen. remove the obturator, and then insert the inner cannula, which can be removed for cleaning as needed. . once the tube is in place, secure the tube around the neck with a length of sterile umbilical tape. postoperative care of the tracheostomy tube is as important as the procedure itself. because the tracheostomy tube essentially bypasses the protective effects of the upper respiratory system, one of the most important aspects of tracheostomy tube care and maintenance is to maintain sterility at all times. any oxygen source should be humidified with sterile water or saline to prevent drying of the respiratory mucosa. if supplemental oxygen is not required, instill to ml of sterile saline every to hours to moisten the mucosa. wearing sterile gloves, remove the internal tube and place it in a sterile bowl filled with sterile hydrogen peroxide and to be cleaned every hours (or more frequently as necessary). if a shiley tube is not available, apply suction to the internal lumen of the tracheostomy tube every to hours (or more frequently as needed) with a sterile f red rubber catheter attached to a vacuum pump to remove any mucus or other debris that potentially could plug the tube. unless the patient demonstrates clinical signs of fever or infection, the prophylactic use of antibiotics is discouraged because of the risk of causing a resistant infection. after the temporary tracheostomy is no longer necessary, remove the tube and sutures, and leave the wound to heal by second intention. primary closure of the wounds could predispose the patient to subcutaneous emphysema and infection. baker gd: trans-tracheal oxygen therapy in dogs with severe respiratory compromise due to tick (i. holocyclus) toxicity, aust vet pract ( ) urohydropulsion is a therapeutic procedure for removal of uroliths from the urethra of the male dog. the technique works best if the animal is heavily sedated or is placed under general anesthesia (figure - ) . to perform urohydropulsion, follow this procedure: . place the animal in lateral recumbency. . clip the fur from the distal portion of the prepuce. . aseptically scrub the prepuce and flush the prepuce with to ml of antimicrobial flush solution. . have an assistant who is wearing gloves retract the penis from the prepuce. . while wearing sterile gloves, lubricate the tip of a rigid urinary catheter as for urethral catheterization. . gently insert the tip of the catheter into the urethra until you meet the resistance of the obstruction. . pinch the tip of the penis around the catheter. . have an assistant insert a gloved lubricated finger into the patient's rectum and press ventrally on the floor of the rectum to obstruct the pelvic urethra. . attach a -ml syringe filled with sterile saline into proximal tip of the catheter. . quickly inject fluid into the catheter and alternate compression and relaxation on the pelvic urethra such that the urethra dilates and suddenly releases the pressure, causing dislodgement of the stone. small stones may be ejected from the tip of the urethra, whereas larger stones may be retropulsed back into the urinary bladder to be removed surgically at a later time. the type of catheter that you choose for vascular access depends largely on the size and species of the patient, the fragility of the vessels to be catheterized, the proposed length of time that the catheter will be in place, the type and viscosity of the fluid or drug to be administered, the rate of fluid flow desired, and whether multiple repeated blood samples will be required (table - ) . a variety of over-the-needle, through-the-needle, and over-the-wire catheters are available for placement in a variety of vessels, including the jugular, cephalic, accessory cephalic, medial saphenous, lateral saphenous, dorsal pedal artery, and femoral artery. one of the most important aspects of proper catheter placement and maintenance is to maintain cleanliness at all times. the patient's urine, feces, saliva, and vomit are common sources of contamination of the catheter site. before placing a peripheral or central catheter in any patient, consider the patient's physical status including whether vomiting, diarrhea, excessive urination, or seizures. in a patient with an oral mass that is drooling excessively or a patient that is vomiting, peripheral cephalic catheterization may not be the most appropriate, to prevent contamination. conversely, in a patient with excessive urination or diarrhea, a lateral or medial saphenous catheter is likely to become contaminated quickly. whenever one places or handles a catheter or intravenous infusion line, the person should wash the hands carefully and wear gloves to prevent contamination of the intravenous catheter and fluid lines. one of the most common sources of catheter contamination in veterinary hospitals is through caretakers' hands. in emergent situations, placement of a catheter may be necessary under less than ideal circumstances. remove those catheters as soon as the patient is more stable, and place a second catheter using aseptic techniques. in general, once the location of the catheter has been decided, set up all equipment necessary for catheter placement before starting to handle and restrain the patient. lists the equipment needed for most types of catheter placement. after setting up all of the supplies needed, clip the fur over the site of catheter placement. make sure to clip all excess fur and long feathers away from the catheter site, to prevent contamination. for catheter placement in limbs, clip the fur circumferentially around the site of catheter placement to facilitate adherence of the tape to the limb and to facilitate catheter removal with minimal discomfort at a later date. next, aseptically scrub the catheter site with an antimicrobial scrub solution such as hibiclens. the site is now ready for catheter insertion. consider using a central venous catheter whenever multiple repeated blood samples will need to be collected from a patient during the hospital stay. central venous catheters also can be used for cvp measurement, administration of hyperoncotic solutions such as parenteral nutrition, and administration of crystalloid and colloid fluids, anesthesia, and other injectable drugs (figures - and - ) . to place a jugular central venous catheter, place the patient in lateral recumbancy and extend the head and neck such that the jugular furrow is straight. clip the fur from the ramus of the mandible caudally to the thoracic inlet and dorsally and ventrally to midline. wipe the clipped area with gauze × -inch squares to remove any loose fur and other debris. aseptically scrub the clipped area with an antimicrobial cleanser. venocaths (abbott laboratories) are a through-the-needle catheter that is contained within a sterile sleeve for placement. alternately, other over-the-wire central venous catheters can be placed by the seldinger technique. sterility must be maintained at all times, regardless of the type of catheter placed. wearing sterile gloves, drape the site of catheter placement with sterile drapes, and occlude the jugular vein at the level of the thoracic inlet. pull the clear ring and wings of emergency diagnostic and therapeutic procedures figure - : lateral thoracic radiograph of a central venous catheter. note that the tip of the catheter is inserted in its proper location, just outside of the right atrium. the catheter cover down toward the catheter itself to expose the needle. remove the guard off of the needle. lift the skin over the proposed site of catheter insertion and insert the needle under the skin, with the bevel of the needle facing up. next, reocclude the vessel and pull the skin tight over the vessel to prevent movement of the vessel as you attempt to insert the needle. in some cases, it may be difficult actually to see the vessel in obese patients. if you cannot visualize or palpate the needle, gently bounce the needle over the vessel with the bevel up. the vessel will bounce in place slightly, allowing a brief moment of visualization to facilitate catheter placement. once the vessel has been isolated and visualized, insert the needle into the vessel at a -to -degree angle. watch closely for a flash of blood in the catheter. when blood is observed, insert the needle a small distance farther, and then push the catheter and stylette into the vessel for the entire length, until the catheter and stylette can be secured in the catheter hub. if the catheter cannot be inserted fully into the vessel for its entire length, the tip of the needle may not be within the entire lumen, the catheter may be directed perivascularly, and the catheter may be caught at the thoracic flexure and may be moving into one of the tributaries that feeds the forelimb. extend the patient's head and neck, and lift the forelimb up to help facilitate placement. do not force the catheter in because the catheter potentially can form a knot and will need to be removed surgically. remove the needle from the vessel, and have an assistant place several × -inch gauze squares over the site of catheter placement with some pressure to control hemorrhage. secure the catheter hub into the needle guard, and remove the stylette from the catheter. immediately insert a -to -ml syringe of heparinized saline and flush the catheter and draw back. if you are in the correct place, you will be able to draw blood from the catheter. to secure the catheter in place, tear a length of -inch white tape that will wrap around the patient's neck. pull a small length of the catheter out of the jugular vein to make a semicircle. the semicircle should be approximately / inch in diameter. let the length of catheter lie on the skin, and then place × -inch gauze squares impregnated with antimicrobial ointment over the site of catheter insertion. secure the proximal end of white tape around the white and blue pieces of the catheter, and wrap the tape around the patient's neck so that the tape adheres to the skin and fur. repeat the process by securing the gauze to the skin with two additional lengths of white tape, starting to secure the gauze in place by first wrapping the tape dorsally over the patient's neck, rather than under the patient's neck. in between each piece of tape and bandage layer, make sure that the catheter flushes and draws back freely, or else occlusion can occur. gently wrap layers of cotton roll gauze, kling, and elastikon or vetrap over the catheter. secure a male adapter or t port that has been flushed with heparinized saline, and then label the catheter with the size and length of catheter, date of catheter placement, and initials of the person who placed the catheter. the catheter is ready for use. monitor the catheter site daily for erythema, drainage, vessel thickening, or pain upon infusion. if any of these signs occur, or if the patient develops a fever of unknown origin, remove the catheter, culture the catheter tip aseptically, and replace the catheter in a different location. as long as the catheter is functional without complications, the catheter can remain in place. central catheters also can be placed via the seldinger or over-the-wire technique. a number of companies manufacture kits that contain the supplies necessary for over-the-wire catheter placement. each kit minimally should contain an over-the-needle catheter to place into the vessel, a long wire to insert through the original catheter placed, a vascular dilator to dilate the hole in the vessel created by the first catheter, and a long catheter to place into the vessel over the wire. additional accessories can include a paper drape, sterile gauze, a scalpel blade, local anesthetic, -gauge needles, and -or -ml syringes. restrain the patient and prepare the jugular furrow aseptically as for the percutaneous through-the-needle catheter placement. the person placing the catheter should wear sterile gloves throughout the process to maintain sterility. pick up the skin over the site of catheter placement, and insert a small bleb of local anesthetic through the skin. the local anesthetic should not be injected into the underlying vessel (figure - ) . make a small nick into the skin through the local anesthetic with a no. or no. scalpel blade. use care to avoid lacerating the underlying vessel. next, occlude the jugular vein as previously described, and insert the over-the-needle catheter into the vessel. watch for a flash of blood in the catheter hub. remove the stylette from the catheter. next, insert the long wire into the catheter and into the vessel (figures - and - ) . never let go of the wire. remove the catheter, and place the vascular dilator over the wire and into the vessel (figure - ) . gently twist to place the dilator into the vessel a short distance, creating a larger hole in the vessel. the vessel will bleed more after creating a larger hole. remove the vascular dilator, and leave the wire in place within the vessel. insert the long catheter over the wire into the vessel (figure - ) . push the catheter into the vessel to the catheter hub (figure - ) . slowly thread the wire through a proximal port in the catheter. once the catheter is in place, remove the wire, and suture the catheter in place to the skin with nonabsorbable suture. cover the catheter site with sterile gauze and antimicrobial ointment, cotton roll bandaging material, gauze, and kling or vetrap. flush the catheter with heparinized saline solution, and then use the catheter for infusion of parenteral nutrition, blood products, crystalloid and colloid fluids, medications, and frequent blood sample collection. examine the catheter site daily for evidence of infection or thrombophlebitis. the catheter can remain in place as long as it functions and no complications occur. place the patient in sternal recumbency as for cephalic venipuncture. clip the antebrachium circumferentially, and wipe the area clean of any loose fur and debris (figure - ) . aseptically scrub the clipped area, and have an assistant occlude the cephalic vein at the crook of the elbow. the person placing the catheter should grasp the distal carpus with the nondominant hand and insert the over-the-needle catheter into the vessel at a -to -degree angle ( figure - ) . watch for a flash of blood in the catheter hub, and then gently push the catheter off of the stylette (figure - ) . have the assistant occlude the vessel over the catheter to prevent backflow. flush the catheter with heparinized saline solution. make sure that the skin and catheter hub are clean and dry to ensure that the tape adheres to the catheter hub and skin. secure a length of / -inch white tape tightly around the catheter and then around the limb. make sure that the catheter hub does not "spin" in the tape, or else the catheter will fall out. next, secure a second length of -inch adhesive tape under the catheter and around the limb and catheter hub (figure - ). this piece of tape helps to stabilize the catheter in place. finally, place a flushed t port or male adapter in the catheter hub and secure to the limb with white tape. make sure that the tape is adhered to the skin securely, but not so tightly as to impede venous outflow (figure - ) . the catheter site can be covered with a cotton ball impregnated with antimicrobial ointment and layers of bandage material. label all catheters with the date of placement, the type and gauge of catheter inserted, and the initials of the person who placed the catheter. the femoral artery can be catheterized for placement of an indwelling arterial catheter. indwelling arterial catheters can be used for continuous invasive arterial blood pressure monitoring and for procurement of arterial blood samples. place the patient in lateral recumbancy, and tape the down leg in an extended position. clip the fur over the femoral artery and aseptically scrub the clipped area. palpate the femoral artery as it courses distally on the medial surface of the femur and anterior to the pectineus muscle. make a small nick incision over the proposed site of catheter placement using the bevel of an -gauge needle. place a long over-the-needle catheter through the nick in the skin and direct it toward the palpable pulse. place the tip of the catheter so that the needle tip rests in the subcutaneous tissue between the artery and the palpating index finger. advance the needle steeply at a -degree angle to secure the superficial wall of the vessel and then the deep wall of the vessel. the spontaneous flow of blood in the catheter hub ensures that the catheter is figure - : catheter is taped in place with a t-port. situated in the lumen of the artery. feed the catheter off of the stylette, and cover the hub with a catheter cap. flush the catheter with sterile heparinized saline solution, and then secure it in place. some persons simply tape the catheter in place with pieces of / -and -inch adhesive tape. others use a "butterfly" piece of tape around the catheter hub and suture or glue the tape to the adjacent skin for added security. the dorsal pedal artery commonly is used for catheter placement. to place a dorsal pedal arterial catheter, place the patient in lateral recumbency. clip the fur over the dorsal pedal artery, and then aseptically scrub the clipped area. tape the distal limb so that the leg is twisted slightly medially for better exposure of the vessel, or the person placing the arterial catheter can manipulate the limb into the appropriate position. palpate the dorsal pedal pulse as it courses dorsally over the tarsus. place an over-the-needle catheter percutaneously at a -to -degree angle, threading the tip of the needle carefully toward the pulse. advance the needle in short, blunt movements, and watch the catheter hub closely for a flash of pulsating blood that signifies penetration into the lumen of the artery. then thread the catheter off of the stylette, and cover the catheter hub with a catheter cap. secure the catheter in place with lengths of / -and -inch adhesive tape as with any other intravenous catheter, and then flush it with heparinized saline solution every to hours. any vessel that can be catheterized percutaneously also can be catheterized with surgical cutdown. restrain the patient and clip and aseptically scrub the limb or jugular vein as for a percutaneous catheterization procedure. block the area for catheter placement with a local anesthetic before cutting the skin over the vessel with a no. scalpel blade. while wearing sterile gloves, pick up the skin and incise the skin over the vessel. direct the sharp edge of the blade upward to avoid lacerating the underlying vessel. using blunt dissection, push the underlying subcutaneous fat and perivascular fascia away from the vessel with a mosquito hemostat. make sure that all tissue is removed from the vessel. using the mosquito hemostat, place two stay sutures of absorbable suture under the vessel. elevate the vessel until it is parallel with the incision, and gently insert the catheter and stylette into the vessel. secure the stay sutures loosely around the catheter. suture the skin over the catheter site with nonabsorbable suture, and then tape and bandage the catheter in place as for percutaneous placement. remove catheters placed surgically as soon as possible and exchange them for a percutaneously placed catheter to avoid infection and thrombophlebitis. the most important aspect of catheter maintenance is to maintain cleanliness and sterility at all times. an indwelling catheter can remain in place for as long as it is functional and no complications occur. change the bandage whenever it becomes wet or soiled to prevent wicking of bacteria and debris from the environment into the vessel. check the bandages and catheter sites at least once a day for signs of thrombophlebitis: erythema, vessel hardening or ropiness, pain upon injection or infusion, and discharge. also closely examine the tissue around and proximal and distal to the catheter. swelling of the paw can signify that the catheter tape and bandage are too tight and are occluding venous outflow. swelling above the catheter site is characteristic of perivascular leakage of fluid and may signify that the catheter is no longer within the lumen of the vessel. remove the catheter if it is no longer functional, if there is pain or resistance upon infusion, if there is unexplained fever or leukocytosis, or if there is evidence of cellulitis, thrombophlebitis, or catheter-related bacteremia or septicemia. aseptically culture the tip of the indwelling catheter for bacteria. animals should wear elizabethan collars or other forms of restraint if they lick or chew at the catheter or bandage. catheter patency may be maintained with constant fluid infusion or by intermittent flushing with heparinized saline ( units of unfractionated heparin per to ml of saline) every hours. flush arterial catheters more frequently (every hours). disconnect intravenous connections only when absolutely necessary. wear gloves whenever handling the catheter or connections. label all fluid lines and elevate them off of the floor to prevent contamination. date each fluid line and replace it once every to hours. if an intravenous catheter cannot be placed because of small patient size, hypovolemia, hypothermia, or severe hypotension, needles can be placed into the marrow cavity of the femur, humerus, and tibia for intraosseous infusion of fluids, drugs, and blood products. this technique is particularly useful in small kittens and puppies and in exotic species. contraindications to intraosseous infusion is in avian species (which have air in their bones), fractures, and sepsis, because osteomyelitis can develop. an intraosseous catheter is relatively easy to place and maintain but can cause patient discomfort and so should be changed to an intravenous catheter as soon as vascular access becomes possible. to place an intraosseous catheter, clip and aseptically scrub the fur over the proposed site of catheter placement. the easiest place for intraosseous placement is in the intertrochanteric fossa of the femur. inject a small amount of a local anesthetic through the skin and into the periosteum where the trocar or needle will be inserted. place the patient in lateral recumbency, and grasp the leg in between your fingers, with the stifle braced against the palm of your hand. push the stifle toward the abdomen (medially) to abduct the proximal femur away from the body. this will shift the sciatic nerve out of the way of catheter placement. insert the tip of the needle through the skin and into the intertrochanteric fossa. gently push with a simultaneous twisting motion, pushing the needle parallel with the shaft of the femur, toward your palm. you may feel a pop or decreased resistance as the needle enters the marrow cavity. gently flush the needle with heparinized saline. if the needle is plugged with bone debris, remove the needle and replace it with a fresh needle of the same type and size in the hole that you have created. a spinal needle with an internal stylette also can be placed. the stylette will prevent the needle from becoming clogged with bone debris during insertion. secure the hub of the needle with a butterfly length of white adhesive tape and then suture it to the skin to keep the catheter in place. the catheter is now ready for use. the patient should wear an elizabethan collar to prevent disruption or removal of the catheter. the intraosseous catheter can be maintained as any peripheral catheter, with frequent flushing and daily evaluation of the catheter site. the definition of pain has been debated philosophically over the ages and has changed as knowledge has increased. pain is defined as an unpleasant sensory or emotional experience associated with actual or perceived tissue damage. until recognition of a noxious stimulus occurs in the cerebral cortex, no response or adaptation results. rational management of pain requires an understanding of the underlying mechanisms involved in pain and an appreciation of how analgesic agents interact to disrupt pain mechanisms. multiple factors and causes produce pain in human beings and domestic animal species. the causes of pain, psychological and physical, may derive from many different mechanisms within emergency medicine, among them trauma, infectious disease, neglect, environmental stress, surgery, and acute decompensation of chronic medical conditions. the two major classes of pain are acute and chronic pain. box - gives specific categories and causes of pain. the pain sensing and response system can be divided into the following categories: nociceptors, which detect and filter the intensity of the noxious stimuli; primary afferent nerves, which transmit impulses to the central nervous system (cns); ascending tracts, which are part of the dorsal horn and the spinal cord that conveys stimuli to higher centers in the brain; higher centers, which are involved in pain discrimination, some memory, and motor control; and modulating or descending systems, which are a means of processing, memorizing, and modifying incoming impulses. current analgesic therapies may inhibit afferent nociceptive transmission within the brain and spinal cord; directly interrupt neural impulse conduction through the dorsal horn, primary afferent nerves, or dorsal root ganglion; or prevent the nociceptor sensitization that accompanies initial pain and inflammation. the physiologic aspects of pain are believed to be produced by the transmission, transduction, and integration of initial nerve endings, peripheral neuronal input, and ascending afferent nerves via the thalamus to the cerebral cortex. ascending afferent nerves to the limbic system are believed to be responsible for the emotional aspects of pain. there are several classification schemes for different types of pain. acute pain, such as that which results from trauma, surgery, or infectious agents, is abrupt in onset, relatively short in duration, and may be alleviated easily by analgesics. in contrast, chronic pain is a long-standing physical disorder or emotional distress that is slow in onset and difficult to treat. both types of pain can be classified further based on site of origin. somatic pain arises from superficial skin, subcutaneous tissue, body wall, or appendages. visceral pain arises from abdominal or thoracic viscera and primarily is associated with serosal irritation. analgesia, then, is the loss of pain without the loss of consciousness. this is in contrast to anesthesia, which is the loss of sensation in the whole body or a part of the body with the loss of consciousness or at least depression of the cns. untreated pain causes immediate changes in the neurohormonal axis, which in turn causes restlessness, agitation, increased heart and respiratory rates, fever, and blood pressure fluctuations, all of which are detrimental to the healing of the animal. a catabolic state is created as a result of increased secretion of catabolic hormones and decreased secretion of anabolic hormones. the net effect the majority of neurohormonal changes produce is an increase in the secretion of catabolic hormones. hyperglycemia is produced and may persist because of production of glucagon and relative lack of insulin. lipolytic activity is stimulated by cortisol, catecholamines, and growth hormone. cardiorespiratory effects of pain include increased cardiac output, vasoconstriction, hypoxemia, and hyperventilation. protein catabolism is a common occurrence and major concern regarding healing. pain associated with inflammation causes increase in tissue and blood levels of prostaglandins and cytokines, both of which promote protein catabolism indirectly by increasing the energy expenditure of the body. powerful evidence indicates that local anesthetic, sympathetic agonist, and opioid neural blockade may produce a modification of the responses to these physiologic changes. variable reduction in plasma cortisol, growth hormone, antidiuretic hormone, β-endorphin, aldosterone, epinephrine, norepinephrine, and renin is based on the anesthetic technique and the drugs selected. prophylactic administration of analgesics blunts the response before it occurs; analgesics administered following perception or pain are not as effective, and higher doses are generally necessary to achieve an equivalent level of analgesia. effective pain control can be achieved only when the signs of pain can be assessed effectively, reliably, and regularly. the experience of pain is unique to each individual, which makes pain assessment difficult, especially in traumatized and critical patients. most attempts to assess clinical pain use behavioral observations and interactive variables in addition to assessment of physiologic responses such as heart rate and respiratory rate, blood pressure, and temperature. but many factors can influence the processing and outward projection of pain, including altered environments, species differences, withinspecies variations (age, breed, sex), and the type, severity, and chronicity of pain. within-species differences (age, breed, and sex) further complicate the pain assessment. most notable is that different breeds of dogs act differently when confronted with pain or fear. labrador retrievers tend to be stoic, whereas greyhounds and teacup breeds tend to react with a heightened state of arousal around even the simplest of procedures (e.g., subcutaneous injections and nail trims). the individual character and temperament of the animal further influences its response. pediatric and neonatal animals seem to have a lower threshold for pain and anxiety than older animals. in any species, the duration and type of pain make it more (acute) or less (chronic) likely to be expressed or exhibited outwardly. unfamiliarity with normal behaviors typical of a particular species or breed makes recognition of their painful behaviors and responses impossible. the definition and recognition of pain in an individual animal is challenging. because of all the differences discussed, there is no straight line from insult, albeit actual or perceived, to degree of pain experienced. nor is there a formula for treating "x" type of pain with "y" type of analgesic. a goal of analgesia is to treat all animals with analgesic drugs and modalities as preemptively as possible and using a multimodal approach. use analgesic treatment as a tool for diagnosis of pain in the event that recognition of these phenomena is difficult for the patient. in other words, with countless drugs and treatment modalities available, analgesic administration should never be withheld in an animal, even if pain is questionable. it is important to remember that no behavior or physiologic variable in and of itself is pathognomonic for pain. interactive and unprovoked (noninteractive) behavior assessments and trending of physiologic data are useful to determine the pain in an individual animal. this is known as pain scoring. baseline observations, especially those observations from someone who has known the animal well, can be helpful to serial behavior and pain assessments. pain scoring systems have been developed and are reviewed elsewhere; the purposes of these systems are to evaluate and to help guide diagnostic and analgesic treatments (table - ) . regardless of the scale or method used to assess pain, the caregiver must recognize the limitations of the scale. if in doubt of whether pain is present or not, analgesic therapy should be used as a diagnostic tool. classic behaviors associated with pain in dogs and cats include abnormal postures, gaits, movements, and behaviors (boxes - and . stoicism is the apparent apathy and pain: assessment, prevention, and management indifference in the presence of pain and is perhaps the no. sign of ineffective pain relief or persistent pain in many animals, because so many display apathy and classically normal physiologic parameters even in the face of severe distress, overt suffering, or blatant trauma and illness. the absence of normal behaviors is also a clinical sign of pain, even when abnormal behaviors are not observed. acute pain results in many of the aforementioned behavioral and physiologic signs, but chronic pain in small animals is an entirely different and distinct entity. chronic pain is often present in the absence of obvious tissue pathology and changes in physical demeanor. again, the severity of the pain may not correlate with the severity of any pathologic condition that may or may not be present. chronic pain, especially if insidious in onset (cancer, dental, or degenerative pain), may well go unnoticed in dogs and cats, even by family members or intermittent caregivers. inappetance, lack of activity, panting in a species classically designed to be nose breathers, decreased interest in surroundings, different activity patterns, and abnormal postures are just a few signs of chronic pain in cats and dogs. cats are a species that in particular are exemplary in their abilities to hide chronic pain. they will exhibit marked familial withdrawal, finding secluded areas where they may remain for days to weeks when they experience acute and chronic pain. when deciding on a pain management protocol for a patient, always perform a thorough physical examination and include a pain score assessment before injury and pain has occurred, whenever possible. form a problem list to guide your choice of anesthesia and analgesia. for example, using a nonsteroidal antiinflammatory drug (nsaid) in an animal with renal failure would not be wise. remember to account for current medications that the patient may be taking that may augment or interfere with the analgesic or anesthetic drugs. use multimodal techniques and regional therapy and drugs to target pain at different sites before it occurs. once a strategy is decided upon, frequently reassess the patient and tailor the protocol to meet each patient's response and needs. drug therapy (in particular, opioids with or without α -agonists) is a cornerstone for acute pain treatment and surgical preemptive pain prevention. however, local anesthetics delivered epidurally, via perineural or plexus injection, intraarticular or trigger point injection, are also effective analgesics for acute and chronic forms of pain and inflammation. the nsaids that classically have been reserved for treatment of more chronic or persistent pain states now are being used regularly for treatment of acute and perioperative pain once blood pressure, coagulation, and gastrointestinal parameters have been normalized. an opioid is any natural or synthetic drug that is derived from the poppy, which interacts with opiate receptors identified on cell membranes. the drugs from this class constitute the most effective means of controlling acute, perioperative, and chronic pain in human and veterinary medicine (table - ) . their physiologic effects result from the interaction with one or more of at least five endogenous opioid receptors (µ, σ, δ, ε, and κ). µ-receptor agonists are noted for their ability to produce profound analgesia with mild sedation. these drugs diminish "wind-up," the hyperexcitable state resulting from an afferent volley of nociceptive impulses. they elevate the pain threshold and are used preemptively to prevent acute pain. as a class, opioids cause cns depression with their intense analgesia. dose-related respiratory depression reflects diminished response to carbon dioxide levels. cardiac depression is secondary only to bradycardia and is more likely with certain opioids such as morphine and oxymorphone. narcotics produce few if any clinically significant cardiovascular effects in dogs and cats; they are considered cardiac soothing or sparing. because opioids increase intracranial and intraocular pressure, use them more cautiously in patients with severe cranial trauma and or ocular lesions. opioids directly stimulate the chemoreceptor trigger zone and may cause nausea and vomiting. most opioids depress the cough reflex via a central mechanism; this may be helpful in patients recovering from endotracheal intubation irritation. a key characteristic of opioids that makes them desirable for use in emergency and critical care situations is their reversibility. antagonists block or reverse the effect of agonists by combining with receptors and producing minimal or no effects. administer all reversal agents, such as naloxone and naltrexone, slowly if given intravenously and to effect. α α -agonists as a class of drugs, α -agonists warrant special attention because most members of the group possess potent analgesic power at doses that are capable of causing sedation, cns depression, cardiovascular depression, and even general anesthetic states. originally developed for antihypertensive use, α -agonists quickly have attained sedative analgesic status in veterinary medicine (table - ) . like the opioids, α -agonists produce their effects by aggravating α-adrenergic receptors in the cns and periphery. emergency care among them cyclooxygenase- (cox- ), the major constitutive enzyme primarily involved in normal physiologic functions, and cox- , the enzyme responsible for most of the hyperalgesia and pain responses experienced after tissue injury or trauma. some nsaids inhibit cyclooxygenase and lipoxygenase activity. most of the currently available oral and parenteral nsaids for small animal medicine and surgery target the cyclooxygenase pathways predominantly, although one (tepoxalin) is thought to inhibit both pathways. inhibition of cox- and cox- can inhibit the protective effects and impair platelet aggregation and lead to gastrointestinal ulceration. there are definite contraindications and relative contraindications for the use of nsaids. nonsteroidal antiinflammatory drugs should not be administered to patients with renal or hepatic insufficiency, dehydration, hypotension or conditions that are associated with low circulating volume (congestive heart failure, unregulated anesthesia, shock), or evidence of ulcerative gastrointestinal disease. trauma patients should be stabilized completely regarding vascular volume, tone, and pressure before the use of nsaids. patients receiving concurrent administration of other nsaids or corticosteroids, or those considered to be cushingoid, should be evaluated carefully for an adequate "washout" period (time of clearance of drug from the system) before use of an nsaid or before switching nsaids. patients with coagulopathies, particularly those that are caused by platelet number or function defects or those caused by factor deficiencies, and patients with severe, uncontrolled asthma or other bronchial disease are probably not the patients in which to use nsaids. other advice is that nsaids not be administered to pregnant patients or to females attempting to become pregnant because cox- induction is necessary for ovulation and subsequent implantation of the embryo. the administration of nsaids should be considered only in the well-hydrated, normotensive dog or cat with normal renal or hepatic function, with no hemostatic abnormalities, and no concurrent steroid administration. nonsteroidal antiinflammatory drugs can be used in many settings of acute and chronic pain and inflammation. among these are the use in well-stabilized musculoskeletal trauma and surgical pain, osteoarthritis management, meningitis, mastitis, animal bite and other wound healing, mammary or transitional cell carcinoma, epithelial (dental, oral, urethral) inflammation, ophthalmologic procedures, and dermatologic or otic disease. whereas opioids seem to have an immediate analgesic effect when administered, most nsaids will take up to minutes for their effect to be recognized. as such, most perioperative or acute nsaids use is part of a balanced pain management scheme, one that uses narcotics and local anesthetic techniques. nonsteroidal antiinflammatory drugs are devoid of many of the side effects of narcotic administration; namely, decreased gastrointestinal motility, altered sensorium, nausea/vomition, and sedation. nonsteroidal antiinflammatory drugs are also devoid of many of the side effects of steroid administration; namely, suppression of the pituitary adrenal axis. the toxic effects of salicylates in cats are well documented. cats are susceptible because of slow clearance and dose-dependent elimination because of deficient glucuronidation in this species. because of this, the dose and the dosing interval of most commonly used nsaids need to be altered in order for these drugs to be used. cats that have been given canine doses of nsaids (twice daily or even once daily repetitively) may show hyperthermia, hemorrhagic or ulcerative gastritis, kidney and liver injury, hyperthermia, respiratory alkalosis, and metabolic acidosis. acute and chronic toxicities of nsaids have been reported in cats, especially after repeat once daily dosing. ketoprofen, flunixin, aspirin, carprofen, and meloxicam have been administered safely to cats, although like most antibiotics and other medications, they are not approved and licensed for use in cats. an important note, though, is that dosing intervals ranging from to hours have been used, and antithrombotic effects often can be achieved at much lower doses than those required to treat fevers and inflammation. i recommend the use of no loading doses, minimum -hour dosing intervals, and assurance of adequate circulating blood volume, blood pressure, and renal function. because many of the nsaids are used off-label in cats, it is imperative that the clinician carefully calculate the dose, modify the dosing interval, and communicate this information to the client before dispensing the drug. even drugs that come in liquid form (meloxicam), if administered to cats via box-labeled directions used for dogs, will be given in near toxic doses. to worsen the misunderstanding about dosages for cats, drops from manufacturer's bottles often are calibrated drops; when these same liquids are transferred into pharmacy syringes for drop administration, the calibration of course is lost, and the animal potentially is overdosed. a more accurate method of dispensing and administering oral nsaids in cats is to calculate the dose in milligrams and determine the exact number of milliliters to administer, rather than use the drop method. ketamine classically was considered a dissociative anesthetic, but it also has potent activity as an n-methyl-d-aspartate (nmda) receptor antagonist. this receptor located in the cns mediates windup and central sensitization (a pathway from acute to chronic pain). blockade of this receptor with microdoses of ketamine results in the ability to provide body surface, somatic, and skin analgesia with potentially lower doses of opioids and α-agonists. loading doses of . to mg/kg are used intravenously with continuous rate infusions of to µg/kg/minute. in and of itself, this drug possesses little to no analgesic ability and indeed in high doses alone often can aggravate, sensitize, or excite the animal in subacute or acute pain. amantadine is another nmda blocker that has been used for its antiviral and parkinson's stabilizing effects. amantadine has been used for neuropathic pain in human beings but is only available in an oral form. suggested starting doses for cats and dogs range from to mg/kg po daily. when the drug is given orally and intravenously, patients are unlikely to develop behavioral or cardiorespiratory effects with ketamine or amantadine. tramadol is an analgesic that possesses weak opioid µ-agonist activity and norepinephrine and serotonin reuptake inhibition. tramadol is useful for mild to moderate pain in small animals. although the parent compound has very weak opioid activity, the metabolites have excellent binding affinity for the µ-receptor. tramadol has been used for perisurgical pain control when given orally in cats and dogs at a dose of to mg/kg po sid to bid. cats appear to require only once daily dosing. regardless of its affinity for the opioid receptors, the true mechanism of action of tramadol in companion animals remains largely unknown. gabapentin is a synthetic analog of γ-aminobutyric acid (gaba). originally introduced as an antiepileptic drug, the mechanism of action of gabapentin remains somewhat unclear in veterinary medicine. the drug is among a number of commonly used antiepileptic medications used to treat central pain in human beings. the rationale for use is the ability of the drugs to suppress discharge in pathologically altered neurons. gabapentin does this through calcium channel modulation without binding to glutamate receptors. chronic, burning, neuropathic, and lancinating pain in small animals responds well to to mg/kg po daily. local anesthetic agents are the major class used as a peripheral-acting analgesic ( table - ) . local anesthetics block the transmission of pain impulses at the peripheral nerve nociceptor regions. local anesthetics may be used to block peripheral nerves or inhibit nerve "zones" using regional techniques. although all local anesthetics are capable of providing pain relief, agents with a longer duration of action are preferred for pain management purposes. bupivacaine is an example of a long-acting local anesthetic drug that is used along with lidocaine for long-acting pain relief. a single dose of bupivacaine injected at a local site will provide local anesthesia and analgesia for to hours. when lidocaine is administered as an intravenous constant rate infusion ( to µg/kg/minute in dogs, to µg/kg/minute in cats) is effective in the treatment of chronic neuropathic pain and periosteal and peritoneal pain (e.g., pancreatitis). mexiletine, an oral sodium channel blocker, can be used as an alternative to injectable lidocaine for provision of background analgesia. many drugs (table - ) are used in combination with opioids, α -agonists, and ketamine to provide anxiolysis and sedation. injection of local anesthetic solution into the connective tissue surrounding a particular nerve produces loss of sensation (sensory blockade) and/or paralysis (motor nerve blockade) in the region supplied by the nerve. local anesthetics also may be administered epidurally, intrathoracically, intraperitoneally, and intraarticularly. lidocaine and bupivacaine are the most commonly administered local anesthetics. lidocaine provides for quick, short-acting sensory and motor impairment. bupivacaine provides for later-onset, longerlasting desensitization without motor impairment. combinations of the two agents diluted with saline are used frequently to provide for quick-onset analgesia that lasts between and hours in most patients. adding narcotic and/or α agent often maximizes the analgesia and increases the pain-free interval to to hours. epinephrine and preservative-free solutions are recommended. precision placement of anesthetic close to nerves, roots, or plexuses is improved with the use of a stimulating nerve locator. cats seem to be more sensitive to the effects of local anesthetics; as such the lower ends of most dosing ranges are used for blockades in this species. unlike most instances of general anesthesia, during which the animal is rendered unconscious and nerve transmission is decreased by virtue of cns depression, local and regional techniques block the initiation of noxious signals, thereby effectively preventing pain from entering the cns. this is an effective means of not only preventing initial pain but also reducing the changes that take place in the dorsal horn of the spinal cord, spinothalamic tracts, limbic and reticular activating centers, and cortex. frequently, the neurohormonal response that is stimulated in pain and stress is blunted as well. overall, the patient has fewer local and systemic adverse effects of pain, disease processes are minimized, chronic pain states are unlikely, and outcome is improved. regional techniques are best used as part of an analgesic regimen that consists of their continuous administration, narcotics, α-agonists, anxiolytics, and good nursing. lidocaine can be added to sterile lubricant in a one-to-one concentration to provide decreased sensation for urinary catheterization, nasal catheter insertion, minor road burn analgesia, and pyotraumatic dermatitis analgesia. proparacaine is a topical anesthetic useful for corneal or scleral injuries. local anesthetics can be used to infiltrate areas of damage or surgery by using long-term continuous drainage catheters and small, portable infusion pumps. this is an effective means of providing days of analgesia for massive surgical or traumatic soft tissue injury. even without the catheter, incisional or regional soft tissue blocking using a combination of to mg/kg lidocaine and . to mg/kg bupivacaine diluted with equal volume of saline and : with sodium bicarbonate is effective for infiltrating large areas of injury. administration of local anesthetic drugs around the infraorbital, maxillary, ophthalmic mental, and alveolar nerves can provide excellent analgesia for dental, orofacial, and ophthalmic trauma and surgical procedures. each nerve may be desensitized by injecting . to . ml of a % lidocaine hydrochloride solution and . to . ml of . % bupivacaine solution using a . -to . -cm, -to -gauge needle. precise placement perineurally versus intraneurally (neuroma formation common) is enhanced by using catheters in the foramen versus needle administration. always perform aspiration before administration to rule out intravascular injection of agents. this block is used to provide analgesia for thoracic, lower cervical, cranial abdominal, and diaphragmatic pain. following aseptic preparation, place a small through-the-needle ( -to -gauge) catheter in the thoracic cavity between the seventh and ninth intercostal space on the midlateral aspect of the thorax. aseptically mix a . to mg/kg lidocaine and a . to . mg/kg bupivacaine dose with volume of saline equal to the volume of bupivacaine, and slowly inject it over a period of to minutes following aspiration to ensure that no intravascular injection occurs. depending on where the lesion is, position the patient to allow the intrapleural infusion to "coat" the area. most effective is positioning the patient in dorsal recumbency for several minutes following the block to make sure local anesthetic occupies the paravertebral gutters and hence the spinal nerve roots. the block should be repeated every hours in dogs and every to hours in cats. secure the catheter to the skin surface for repetitive administration. administration of local anesthetic around the brachial plexus provides excellent analgesia for forelimb surgery, particularly that distal to the shoulder, and amputations. nerve locator-guided techniques are much more accurate and successful than blind placement of local anesthetic; however, even the latter is useful. to administer a brachial plexus blockade, follow this procedure: . aseptically prepare a small area of skin over the point of the shoulder. . insert a -gauge, / -to -inch spinal needle medial to the shoulder joint, axial to the lesser tubercle, and advance it caudally, medial to the body of the scapula, and toward the costochondral junction of the first rib. aspirate first before injection to make sure that intravenous injection does not occur. . inject one third of the volume of local anesthetic mix, and then slowly withdraw the needle and fan dorsally and ventrally while infusing the remaining fluid. . local anesthetic doses are similar to those for intrapleural blockade. epidural analgesia refers to the injection of an opioid, a phencyclidine, an α-agonist, or an nsaid into the epidural space. epidural anesthesia refers to the injection of a local anesthetic. in most patients a combination of the two is used. epidural analgesia and anesthesia are used for a variety of acute and chronic surgical pain or traumatically induced pain in the pelvis, tail, perineum, hind limbs, abdomen, and thorax (table - ) . procedures in which epidural analgesia and anesthesia are useful include forelimb and hind limb amputation, tail or perineal procedures, cesarean sections, diaphragmatic hernia repair, pancreatitis, peritonitis, and intervertebral disk disease. epidural blocks performed using opioids or bupivacaine will not result in hind limb paresis or decreased urinary or anal tone (incontinence), unlike lidocaine or mepivicaine epidural blocks. morphine is one of the most useful opioids for administration in the epidural space because of its slow systemic absorption. epidural catheters used for the instillation of drugs through constant rate infusion or intermittent injection can be placed in dogs and cats. routinely placed at the lumbosacral junction, these catheters are used with cocktails including preservative-free morphine, bupivacaine, medetomidine, and ketamine. extremely effective for preventing windup pain in the peritoneal cavity or caudal half of the body, the catheters may be maintained if placed aseptically for to days. to provide epidural analgesia or anesthesia, follow this procedure: . position the animal in lateral or sternal recumbency. . clip and aseptically scrub over the lumbosacral site. . palpate the craniodorsal-most extent of the wings of the ileum bilaterally and draw an imaginary line through them to envision the spine of l located immediately behind the imaginary line. . advance a -to -gauge, / -to -inch spinal or epidural needle through the skin just caudal to the spine of l . . the needle will lose resistance as it is introduced into the epidural space. drop saline into the hub of the needle, and the saline will be pulled into the epidural space as the needle enters. discrete intercostal nerve blocks can provide effective analgesia for traumatic or postsurgical pain. identify the area of the injury, and infiltrate three segments on either side of the injury with analgesic. to perform an intercostal nerve block, follow this procedure: . clip and aseptically scrub the dorsal and ventral third of the chest wall. . palpate the intercostal space as far dorsally as possible. . use a -gauge, . -inch needle at the caudolateral aspect of the affected rib segments and those cranial and caudal. . direct the tip of the needle caudally such that the tip of the needle "drops" off of the caudal rib. (this places the needle tip in proximity to the neuromuscular bundle that contains the intercostal nerve that runs in a groove on the caudomedial surface of the rib.) . aspirate to confirm that the drug will not go intravenously. . inject while slowly withdrawing the needle. inject . to . ml at each site, depending on the size of the animal. gaynor js, an acute condition in the abdomen is defined as the sudden onset of abdominal discomfort or pain caused by a variety of conditions involving intraabdominal organs. many animals have the primary complaint of lethargy, anorexia, ptyalism, vomiting, retching, diarrhea, hematochezia, crying out, moaning, or abnormal postures. abnormal postures can include generalized rigidity, walking tenderly or as if "on eggshells," or a prayer position in which the front limbs are lowered to the ground while the hind end remains standing. in some cases, it may be difficult initially to distinguish between true abdominal pain or referred pain from intervertebral disk disease. rapid progression and decompensation of the patient's cardiovascular status can lead to stupor, coma, and death in the most extreme cases, making rapid assessment, treatment, and definitive care extremely challenging. often the patient's signalment and history can increase the index of suspicion for a particular disease process. a thorough history often is overlooked or postponed in the initial stages of resuscitation of the patient with acute abdominal pain. often, asking the same question in a variety of methods can elicit an answer from the client that may lead to the source of the problem and the reason for acute abdominal pain. important questions to ask the client include the following: • what is your chief complaint or reason that you brought your animal in on emergency? • when did the signs first start, or when was your animal last normal? • do you think that the signs have been the same, better, or getting worse? • does your animal have any ongoing or past medical problems? • have similar signs occurred in the past? • does your animal have access to any known toxins, or does he or she run loose unattended? as with any other emergency, the clinician must follow the abcs of therapy, treating the most life-threatening problems first. first, perform a perfunctory physical examination. examination of the abdomen ideally should be performed last, in case inciting a painful stimulus precludes you from evaluating other organ systems more thoroughly. briefly observe the patient from a distance. are there any abnormal postures? is there respiratory distress? is the animal ambulatory, and if so, do you observe any gait abnormalities? do you observe any ptyalism or attempts to vomit? auscultate the patient's thorax for crackles that may signify aspiration pneumonia resulting from vomiting. examine the patient's mucous membrane color and capillary refill time, heart rate, heart rhythm, and pulse quality. many patients in pain have tachycardia that may or may not be accompanied by dysrhythmias. if a patient's heart rate is inappropriately bradycardic, consider hypoadrenocorticism, whipworm infestation, or urinary obstruction or trauma as a cause of hyperkalemia. assess the patient's hydration status by evaluating skin turgor, mucous membrane dryness, and whether the eyes appear sunken in their orbits. a brief neurologic examination should consist of whether the patient is actively having a seizure, or whether mental dullness, stupor, coma, or nystagmus are present. posture and spinal reflexes can assist in making a diagnosis of intervertebral disk disease versus abdominal pain. perform a rectal examination to evaluate for the presence of hematochezia or melena. finally, examination of the abdomen should proceed first with superficial and then deeper palpation. visually inspect the abdomen for the presence of external masses, bruising, or penetrating injuries. reddish discoloration of the periumbilical area often is associated with the presence of intraabdominal hemorrhage. it may be necessary to shave the fur to inspect the skin and underlying structures visually for bruising and ecchymoses. auscultate the abdomen for the presence or absence of borborygmi to characterize gut sounds. next, perform percussion and ballottement to evaluate for the presence of a gas-distended viscus or peritoneal effusion. finally, perform first superficial and then deep palpation of all quadrants of the abdomen, noting abnormal enlargement, masses, or whether focal pain is elicited in any one area. once the physical examination has been performed, implement initial therapy in the form of analgesia, fluid resuscitation, and antibiotics. treatment for any patient with an acute condition in the abdomen and shock is to treat the underlying cause, maintain tissue oxygen delivery, and prevent end-organ damage and failure. a more complete description of shock and oxygen delivery is given in the section on shock. emergency care the administration of analgesic agents to any patient with acute abdominal pain is one of the most important therapies in the initial stages of case management. many patients with acute abdominal pain are clinically dehydrated or are in hypovolemic shock because of hemorrhage. careful titration of intravenous crystalloid and colloid fluids including blood products is necessary based on the patient's perfusion parameters including heart rate, capillary refill time, blood pressure, urine output, and pcv. fluid therapy also should be based on the most likely differential diagnoses, with specific fluid types administered according to the primary disease process. in dogs, a shock volume of fluids is calculated based on the total blood volume of ml/kg/hour. in cats, shock fluid rate is based on plasma volume of ml/kg/hour. in most cases, any crystalloid fluid can be administered at an initial volume of one fourth of a calculated shock dose and then titrated according to whether the patient's cardiovascular status responds favorably or not. in cases of an acute condition in the abdomen from known or suspected hypoadrenocorticism, severe whipworm infestation, or urinary tract obstruction or rupture, . % sodium chloride fluid without added potassium is the fluid of choice. when hemorrhage is present, the administration of whole blood or packed rbcs may be indicated if the patient has clinical signs of anemia and shows clinical signs of lethargy, tachypnea, and weakness. fresh frozen plasma is indicated in cases of hemorrhage resulting from vitamin k antagonist rodenticide intoxication or hepatic failure or in cases of suspected disseminated intravascular coagulation (dic). a more thorough description of fluid therapy is given under the sections on shock and fluid therapy. the empiric use of broad-spectrum antibiotics is warranted in cases of suspected sepsis or peritonitis as a cause of acute abdominal pain. ampicillin sulbactam ( mg/kg iv q - h) and enrofloxacin ( mg/kg once daily) are the combination treatment of choice to cover gram-negative, gram-positive, aerobic, and anaerobic infections. alternative therapies include a second-generation cephalosporin such as cefotetan ( mg/kg iv tid) or cefoxitin ( mg/kg iv tid) or added anaerobic coverage with metronidazole ( to mg/kg iv tid). tissue oxygen delivery depends on a number of factors, including arterial oxygen content and cardiac output. if an animal has had vomiting and subsequent aspiration pneumonitis, treatment of hypoxemia with supplemental oxygen in the form of nasal, nasopharyngeal, hood, or transtracheal oxygen administration is important (see oxygen supplementation under emergency diagnostic and therapeutic procedures). perform a complete blood count in all cases of acute abdominal pain to determine if lifethreatening infection or coagulopathy including dic is present. in cases of sepsis, infection, or severe nonseptic inflammation, the white blood cell count may be normal, elevated, or low. examine a peripheral blood smear for the presence of toxic neutrophils, eosinophils, atypical lymphocytes, nucleated rbcs, platelet estimate, anisocytosis, and blood parasites. a falling pcv in the face of rbc transfusion suggests ongoing hemorrhage. perform a biochemistry panel to evaluate organ system function. azotemia with elevated bun and creatinine may be associated with prerenal dehydration, impaired renal function, or postrenal obstruction or leakage. the bun also can be elevated when gastrointestinal hemorrhage is present. serum amylase may be elevated with decreased renal function or in cases of pancreatitis. a normal serum amylase, however, does not rule out pancreatitis as a source of abdominal pain. serum lipase may be elevated with gastrointestinal inflammation or pancreatitis. like amylase, a normal serum lipase does not rule out pancreatitis. total bilirubin, alkaline phosphatase, and alanine transaminase may be elevated with primary cholestatic or hepatocellular diseases or may be due to extrahepatic causes including sepsis. obtain a urinalysis via cystocentesis whenever possible, except in cases of suspected pyometra or transitional cell carcinoma. azotemia in the presence of a nonconcentrated (isosthenuric or hyposthenuric) urine suggests primary renal disease. secondary causes of apparent renal azotemia and lack of concentrating ability also occur in cases of hypoadrenocorticism and gram-negative sepsis. renal tubular casts may be present in cases of acute renal ischemia or toxic insult to the kidneys. bacteriuria and pyuria may be present with infection and inflammation. when a urinalysis is obtained via free catch or urethral catheterization, the presence of bacteriuria or pyuria also may be associated with pyometra, vaginitis, or prostatitis/prostatic abscess. serum lactate is a biochemical indicator of decreased organ perfusion, decreased oxygen delivery or extraction, and end-organ anaerobic glycolysis. elevated serum lactate greater than mmol/l has been associated with increased morbidity and need for gastric resection in cases of gdv and increased patient morbidity and mortality in other disease processes. rising serum lactate in the face of adequate fluid resuscitation is a negative prognostic sign. obtain abdominal radiographs as one of the first diagnostic tests when deciding whether to pursue medical or surgical management. the presence of gdv, linear foreign body, pneumoperitoneum, pyometra, or splenic torsion warrants immediate surgical intervention. if a loss of abdominal detail occurs because of peritoneal effusion, perform additional diagnostic tests including abdominal paracentesis (abdominocentesis) and abdominal ultrasound to determine the cause of the peritoneal effusion. abdominal ultrasonography is often useful in place of or in addition to abdominal radiographs. the sensitivity of abdominal ultrasonography is largely operator dependent. indications for immediate surgical intervention include loss of blood flow to an organ, linear bunching or placation of the intestinal tract, intussusception, pancreatic phlegmon or abscess, a fluid-filled uterus suggestive of pyometra, gastrointestinal obstruction, intraluminal gastrointestinal foreign body, dilated bile duct, or gallbladder mucocele, or gas within the wall of the stomach or gallbladder (emphysematous cholecystitis). the presence of peritoneal fluid alone does not warrant immediate surgical intervention without cytologic and biochemical evaluation of the fluid present. see also abdominal paracentesis and diagnostic peritoneal lavage. abdominal paracentesis (abdominocentesis) often is the deciding factor in whether to perform immediate surgery. abdominocentesis is a sensitive technique for detecting peritoneal effusion when more than ml/kg of fluid is present within the abdominal cavity. abdominal effusion collected should be saved for bacterial culture and evaluated biochemically and cytologically based on your index of suspicion of the primary disease process. if creatinine, urea nitrogen (bun) or potassium is elevated compared with that of serum, uroabdomen is present. elevated abdominal fluid lipase or amylase compared with serum supports a diagnosis of pancreatitis. elevated lactate compared with serum lactate or an abdominal fluid glucose less than mg/dl is highly sensitive and specific for bacterial/ septic peritonitis. the presence of bile pigment or bacteria is supportive of bile and septic peritonitis, respectively. free fibers in abdominal fluid along with clinical signs of abdominal pain strongly support gastrointestinal perforation, and immediate surgical exploration is required. text continued on p. the following are clinical conditions, patient signalment, common history, physical examination, and characteristic findings of various diagnostic tests. a blank column next to a condition indicates no specific signalment, history, physical examination, or diagnostic test characteristic for a particular disease process. lack of contiguity of body wall surgical ( medical unless perforation present present c-shaped abnormal gas pattern with plication on radiographs surgical (immediate) dilation of bowel cranial to foreign object, radiopaque object in surgical (immediate) stomach or intestines, hypochloremic metabolic acidosis on bloodwork if pyloric outflow obstruction is present elevated or decreased wbc; foreign material, wbcs and medical unless perforation bacteria on abdominal fluid, elevated lactate and decreased present glucose on abdominal fluid target shaped soft tissue density on abdominal u/s, soft tissue surgical (immediate): density with gas dilation cranially on abdominal radiographs medical management of primary cause colonic distension with hard feces on radiographs medical increased or decreased wbc, septic abdominal effusion surgical (immediate) elevated t bili, alt, alk phos, and wbc hypoechoic hepatic medical after biopsy parenchyma on ultasound hepatomegaly elevated t bili, alt, alk phos, and wbc hyperechoic foci in surgical (immediate) gallbladder or sludge on u/s, free gas in wall of gall bladder abdominal effusion, bile pigment in effusion surgical (immediate) elevated t bili, alk phos, alt surgical (immediate) elevated or decreased wbc, elevated t bili, alk phos and surgical (immediate) alt, free gas in hepatic parenchyma on rads, hypoechoic mass with hyperechoic material in hepatic parenchyma on u/s heteroechoic liver with hyperechoic center on ultrasound surgical (immediate) mixed echogenic mass on ultrasound, soft tissue mass surgical (immediate or density on radiographs, elevated alk phos, alt, delayed) t bili, hypoglycemia pain-cont'd elevated t bili, alk phos, alt, amylase and/or lipase, elevated medical in most cases or decreased wbc, hypocalcemia, focal loss of detail in right unless abscess or cranial quadrant on radiographs hypo-to hyperechoic phlegmon is present pancreas with hyperechoic peri-pancreatic fat on ultrasound, abdominal and/or pleural effusion on radiographs and ultrasound pancreatic soft tissue mass effect on radiographs and surgical if mass identified, ultrasound, elevated amylase and lipase, hypoglycemia, otherwise medical elevated serum insulin management of hypoglycemia splenomegaly on radiographs, hyperechoic spleen with no surgical (immediate) blood flow on ultrasound soft tissue mass effect and loss of abdominal detail on surgical (immediate) radiographs, cavitated mass with abdominal effusion on u/s hyperechoic spleen with no blood flow on abdominal u/s, surgical (immediate) abdominal effusion, thrombocytopenia loss of abdominal detail on radiographs, peritoneal effusion medical unless refractory on u/s, hemoabdomen on abdominocentesis hypotension diagnosis based primarily on clinical signs medical fracture of the os penis on radiographs largely medical unless urethral tear diagnosis based primarily on clinical signs medical, although prepuce may need to be incised to allow replacement of penis into sheath prostatomegaly on radiographs and ultrasound hypoechoic medical prostate on u/s, pyuria and bacteriuria and u/a prostatomegaly on radiographs and ultrasound hypo-to surgical (delayed) hyperechoic prostate on u/s, bacteriuria and pyuria on u/a prostatomegaly on radiographs and ultrasound, prostatic medical/surgical mineralization on radiographs and ultrasound hypoechoic kidneys on u/s, pyuria on u/a, elevated wbc, medical azotemia pyuria, bacteriuria on u/a medical pyelectasia in abdominal u/s, azotemia surgical (immediate) renomegaly on radiographs, azotemia renal mass on u/s, renomegaly on radiographs surgical (immediate) renal mass on u/s, azotemia, lack of renal blood flow surgical (delayed) on u/s calculi in renal pelvis on radiographs and ultrasound, azotemia medical unless both kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, medical unless both azotemia kidneys affected ureteral calculi on radiographs and ultrasound, hydronephrosis, surgical (delayed until fluid or soft tissue density on u/s, azotemia electrolyte stabilization) diagnosis largely based on physical examination medical unless cannot pass findings urethral catheter azotemia, no peritoneal effusion, lack of urine output or surgical (delayed until outflow with ureteral catheterization, double contrast electrolyte stabilization) cystourethrogram indicated transitional cellular casts on u/a, hematuria, mass effect or surgical and medical thickened irregular urethra on ultrasound or management cystourethrogram hypoechoic swollen testicle on testicular ultrasound surgical (immediate) fluid or gas-filled tubular structure on abdominal ultrasound or surgical (immediate) abdominal radiographs soft tissue tubular structure on radiographs, fluid-filled uterus surgical ( in the event of a negative abdominocentesis, but peritoneal effusion or bile or gastrointestinal perforation are suspected, perform a diagnostic peritoneal lavage. peritoneal dialysis kits are commercially available but are often expensive and impractical (see p. ). animals that have acute abdominal pain can be divided into three broad categories, depending on the primary cause of pain and the initial definitive treatment (table - ) . some diseases warrant a nonsurgical, medical approach to case management. other conditions require immediate surgery following rapid stabilization. other conditions initially can be managed medically until the patient is hemodynamically more stable and then may or may not require surgical intervention at a later time. specific management of each disease entity is listed under its own subheading. box - lists specific indications for exploratory laparotomy. the best means to explore the abdominal cavity accurately and thoroughly is to open the abdomen on midline from the level of the xyphoid process caudally to the pubis for full exposure and then to evaluate all organs in every quadrant in a systematic manner. address specific problems such as gastric or splenic torsion, enteroplication, and foreign body removal, and then copiously lavage the abdomen with warmed sterile saline solution. suction the saline solution thoroughly from the peritoneal cavity so as to not impair macrophage function. in cases of septic peritonitis, the abdomen may be left open, or a drain may be placed for further suction and lavage. the routine use of antibiotics in irrigation solutions is contraindicated because the antibiotics can irritate the peritoneum and delay healing. when the abdominal cavity is left open, secure sterile laparotomy towels and water-impermeable dressings over the abdominal wound with umbilical tape, and then change these daily or as strike-through occurs. open abdomen cases are often effusive and require meticulous evaluation and management of electrolyte imbalances and hypoalbuminemia. the abdomen can be closed and/or the abdominal drain removed when the volume of the effusion decreases, when bacteria are no longer present, and when the neutrophils become more healthy in appearance. bischoff mg: radiographic techniques and interpretation of the acute abdomen, clin tech small anim pract ( ) anaphylactic shock occurs as an immediate hypersensitivity reaction to a variety of inciting stimuli (box - ). in animals, the most naturally occurring anaphylactic reaction results from wasp or bee stings. most other reactions occur as a result of an abnormal sensitivity to items used in making medical diagnoses or treatment. during an anaphylactic reaction, activation of c a and the complement system results in vascular smooth muscle dilation and the release of a cascade of inflammatory mediators, including histamine, slow-reacting substance of anaphylaxis, serotonin, heparin, acetylcholine, and bradykinin. clinical signs associated with anaphylaxis differ between dogs and cats. in dogs, clinical signs may include restlessness, vomiting, diarrhea, hematochezia, circulatory collapse, coma, and death. in cats, clinical signs often are associated with respiratory system abnormalities. clinical signs may include ptyalism, pruritus, vomiting, incoordination, bronchoconstriction, pulmonary edema and hemorrhage, laryngeal edema, collapse, and death. the most important steps to remember in any emergency is to follow the abcs of airway, breathing, and circulation. first, establish an airway through endotracheal intubation or emergency tracheostomy, if necessary. concurrently, an assistant should establish vascular or intraosseous access to administer drugs and fluids (box - ). the patient should be hospitalized until complete resolution of clinical signs. after initial stabilization and treatment, it is important to maintain vascular access and continue intravenous fluid therapy until the patient is no longer hypotensive, and vomiting and diarrhea have resolved. in cases of fulminant pulmonary hemorrhage and edema, administer supplemental oxygen until the patient is no longer hypoxemic or orthopneic on room air. normalize and maintain blood pressure using positive inotropes (dobutamine, - µg/kg/ minute cri) or pressors (dopamine, to µg/kg/minute iv cri; see shock). if bloodtinged vomitus or diarrhea has been observed, administer antibiotics to decrease the risk of bacterial translocation and sepsis (cefoxitin, mg/kg iv tid; metronidazole, mg/kg iv tid). also consider using gastroprotectant drugs (famotidine, . to . mg/kg iv; ranitidine, . to . mg/kg po, iv, im bid; sucralfate, . to . g po tid; omeprazole, . to . mg/kg po sid). a second and less serious form of allergic reaction is manifested as angioneurotic edema and urticaria. in most cases, clinical signs develop within minutes of an inciting allergen. although this type of reaction causes patient discomfort, it rarely poses a life-threatening problem. most animals have mild to severe swelling of the maxilla and periorbital regions. the facial edema also may be accompanied by mild to severe generalized urticaria. some animals may paw at their face, rub at their eyes, or have vomiting or diarrhea. the treatment for angioneurotic edema involves suppressing the immune response by administration of short-acting glucocorticoid drugs and blocking the actions of histamine by the synergistic use of histamine and histamine receptor blockers (box - ). in some cases, the inciting cause is a known recent vaccination or insect sting. many times, however, the inciting cause is not known and is likely an exposure to a stinging insect or arachnid. differential diagnoses for acute facial swelling and/or urticaria include acetaminophen toxicity (cats), anterior caval syndrome, lymphadenitis, vasculitis, hypoalbuminemia, and contact dermatitis. observe animals that have presented for angioneurotic edema for a minimum of to minutes after injection of the short-acting glucocorticoids and antihistamines. monitor blood pressure to make sure that the patient does not have concurrent anaphylaxis and hypotension. after partial or complete resolution of clinical signs, the animal can be discharged to its owner for observation. in dogs, mild vomiting or diarrhea may occur within to days after this type of reaction. wherever possible, exposure to the inciting allergen should be avoided. • administer short-acting glucocorticoid: complications observed while a patient is under anesthesia can be divided into two broad categories: ( ) those related to equipment malfunction or human error and ( ) the patient's physiologic response to the cardiorespiratory effects of the anesthetic drugs. careful observation of the patient and familiarity with anesthetic equipment, drug protocols, and monitoring equipment is necessary for the safest anesthesia to occur. despite this, however, anesthetic-related complications are frequent and need to be recognized and treated appropriately. many anesthetic drugs have a dose-dependent depressive effect on the respiratory system and cause a decrease in respiratory rate and tidal volume, leading to hypoventilation. respiratory rate alone is not a reliable indicator of the patient's oxygenation and ventilatory status. the respiratory tidal volume can be measured with a wright's respirometer. perform pulse oximetry and capnography as noninvasive measures of the patient's oxygenation and ventilation. ventilation can be impaired as a result of anesthetic drugs, patient position, pneumothorax, pleural effusion (chylothorax, hemothorax, pyothorax), equipment malfunction, rebreathing of carbon dioxide, thoracic wall injury, or alveolar fluid (pulmonary edema, hemorrhage, or pneumonia). problems such as a diaphragmatic hernia, gdv, or gravid uterus can impede diaphragmatic excursions once the patient is placed on its back and can lead to impaired ventilation. the work of breathing also may be increased because of increased resistance of the anesthesia circuit and increased dead space ventilation. this is particularly important in small toy breeds. clinical signs of inadequate ventilation and respiratory complications include abnormal respiratory pattern, sudden changes in heart rate, cardiac dysrhythmias, cyanosis, and cardiopulmonary arrest. end-tidal carbon dioxide, or capnography, gives a graphic display of adequacy of ventilation. rapid decreases in end-tidal carbon dioxide can be caused by disconnection or obstruction of the patient's endotracheal tube or poor perfusion, namely, cardiopulmonary arrest (see capnometry [end-tidal carbon dioxide monitoring]). postoperatively, hypoventilation can occur because of the residual effects of the anesthetic drugs, hypothermia, overventilation during intraoperative support, surgical techniques that compromise ventilation (thoracotomy, cervical disk surgery, atlantooccipital stabilization), postoperative bandaging of the abdomen or thorax, ventilatory muscle fatigue, or injury to the cns. cardiac output is a function of heart rate and stroke volume. factors that influence stroke volume include vascular and cardiac preload, cardiac afterload, and cardiac contractility. the patient's cardiac output can be affected adversely by the negative inotropic and chronotropic and vasodilatory effects of anesthetic drugs, all leading to hypotension. emergency care bradycardia, tachycardia, cardiac dysrhythmias, and vascular dilation can lead to hypotension and inadequate organ perfusion. table - lists the normal heart rate and blood pressure in dogs and cats. bradycardia is defined as a heart rate below normal values. many anesthetic drugs can cause bradycardia. causes of bradycardia include the use of narcotics or α -agonist drugs, deep plane of anesthesia, increased vagal tone, hypothermia, and hypoxia. table - lists the causes of bradycardia and the necessary immediate action or treatment. tachycardia is defined as a heart rate above normal values. common causes of tachycardia include vasodilation, drugs, inadequate anesthetic depth and perceived pain, hypercapnia, hypoxemia, hypotension, shock, or hyperthermia. table - lists the causes and immediate action or treatment for tachycardia. hypotension is defined as physiologically low blood pressure (mean arterial pressure less than mm hg). a mean arterial blood pressure less than mm hg can result in inadequate tissue perfusion and oxygen delivery. the coronary arteries are perfused during diastole. inadequate diastolic blood pressure, less than mm hg, can cause decreased coronary artery perfusion and myocardial hypoxemia that can predispose the heart to dysrhythmias. causes of perianesthetic hypotension include peripheral vasodilation by anesthetic drugs, bradycardia or tachyarrhythmias, hypothermia, inadequate cardiac preload from vasodilation or hemorrhage, decreased venous return from patient position or surgical manipulation of viscera, and decreased cardiac contractility. electrocardiogram monitoring is useful for the early detection of cardiac dysrhythmias during the perianesthetic period. clinical signs of cardiac dysrhythmias include irregular pulse rate or pressure, abnormal or irregular heart sounds, pallor, cyanosis, hypotension, and an abnormal ecg tracing. remember that the single best method of detecting cardiac emergency care vagolytic drugs atropine allow time for the drug to wear off. glycopyrrolate allow time for the drug to wear off. sympathomimetic drugs epinephrine allow time for the drug to wear off; administer a β-blocker; turn off infusion. isoproterenol administer a β-blocker. turn off infusion; administer a β-blocker. allow time for drug to wear off. inadequate anesthetic depth increase anesthetic depth. hypercapnia increase ventilation (assisted ventilation). hypoxemia increase gas flow and oxygenation. hypotension decrease anesthetic depth; administer an intravenous crystalloid or colloid bolus, positive inotrope drug, positive chronotrope drug, or pressor. hyperthermia apply ambient or active cooling measures; administer dantrolene sodium if malignant hyperthermia is suspected. hypothermia provide ambient rewarming. hypocalcemia * administer calcium chloride ( mg/kg iv) or calcium gluconate ( mg/kg). decrease vaporizer setting/anesthetic depth. reverse with opioids or a -agonists. vasodilation administer an intravenous crystalloid bolus ( ml/kg). administer an intravenous colloid bolus ( ml/kg). administer a pressor (epinephrine, phenylephrine dysrhythmias is with your fingertips (palpate a pulse or apex heartbeat) and ears (auscultate the heart). confirm the dysrhythmia by auscultating the heart rate and rhythm, identify the p waves and the qrs complexes, and evaluate the relationship between the p waves and qrs complexes. is there a p wave for every qrs, and a qrs for every p wave? during anesthesia, fluid, acid-base, and electrolyte imbalances can predispose the patient to dysrhythmias. sympathetic and parasympathetic stimulation, including the time of intubation, can predispose the patient to dysrhythmias. if the patient's plane of anesthesia is too light, perception of pain can cause catecholamine release, sensitizing the myocardium to ectopic beats. atrioventricular blockade can be induced with the administration of α -agonist medications, including xylazine and medetomidine. thiobarbiturates (thiopental) can induce ventricular ectopy and bigeminy. although these dysrhythmias may not be harmful in the awake patient, anesthetized patients are at a particular risk of dysrhythmia-induced hypotension. carefully monitor and treat all dysrhythmias (see cardiac dysrhythmias). box - lists steps to take to prevent perianesthetic dysrhythmias. awakening during anesthesia can occur and can be caused by equipment failure and simply, although no one likes to admit it, human error. table - lists causes of arousal during anesthesia and appropriate immediate actions. awaken patient, and administer dantrolene arousal (e.g., malignant hyperthermia) sodium. • stabilize acid-base and electrolyte balance before anesthetic induction, whenever possible. • rehydrate patient before anesthetic induction. • select anesthetic agents appropriate for the particular patient. • be aware of the effects of the drugs on the myocardium. • ensure adequate anesthetic depth and oxygenation before anesthetic induction. • ensure ventilatory support during anesthesia. • monitor heart rate, rhythm, blood pressure, pulse oximetry, and capnometry during anesthesia. • ensure adequate anesthetic depth before surgical stimulation. • avoid surgical manipulation to the heart or great vessels, whenever possible. • avoid changes in perianesthetic depth. • avoid hypothermia. delayed recovery can be caused by a number of factors, including excessive anesthetic depth, hypothermia, residual action of narcotics or tranquilizers, delayed metabolism of anesthetic drugs, hypoglycemia, hypocalcemia, hemorrhage, and breed or animal predisposition. careful monitoring of the patient's blood pressure, acid-base and electrolyte status, anesthetic depth, pcv, and vascular volume intraoperatively and taking care with supportive measures to prevent abnormalities can hasten anesthetic recovery and avoid postoperative complications. gaynor the presentation of a patient with a bleeding disorder often is a diagnostic challenge for the veterinary practitioner (boxes - and - ). in general, abnormal bleeding can be caused by five major categories: ( ) vascular trauma, ( ) circulating inhibitors of coagulation heparin fibrin degradation products development of spontaneous deep hematomas, unusually prolonged bleeding after traumatic injury, bleeding at multiple sites throughout the body involving multiple organ systems, delayed onset of severe hemorrhage after bleeding, and an inability on the practitioner's part to find an organic cause of bleeding. the signalment, history, clinical signs, and results of coagulation often can aid in making a rapid diagnosis of the primary cause of the disorder and in the selection of appropriate case management. when taking a history, ask the following important questions: • what is the nature of the bleeding? • what sites are affected? • how long has the bleeding been going on? • has your animal had any previous or similar episodes? • is there any possibility of any toxin exposure? • if so, when and how much did your animal consume? • is there any possibility of trauma? • does your animal run loose outdoors unattended? • have you ever traveled, and if so, where? • has your animal been on any medications recently or currently? • has your animal been vaccinated recently? • have any known relatives of your animal had any bleeding disorders? • are there any other abnormal signs that you have seen? abnormalities found on physical examination may aid in determining whether the hemorrhage is localized or generalized (i.e., bleeding from a venipuncture site versus bleeding diathesis). note whether the clinical signs are associated with a platelet problem and superficial hemorrhage or whether deep bleeding can be associated with abnormalities of the coagulation cascade. also, make an attempt to identify any concurrent illness that can predispose the patient to a bleeding disorder (i.e., pancreatitis, snakebite, sepsis, immunemediated hemolytic anemia, or severe trauma and crush or burn injury). abnormalities associated with coagulopathies include petechiae and ecchymoses, epistaxis, gingival bleeding, hematuria, hemarthrosis, melena, and hemorrhagic cavity (pleural and peritoneal or retroperitoneal) effusions. disseminated intravascular coagulation is a complex syndrome that results from the inappropriate activation of the clotting cascade, leading to disruption of the normal balance between thrombosis and fibrinolysis. the formation of diffuse microthrombi with concurrent consumption of platelets and activated clotting factors leads to end-organ thrombosis with various degrees of clinical hemorrhage. in animals, dic always results from some other pathologic process, including various forms of neoplasia, crush and heat-induced injury, sepsis, inflammation, and immune-mediated disorders (box - ). the pathophysiologic mechanisms involved in dic include vascular endothelial damage, activation and consumption of platelets, release of tissue procoagulants, and consumption of endogenous anticoagulants. because dic always results from some other disease process, diagnosis of dic is based on a number of criteria when evaluating various coagulation tests, peripheral blood smears, platelet count, and end products of thrombosis and fibrinolysis. there is no one definitive criterion for the diagnosis of dic (box - ). thrombocytopenia occurs as platelets are consumed during thrombosis. it is important to remember that trends in decline in platelet numbers are just as important as thrombocytopenia when making the diagnosis. in some cases the platelet count still may be within the normal reference range but has significantly decreased in the last hours. early in dic the procoagulant cascade dominates, with hypercoagulability. activated clotting time, aptt, and pt may be rapid and shorter than normal. in most cases, we do not recognize the hypercoagulable state in our critically ill patients. later in dic, as platelets and activated clotting factors become consumed, the act, aptt, and pt become prolonged. antithrombin, a natural anticoagulant, also becomes consumed, and antithrombin levels decline. antithrombin levels can be measured at commercial laboratories and in some large veterinary institutions. the end products of thrombosis and subsequent fibrinolysis also can be measured. fibrinogen levels may decline, although this test is not sensitive or specific for dic. fibrin degradation (split) products also become elevated. fibrin degradation products are normally cleared by the liver, and these also become elevated in cases of hepatic failure because of lack of clearance. more recently, cageside d-dimer tests have become available to measure the breakdown product of cross-linked fibrin as a more sensitive and specific monitor of dic. management of dic first involves treating the primary underlying cause. by the time dic becomes evident, rapid and aggressive treatment is necessary. if you are suspicious of dic in any patient with a disease known to incite dic, then ideally, you should begin treatment before the hemostatic abnormalities start to occur for the best possible prognosis. treatment involves replacement of clotting factors and antithrombin and prevention of further clot formation. to replenish clotting factors and antithrombin, administer fresh whole blood or fresh frozen plasma. heparin requires antithrombin as a cofactor to inactivate thrombin and other activated coagulation factors. administer heparin ( to units/kg sq q - h of unfractionated heparin; or fractionated enoxaparin [lovenox], mg/kg sq bid). aspirin ( mg/kg po bid in dogs; every third day in cats) also can be administered to prevent platelet adhesion. management of dic also involves the rule of twenty monitoring and case management to maintain end-organ perfusion and oxygen delivery (see the rule of ). hemophilia a is a sex-liked recessive trait that is carried by females and manifested in males. female hemophiliacs can occur when a hemophiliac male is bred with a carrier female. hemophilia a has been reported in cats and a number of dog breeds, including miniature schnauzer, saint bernard, miniature poodle, shetland sheepdog, english and irish setters, labrador retriever, german shepherd, collie, weimaraner, greyhound, chihuahua, english bulldog, samoyed, and vizsla. mild to moderate internal or external bleeding can occur. clinical signs of umbilical cord bleeding can become apparent in some animals shortly after weaning. gingival hemorrhage, hemarthrosis, gastrointestinal hemorrhage, and hematomas may occur. clotting profiles in animals with factor viii deficiency include prolonged aptt and act. the pt and buccal mucosa bleeding time are normal. affected animals have low factor viii activity but normal to high levels of factor viii-related antigen. carrier females can be detected by low ( % to % of normal) factor viii activity and normal to elevated levels of factor vii-related antigen. von willebrand's disease is a deficiency or defect in von willebrand's protein. a number of variants of the disease have been described: von willebrand's disease type i is associated with a defect in factor viir/protein concentration, and von willebrand's disease type ii is associated with a defect in viiir:vwf. type i von willebrand's disease is most common in veterinary medicine. von willebrand's disease has been identified in more than breeds of dogs, with an incidence that varies from % to % depending on the breed of origin. affected breeds include doberman pinchers, german shepherd dogs, scottish terriers and standard manchester terriers, golden retrievers, chesapeake bay retrievers, miniature schnauzers, and pembroke welsh corgis. two forms of genetic expression occur: ( ) autosomal recessive disease in which homozygous von willebrand's disease individuals have a bleeding disorder, whereas heterozygous individuals carry the trait but are clinically normal. the second variant of genetic expression involves an autosomal dominant disease with incomplete expression such that heterozygous individuals are affected carriers and homozygous individuals are severely affected. von willebrand's disease has high morbidity, but fortunately a low mortality. dogs with % or less than normal vwf tend to hemorrhage. platelet counts are normal, but bleeding times can be prolonged. the aptt can be slightly prolonged when factor viii is less than % of normal. routine screening tests are nondiagnostic for this disease, although in a predisposed breed with a normal platelet count, a prolonged buccal mucosa bleeding time strongly supports a diagnosis of von willebrand's disease. documentation of clinical bleeding with low or undetectable levels of factor viii antigen or platelet-related activities of vwf support a diagnosis of von willebrand's disease. recessive animals have zero vwf:antigen (a subunit of factor iii); heterozygotes have % to % of normal. in the incompletely dominant form, levels of vwf antigen are reduced (less than % to %). clinical signs in affected animals include epistaxis, hematuria, diarrhea with melena, penile bleeding, lameness, hemarthrosis, hematoma formation, and excessive bleeding with routine procedures such as nail trimming, ear cropping, tail docking, surgical procedures (spay, neuter), and lacerations. estrous and postpartum bleeding may be prolonged. a dna test to detect carriers of the vwf gene is available through vetgen (ann arbor, michigan) and michigan state university. patients with von willebrand's disease should avoid drugs known to affect platelet function adversely (sulfonamide, ampicillin, chloramphenicol, antihistamines, theophylline, phenothiazine tranquilizers, heparin, and estrogen). hemophilia b is an x-linked recessive trait that occurs with less frequency that hemophilia a. the disease has been reported in scottish terriers, shetland and old english sheepdogs, saint bernards, cocker spaniels, alaskan malamutes, labrador retrievers, bichon frises, airdale terriers, and british shorthair cats. carrier females have low ( % to % of normal) factor ix activity. clinical signs are more severe than for hemophilia a. congenital deficiencies of factor vii have been reported as an autosomal, incompletely dominant characteristic in beagles. heterozygotes have % factor vii deficiency. bleeding tends to be mild. the pt is prolonged in affected individuals. factor x deficiency has been documented in cocker spaniels and resembles fading-puppy syndrome in newborn dogs. internal or umbilical bleeding can occur, and affected dogs typically die. bleeding may be mild in adult dogs. in severe cases, factor x levels are reduced to % of normal; in mild cases, factor x levels are % to % of normal. factor xii deficiency has been documented as an inherited autosomal recessive trait in domestic cats. heterozygotes can be detected because they have a partial deficiency ( % of normal) of factor xii. homozygote cats have less than % factor xii activity. deficiency of hageman factor usually does not result in bleeding or other disorders. factor xi deficiency is an autosomal disease that has been documented in kerry blue terriers, great pyrenees, and english springer spaniels. in affected individuals, protracted bleeding may be observed. homozygotes have low factor xi activity (< % of normal), and heterozygotes have % to % of normal. the management of congenital defects of hemostasis typically involves replenishing the clotting factor that is present. usually, this can be accomplished in the form of fresh frozen plasma transfusion ( ml/kg). if anemia is present because of severe hemorrhage, fresh whole blood or packed rbcs also can be administered. recent research has investigated the use of recombinant gene therapy in the treatment of specific factor deficiencies in dogs; however, the therapy is not yet available for use in clinical practice. in cases of von willebrand's disease, administration of fresh frozen plasma ( to ml/kg) or cryoprecipitate ( unit/ kg body mass) provides vwf, factor viii, and fibrinogen. doses can be repeated until hemorrhage ceases. -desamino- -d-arginine vasopressin (ddavp) also can be administered ( µg/kg sc or iv diluted in . % saline given over to minutes) to the donor and patient to increase the release of stored vwf from endothelial cells. a fresh whole blood transfusion can be obtained from the donor and immediately administered to the patient, or spun down and the fresh plasma administered if rbcs are not needed. administer a dose of ddavp to any affected dog before initiating any elective surgical procedures. a supply of fresh frozen plasma and rbcs should be on hand, should uncontrolled hemorrhage occur. platelets are essential to normal blood coagulation. after a vessel is damaged, release of vasoactive amines causes vasoconstriction and sluggish flow of blood in an attempt to squelch hemorrhage. platelets become activated by platelet activating factor, and attach to the damaged vascular endothelium. normal platelet adhesion depends on mediators such as calcium, fibrinogen, vwf:antigen, and a portion of factor viii. after adhesion, the platelets undergo primary aggregation and release a variety of chemical mediators including adenosine diphosphate, prostaglandins, serotonin, epinephrine, thromboplastin, and thromboxane a that promote secondary aggregation and contraction. platelet abnormalities can include decreased platelet production (thrombocytopenia), decreased platelet function (thrombocytopathia), increased platelet destruction, increased platelet consumption, and platelet sequestration. thrombocytopathia refers to platelet function abnormalities. alterations in platelet function can affect platelet adhesion, aggregation, or release of vasoactive substances that help form a stable clot (box - ). in von willebrand's disease there is a deficiency in vwf:antigen that results in altered platelet adhesion. vascular purpuras are reported and have been seen in collagen abnormalities such as ehlers-danlos syndrome, which can be inherited as an autosomal dominant trait with complete penetrance and has been recognized in german shepherd dogs, dachshunds, saint bernards, and labrador retrievers. thrombasthenic thrombopathia is a hereditary autosomal dominant abnormality that has been described in otterhounds, foxhounds and scottish terriers. in this condition, platelets do not aggregate normally in response to adenosine diphosphate and thrombin stimulation. evaluation of platelet function is based on a total platelet count, buccal mucosa bleeding time, and thromboelastography. platelet function defects (thrombocytopenia and thrombocytopathia) can affect both sexes. clinical signs can resemble von willebrand's disease. in most cases, buccal mucosa bleeding time will be prolonged, but platelet count and clotting tests will be normal. platelet count can be decreased because of problems with production, increased consumption, sequestration, or destruction. causes of accelerated platelet destruction are typically immune-mediated autoantibodies, drug antibodies, infection, and isoimmune destruction. consumption and sequestration usually are caused by dic, vasculitis, microangiopathic hemolytic anemia, severe vascular injury, hemolytic uremic syndrome, and gram-negative septicemia. primary thrombocytopenia with no known cause has been called idiopathic thrombocytic purpura. in approximately % of the cases, thrombocytopenia is associated with immune-mediated destruction caused by immune-mediated hemolytic anemia, systemic lupus erythematosus, rheumatoid arthritis, dic, and diseases that affect the bone marrow. in systemic lupus erythematosus, % to % of the affected dogs have concurrent idiopathic thrombocytic purpura. when immune-mediated hemolytic anemia and idiopathic thrombocytic purpura are present in the same patient, the disease is called evans syndrome. pf- is a non-complement-fixing antibody that is produced in the spleen and affects peripheral and bone marrow platelets and megakaryocytes. antibodies directed against platelets are usually of the igg subtype in animals. antiplatelet antibodies can be measured by a pf- release test. platelet counts with immune-mediated destruction typically are less than , platelets/µl. infectious causes of thrombocytopenia include ehrlichia canis, anaplasma phagocytophilum (formerly, ehrlichia equi), and rickettsia rickettsii (rocky mountain spotted fever). primary immune-mediated thrombocytopenia has an unknown cause and most frequently is seen in middle-to older-aged female dogs. breed predispositions include cocker spaniels, german shepherd dogs, poodles (toy, miniature, standard), and old english sheepdogs. thrombocytopenia usually is manifested as petechiae, ecchymoses of skin and mucous membranes, hyphema, gingival and conjunctival bleeding, hematuria, melena, and epistaxis. to make a diagnosis of idiopathic thrombocytic purpura, measure the severity of thrombocytopenia (< , platelets/µl), analyze the peripheral blood smear for evidence of platelet fragmentation or microthrombocytosis, normal to increased numbers of megakaryocytes in the bone marrow, detection of antiplatelet antibody, increased platelet counts after starting glucocorticoid therapy, and elimination of other causes of thrombocytopenia. if tick-borne illnesses are suspected, antibody titers for e. canis, a. phagocytophilum (formerly e. equi), and r. rickettsii should be performed. treatment of immune-mediated thrombocytopenia involves suppression of the immune system to stop the immune-mediated destruction and to stimulate platelet release from the bone marrow. traditionally, the gold standard to suppress the immune system is to use glucocorticoids (prednisone or prednisolone, to mg/kg po bid divided, or dexamethasone, . to . mg/kg iv or po q h). more recently human serum immunoglobulin (igg) also has been used ( . to . g/kg iv in saline over hours; pretreat with mg/kg diphenhydramine minutes before starting infusion). vincristine ( . mg/m iv once) can stimulate the release of platelets from the bone marrow if megakaryocytic precursors are present; however, the platelets released may be immature and potentially nonfunctional. treatment with fresh whole blood or packed rbcs is appropriate if anemia is present; however, unless specific platelet-rich plasma has been purchased from a blood bank, fresh whole blood contains relatively few platelets, which are shortlived ( hours) and will not effectively raise the platelet count at all. finally, long-term therapy is usually in the form of azathioprine ( mg/kg po once daily, tapered to mg/kg daily to every other day after week) and cyclosporine ( to mg/kg po divided). if a tickborne illness is suspected, administer doxycycline ( to mg/kg po bid) for weeks or if titers come back negative. thrombocytopenia also can occur in the cat. causes for thrombocytopenia in cats include infections ( %), neoplasia ( %), cardiac disease ( %), primary immune-mediated disease ( %), and unknown causes ( %). in one study of cats with feline leukemia and myeloproliferative disease, % of cases had thrombocytopenia. warfarin and coumarin derivatives are the major class of rodenticides used in the united states. vitamin k antagonist rodenticides inhibit the epoxidase reaction and deplete active vitamin k, causing a depletion of vitamin k-dependent coagulation factors (ii, vii, ix, x) within hours to week of ingestion, depending on the ingested dose. affected animals can spontaneously hemorrhage anywhere in the body. clinical signs can include hemoptysis, respiratory difficulty, cough, gingival bleeding, epistaxis, hematuria, hyphema, conjunctival bleeding, petechiae and ecchymoses, cavity hemorrhage (pleural, peritoneal, retroperitoneal) with acute weakness, lethargy or collapse, hemarthrosis with lameness, deep muscle bleeds, and intracranial or spinal cord hemorrhage. diagnosis of vitamin k antagonism includes prolonged pt. a pivka (protein induced by vitamin k absence or antagonism) test also can be performed, if possible. treatment of vitamin k antagonist rodenticide intoxication and other causes of vitamin k deficiency involves supplementation with vitamin k (phytonadione, mg/kg sq once with -gauge needle in multiple sites, and then . mg/kg po bid to tid for days). never administer injections of vitamin k intramuscularly, because of the risk of causing deep muscle hematomas, or intravenously, because of the risk of anaphylaxis. the pt should be rechecked days after the last vitamin k capsule is administered, for some of the secondgeneration warfarin derivates are fat-soluble, and treatment may be required for an additional weeks. act, activated clotting time; aptt, activated partial thromboplastin time; bmbt, buccal mucosa bleeding time; fdp, fibrin degradation products; n, normal; pt, prothrombin time. thermal burns are fortunately a relatively infrequent occurrence in veterinary patients. box - lists various causes of malicious and accidental burns. the location of the burn is also important in assessing its severity and potential to lose function. burns on the perineum, feet, face, and ears are considered to be the most severe because of loss of function and severe pain. often the severity of thermal injury is difficult to assess in animals because hair coat potentially can mask clinical signs and because the thermal injury can continue after the animal has been removed from the heat source. the skin cools slowly and warms slowly, considerations that become important when initiating therapy for burns. the severity of thermal injury is associated with the temperature to which the animal is exposed, the duration of contact, and the ability of the tissue to dissipate heat. the tissue closest to the heat source undergoes necrosis and has decreased blood flow. the severity of thermal burn injury is associated directly with the temperature to which the animal is exposed, the percentage of total body surface area affected, the thickness of injured tissue, and whether underlying complications with other body systems occur. prognosis largely depends on the total body surface area affected (table - ) . superficial partial thickness, or first-degree, burns offer the most favorable prognosis. the affected epidermis initially appears erythematous and then quickly desquamates within to days. in most cases, fur grows back without leaving a scar. deep partial thickness, or second-degree, burns involve the epidermis and dermis and are associated with subcutaneous edema, inflammation, and pain. deep partial thickness burns heal from deeper adnexal tissues and from the wound edges and are associated with an increased chance of scarring and depigmentation. the most severe type is known as full thickness, or third-degree, burns, in which thermal injury destroys the entire thickness of the skin and forms an eschar. thrombosis of superficial and deeper skin vasculature and gangrene occurs. treatment involves sequential wound debridement. healing occurs by second intention and reepithelialization or by wound reconstruction. in most cases, scarring is extensive in affected areas. burns greater than % of total body surface area will have systemic effects, including impaired cardiovascular function, pulmonary dysfunction, and impaired immune function. burned tissue, with capillary damage, has increased permeability. the release of inflammatory cytokines, oxygen-derived free radical species, prostaglandins, leukotrienes, emergency care histamine, serotonin, and kinins results in increased vascular permeability and leakage of plasma proteins into the interstitium and extravascular space. at the time of presentation, first examine the patient and ascertain whether airway obstruction, impaired ventilatory function, circulatory shock, or pain are present. if necessary, establish an airway with endotracheal intubation or emergency tracheostomy. next, cool the burned area(s) with topical cool water. use care to avoid overcooling and iatrogenic hypothermia. the best approach is to cool only one portion of the patient's body at a time, then dry, and repeat the process for all affected areas to avoid overcooling and iatrogenic hypothermia. establish vascular access and administer appropriate and judicious analgesic drugs and intravenous fluid therapy. whenever possible, avoid placing a catheter through an area of burned or damaged skin. in the early stages of burn injury, shock doses of intravenous crystalloid fluids usually are not required. later, however, as severe tissue exudation occurs, protein and fluid losses can become extensive, requiring aggressive crystalloid and colloid support to treat hypovolemia and hypoproteinemia. flush the eyes with sterile saline and examine behind the third eyelids for any particulate matter. stain the corneas to make sure that superficial corneal burns are not present. treat superficial corneal burns with triple antibiotic ophthalmic ointment. next, assess the total body surface area affected, as this will gauge prognosis. depending on the extent of the damage, decide whether the burn is superficial and local therapy is indicated or whether more severe injuries exist that may involve systemic therapy or possibly euthanasia. in most cases the diagnoses of thermal burns are based on a clinical history of being in a house fire, clothes dryer, or under a heating lamp. too frequently, however, thermal burns become apparent days after an elective surgical procedure in which the patient was placed on a faulty heating pad rather than a circulating warm water or warm air blanket. superficial burns appear as singed fur with desquamating, easily epilated hair. this condition also can resemble a superficial or deeper dermatophytosis if history is unknown. other differential diagnoses include immune-mediated vasculitis or erythema multiforme. unless the superficial dermis is blistered, it may be difficult to distinguish between a thermal burn, chemical burn, or electrical burn if the trauma went unnoticed. management of burn injury largely depends on the depth of injury and the total body surface area affected. partial thickness burns and those affecting less than % of the total body surface area will require support in the form of antibiotic ointment and systemic analgesic drugs. burns affecting greater than % of total body surface area or deep thickness burns require more aggressive therapy. central venous catheters can be placed to administer crystalloid and colloid fluids, parenteral nutrition if necessary, antibiotics, and analgesic drugs. monitor perfusion parameters closely, including heart rate, blood pressure, capillary refill time, and urine output. respiratory function can be impaired because of concurrent smoke inhalation, thermal damage to the upper airways and alveoli, and carboxyhemoglobin or methemoglobin intoxication. respiratory function also can be impaired because of burn injury to the skin around the thoracic cage. thoracic radiographs may reveal patchy interstitial to alveolar infiltrates associated with pulmonary edema, pneumonia, and atelectasis. bronchoscopy often reveals edema, inflammation, particulate matter, and ulceration of the tracheobronchial tree. in some cases, upper airway inflammation is so severe that an emergency tracheostomy must be performed to treat airway obstruction. administer supplemental humidified oxygen at to ml/kg/minute via endotracheal tube, tracheostomy, nasal or intratracheal tube, or hood oxygen if respiratory function and hypoxemia are present. perform blood work including a hematocrit, albumin, bun, creatinine, and glucose at the time of presentation. monitor serum electrolytes, albumin, and colloid oncotic pressure closely because derangements can be severe as burns become exudative. the goal of fluid therapy in the burn patient is to establish and maintain intravascular and interstitial fluid volume, normalize electrolyte and acid-base status, and maintain serum albumin and oncotic pressure. in the first hours following burn injury, direct fluid therapy to maintaining the patient's metabolic fluid requirements. crystalloid fluids in the form of normosol-r, plasmalyte-m, or lactated ringer's solution can be administered according to the patient's electrolyte and acid-base status (see fluid therapy). monitor urine output, and keep it at to ml/kg/hour. avoid overhydration in the early stages of burn injury. in affected burn patients, calculate the amount of fluid that should be administered over a -hour period from the formula − ml/kg × percent total body surface area. administer half of this calculated dose over the first hours and then the remaining half over the next hours. in cats, administer only % to % of this calculated volume. to administer this volume and also avoid fluid overload is often difficult in critically ill patients with pulmonary involvement associated with smoke inhalation injury. avoid colloids in the first hours after burn injury. monitor the patient closely for serous nasal discharge, chemosis, and rales that may signify pulmonary edema. as burns become exudative, weigh the patient at least twice daily. infused fluid should equal fluid output in the form of urine and wound exudates. acute weight loss signifies acute fluid loss and that crystalloid fluid infusion should be more aggressive. ideally, keep the patient's serum albumin equal to or greater than . g/dl and total protein between . and . g/dl using a combination of fresh frozen plasma or concentrated human albumin. adjunct colloidal support can be provided with synthetic colloids including hetastarch or hbocs. keep serum potassium within . to . meq/l using potassium chloride or potassium phosphate supplementation. if potassium supplementation exceeds to meq/l and the patient continues to have severe refractory hypokalemia, administer magnesium chloride ( . meq/kg/day) to enhance potassium retention. if anemia occurs, administer packed rbcs or whole blood (see blood component therapy). lavage wounds daily with lactated ringer's solution or . % sodium chloride solution. place wet-to-dry bandages or bandages soaked in silver sulfadiazine or nitrofurazone ointment over the wounds. depending on the thickness of the burn, epilation and eschar formation and separation may take to days. at each bandage change, debride devitalized tissue to normal tissue. perform staged partial or total escharectomy, and leave the wound to heal by second intention or by reconstruction using skin advancement flaps or grafts. maintain meticulous sterility at all times, given that burn patients are at high risk for infection. administer broad-spectrum antibiotics including cefazolin and enrofloxacin. perform wound culture if a resistant bacterial infection is suspected. the most common cause of electrical injury is associated with an animal chewing on low-voltage alternating current electrical cords in the household. damage is caused by the current flowing through the path of least resistance, causing heat and thrombosis of vessels and neurons. in some cases, the owner witnesses the event. in other cases, the owner presents the patient because of vague nonspecific signs, and characteristic abnormalities on physical examination support a diagnosis of electrocution. burns on the face, paws, commissures of the mouth, tongue, and soft palate may be present. electrocution causes a massive release of catecholamines and can predispose the patient to noncardiogenic pulmonary edema within hours of the incident. clinical signs may be isolated to the pulmonary system, including orthopnea, pulmonary crackles, and cyanosis. assess the patient's lips, tongue, soft palate, gingivae, and commissures of the mouth. early after electrocution, the wound may appear small and white, black, or yellow. later, the wound may become larger as tissue sloughs because of damaged vascular supply. assess the patient's respiratory status. auscultate the lungs to determine whether pulmonary crackles emergency care are present. if the patient is stable, thoracic radiographs may demonstrate an interstitial to alveolar lung pattern in the dorsocaudal lung fields. measure the patient's heart rate, blood pressure, oxygenation as determined by pulse oximetry or arterial blood gas and urine output. immediate treatment consists of judicious use of analgesics for the burn injury, antibiotics (cefazolin, mg/kg q h; cephalexin, mg/kg q h), and humidified supplemental oxygen ( to ml/kg/minute). direct fluid therapy at providing the patient's metabolic fluid requirements. because of the risk of development of noncardiogenic pulmonary edema, avoid overzealous administration of crystalloid fluids. differential diagnoses for the patient with electrical burn injury and electrocution include chemical or thermal burn, immune-mediated glossitis, cardiogenic pulmonary edema, and pneumonia. management of the patient with electrical burn injury and electrocution primarily involves the administration of analgesic agents, supplemental humidified oxygen, and topical treatment of electrical burns. the noncardiogenic pulmonary edema is typically unresponsive to diuretics (i.e., furosemide), bronchodilators (i.e., aminophylline), and splanchnic vascular dilators (i.e., low-dose morphine). the use of glucocorticoids has no proven benefit and may impair respiratory immune function and is therefore contraindicated. oral burns may require debridement and advancement flaps if large defects or oronasal fistulas develop. if oral injury is severe, place an esophagostomy or percutaneous gastrostomy tube to ensure adequate nutrition during the healing process. if an animal survives the initial electrocution, prognosis is generally favorable with aggressive supportive care. chemical burns are associated with a number of inciting causes, including oxidizing agents, reducing agents, corrosive chemicals, protoplasmic poisons, desiccants, and vesicants. the treatment for chemical burns differs slightly from that for thermal burns, so it remains important to investigate the cause of the burn when providing initial treatment, whenever possible. at the scene, advise the owner to wrap the patient in a clean towel for transport. chilling can be avoided by then wrapping the patient in a second or third blanket. placement of ointments by well-doers should be avoided. encourage immediate transport to the nearest triage facility. the first and foremost consideration when treating a patient with chemical burn is to remove the animal from the inciting cause or offending agent. make no attempt to neutralize alkaline or acid substances because the procedure potentially could cause an exothermic reaction, leading to thermal injury in addition to the chemical injury. remove collars or leashes that may act as tourniquets or constricting devices. flush affected areas with copious amounts of cool water for several minutes, not cooling more than % to % of the body at any one time to prevent iatrogenic hypothermia. support breathing by extending the patient's head and neck. carefully clip the fur over affected areas for further evaluation of the extent of the injury. lavage exposed eyes with sterile saline, and stain the cornea to evaluate for any corneal burns. debride any wounds carefully, knowing that the full extent of the wound may not manifest itself for several days. then cover the wounds with antibiotic burn ointment such as silver sulfadiazine and an occlusive dressing. without a history of exposure, the differential diagnosis for any chemical burn includes thermal burn, necrotizing vasculitis, erythema multiforme, or superficial or deep pyoderma. contact local or national animal poison control regarding whether to attempt neutralization. perform daily bandage changes with staged debridement as the full extent of the wound manifests itself. place antimicrobial ointment and silver sulfadiazine ointment over the wound to prevent infection. the routine use of antibiotics may promote the development of a resistant bacterial infection. first-generation cephalosporin can be administered. if a more serious infection develops, perform culture and susceptibility testing to direct appropriate antibiotic therapy. the wound can heal by second intention or may require reconstructive repair for definitive closure. the primary cause of radiation injury in small animal patients is radiation therapy for neoplastic conditions. the goal of radiation therapy is to kill neoplastic cells. an unfortunate side effect is damage to adjacent normal tissue that results in necrosis, fibrosis, and impaired circulation to the affected area. radiation burns result in dermatitis, mucositis, impaired surgical wound healing, and chronic nonhealing wounds. in many cases, the degree of secondary radiation injury to normal tissue can be prevented or decreased with careful radiation planning and mapping of the radiation field, such that radiation exposure to normal tissue is limited to the smallest extent possible. with the advent of three-dimensional imaging modalities such as computed tomography (ct) and magnetic resonance imaging (mri), this has become more routine in veterinary oncology to date. radiation injury can be early and appear at the later stage of the course of radiation therapy. late effects can be delayed and occur months to years after treatment. the degree of radiation injury is categorized based on the depth of tissue affected. first-degree changes cause cutaneous erythema. second-degree changes cause superficial desquamation. thirddegree changes cause deeper moist desquamation, and fourth-degree changes are associated with complete dermal destruction and ulceration. during the early stages of radiation injury, affected tissues may appear erythematous and edematous. wound exudates may be moist, or the skin may appear dry and scaly with desquamation or ulceration. later, the area may scar and depigment or may have induration, atrophy, telangiectasia, keratosis, and decreased adnexal structures. treatment for radiation dermatitis is to irrigate the area with warmed saline and to protect the area from self-mutilation. no-bite, or elizabethan, collars or loose clothing can be used to protect the area for patient-induced injury. mucositis can be treated with topical green tea baths and the administration of an oral solution of l-glutamine powder ( g/m ). local irrigation of xylocaine or lidocaine viscous jelly can be used in dogs but should be avoided in cats because of the risk of inducing hemolytic anemia and neurotoxicity. topical and systemic antibiotics (cephalexin, mg/kg po tid) also can be administered. avoid antibiotics that can be sensitized by radiation (i.e., metronidazole). because most radiation burns are associated with a known exposure to radiation therapy, the cause of the patient's injury usually is known. if an animal presents to you with a scar, however, differential diagnoses may include nasal planum solar dermatitis, pemphigus foliaceus, discoid lupus, superficial necrolytic dermatitis, superficial or deep pyoderma, chemical burn, or thermal burn. treatment of radiation injury involves making the patient as comfortable as possible with analgesic drugs, prevention of self-mutilation, and staged debridement techniques. wounds can heal by second intention or may require reconstructive surgery. distress syndrome (ards), and anesthetic agents. the acute onset of bradycardia, change in mucous membrane color and capillary refill time, change in respiratory pattern, and change in mentation are signs of possible deterioration and impending cardiopulmonary arrest. the diagnosis of cardiopulmonary arrest is based on the absence of effective ventilation, severe cyanosis, absence of a palpable pulse or apex heartbeat, absence of heart sounds, and ecg evidence of asystole or other nonperfusing rhythm such as electricalmechanical dissociation (aka pulseless electrical activity) or ventricular fibrillation. the goals of cpcr are to obtain airway access, provide artificial ventilation and supplemental oxygen, implement cardiac compressions and cardiovascular support, recognize and treat dysrhythmias and arrhythmias, and provide stabilization and treatment for cardiovascular, pulmonary, and cerebral function in the event of a successful resuscitation. even with aggressive treatment and management, the overall success of cpcr is less than % in critically ill or traumatized patients and % to % in anesthetized patients. basic life support involves rapid intubation to gain airway access, artificial ventilation, and cardiac compressions to promote blood flow and delivery of oxygen to the brain and other important tissues (figure - ). perform the abcs or cabs of cpcr, where a is airway, b is breathing, and c is compression and circulation. recently, the paradigm has shifted to cabs. while a team member is grabbing an endotracheal tube, clearing the airway of foreign debris, and establishing airway access through endotracheal intubation, a second person starts external cardiac compressions to deliver oxygen that is in the bloodstream to the vital organs. the patient should be positioned in dorsal (> kg) or lateral (< kg) recumbency for external cardiac compressions. approximately to external compressions should be performed over the patient's sternum. a team member should palpate for a peripheral pulse to determine whether cardiac compressions are actually effective. if a peripheral pulse cannot be palpated for every chest compression, change the patient's position and have a larger individual perform compressions, or initiate open-chest cardiac resuscitation. once the patient is intubated, tie in the endotracheal tube and attach it to an oxygen source (anesthetic machine or mechanical ventilator or ambu bag) for artificial ventilation. the oxygen flow rate should be ml/kg/minute. give two long breaths, and then to breaths per minute. simultaneous ventilation with thoracic compression increases the pressure difference in the thorax and allows more forward flow of oxygenated blood through the great vessels into the periphery. if possible, a third team member can initiate interposed abdominal compressions, compressing the abdomen when the thoracic cage is relaxed, to improve forward flow. if only one person is available to perform the thoracic compressions and ventilation, give two breaths for every compressions (i.e., thoracic compressions followed by two long breaths, and then start thoracic compressions again). the jen chung maneuver can be performed by placing a -to -gauge hypodermic needle through the skin of the nasal philtrum and twisting the needle into the periosteum to stimulate respirations. this maneuver appears to work better in cats than dogs at return to spontaneous respiration. advanced life support during cpcr involves ecg, pulse oximetry and capnometry monitoring, administration of drugs, and the administration of intravenous fluids (in select cases). most of the drugs used during cpcr can be administered directly into the lungs from the endotracheal tube (intratracheal tube). therefore, only in select instances is it necessary to establish vascular or intraosseous access during cpcr (figure - ) . if an animal experiences cardiopulmonary arrest because of extreme hemorrhage or hypovolemia, inappropriate vasodilation caused by sepsis or systemic inflammation, or vasodilation resulting from anesthesia, the administration of shock volumes ( ml/kg/hour in dogs and ml/kg/hour in cats) is appropriate. if a patient is euvolemic and experiences cardiopulmonary arrest, however, an increase in circulating fluid volume actually can impair coronary artery perfusion by increasing diastolic arterial blood pressure and is asystole is one of the most common rhythm disturbances that causes cardiac arrest in small animal patients. one of the most important things to do when the ecg looks like asystole is to make sure that the ecg monitor is working properly and that all ecg leads are attached properly to the patient. if asystole is truly present, reverse any opiate, α -agonist, or benzodiazepine drugs with their appropriate reversal agents. lowdose epinephrine ( . to . mg/kg diluted with ml sterile saline) can be administered directly into the endotracheal tube via a rigid or red rubber catheter. if vascular access is available, epinephrine ( . to . mg/kg) can be administered intravenously. no drug should ever be administered directly into the heart by intracardiac injection. unless the heart is in the veterinarian's hand during open-chest cpcr, intracardiac injection is risky and potentially could lacerate a coronary artery or cause the myocardium to become more irritable and refractory to other therapies, if a drug is delivered into the myocardium and not into the ventricle. for these reasons, intracardiac injections are contraindicated. administer atropine ( . mg/kg iv, io, or . mg/kg it) immediately after the epinephrine. atropine, a vagolytic drug, serves to decrease tonic vagal inhibition of the sinoatrial and atrioventricular node and increase heart rate. administer atropine and epinephrine every to minutes during asystole while cardiac compressions, interposed abdominal compressions, and artificial ventilation are continued. although discontinuation of thoracic compressions can decrease the chance of success during cpcr, you must intermittently evaluate the ecg monitor for any rhythm change that may require different drug therapies. if the cardiac arrest was not witnessed or more than to minutes have passed without successful return to a perfusing rhythm, perform open-chest cpcr, if the client wishes. administer sodium bicarbonate ( to meq/kg iv) every to minutes during cpcr. sodium bicarbonate is the only drug used in cpcr that should not be administered intratracheally because of inactivation of pulmonary surfactant. electrical-mechanical dissociation also is known as pulseless electrical activity and is an electrical rhythm that may look wide and bizarre and irregular with no associated mechanical contraction of the ventricles. the rhythm can appear different from patient to patient. electrical-mechanical dissociation is one of the more common nonperfusing rhythms observed during cardiopulmonary arrest in small animal patients (figure - ) . when electrical-mechanical dissociation is identified, first confirm the rhythm and proceed with cpcr as previously described. electrical-mechanical dissociation is thought to be associated with high doses of endogenous endorphins and high vagal tone. the treatment of choice for electrical-mechanical dissociation is high-dose atropine ( mg/kg iv, it [ times the normal dose]) and naloxone hydrochloride ( . mg/kg iv, io, it). administer epinephrine ( . to . mg/kg diluted in ml sterile . % saline it). if the rhythm does not change within minutes, consider open-chest cardiac massage. ventricular fibrillation can be coarse (figure - ) . patients with coarse ventricular fibrillation are easier to defibrillate than those with fine defibrillation. if ventricular fibrillation is identified, initiate cpcr as described previously (figure - ) . if an electrical defibrillator is available, administer j/kg of direct current externally. when a patient in cardiopulmonary arrest is attached to ecg leads, it is important to use contact electrode paste, water-soluble gel such as ky jelly, or water, rather than any form of alcohol. electrical defibrillation of a patient who has alcohol on the ecg leads can lead to fire and thermal burns. reverse any opioid, α -agonist, and phenothiazine drugs that have been administered to the patient. if fine ventricular fibrillation is identified, administer epinephrine figure - : electrical-mechanical dissociation (emd), also known as pulseless electrical activity (pea). the complexes often appear wide and bizarre without a palpable apex beat or functional contraction of the heart. this is just one example of emd, as many shapes and complexes may be observed. organized according to whether an electrical defibrillator is available. after each intervention step, the ecg should be reevaluated and the next step initiated if v-fib is still seen. if a new arrhythmia develops, the appropriate therapy for that rhythm should be inititated. if a sinus rhythm is seen with a palpable apex beat, postresuscitation measures should be implemented. perform open-chest cpcr immediately if a pathologic condition exists that prevents enough of a change in intrathoracic pressure that closed-chest cpcr will not be effective in promoting forward blood flow (box . to perform open-chest cpcr, place the patient in right lateral recumbency. clip a wide strip of fur over the left fifth to seventh intercostal space and quickly aseptically scrub over the clipped area. using a no. scalpel blade, incise over the fifth intercostal space through the skin and subcutaneous tissue to the level of the intercostal muscles. with a mayo scissors, make a blunt stab incision through the intercostal muscles in the left sixth intercostal space. make sure that the person who is breathing for the patient deflates the lungs as you make the stab incision to avoid iatrogenic lung puncture. after the stab incision, open the tips of the mayo scissors and quickly open the muscle dorsally and ventrally to the sternum with a sliding motion. avoid the internal thoracic artery at the sternum and the intercostal arteries at the caudal aspect of each rib. cut the rib adjacent to the sternum and push it behind the rib in front of and at the caudal aspect of the incision to allow more room and better visualization if a rib spreading retractor is not available. visualize the heart in the pericardial sac. visualize the phrenic nerve, and incise the pericardium just ventral to the phrenic nerve. make sure to not cut the phrenic nerve. grasp the heart in your hand(s) and gently squeeze it from apex to base, allowing time for the ventricle to fill before the next "contraction." if the heart does not seem to be filling, administer fluids intravenously or directly into the right atrium. the descending aorta can be cross-clamped with a rummel tourniquet or red rubber catheter to improve perfusion to the brain and heart. postresuscitation care and monitoring (prolonged life support) postresuscitation care involves careful monitoring and management of the adverse effects of hypoxia and reperfusion injury on the brain and other vital organs. the first hours after an arrest are most critical, because this is the time period in which an animal is most likely to rearrest unless the underlying cause of the initial arrest has been determine and treated (table - ) . until an animal is adequately ventilating on its own, artificial ventilation by manual bagging or attaching the patient to a mechanical ventilator with supplemental oxygen must continue. the efficacy of oxygenation and ventilation can be monitored using a wright's respirometer, pulse oximetry, capnometry, and arterial blood . once an animal is extubated, administer supplemental oxygen ( to ml/ kg/minute) (see oxygen supplementation). the brain is sensitive to ischemia and reperfusion injury. the effects of cellular hypoxia and reperfusion include the development of oxygen-derived free radical species that contribute to cerebral edema. administer mannitol ( . to g/kg iv over to minutes), followed by furosemide ( mg/kg iv) minutes later, to all patients that have experienced cardiopulmonary arrest and have had successful resuscitation. mannitol and furosemide work synergistically to decrease cerebral edema formation and scavenge oxygen-derived free radical species. the combination of cardiac arrest, myocardial ischemia and acidosis, and external or internal cardiac compressions often make the myocardium irritable and predisposed to dysrhythmias following successful cpcr. start lidocaine ( to mg/kg iv, followed by to µg/kg/minute iv cri) in all patients following successful resuscitative efforts. monitor the ecg continuously for the presence of cardiac dysrhythmias and recurrence of nonperfusing rhythms. perform direct or indirect blood pressure monitoring. if a patient's systolic blood pressure is less than mm hg, diastolic pressure is less than mm hg, or mean arterial blood pressure is less than mm hg, administer positive inotropic drugs (dobutamine, to µg/kg/minute) and pressor agents (epinephrine, . to . mg/kg iv, io, it) to improve cardiac contractility, cardiac output, and core organ perfusion. the kidneys are sensitive to decreased perfusion and cellular hypoxia. place a urinary catheter and monitor urine output. in a euvolemic patient, normal urine output should be no less than to ml/kg/hour. if urine output is low, administer low-dose dopamine ( to µg/kg/minute iv cri) in an attempt to dilate afferent renal vessels and improve renal perfusion. maintain acid-base and electrolyte status within normal reference ranges. monitor serum lactate as a rough indicator of organ perfusion and cellular oxygen extraction. the presence of elevated or rising serum lactate in the face of aggressive cardiorespiratory and cerebral support makes prognosis less favorable. cole sg, otto cm, hughes d: cardiopulmonary cerebral resuscitation: a clinical practice review part i, j vet emerg crit care ( ) immediate action depends largely on recognition of the primary or secondary cause of the dysrhythmia and treating the dysrhythmia and underlying cause. diagnosis of cardiac dysrhythmias is based on physical examination findings of abnormal thoracic/cardiac auscultation, the presence of abnormal pulse rhythm and quality, and recognition of ecg abnormalities. the ecg is critical to the accurate diagnosis of dysrhythmias. ventricular dysrhythmias arise from ectopic foci in the ventricles that cause the wave of depolarization to spread from cell to cell rather than spread through fast-conducting tissue. this causes the qrs complex to appear wide and bizarre, unless the ectopic focus originates close to the atrioventricular node high in the ventricle. other ecg features of ventricular dysrhythmias include a t wave polarity that is opposite to the qrs complex and nonrelated p waves. ventricular dysrhythmias may manifest as isolated ventricular premature complexes, couplets, or triplets; bigeminy; or ventricular tachycardia. relatively slow ventricular tachycardia is known as an idioventricular rhythm and is not as hemodynamically significant as faster ventricular tachycardia. idioventricular rhythm usually is less than beats per minute and may alternate spontaneously with sinus arrhythmias (figures - to . supraventricular dysrhythmias arise from ectopic foci in the atria and are commonly associated with atrial dilatation and structural heart disease such as advanced acquired or congenital heart disease, cardiomyopathies, cardiac neoplasia, or advanced heartworm disease. occasionally, supraventricular dysrhythmias may be associated with respiratory or other systemic illness. sustained supraventricular tachycardia in the absence of underlying structural heart or systemic disease is disturbing and should alert the clinician that an accessory pathway conduction disturbance may be present, particularly in labrador retrievers. supraventricular dysrhythmias can manifest as isolated premature complexes (atrial premature complexes or contractions), sustained or paroxysmal supraventricular tachycardia (atrial tachycardia), or atrial fibrillation or flutter. in the dog, atrial fibrillation most commonly is associated with dilative cardiomyopathy. rarely and primarily in giant breed dogs, lone atrial fibrillation can occur with no underlying heart disease. atrial fibrillation and the resultant sustained elevation in ventricular rate are presumed to progress to dilative cardiomyopathy in such breeds. by comparison, atrial fibrillation is relatively uncommon in cats because of the small size of their atria but is associated most commonly with hypertrophic and restrictive cardiomyopathy. the ecg is critical to the diagnosis of a supraventricular dysrhythmia. the ecg usually demonstrates a normal appearance to the qrs complex unless aberrant conduction occurs in the ventricles, in which case the qrs can be wide but still originate from above the atrioventricular node. in most cases of a supraventricular dysrhythmia, some evidence of atrial activity including p waves, atrial flutter, or atrial fibrillation is apparent. in some cases, it may be difficult to diagnose the exact rhythm without slowing the rate down mechanically or through pharmacologic intervention. once a rhythm diagnosis is made, appropriate treatment strategies can be implemented (figures - and - ). treatment of ventricular dysrhythmias largely depends on the number of ectopic foci discharging, the rate and character of the dysrhythmia, and whether the presence of the abnormal beats is of adverse hemodynamic consequence, including risk of sudden death. many ventricular dysrhythmias, including slow idioventricular rhythms, ventricular bigeminy, or intermittent ventricular premature complexes, do not warrant antiarrhythmic therapy unless the patient is hypotensive and the dysrhythmia is thought to be contributing to the hypotension. in such cases, correction of the underlying disease process including hypoxia, pain, or anxiety often alleviates or decreases the incidence of the dysrhythmia. more serious ventricular dysrhythmias that warrant antiarrhythmic therapy (table - ) include sustained ventricular tachycardia (> beats/minute in dogs; > beats/minute in cats), multifocal ventricular premature complexes originating from more than one place in the ventricles, and the presence of r-on-t phenomena where the t wave of the preceding complex is superimposed on the qrs of the next complex with no return to isoelectric shelf in between complexes. treat these ventricular dysrhythmias immediately and aggressively. in dogs, the mainstay of emergency treatment for ventricular dysrhythmias is lidocaine therapy. administer lidocaine ( to mg/kg iv bolus) over a period of minutes to prevent the adverse side effects of seizures or vomiting. the bolus can be repeated an additional times (total dose mg/kg) over minutes, or the patient can be placed on a constant rate infusion ( to µg/kg/minute) if control of ventricular tachycardia is accomplished. also correct the patient's magnesium and potassium deficiencies to maximize the success of lidocaine therapy in the treatment of ventricular tachycardia. procainamide ( mg/kg iv slowly over to minutes) also can be used to control ventricular tachycardia. if procainamide is successful at controlling ventricular tachycardia, administer it as a constant rate infusion ( to µg/kg/minute). side effects of procainamide include vomiting, diarrhea, and hypotension. chronic oral therapy may or may not be necessary in the treatment of acute ventricular tachycardia. the decision to continue antiarrhythmic therapy depends on the underlying disease process and the expectation of persistent arrhythmogenesis of the underlying disease process. oral antiarrhythmic therapy is warranted in cases in which a serious ventricular dysrhythmia is recognized but the animal does not require hospitalization, such as the syncopal boxer with intermittent ventricular dysrhythmias and no evidence of structural heart disease. it deserves emphasis that asymptomatic, low-grade ventricular dysrhythmias probably do not require treatment. if maintenance therapy for ventricular dysrhythmias is needed, use an oral drug based on the underlying disease process, clinical familiarity, class of drug, dosing frequency, owner compliance, concurrent medications, cost, and potential adverse side effects. in the cat the mainstay of antiarrhythmic therapy is the use of a β-adrenergic antagonist. in the acute management of ventricular dysrhythmias in cases of hypertrophic, restrictive, or unclassified cardiomyopathies, consider using injectable esmolol ( . to . mg/kg iv slowly to effect) or propranolol ( . to . mg/kg iv slowly to effect), particularly if the dysrhythmia results from hyperthyroidism. for chronic oral ventricular antiarrhythmic therapy in cats, propranolol ( . to . mg po per cat q h) or atenolol ( . to . mg po per cat q - h) can be used. the decision to treat supraventricular dysrhythmias depends on the ventricular rate and the hemodynamic consequences of the dysrhythmia. for intermittent isolated atrial emergency care procainamide - mg/kg po q - h tocainide* - mg/kg po q h sotalol - mg per dog q h (start low, then titrate up to effect) mexiletine - mg/kg po q h atenolol . - . mg/kg po q - h (start low, titrate upward to effect) *do not use for longer than weeks because of idiosyncratic blindness. premature contractions, couplets, and triplets, usually no treatment is required. when the ventricular rate exceeds beats/minute, diastolic filling time is shortened, causing the heart to not fill adequately. the consequence is decreased cardiac output and decreased coronary artery perfusion. the goal of therapy is rhythm control or, in most cases, rate control. in cases of atrial fibrillation and congestive heart failure, conversion to a normal sinus rhythm rarely can be achieved, although electrocardioversion or pharmacoconversion can be attempted. in the dog a vagal maneuver can be attempted by pressing on the eyeballs or massaging the carotid body. for sustained supraventricular tachycardia, diltiazem ( . mg/kg iv), esmolol ( . to . , titrated upward to a cumulative dose of . mg/kg iv), or propranolol ( . to . mg/kg iv slowly to effect) can be administered in an attempt to slow the ventricular rate in emergent situations. administer oral diltiazem ( . mg/kg po q h), diltiazem (dilacor-xr) ( . to mg/kg po q - h), propranolol ( . to . mg/kg tid, titrated up to a maximum of . mg/kg po q h), atenolol ( . to mg/kg q - h), or digoxin ( . to . mg/kg bid or . mg/m for dogs greater than kg). in the cat a vagal maneuver can be attempted by ocular or carotid massage. (diltiazem [dilacor] to po q - h), propranolol ( . to mg/kg q - h), or atenolol ( . mg q - h) also can be administered. if structural heart disease is present, treat pulmonary edema and start angiotensin-converting enzyme inhibitor therapy. table - summarizes the drugs used in the management of supraventricular dysrhythmias. severe bradycardia often results from systemic disease, drug therapy, anesthetic agents, or hypothermia and thus rarely requires specific therapy except to treat or reverse the underlying mechanisms promoting bradycardia. hemodynamically significant bradyarrhythmias that must be treated include atrial standstill, atrioventricular block, and sick sinus syndrome. atrial standstill most commonly is associated with hyperkalemia and is seen most often in urinary obstruction, renal failure, urinary trauma with uroabdomen, and hypoadrenocorticism. characteristic ecg abnormalities observed in atrial standstill are an absence of p waves, widened qrs complexes, and tall spiked t waves (figure - ). the treatment for hyperkalemia-induced atrial standstill is to correct the underlying cause and to drive potassium intracellularly and protect the myocardium from the adverse effects of hyperkalemia. regular insulin ( . to . units/kg iv) followed by dextrose ( g/unit insulin iv, followed by . % dextrose cri to prevent hypoglycemia) or sodium bicarbonate ( meq/kg iv) can be administered to drive potassium intracellularly. calcium gluconate ( . ml/kg of % solution iv over minutes) also can be administered as a cardioprotective drug until the cause of hyperkalemia has been identified and resolved. also administer sodium chloride fluids ( . % sodium chloride iv) to promote kaliuresis. less commonly, atrial standstill is associated with atrial cardiomyopathy or silent atrium syndrome. persistent atrial standstill has been recognized without electrolyte abnormalities in the english springer spaniel and the siamese cat. short-term therapy for persistent atrial standstill includes atropine ( . mg/kg sq) until definitive treatment by implantation of a cardiac pacemaker can be performed. complete or third-degree atrioventricular block or high-grade symptomatic seconddegree atrioventricular block can be hemodynamically significant when ventricular rates are less than beats/minute in the dog. classic clinical signs include weakness, exercise intolerance, lethargy, anorexia, syncope, and occasionally seizures. advanced atrioventricular block usually is caused by advanced idiopathic degeneration of the atrioventricular node. less commonly, atrioventricular block has been associated with digoxin toxicity, magnesium oversupplementation, cardiomyopathy, endocarditis, or infectious myocarditis (lyme disease). an accurate diagnosis is made based on the ecg findings of nonconducted p waves with ventricular escape beats. first-and second-degree atrioventricular block may not be hemodynamically significant and therefore may not require therapy. initially treat third-degree (complete) or symptomatic high-grade second-degree atrioventricular block (< beats/minute) with atropine ( . mg/kg sq or im). perform a follow-up ecg in to minutes. atropine is rarely successful in treating complete atrioventricular block. also attempt treatment with isoproterenol ( . to . µg/kg/minute iv cri or . mg in ml % dextrose in water iv slowly), a pure β-agonist. definitive treatment requires permanent pacemaker implantation. consultation with a veterinary cardiologist who implants pacemakers is suggested. never attempt to convert or treat the observed ventricular escape beats with lidocaine ( figure - ) . sick sinus syndrome most commonly is recognized in the miniature schnauzer, although any dog can be affected. sick sinus syndrome usually results from idiopathic degeneration of the sinus node in the dog. in the cat, sinus node degeneration usually is associated with cardiomyopathy. dysfunction of the sinus node may manifest as marked bradycardia with periods of sinus arrest followed by junctional or ventricular escape complexes. a variant of sick sinus syndrome is the presence of severe bradycardia followed by periods of supraventricular tachycardia, often termed bradycardia-tachycardia syndrome. the most common clinical signs are syncope, exercise intolerance, and lethargy. in cats, hypertrophic cardiomyopathy is the most common form of acquired cardiac disease observed. congestive heart failure resulting from hypertrophic cardiomyopathy can occur in animals as young as to months of age. hypertrophic cardiomyopathy is characterized by stiff, noncompliant ventricles that do not relax during diastole, causing an increase in left atrial pressures and left atrial enlargement. other cardiomyopathies, including unclassified, restrictive, and dilated, are less common but also can occur in the cat. cats often develop acute exacerbation of clinical signs because of stress or arterial embolization. the rapid diagnosis of chf often is made on owner history, signalment, and physical examination findings (box - ). typical physical examination findings include a cardiac murmur or gallop dysrhythmia, abnormal breath sounds, respiratory difficulty and orthopnea, tachycardia, weak pulse quality, cool peripheral extremities, and pale or cyanotic mucous membrane. initiate immediate treatment based on physical examination findings and index of suspicion. in some cases, it is difficult to distinguish between chf and feline lower airway disease (asthma) without performing thoracic radiographs. let the animal rest and become stabilized before attempting any stressful procedures, including thoracic radiographs. immediate treatment consists of administering supplemental oxygen, decreasing circulating fluid volume with furosemide, dilating pulmonary and splanchnic capacitance vessels with topical nitroglycerine and morphine, and alleviating patient anxiety and stress (box - ). primary differential diagnoses are made based primarily on the patient's breed, age, clinical signs, history, and physical examination abnormalities. the most common differential diagnoses in a patient with chf are cardiac abnormalities and respiratory disease (chronic bronchitis [asthma], pulmonary hypertension, cor pulmonale, neoplasia). postpone diagnostic tests in any patient with suspected chf until the immediate treatments have taken effect and the patient is cardiovascularly more stable. in most cases, lateral and dorsoventral thoracic radiographs are one of the most important diagnostic tools in helping make a diagnosis of chf. increased perihilar interstitial to alveolar infiltrates are characteristic of pulmonary edema. left atrial enlargement may be observed as a "backpack" sign at the caudal cardiac waist. cardiomegaly of the right or left side also may be present in cases of valvular insufficiency. in cats, increased sternal contact and a classic valentine-shaped heart may be observed in cases of hypertrophic cardiomyopathy. perform a vertebral heart score (sum) to measure cardiac size and determine whether cardiomegaly is present (box - ). also obtain arterial blood pressure and ecg readings to determine whether hypotension and dysrhythmias are present. atrial fibrillation, ventricular premature contractions, and supraventricular tachycardia are common rhythm disturbances that can affect cardiac output adversely and influence treatment choices. the echocardiogram is a useful noninvasive and nonstressful method to determine the degree of cardiac disease present. the echocardiogram is largely user-dependent. the quality of the study is based on the experience of the operator and the quality of the ultrasound machine. echocardiography can be a useful tool in making a diagnosis of pericardial effusion, dilated or hypertrophic cardiomyopathy, cardiac neoplasia, and endocarditis. the medical management of chf is designed to improve cardiac output and relieve clinical signs. the immediate goal of therapy is to reduce abnormal fluid accumulation and provide adequate cardiac output by increasing contractility, decreasing preload and ventricular afterload, and/or normalizing cardiac dysrhythmias. strict cage rest is of utmost importance when managing a patient with chf. after initial administration of furosemide, morphine, oxygen, and nitroglycerine paste, clinical signs of respiratory distress should show improvement within minutes. if no improvement is observed, administer repeated doses of furosemide. reevaluate severe cases that are refractory to this standard treatment protocol. vasodilation should be the next step in the management of refractory cases, provided that a normal blood pressure is present. sodium nitroprusside is a potent balanced vasodilator that should be administered ( to µg/kg/minute iv cri), taking care to monitor blood pressure continuously because severe vasodilation and hypotension can occur. the goal of nitroprusside therapy is to maintain a mean arterial blood pressure of mm hg. sodium nitroprusside should not be considered in cases of refractory chf with severe hypotension. for more long-term management of chf, the use of angiotensin-converting enzyme (ace) inhibitors including enalapril ( . mg/kg po q - h), benazepril ( . mg/kg po q h), and lisinopril ( . mg/kg po q h) have become the mainstay of therapy to reduce sodium and fluid retention and decrease afterload. start angiotensin-converting enzyme inhibition as soon as a patient is able to tolerate oral medications. dobutamine ( . to µg/kg/minute cri diluted in % dextrose in water) can be administered to improve cardiac contractility, particularly in cases of dilated cardiomyopathy. at low doses, dobutamine, primarily a β-adrenergic agonist, will improve cardiac output with minimal effects on heart rate. dobutamine must be given as a constant rate infusion with careful, continuous ecg monitoring. despite minimal effects on heart rate, emergency management of specific conditions the vertebral heart sum can be calculated by performing the following steps: . measure the long axis of the heart from the apex to the carina on the lateral view and mark the distance on a sheet of paper. . measure the length of the long axis of the heart in terms of vertebral bodies, starting by counting caudally from the fourth thoracic vertebra; count the number of vertebrae that are covered by the length of the long axis of the heart. . measure the short axis of the heart at the caudal vena cava, perpendicular to the long axis of the heart. . count the number of thoracic vertebrae covered by the short axis of the heart, starting at t . . add the two numbers together to yield the vertebral heart sum; a vertebral heart sum greater than . is consistent with cardiomegaly. sinus tachycardia or ventricular dysrhythmias may develop during infusion. cats are more sensitive to the effects of dobutamine than dogs. monitor carefully for seizures and facial twitching. digoxin is a cardiac glycoside that acts as a positive inotrope and negative chronotrope in the long-term management of chf. digoxin has a long ( hours in dogs, and hours in cats) half-life and so has minimal use in the emergency management of chf. in chronic management of chf resulting from dilated cardiomyopathy or advanced mitral disease, however, digoxin is extremely useful. oral digitalization protocols have been developed but are risky in that dysrhythmias and severe gastrointestinal side effects can occur. cats with chf often have fulminant pulmonary edema, pleural effusion, arterial thromboembolism, or some combination of all three. if the pleural effusion is significant, perform therapeutic thoracocentesis to relieve pulmonary atelectasis and improve oxygenation. once the diagnosis and initial management of chf has been made, formulate a plan for continued management and monitoring. tailor the therapeutic plan to the patient based on the cause of the chf, the presence of concurrent diseases, and response to therapy. an important and often overlooked part of the successful emergency management of chf is the open communication with the owner regarding the owner's emotional and financial commitment for immediate and long-term management to ensure appropriate quality of life for each patient. pathophysiology and treatment, vet j ( ) caval syndrome resulting from severe heartworm disease is caused by the rapid maturation of a large quantity of adult worms in the right atrium and cranial and caudal venae cavae. most cases of caval syndrome occur in regions of the world where heartworm disease is highly endemic and dogs spend a large portion of time living outdoors. caval syndrome is recognized by the following clinical signs and results of biochemical analyses: acute renal and hepatic failure, enlarged right atrium and posterior vena cava, ascites, hemoglobinuria, anemia, acute collapse, respiratory distress, dic, jugular pulses, circulating microfilariae, and sometimes tricuspid insufficiency. immediate action in cases of caval syndrome in dogs involves immediate stabilization of the cardiovascular and respiratory systems with supplemental oxygen, furosemide ( mg/kg iv), and careful crystalloid fluid infusion. diagnosis of caval syndrome is based on clinical signs of cardiogenic shock with right ventricular heart failure, intravascular hemolysis, and renal and hepatic failure. thoracic radiographs reveal cardiomegaly of the right side and enlarged tortuous pulmonary arteries. a right axis deviation may be seen on ecg tracings. clinicopathologic changes observed include azotemia, inflammatory leukogram, regenerative anemia, eosinophilia, elevated hepatocellular enzyme activities, hemoglobinuria, and proteinuria. circulating microfilariae may be observed on peripheral blood smears or in the buffy coat of microhematocrit tubes. heart worm antigen tests will be strongly positive. echocardiographic changes include visualization of a large number of heartworms in the right atrium, pulmonary arteries, and vena cava, tricuspid insufficiency, and right atrial and ventricular enlargement. treatment involves surgical removal of as many of the adult heartworms as possible from the right jugular vein and right atrium. glucocorticosteroids are recommended to decrease inflammation and microangiopathic disease associated with heartworm infection. for more long-term management, administer adulticide therapy several weeks following surgery, followed by routine microfilaricide therapy and then prophylaxis. calvert pericardial effusion often develops as a consequence of neoplasia in the older dog and cat. the most common types of neoplasia that affect the heart and pericardium include hemangiosarcoma, chemodectoma, mesothelioma, and metastatic neoplasia. more rarely, other causes of pericardial effusion include benign idiopathic pericardial effusion, coagulopathy, left atrial rupture in dogs with chronic mitral valvular insufficiency, infection, or pericardial cysts. regardless of the cause of the effusion, the development of pericardial tamponade adversely affects cardiac output. cardiac output is a function of heart rate and stroke volume. stroke volume depends on cardiac preload. the presence of pericardial effusion can impede venous return to the heart and thus adversely affect preload. in addition, as preload decreases, heart rate reflexively increases in an attempt to maintain normal cardiac output. as heart rate increases more than beats/minute, diastolic filling is impaired further, and cardiac output further declines. animals with pericardial effusion often demonstrate the classic signs of hypovolemic or cardiogenic shock: anorexia, weakness, lethargy, cyanosis, cool peripheral extremities, tachycardia, weak thready pulses, hypotension, and collapse. physical examination abnormalities may include muffled heart sounds, thready femoral pulses, pulsus paradoxus, jugular venous distention, weakness, tachycardia, cyanosis, and tachypnea. electrocardiogram findings may include low amplitude qrs complexes (< . mv), sinus tachycardia, ventricular dysrhythmias, or electrical alternans (figure - ) . thoracic radiographs often demonstrate a globoid cardiac silhouette, although the cardiac silhouette rarely may appear normal with concurrent clinical signs of cardiogenic shock in cases of acute hemorrhage. in such cases the removal of even small amounts of pericardial effusion by pericardiocentesis can increase cardiac output exponentially and alleviate clinical signs (table - ) . unless an animal is dying before your eyes, ideally perform an echocardiogram to attempt to determine whether a right atrial, right auricular, or heart base mass is present before pericardiocentesis. before attempting pericardiocentesis, assemble all of the required supplies (box - ) . to perform pericardiocentesis, follow this procedure: . place the patient in sternal or lateral recumbency. . attach ecg leads to monitor the patient for dysrhythmias during the procedure. . clip a -cm square caudal to the right elbow over the fifth to seventh intercostal space. . aseptically scrub the clipped area, and infuse to mg/kg of % lidocaine mixed with a small amount of sodium bicarbonate just dorsal to the sternum at the sixth intercostal space. bury the needle to the hub, and inject the lidocaine as you withdraw the needle. . while the local anesthetic is taking effect, assemble the intravenous extension tubing, three-way stopcock, and -ml syringe. . wearing sterile gloves, make a small nick incision in the skin to decrease drag on the needle and catheter during insertion. . slowly insert the needle and catheter, watching for a flash of blood in the hub of the needle, and simultaneously watching for cardiac dysrhythmias on the ecg monitor. . once a flash of blood is observed in the hub of the needle, advance the catheter off of the stylette further into the pericardial sac, and remove the stylette. . attach the length of intravenous extension tubing to the catheter, and have an assistant withdraw the fluid slowly. . place a small amount of fluid in a red-topped tube, and watch for clots. clot formation could signify that you have penetrated the right ventricle inadvertently or that active hemorrhage is occurring. withdraw as much of the fluid as possible, and then remove the catheter. monitor the patient closely for fluid reaccumulation and recurrence of clinical signs of cardiogenic shock. less rd, bright jm, orton ec: intrapericardial cyst causing cardiac tamponade in a cat, j am anim hosp assoc ( ) foreign bodies within the ear canal (e.g., foxtails) can present as emergencies because of acute inflammation and pressure necrosis of the tissue of the external auditory meatus causing pain and discomfort. clinical signs may be limited to incessant head shaking or scratching of the ear canal. complete examination of the ear canal and removal of any foreign body often requires administration of a short-acting anesthetic agent. once the animal has been restrained sufficiently and placed under anesthesia, carefully examine the ear canal and remove any foreign material with an alligator forceps. stimulation of the ear canal can cause awakening after removal of all debris and detritus, gently wipe the internal and external ear canal with a sterile gauze. place a topical antimicrobial-antifungal-steroid ointment such as otomax in the ear every to hours. if pain and discomfort is severe, systemically effective opioids or nsaids may be required. otitis externa is a common emergency that causes excessive head shaking, scratching, and purulent malodorous aural discharge. clean the ear canal with an irrigating solution such as epiotic and wipe it clean of debris. perform a complete aural examination to determine whether a foreign body or tumor is present and whether the tympanic membrane is intact. heat-fix any discharge and examine it cytologically for bacteria and fungal organisms. following careful cleansing, instill a topical antibiotic-antifungal-steroid ointment. in severe cases in which the ear canal has scarred and closed down with chronicity, consider administering systemically effective antibiotics (cephalexin, mg/kg po tid) and antifungal agents (ketoconazole, mg/kg po q h) instead of topical therapy. systemically effective steroids (prednisone or prednisolone, . mg/kg po q h) may be indicated in cases of severe inflammation to decrease pruritus and patient discomfort. presentation of a patient with otitis interna often is characterized by torticollis, head tilt, nystagmus, circling to the affected side, or rolling. fever, pain, vomiting, and severe depression may accompany clinical signs. most cases of severe otitis interna are accompanied by severe otitis media. both conditions must be treated simultaneously. the most common causes of otitis interna are staphylococcus aureus, pseudomonas, escherichia coli, or proteus spp. otitis interna can develop by infection spreading across the tympanic membrane, through the eustachian tubes, or by hematogenous spread from the blood supply to the middle ear. in most cases of otitis media, the tympanic membrane is ruptured. perform a culture and susceptibility test of the debris behind the tympanic membrane and within the aural canal. carefully clean the external ear canal. medicate with a topical combination antibiotic, antifungal, and antibiotic ointment. administer high-dose antibiotics (cephalexin, mg/kg po q h, or enrofloxacin, to mg/kg po q h). if the tympanic membrane is not ruptured but appears swollen and erythematous, a myringotomy may need to be performed. if clinical signs of otitis media persist despite topical and systemic therapy, radiographic or ct/mri examination of the tympanic bullae may be required. chronic shaking of the head and ears or aural trauma (bite wounds) causes disruption of the blood vessels and leads to the development of unilateral or bilateral aural hematomas. aural hematomas are clinically significant because they cause patient discomfort and are often due to the presence of some other underlying problem such as otitis externa, atopy, or aural foreign bodies. acute swelling of the external ear pinna with fluid is characteristic of an aural hematoma. in some cases, swelling can be so severe that the hematoma breaks open, bathing the patient and external living environment in blood. when a patient has an aural hematoma, investigate the underlying cause. perform a complete aural examination to determine whether an aural foreign body, otitis externa, or atopy are present. carefully examine and gently clean the inner ear canal. treat underlying causes. management of an aural hematoma involves draining the hemorrhagic fluid from the aural tissue and tacking the skin down in multiple places to prevent reaccumulation of fluid until the secondary cause is resolved. many techniques have been described to surgically tack down the skin overlying the hematoma. after the animal has been placed under general anesthesia, lance the hematoma down the middle with a scalpel blade and remove the fluid and blood clot. tack down the skin with multiple through-and-through interrupted or mattress sutures through the ear. some clinicians prefer to suture through and attach a sponge or length of x-ray film to the front and back of the ear for stabilization and support. more recently, a laser can be used to drill holes in the hematoma and tack the skin down in multiple areas. compress the ear against the head with a compression bandage, whenever possible, for to days after the initial surgery, and then recheck the ear. the patient must wear an elizabethan collar until the surgical wound and hematoma heal to prevent selfmutilation. also systemically treat underlying causative factors such as otitis externa with antibiotics, antifungals, and steroids as indicated. investigate and treat other underlying causes such as hypothyroidism or allergies. bass electrocution usually is observed in young animals after they have chewed on an electric cord. other causes of electrocution include use of defective electrical equipment or being struck by lightning. electric current passing through the body can produce severe dysrhythmias, including supraventricular or ventricular tachycardia and first-and thirddegree atrioventricular block. the electric current also can produce tissue destruction from heat and electrothermal burns. electrocution also commonly results in noncardiogenic pulmonary edema caused by massive catecholamine release and increase in pulmonary vascular pressures during the event. ventricular fibrillation can occur, although that depends on the intensity and path of the electrical current and duration of contact. clinical signs of electrocution include acute onset of respiratory distress with moist rales, and localized necrosis or thermal burns of the lips and tongue. often the skin at the commissures of the mouth appears white or yellow and firm to the touch. muscle fasciculations, loss of consciousness, and ventricular fibrillation may occur. thoracic radiographs often reveal an increased interstitial to alveolar lung pattern in the dorsocaudal lung fields. noncardiogenic pulmonary edema can develop up to to hours after the initial incident. the first hours are most critical for the patient, and then prognosis improves. the most important aspect in the treatment of the patient with noncardiogenic pulmonary edema is to minimize stress and to provide supplemental oxygen, with positive pressure ventilation, when necessary. although treatment with vasodilators (low-dose morphine) and diuretics (furosemide) can be attempted, noncardiogenic pulmonary edema is typically resistant to vasodilator and diuretic therapy. positive inotropes and pressor drugs may be necessary to treat shock and hypotension. opioid drugs (morphine, hydromorphone, oxymorphone) may be useful in controlling anxiety until the pulmonary edema resolves. administer broad-spectrum antibiotics (cefazolin; amoxicillin and clavulanic acid [clavamox]) to treat thermal burns. use analgesic drugs to control patient discomfort. if thermal burns are extensive and prohibit adequate food intake, place a feeding tube as soon as the patient's cardiovascular and respiratory function are stable and the patient can tolerate anesthesia. prolapse of the uterus occurs in the immediate postparturient period in the bitch and queen. excessive straining during or after parturition causes the uterus to prolapse caudally through the vagina and vulva. immediate intervention is necessary. examine the bitch or queen for a retained fetus. treatment consists of general anesthesia to replace the prolapsed tissue. if the uterus is edematous, physical replacement may be difficult or impossible. application of a hypertonic solution such as hypertonic ( %) saline or dextrose ( %) to the exposed endometrium can help shrink the tissue. that, combined with gentle massage to stimulate uterine contraction and involution and lubrication with sterile lubricating jelly, can aid in replacement of the organ into its proper place. to ensure proper placement in the abdominal cavity and to prevent recurrence, perform an exploratory laparotomy and hysteropexy. postoperatively, administer oxytocin ( to units im) to cause uterine contraction. if the uterus contracts, it is usually not necessary to suture the vulva. administer antibiotics postoperatively. recurrence is uncommon, even with subsequent pregnancies. if the tissue is damaged or too edematous to replace or if the tissue is devitalized, traumatized or necrotic, perform an ovariohysterectomy. in some instances, replacement of the damaged tissue is not necessary before removal. pyometra occurs in dogs and cats. the disease process occurs as a result of infection overlying cystic endometrial hyperplasia under the constant influence of progesterone. during the -month luteal phase after estrus or following copulation, artificial insemination, or administration of hormones (particularly estradiol or progesterone), the myometrium becomes relaxed and favors a quiescent environment for bacterial proliferation. clinical signs of pyometra are associated with the presence of bacterial endotoxin and sepsis. early, affected animals become lethargic and anorectic. polyuria with secondary polydipsia is often present because of the influence of bacterial endotoxin on renal tubular concentration. if the cervix is open, purulent or mucoid vaginal discharge may be observed. later in the course of pyometra, vomiting, diarrhea, and progressive debilitation resulting from sepsis occur. diagnosis is based on clinical signs in an intact queen or bitch and radiographic or ultrasonographic evidence of a fluid-filled tubular density in the ventrocaudal abdomen, adjacent to the urinary bladder (figures - and - ) . treatment of open and closed pyometra is correction of fluid and electrolyte abnormalities, administration of broad-spectrum antibiotics, and ovariohysterectomy. close pyometra is a life-threatening septic condition. open pyometra also can become life-threatening and so should be treated aggressively. in closed pyometra, conservative medical therapy is not advised. administration of prostaglandins and oxytocin do not reliably cause the cervix to open and can result in ascending infection from the uterus into the abdomen or uterine rupture, both of which can result in severe peritonitis. for animals with an open pyometra, ovariohysterectomy is the most reliable treatment for chronic cystic endometrial hyperplasia. although less successful than ovariohysterectomy, medical therapy may be attempted in breeding bitches as an alternative to surgery. the most widely used medical therapy in the breeding queen and bitch is administration of prostaglandin f α . this drug has not been approved for use in the queen or bitch in the united states. to proceed with medical management of pyometra, first determine the size of the uterus. start the patient on antibiotic therapy (ampicillin, mg/kg iv q h, or enrofloxacin, mg/kg po q h). administer the prostaglandin f α ( µg/kg sq q h) for to days until the size of the uterus approaches normal. measure serum progesterone concentrations if the bitch is in diestrus. as the corpus luteum degrades under the influence of prostaglandin f α , serum progesterone levels will decline. prostaglandin f α is an abortifacient and thus should not be administered to the pregnant bitch or queen. clinical signs of a reaction to prostaglandin f α can occur within to minutes in the bitch and can last for as long as minutes. clinical signs of a reaction include restlessness, hypersalivation, panting, vomiting, defecation, abdominal pain, fever, and vocalization. in a very ill animal, death can occur. the efficacy of prostaglandin f α is limited and may require more than one treatment. the bitch should be bred on the next heat cycle and then spayed because progressive cystic endometrial hyperplasia will continue to occur. acute metritis is an acute bacterial infection of the uterus that typically occurs within to weeks after parturition. the most common organism observed in metritis is e. coli ascending from the vulva and vaginal vault. sepsis can progress rapidly. clinical signs of acute metritis include inability to nurse puppies, anorexia, lethargy, foul-smelling purulentsanguineous vaginal discharge, vomiting, or acute collapse. physical examination may reveal fever, dehydration, and a turgid distended uterus. septic inflammation will be observed on vaginal cytologic examination. an enlarged uterus can be observed with abdominal radiographs and ultrasonography. treatment of acute metritis is directed at restoring hydration status with intravenous fluids and treating the infection with antibiotics. because the primary cause of metritis is e. coli infection, start enrofloxacin ( mg/kg iv or po once daily) therapy. as soon as the patient's cardiovascular status is stable enough for anesthesia, perform an ovariohysterectomy. if the patient is not critical and is a valuable breeding bitch, medical therapy can be attempted. medical management of acute bacterial metritis includes administration of oxytocin ( to units q h for three treatments) or administration of prostaglandin f α ( µg/kg/day for to days) to evacuate the uterine exudate and increase uterine blood flow. either drug should be used concurrently with antibiotics. rupture of the gravid uterus is rare in cats and dogs but has been reported. uterine rupture may occur as a consequence of parturition or result from blunt abdominal trauma. feti expelled into the abdominal cavity may be resorbed but more commonly cause the development of peritonitis. if fetal circulation is not disrupted, the fetus actually may live to term. uterine rupture is an acute surgical emergency. an ovariohysterectomy with removal of the extrauterine puppies and membranes is recommended. if only one horn of the uterus is affected, a unilateral ovariohysterectomy can be performed to salvage the remaining unaffected puppies and preserve the breeding potential for the valuable bitch. if uterine rupture occurs because of pyometra, peritonitis is likely, and copious peritoneal lavage should be performed at the time of surgery. the patient should be placed on to days of antibiotic therapy (amoxicillin or amoxicillin and clavulanic acid [clavamox] with enrofloxacin). vaginal prolapse occurs from excessive proliferation and hyperplasia of vaginal tissue while under the influence of estrogen during proestrus (figure - ) . the hyperplastic tissue usually recedes during diestrus but reappears with subsequent heat cycles. vaginal prolapse can be confused with vaginal neoplasia. the former condition occurs primarily in younger animals, whereas the latter condition occurs primarily in older animals. treatment for vaginal hyperplasia or prolapse generally is not required if the tissue remains within the vagina. the proliferation can lead to dysuria or anuria, however. in some cases, the tissue becomes emergency care dried out and devitalized or becomes traumatized by the animal. such extreme cases warrant immediate surgical intervention. the treatment for vaginal prolapse consists of ovariohysterectomy to remove the influence of estrogen, placement of an indwelling urinary catheter if the patient is dysuric, and protection of the hyperplastic tissue until it recedes on its own. although surgical resection of the hyperplastic tissue has been recommended, excessive hemorrhage after removal can occur, and so the procedure should not be attempted. the patient should wear an elizabethan collar at all times to prevent selfmutilation. administer broad-spectrum antibiotics for a minimum of to days or until the hyperplastic tissue recedes. keep the tissue clean with saline solution. dystocia, or difficult birth, can occur in the dog and cat but is more common in the dog. a diagnosis of dystocia is made based on the time of onset of visible labor and the time in which the last puppy or no puppy has been born, the intensity and timing of contractions, the timing of when the amniotic membranes first appear, the condition of the bitch, and the timing of gestation. causes of dystocia can be maternal or fetal and include primary or secondary uterine inertia, narrowing of the pelvic canal, hypocalcemia, psychological disturbances, or uterine torsion. maternal-fetal disproportion, or large fetus size in relation to the bitch or queen, also can result in dystocia (box - ). obtain an abdominal radiograph for all cases of suspected dystocia at the time of presentation to determine the size of the fetus, presentation of the fetus (both anterior or posterior presentation can be normal in the bitch or queen, but fetal malpositioning can cause dystocia), and whether there is radiographic evidence of a uterine rupture or torsion. if maternal-fetal disproportion, uterine torsion, or uterine rupture is observed, take the patient immediately to surgery. if the puppies or kittens are in a normal position for birth, medical management can be attempted. clip the perineum and aseptically scrub it. wearing sterile gloves, insert a lubricated finger into the vagina and palpate the cervix. massage (or "feather") the dorsal wall of the vagina to stimulate contractions. place an intravenous catheter, and administer oxytocin ( to units im), repeating up to times at -minute intervals. in some cases, hypoglycemia or hypocalcemia can contribute to uterine inertia. administration of a calciumcontaining solution (lactated ringer's solution) with . % dextrose is advised. alternately, administer % calcium gluconate ( mg/ kg iv slowly). if labor has not progressed after hour, immediately perform a cesarean section. uterine torsion is an uncommon emergency seen in the gravid and nongravid uterus and has been reported in dogs and cats. the onset of clinical signs of abdominal pain and straining as if to whelp/queen or defecate is usually acute and constitutes a surgical emergency. in some cases, there may have been a history of delivery of a live or dead fetus. vaginal discharge may or may not be present. radiographs or ultrasound examination reveal a fluid-filled or air-filled tubular density in the ventral abdomen. treatment consists of placing an intravenous catheter, stabilizing the patient's cardiovascular status with intravenous fluids and sometimes blood products, and performing an immediate ovariohysterectomy. if there are viable feti, the uterus should be delivered en mass and the puppies or kittens delivered. the expulsion of one or more fetus before term is known as spontaneous abortion. in dogs and cats, it is possible to expel or abort one or more fetuses and still carry viable fetuses to term and deliver normally. clinical signs of spontaneous abortion include vaginal discharge and abdominal contractions. in some cases, the fetus is found, or there may be evidence of fetal membranes or remnants. causes of spontaneous abortion in dogs include brucella canis, herpesvirus, coronavirus, and toxoplasmosis. in cats, herpesvirus, coronavirus, and feline leukemia virus can cause spontaneous abortion. in both species, trauma, hormonal factors, environmental pathogens, drugs, and fetal factors also can result in spontaneous abortion. the safest method of pregnancy termination in the bitch or queen is by performing an ovariohysterectomy. oral diethylstilbesterol is not an effective mechanism of pregnancy termination in the bitch. a so-called mismating shot, an injection of estradiol cypionate ( . mg/lb im) is effective at causing termination of an early pregnancy but can be associated with severe side effects, including bone marrow suppression and pyometra. estradiol cypionate is not approved for use in the bitch or queen and is not recommended. prostaglandin f α is a natural abortifacient in the bitch if treatment is started within days of cytologic evidence of diestrus (noncornified epithelium on a vaginal smear). the prostaglandin f α causes lysis of the corpora lutea and a rapid decline in progesterone concentration. the prostaglandin f α is administered for a total of eight injections ( µg/kg q h for days), along with atropine ( to µg/kg sq). side effects can occur within to minutes of injection and include restlessness, panting, salivation, abdominal pain, urination, vomiting, and diarrhea. walking the patient for to minutes after each treatment sometimes decreases the intensity of the reactions. bitches in the first half of the pregnancy often resorb the embryos. if prostaglandin f α is administered in the second half of the pregnancy, the fetuses are aborted within to days of treatment. measure serum progesterone concentrations at the end of treatment to ensure complete lysis of the corpus luteum. prostaglandin f α is not approved for pregnancy termination in the bitch. in cats, prostaglandin f α can terminate pregnancy after day of gestation. prostaglandin f α should be used only in healthy queens ( to µg/kg sq q h for days). side effects in the queen are similar to those observed in the bitch but typically have a shorter duration ( to minutes). prostaglandin f α is not approved for use in cats in the united states. the use of prostaglandin f α does not preclude breeding and pregnancy at a later date. biddle d, macintire dk: obstetrical emergencies, clin tech small anim pract ( ) in the dog and cat the majority of injuries to the scrotum are associated with animal fights or shearing and abrasive injuries sustained in accidents involving automobiles. scrotal injuries should be categorized as superficial or penetrating. treatment of superficial injuries to the scrotum includes cleaning the wound with dilute antimicrobial cleanser and drying it. administer antiinflammatory doses of steroids (prednisolone, . to . mg/kg po q - h) or nsaids (carprofen, . mg/kg po q h in dogs) for the first several days after scrotal injury to prevent or treat edema. administer topical antibiotic ointment until the wound heals. in most cases, place an elizabethan collar to prevent self-mutilation. prognosis is generally favorable; however, semen quality may be affected for months after injury because of scrotal swelling and increased scrotal temperature. penetrating injuries to the scrotum are more serious and are associated with severe swelling and infection. surgically explore and debride penetrating scrotal wounds. administer systemically effective antibiotics and analgesics. in extreme cases, particularly those that involve the testicle, consider castration and scrotal ablation. scrotal dermatitis is common in intact male dogs and can be associated with direct physical injury, self-infliction from licking, chemical irritation, burns, or contact dermatitis. in affected animals, the scrotum can become extremely inflamed, swollen, and painful. if left untreated, pyogranulomatous dermatitis can develop. make an attempt to determine whether an underlying systemic illness is present that could predispose the animal to scrotal dermatitis. widespread vasculitis with scrotal edema, pain, fever, and dermatitis has been associated with rickettsia rickettsii (rocky mountain spotted fever) infection. brucella canis also has been associated with scrotal irritation and dermatitis. if scrotal dermatitis follows from an infectious cause, empiric use of glucocorticosteroids potentially can make the condition worse by suppressing immune function. empiric treatment with antibiotics also potentially can confound making an accurate diagnosis. treatment of scrotal dermatitis is to eliminate predisposing causes, if possible. place an elizabethan collar at all times to prevent self-mutilation. bathe the scrotum with a mild antimicrobial soap and dry it to remove any offending chemical irritants. topical medications including tar shampoo, tetracaine, neomycin, and petroleum can cause further irritation and are contraindicated. use oral or parenteral administration of glucocorticosteroids or nsaids to control discomfort and inflammation. scrotal hernias occur when the contents of the abdomen (intestines, fat, mesentery, omentum) protrude through the inguinal ring into the scrotal sac. like inguinal hernias, scrotal definitive therapy for a scrotal hernia involves exploratory laparotomy and surgical reduction of the contents of the hernia, surgical correction of the rent in the inguinal ring, and castration. trauma to the epididymis or testicle can cause testicular pain and swelling of one or both testes. treat penetrating trauma to the testicle by castration to prevent infection and selfmutilation. administer oral antibiotics (amoxicillin or amoxicillin-clavulanate) for to days after the injury. nonpenetrating injuries to the scrotum and testicle rarely may cause acute testicular hemorrhage or hydrocele formation. palpation of the affected area often reveals a peritesticular, soft, compliant area. treatment consists of cool compresses on the scrotum and testicle and administration of antiinflammatory doses of glucocorticosteroids or nsaids. if the swelling does not resolve spontaneously in to days, consider surgical exploration and drainage. increased scrotal temperature and testicular inflammation can affect semen quality for months after the initial incident. testicular torsion, or torsion of the spermatic cord, causes rotation of the testicle, ultimately causing obstruction to venous drainage. testicular torsion often is associated with a neoplastic mass of a retained testicle within the abdomen but also can be observed with nonneoplastic testes located within the scrotum. the predominant clinical signs are pain, stiff stilted gait, and the presence of an abnormally swollen testicle (if located within the scrotum). if an intraabdominal testicular torsion is present, pain, lethargy, anorexia, and vomiting can occur (see acute condition in the abdomen). an intraabdominal mass may be palpable. perform an abdominal or testicular ultrasound, preferably with color flow doppler to evaluate perfusion to the testicle. treatment involves surgical removal of the involved testes. bacterial infections of the testicle or epididymis most commonly are caused by ascending infections of the normal bacterial flora of the prepuce or urethra. common inhabitants include escherichia coli, staphylococcus aureus, streptococcus spp., and mycobacterium canis. brucella canis and r. rickettsii are also capable of causing orchitis and epididymitis in the dog. clinical signs of orchitis or epididymitis include testicular enlargement, stiff stilted gait, and reluctance to walk. physical examination often reveals a fever and self-induced trauma to the scrotum from licking or chewing at the inflamed area. collect a semen sample by ejaculation, and culture it to identify the causative organism. alternately, collect samples by needle aspiration of the affected organ(s) and test serologically for b. canis. treatment of infectious orchitis involves a minimum of to weeks of specific antimicrobial therapy, based on culture and susceptibility testing, whenever possible. if a bacterial culture cannot be obtained, initiate fluoroquinolone therapy (enrofloxacin, mg/kg po q h). doxycycline ( mg/kg po bid for days) has been shown to suppress but not eradicate b. canis infection. testicular inflammation and increased temperature can affect sperm quality for months after infection. the most common causes of acute prostatitis are associated with acute bacterial infection (e. coli, proteus spp., pseudomonas spp., and mycoplasma spp.). less common causes include fungal infection (blastomyces dermatitidis) or anaerobic bacterial infection. acute prostatitis is characterized by fever, caudal abdominal pain, lethargy, anorexia, blood in the ejaculate, hematuria, dyschezia, and occasionally stranguria or dysuria. the patient often appears painful and depressed and may be dehydrated on physical examination. symmetric or asymmetric prostatomegaly and prostate pain may be evident on rectal palpation. in severely affected dogs, clinical signs of tachycardia, hyperemic or injected mucous membranes, bounding pulses, lethargy, dehydration, and fever may be present because of sepsis. death can occur within days if a prostatic abscess ruptures. diagnosis of acute prostatitis is confirmed based on the presenting clinical signs, neutrophilic leukocytosis (with or without a left shift), and positive urine culture results. prostatic samples may be obtained from the prostatic portion of the ejaculate, prostatic massage, urethral discharge, urine, or (less commonly) prostatic aspirate. although semen samples can yield positive bacterial cultures, dogs with acute prostatitis are often unwilling to ejaculate. radiography may reveal an enlarged prostate, but this alone does not confirm the diagnosis of prostatitis. an abdominal ultrasound often reveals prostatic abscessation and allows for the collection of samples from the affected area(s) via prostatic aspirate. aspiration of the affected tissue potentially can wick infection into periprostatic tracks. cytologic examination of the patient's ejaculate or prostatic wash from a dog with acute prostatitis reveals numerous inflammatory cells and may contain bacterial organisms. the treatment of a patient with acute prostatitis is directed at correcting dysuria and constipation associated with prostatic enlargement. enrofloxaxin ( mg/kg po sid) can penetrate the inflamed prostatic tissue and is effective in treating gram-negative and mycoplasma spp. infections. ciprofloxacin does not appear to penetrate prostatic tissue as readily. alternatives to enrofloxacin therapy are trimethoprim-sulfamethoxazole ( mg/kg po q h) or chloramphenicol ( - mg/kg po q h) for a minimum of to weeks. castration is recommended because benign prostatic hyperplasia may be a predisposing factor in the development of acute prostatitis. do not perform castration until the patient has been on antibiotic therapy for a minimum of days, to prevent the surgical complication of schirrous cords. finasteride (proscar, mg/kg po q h), an antiandrogen α-reductase inhibitor, may help reduce the size of prostatic tissue until the effects of castration are observed. if a prostatic abscess is present, perform marsupialization, surgical drainage, or ultrasonographic drainage. surgical therapy is associated with a large incidence of complications, including incontinence, chronic drainage from fistulas and stomas, septic shock, and death. fracture of the os penis is an uncommon condition encountered in male dogs. os penis fractures can occur with minimal soft tissue damage but cause hematuria and dysuria. on physical examination, urethral obstruction and crepitus in the penis are found. a lateral abdominal radiograph is usually sufficient to document the fracture. treatment consists of conservative therapy, in most cases, and consists primarily of analgesia administration. if the urethra also is damaged, place a urethral catheter for to days to allow the urethral mucosa to heal. fractures of the os penis that are comminuted or severe enough to cause urethral obstruction require open reduction and fixation, partial penile amputation, or antescrotal (prescrotal) urethrostomy. lacerations of the penis cause significant bleeding because of the extensive vascular supply to the penis. dogs and cats tend to lick penile lacerations and prevent adequate clot formation. sedation or general anesthesia often is required to evaluate and treat the laceration. after sedation or general anesthesia, place a urinary catheter and examine the penis under a stream of cold water. small lacerations can be managed with cold compresses and one to several absorbable sutures. extensive suturing usually is not required. prevent erection by isolating the patient from females in estrus or allowing excitement or excessive activity. place an elizabethan collar to prevent self-mutilation. initiate systemic antibiotic therapy to prevent infection. the inability to withdraw the penis into the prepuce in male dogs or cats is known as paraphimosis. paraphimosis usually develops following an erection in young male dogs and in emergency care older dogs after coitus. mucosal edema, hemorrhage, self-mutilation, and necrosis requiring penile amputation can occur if left untreated. treatment consists of applying cold water to the penis and reducing edema with application of an osmotic substance such as sugar. examine the base of the penis for hair rings that can prevent retraction of the penis into the prepuce. rinse the penis carefully with cold water and lubricate it with sterile lubricant and replace it into the prepuce. if the penis cannot be reduced easily into the prepuce, anesthetize the patient and make a small incision at the lateral aspect of the preputial opening. replace the penis and close the incision with absorbable suture. place a purse-string suture and leave it in place for several days to prevent recurrence. instill topical antimicrobial ointment with steroids into the prepuce several times a day. in severe cases, a urinary catheter may need to be placed to prevent urethral obstruction, until penile swelling and edema resolve. place an elizabethan collar to prevent excessive licking during the healing process. prolapse of the distal urethra is a condition usually confined to intact male english bulldogs, although isolated incidences also have been reported in yorkshire and boston terriers. the exact cause of this condition is unknown but usually is associated with a condition that causes increased intraabdominal pressure or urethral straining, including sexual excitement, coughing, vomiting, obstructed airway or brachycephalic airway syndrome, urethral calculi, genitourinary tract infection, and masturbation. the urethral prolapse usually appears as a mushroom-tip congested, irritated mass at the end of the penis that may or may not bleed (figure - ) . in some cases, bleeding occurs or worsens with sexual excitement. clinical signs associated with the prolapsed urethra include excessive licking of the prepuce, stranguria, and preputial bleeding. once the mass is observed, other differential diagnoses include transmissible venereal tumor, urethral polyp, trauma, urethritis, and neoplasia. in most cases, however, the prolapse occurs in intact young dogs, making neoplastic conditions less likely. treatment for urethral prolapse should occur at the time of diagnosis to prevent selfinduced trauma and infection. immediate therapy includes manual reduction of the prolapsed tissue and placement of a purse-string suture around an indwelling urinary catheter. the purse-string suture can remain in place for up to days until definitive repair. until the time of surgery, place an elizabethan collar on the patient to prevent self-mutilation. several forms of surgical correction have been described. in some cases, surgical resection of the prolapsed tissue with apposition of the urethral and penile mucosa can be attempted. more recently, a technique involving placement of several mattress sutures to reduce and secure the prolapsed tissue has been described. recurrence of prolapse can occur with either technique, particularly if the inciting event recurs. because there may be a genetic predisposition in this breed and because the prolapse can recur with sexual excitement, neutering should strongly be recommended. local freezing or frostbite most commonly affects the peripheral tissues of the ears, tail, paws, and genitalia that are sparsely covered with fur, are poorly vascularized, and may have been traumatized previously by cold. clinical signs of frostbite are paleness and appearance of a blanched pink to white discoloration to the skin. the skin also may appear black and necrotic. immediate treatment consists of slowly rewarming the affected area with moist heat at . ° c ( °f) or by immersion in warm water baths. analgesics may be required to alleviate patient discomfort. carefully dry the injured areas and protect them from further trauma. the use of prophylactic antibiotics is controversial because it can promote resistant bacterial infection. use of antibiotics should be based on the presence of infection. treatments that are ineffective and may be harmful include rubbing the affected areas, pressure bandages, and ointments. corticosteroids can decrease cellular immunity and promote infection and are therefore contraindicated. many frostbitten areas that appear nonviable can regain function gradually. use care when removing areas of necrotic tissue. affected areas may take several days to a week before fully manifesting areas of demarcation between healthy viable and necrotic nonviable tissue. chilling of the entire body from exposure or immersion in extremely cold water results in a decrease in core body temperature and physiologic processes that become irreversible when the body temperature falls below °c ( °f). mild hypothermia can be °to °c, moderate hypothermia from °to °c, and severe hypothermia below °c. the duration of exposure and the general condition of the animal influences its ability to survive. clinical signs and consequences associated with hypothermia include shivering, vasoconstriction, mental depression, hypotension, sinus bradycardia, hypoventilation with decreased respiratory rate, increased blood viscosity, muscle stiffness, atrial and ventricular irritability, decreased level of consciousness, decreased oxygen consumption, metabolic (lactic) acidosis, respiratory acidosis, and coagulopathies including dic. if the animal is breathing, administer warm, humidified oxygen at to breaths per minute. if the animal is not breathing or is severely hypoventilating, endotracheal intubation with mechanical ventilation may be necessary. place an intravenous catheter and infuse warmed crystalloid fluids. if the blood glucose is less than mg/dl, add supplemental dextrose ( . %) to the crystalloid fluids. monitor the core body temperature and ecg closely. rewarming should occur in the form of external circulating warm water blankets, radiant heat, and circulating warm air blankets (bair hugger). never use a heating pad, to avoid iatrogenic thermal burn injury. severe hypothermia may require core rewarming in the form of intraperitoneal fluids ( to ml/kg of lactated ringer's solution warmed to . °c [ °f]). place a temporary peritoneal dialysis catheter, and repeat the dialysis every minutes until the patient's body temperature reaches . °to . °c ( °to °f). the body temperature should rise slowly, ideally no more than °f per hour. because the response of the body to drugs is unpredictable, avoid administering drugs whenever possible, until the body temperature returns to normal. complications observed during rewarming include dic, cardiac dysrhythmias including cardiac arrest, pneumonia, pulmonary edema, cns edema, ards, and renal failure. heat stroke and heat-induced illness in dogs can be associated with excessive exertion, exposure to high environmental temperatures, stress, and other factors that cause an inability to dissipate heat. brachycephalic breeds, obesity, laryngeal paralysis, and older animals with cardiovascular disease can be particularly affected. hyperthermia is defined as a rectal temperature of °to °c ( °to °f). clinical signs of hyperthermia include congested hyperemic mucous membranes, tachycardia, and panting. more severe clinical signs include collapse (heat prostration), ataxia, vomiting, diarrhea, hypersalivation, muscle tremors, loss of consciousness, and seizures. heat-induced illness can affect all major organ systems in the body because of denaturation of cellular proteins and enzyme activities, inappropriate shunting of blood, hypotension, decreased oxygen delivery, and lactic acidosis. cardiac dysrhythmias, interstitial and intracellular dehydration, intravascular hypovolemia, central nervous dysfunction, slough of gastrointestinal mucosa, oliguria, and coagulopathies can be seen as organ function declines. excessive panting can result in respiratory alkalosis. poor tissue perfusion results in a metabolic acidosis. loss of water in excess of solutes such as sodium and chloride can lead to a free water deficit and severe hypernatremia. a marked increase in pcv occurs because of the free water loss. severe abnormalities in electrolytes and ph can lead to cerebral edema and death. treatment goals for the patient with heat-induced illness are to lower the core body temperature and support cardiovascular, respiratory, renal, gastrointestinal, neurologic, and hepatic functions. at the scene the veterinarian or caretaker can spray the animal with tepid (not cold) water. immersion in cold water or ice baths is absolutely contraindicated. cold water and ice will cause extreme peripheral vasoconstriction, inhibiting the patient's ability to dissipate heat through conductive and convective cooling mechanisms. as a result, core body temperature will continue to rise despite the good intentions of well-doers at the scene. animals that present to the veterinarian that have been cooled to the point of hypothermia have a worse prognosis. once the animal has presented to the veterinarian, the goal is to cool the animal's body temperature with towels soaked in tepid water, cool intravenous fluids, and fans until the temperature has decreased to °f. organ system monitoring and support is based on the severity and duration of the heat stroke and the ability of the body to compensate and respond to treatment. management of the patient with heat-induced illness involves prompt aggressive cooling without being overzealous and creating iatrogenic hypothermia. administer cool intravenous crystalloid fluids to replenish volume and interstitial hydration and correct the patient's acid-base and electrolyte abnormalities. management consists of rule of twenty monitoring (see rule of ), taking care to evaluate, restore, and maintain a normal cardiac rhythm, blood pressure, urine output, and mentation. administer antibiotics if there are any signs of gastrointestinal bleeding that will predispose the patient to bacterial translocation. monitor baseline chemistry tests including a complete blood count, biochemical panel, platelet count, coagulation tests, and urinalysis. treat coagulopathies including dic aggressively and promptly (see also disseminated intravascular coagulation). severe changes in mentation including stupor or coma worsen a patient's prognosis. following initial therapy, monitor the patient for a minimum of to hours for secondary organ damage, including renal failure, myoglobinuria, cerebral edema, and dic. dogs that are going to die of heat-induced illness usually die within the first hours. animals that survive longer than hours have a more favorable prognosis. immediate treatment consists of cooling the patient with cooling measures as for hyperthermia and heat-induced illness (see the previous discussion), and eliminating the cause (i.e., exertion, anesthesia, or neuromuscular blockers such as succinylcholine). if the patient is under general anesthesia, hyperventilate the patient to help eliminate carbon dioxide and respiratory acidosis. administer dantrolene sodium ( to mg/kg iv) to stabilize the sarcoplasmic reticulum and decrease its permeability to calcium. animals with malignant hyperthermia should avoid any predisposing factors, including exertion, hyperthermia, and anesthesia. after an episode of malignant hyperthermia, administer crystalloid fluids intravenously to aid in the elimination of myoglobin. monitor renal function closely for myoglobinuria and pigment damage to the renal tubular epithelium. monitor and correct acid-base and electrolyte changes. walters jm: hyperthermia. in wingfield we, editor: the veterinary icu book, jackson, wyo, , teton newmedia. sometimes it is difficult to assess whether an animal has been bitten by a poisonous or nonpoisonous snake. in colorado, the bull snake closely resembles the prairie rattlesnake. both snakes make similar noise and can be alarming if noticed on a hike or in the backyard. whenever possible, identify the offending reptile but never risk being bitten. know what types of venomous creatures are in the geographic area of the practice. if an animal has been bitten by a nonpoisonous snake, usually the bite marks are small with multiple small tooth punctures, and the bite is relatively nonpainful. usually local reaction is negligible. however, large boas or pythons also can inflict large crushing injuries that can cause severe trauma, including bony fractures. treatment for a nonpoisonous snakebite involves clipping the bite wound and carefully cleaning the area with antimicrobial scrub solution. broad-spectrum antibiotics (e.g., amoxicillin-clavulanate, . mg/kg po q h) are indicated because of the extensive bacterial flora in the mouths of snakes. monitor all snakebite victims for a minimum of hours after the incident, particularly when the species of the offending reptile is in question. if clinical signs of envenomation occur, modify the patient's treatment appropriately and aggressively. the two major groups of venomous snakes in north america are the pit viper and the coral snake. all venomous snakes are dangerous. the severity of any given bite depends on the toxicity of the venom, the amount of venom injected, the site of envenomation, the size of the animal bitten, and the time from bite/envenomation to seeking appropriate medical intervention. the majority of reptile envenomations in the united states are inflicted by pit vipers, including the water moccasin (cottonmouth), copperhead, and numerous species of rattlesnakes. pit vipers are characterized by a deep pit located between the eye and nostril, elliptic pupils, and retractable front fangs (figure - ) . localized clinical signs of pit viper envenomation may include the presence of bleeding puncture wounds, local edema close to puncture wounds, immediate severe pain or collapse, edema, petechiae, and ecchymosis with subsequent tissue necrosis. systemic signs of pit viper envenomation may include hypotension, shock, coagulopathies, lethargy, weakness, muscle fasciculations, lymphangitis, rhabdomyolysis, and neurologic signs including respiratory depression and seizures. neurologic signs largely are associated with envenomation emergency management of specific conditions by the mojave and canebrake rattlesnakes, although a potent neurotoxin, mojave toxin a, also has been identified in other subspecies of rattlesnake. clinical signs of envenomation may take several hours to appear. hospitalize all suspected victims and monitor them for a minimum of hours. the severity of envenomation cannot be judged solely on the basis of local tissue reaction. first aid measures by animal caretakers do little to prevent further envenomation. the most important aspect of initiating therapy is to transport the animal to the nearest veterinary emergency facility. to determine whether an animal has been envenomated by a pit viper, examine a peripheral blood smear for the presence of echinocytes. echinocytes will appear within minutes of envenomation and may disappear within hours. other treatment should be initiated as rapidly and aggressively as possible, although controversy exists whether some therapies are warranted. the mainstay of therapy is to improve tissue perfusion with intravenous crystalloid fluids, prevent pain with judicious use of analgesic drugs, and when necessary, reverse or negate the effects of the venom with antivenin. because pit viper venom consists of multiple fractions, treat each envenomation as a complex poisoning. obtain vascular access and administer intravenous crystalloid fluids (one fourth of a calculated shock dose) according to the patient's perfusion parameters of heart rate, blood pressure, and capillary refill time (see also shock and fluid therapy). opioid analgesics are potent and should be administered at the time of presentation. (see also pharmacologic means to analgesia: major analgesics). diphenhydramine ( . to mg/kg im or iv) also can be administered to decrease the effects of histamine. famotidine, a histamine receptor antagonist, also can be administered ( . to mg/kg iv) to work synergistically with diphenhydramine. although antihistamines have no effect on the venom per se, they may have an effect on the tissue reaction to the venom and may prevent an adverse reaction to antivenin. the use of glucocorticosteroids is controversial. glucocorticosteroids (dexamethasone sodium phosphate [dex-sp], . to . mg/kg iv) may stabilize cellular membranes and inhibit phospholipase, an active component of some pit viper toxins. polyvalent antivenin is necessary in many cases of pit viper envenomation, except in most cases of prairie rattlesnake (crotalus viridis viridis) envenomation in colorado. a recent study demonstrated no difference in outcome with or without the use of antivenin in cases of prairie rattlesnake envenomation. clinically, however, patients that receive antivenin are more comfortable and leave the hospital sooner than those that do not receive antivenin. the exact dose of antivenin is unknown in small animal patients. administer a dose of at least vial of antivenin to neutralize circulating venom. mix antivenin with a swirling, rather than a shaking motion, to prevent foaming. mix the antivenin with a -ml bag of . % saline, and then administer it slowly over a period of hours. pretreat animals with diphenhydramine ( . to mg/kg im) before the administration of antivenin, and then monitor the animal closely for clinical signs of angioneurotic edema, urticaria, tachyarrhythmias, vomiting, diarrhea, and weakness during the infusion. administration of antivenin into the bite site is relatively contraindicated and ineffective because uptake is delayed, and systemic effects are the more life-threatening. management of pit viper envenomation largely involves maintenance of normal tissue perfusion with intravenous fluids, decreasing patient discomfort with analgesia, and negating circulating venom with antivenin. hydrotherapy to the affected bite site with tepid water is often soothing to the patient. the empiric use of antibiotics is controversial but is recommended because of the favorable environment created by a snakebite (i.e., impregnation of superficial gram-positive bacteria and gram-negative bacteria from the mouth of the snake into a site of edematous necrotic tissue). administer amoxicillin-clavulanate ( . mg/kg po q h, or cephalexin, mg/kg po q h). also consider administration of nsaids (carprofen, . mg/kg po q h). monitor the patient closely for signs of local tissue necrosis and the development of thrombocytopenia and coagulopathies including dic (see management of disseminated intravascular coagulation). treat coagulopathies aggressively to prevent end-organ damage. coral snakes are characterized by brightly colored bands encircling the body, with red and black separated by yellow. "red on black, friend of jack; red on yellow, kill a fellow." types of coral snakes include the eastern coral, texas coral, and sonoran coral snakes. clinical signs of coral snake envenomation may include small puncture wounds, transient initial pain, muscle fasciculations, weakness, difficulty swallowing/dysphagia, ascending lower motor neuron paralysis, miotic pinpoint pupils, bulbar paralysis, respiratory collapse, and severe hemolysis. clinical signs may be delayed for as long as hours after the initial bite. immediate treatment with antivenin is necessary in cases of coral snake envenomation before the clinical signs become apparent, whenever possible. support respiration during paralysis with mechanical ventilation. secure the patient's airway with a cuffed endotracheal tube to prevent aspiration pneumonia. clinical signs will progress rapidly once they develop. rapid administration with antivenin is the mainstay of therapy in suspected coral snake envenomation. respiratory and cardiovascular support should occur with mechanical ventilation and intravenous crystalloid fluids. keep the patient warm and dry in a quiet place. turn the patient every to hours to prevent atelectasis and decubitus ulcer formation. maintain cleanliness using a urinary catheter and closed urinary collection system. perform passive range of motion and deep muscle massage to prevent disuse atrophy of limb muscles and function. treat aspiration pneumonia aggressively with broad-spectrum antibiotics (ampicillin, mg/kg iv q h, with enrofloxacin, mg/kg iv q h, and then change to oral once tolerated and the patient is able to swallow) for weeks past the resolution of radiographic signs of pneumonia, intravenous fluids, and nebulization with sterile saline and coupage chest physiotherapy. several weeks may elapse before a complete recovery. the adult black widow spider (latrodectus spp.) can be recognized by a red to orange hourglass-shaped marking on the underside of a globous, shiny, black abdomen. the immature female can be recognized by a colorful pattern of red, brown, and beige on the dorsal surface of the abdomen. adult and immature females are equally capable of envenomation. the male is unable to penetrate the skin because of its small size. black widow spiders are found throughout the united states and canada. black widow spider venom is neurotoxic and acts presynaptically, releasing large amounts of acetylcholine and norepinephrine. there appears to be a seasonal variation in the potency of the venom, lowest in the spring and highest in the fall. in dogs, envenomation results in hyperesthesia, muscle fasciculations, and hypertension. muscle rigidity without tenderness is characteristic. affected animals may demonstrate clinical signs of acute abdominal pain. tonic-clonic convulsions may occur but are rare. in cats, paralytic signs predominate and appear early as a ascending lower motor neuron paralysis. increased salivation, vomiting, and diarrhea may occur. serum biochemistry profiles often reveal significant elevations in creatine kinase and hypocalcemia. myoglobinemia and myoglobinuria can occur because of extreme muscle damage. management of black widow spider envenomation should be aggressive in the cat and dog, particularly when the exposure is known. in many cases, however, the diagnosis is made based on clinical signs, biochemical abnormalities, and lack of other apparent cause. antivenin (one vial) is available and should be administered after pretreatment with diphenhydramine. if antivenin is unavailable, administer a slow infusion of calcium-containing fluid such as lactated ringer's solution with calcium gluconate while carefully monitoring the patient's ecg. the small brown nonaggressive spider is characterized by a violin-shaped marking on the cephalothorax. the neck of the violin points toward the abdomen. brown spiders are found primarily in the southern half of the united states but have been documented as far north as michigan. the venom of the brown spider has a potent dermatonecrolytic effect and starts with a classic bull's-eye lesion. the lesion then develops into an indolent ulcer into dependent tissues promoted by complement fixation and influx of neutrophils into the affected area. the ulcer can take months to heal and often leaves a disfiguring scar. systemic reactions are rare but can include hemolysis, fever, thrombocytopenia, weakness, and joint pain. fatalities are possible. immediate management of an animal with brown spider envenomation is difficult because there is no specific antidote and because clinical signs may be delayed until necrosis of the skin and underlying tissues becomes apparent through the patient's fur to days after the initial bite. dapsone has been recommended at a dose of mg/kg for days. surgical excision of the ulcer may be helpful if performed in the early stages of wound appearance. glucocorticosteroids may be of some benefit if used within hours of the bite. the ulcer should be left to heal by second intention. deep ulcers should be treated with antibiotics. bufo toad species (b. marinus, aka cane toad, marine toad, giant toad; and the colorado river toad or sonoran desert toad b. alvarius) can be associated with severe cardiac and neurotoxicity if an animal licks its skin. the severity of toxicity depends largely on the size of the dog. toxins in the cane toad, b. marinus, include catecholamines and vasoactive substances (epinephrine, norepinephrine, serotonin, dopamine) and bufo toxins (bufagins, bufotoxin, and bufotenine), the mechanism of which is similar to cardiac glycosides. clinical signs can range from ptyalism, weakness, ataxia, extensor rigidity, opisthotonus, and collapse to seizures. clinical signs associated with b. alvarius toxicity are limited largely to cardiac dysrhythmias, ataxia, and salivation. the animal should have its mouth rinsed out thoroughly with tap water even before presentation to the veterinarian. if the animal is unconscious or actively seizing and cannot protect its airway, flushing the mouth is contraindicated. once an animal presents to the veterinarian, the veterinarian should place an intravenous catheter and monitor the patient's ecg and blood pressure. attempt seizure control with diazepam ( . mg/kg iv) or pentobarbital ( to mg/kg iv to effect). ventricular dysrhythmias can be controlled first with esmolol ( . mg/kg). if esmolol is ineffective, administer a longer-acting parenteral β-antagonist such as propranolol ( . mg/kg iv). ventricular tachycardia also can be treated with lidocaine ( to mg/kg iv, followed by to µg/kg/minute iv cri). case management largely depends on supportive care and treating clinical signs as they occur. monitor baseline acid-base and electrolyte balance because severe metabolic acidosis may occur that should be treated with intravenous fluids and sodium bicarbonate ( . to meq/kg iv). monitor ecg, blood pressure, and mentation changes closely. control seizures and cardiac dysrhythmias. eubig pa: bufo species intoxication: big toad, big problem, vet med ( ) lizards of the family hemodermatidae are the only two poisonous lizards in the world. they are found in the southwestern united states and mexico. the venom glands are located on either side of the lower jaw. because these lizards are typically lethargic and nonaggressive, bite wounds are rare. the lizards have grooved teeth that introduce the venom with a chewing motion as the lizard holds tenaciously to the victim. the majority of affected dogs are bitten on the upper lip, which is very painful. there are no proven first aid measures for bites from gila monsters or mexican bearded lizards. the lizard can be disengaged by inserting a prying instrument in between the jaws and pushing at the back of the mouth. the teeth of the lizard are brittle and break off in the wound. topical irrigation with lidocaine and probing with a needle will aid in finding and removing the teeth from the victim. bite wounds will bleed excessively. irrigate wounds with sterile saline or lactated ringer's solution, and place compression on the affected area until bleeding ceases. monitor the patient for hypotension. establish intravenous access, and administer intravenous fluids according to the patient's perfusion parameters. antibiotic therapy is indicated because of the bacteria in the lizard's mouth. because no antidote is available, treatment is supportive according to patient signs. the majority of musculoskeletal emergencies are the result of external trauma, most commonly from motor vehicle accidents. blunt trauma invokes injury to multiple organ systems as a rule, rather than an exception. because of this, massive musculoskeletal injuries are assigned a relatively low priority during the initial triage and treatment of a traumatized animal. perform a rapid primary survey and institute any lifesaving emergency therapies. adhere to a crash plan or the abcs of resuscitation (see initial emergency examination, management, and triage). although musculoskeletal injuries are assigned a relatively lower priority, the degree of recovery from these injuries and financial obligation for fracture repair sometimes becomes a critical factor in a client's decision whether to pursue further therapy. one of the most important deciding factors is the long-term prognosis for the patient to have a good quality of life following fracture repair. the initial management of musculoskeletal injuries is important in ensuring the best chance for maximal recovery with minimal complications after definitive surgical fracture repair. this is particularly important for open fractures, spinal cord compromise, multiple fractures, open joints, articular fractures, physeal fractures, and concomitant ligamentous or neurologic compromise (box - ). immediately after the initial primary survey of a patient, perform a more thorough examination, including an orthopedic examination. multiple injuries often are observed in the patient that falls from height (e.g., "high-rise syndrome"), motor vehicle accidents, gunshot wounds, and encounters with other animals (e.g., "big-dog-little-dog"). address the most life threatening injuries, and palliate musculoskeletal injuries until more definitive repair can be attempted when the patient is more stable. in animals with the history of potential for multiple injuries, search thoroughly and meticulously for areas of injury to the spinal column, extremities, and for small puncture wounds. helpful signs that can provide a clue as to an underlying injury include swelling, bruising, abnormal motion, and crepitus (caused by subcutaneous emphysema or bony fracture). if the patient is alert, look for areas of tenderness or pain. in unconscious or depressed patients, reexamine the patient after the patient becomes more mentally alert. injuries often are missed during the initial examination in obtunded patients because of the early response and attenuation of pain. unconscious or immobile patients must have radiographic examination of the spinal column following stabilization and support. palpate the skull carefully for obvious depressions or crepitus that may be associated with a skull fracture. localization of the injury can be determined by motion in abnormal locations, swelling caused by hemorrhage or edema, pain during gentle movement or palpation, deformity, angular change, or a significant increase or decrease in normal range of motion of bones and joints. perform a rectal examination in all cases to palpate for pelvic fractures and displacement. once the diagnosis of a fracture or luxation has been confirmed, look for any evidence of skin lacerations or punctures near the fracture site. in long-haired breeds, clipping the fur near the fracture site often is necessary to perform a thorough examination of the area. if any wounds are found, the fracture is classified as an open fracture until proven otherwise. in some cases, the open fracture is obvious, with a large section of bone fragment protruding through the skin. in other cases, the puncture wound may be subtle, with only a small amount of blood or pinpoint hole in the skin surface. characteristics observed with open fractures include bone penetration, fat droplets or marrow elements in blood coming from the wound, subcutaneous emphysema on radiographs, and lacerations in the area of a fracture. protect the patient from further injury or contamination of wounds. excessive palpation to intentionally produce crepitus is inappropriate because it causes severe patient discomfort and has the potential to cause severe soft tissue and neurologic injury at the fracture site. sedation and analgesia aids in making the examination more comfortable for the patient and allows localization of the injury and comparison with the opposite extremity. higher-quality radiographs can be performed to determine the extent of the injury when the animal is sedated adequately and pain is controlled. sedate the patient judiciously with analgesic drugs. opioid drugs work well for orthopedic pain, produce minimal cardiorespiratory depression, and can be reversed with naloxone if necessary. handle the fracture site gently to avoid causing further pain and soft tissue injury at the fracture site. rough or careless handling of a fracture site can cause a closed fracture to penetrate through the skin and become an open fracture. cover open fractures immediately to prevent contamination of the fracture with nosocomial infection from the hospital. administer a first-generation cephalosporin (cephalexin, mg/kg po q h, or cefazolin, mg/kg iv q h). the bandage also serves to control hemorrhage and prevent desiccation of the bones and surrounding soft tissue structures. leave the initial bandages in place until the patient's cardiorespiratory status has been determined to be stable and more definitive wound management can occur in a clean, preferably sterile location. examine the neurologic status and cardiovascular status of the limb before and after treatment. determine the vascular status of the limb by checking the color and temperature of the limb, the state of distal pulses, and the degree of bleeding from a cut nail bed. in patients with severe cardiovascular compromise and hypotension caused by hemorrhagic shock, the viability of the limb may be in question until the cardiovascular status and blood pressure are normalized. reduction of the fracture or straightening of gross deformities may return normal vascularity to the limb. when checking neurologic status, examine for motor and sensory function to the limb. swelling may increase pressure on the nerves as they run through osteofascial compartments, resulting in decreased sensory or motor function, or neurapraxia. diminished function often returns to normal once the swelling subsides. serial physical examinations in the patient and response to initial stabilization therapy can lead to a higher index of suspicion that more occult injuries are present, such as a diaphragmatic hernia, perforated bowel, lacerated liver or spleen, or uroabdomen. to prevent ongoing trauma, reduce any fracture and then stabilize the site above and below the fracture. a modified robert jones splint or bandage often works well for fractures emergency management of specific conditions involving the distal extremities. fractures of the humerus or femur are difficult to immobilize without the use of spica or over-the-hip coaptation splints to prevent mobility. inappropriate bandaging of humerus or femur fractures can result in a fulcrum effect and worsen the soft tissue and neurologic injuries. further displacement of vertebral bodies or luxations can cause cord compression or laceration such that return to function becomes impossible. immediately place any patient with a suspected spinal injury on a flat surface, and tape down the animal to prevent further movement until the spine has been cleared by a minimum or two orthogonal radiographic views (lateral and ventrodorsal views performed as a cross-table x-ray technique). wounds associated with musculoskeletal trauma are common and include injury to the bones, joints, tendons, and surrounding musculature (box - ). major problems associated with these cases are the presence of soft tissue trauma that makes wound closure hazardous or impossible, because of the risk of infection. chronic deep infection of traumatized wounds can cause delayed healing and sequestrum to develop, particularly if there is avascular bone or cartilage within the wound. in the early management of an open fracture, the areas should be splinted without pulling any exposed bone back into the soft tissue. the wound should not be probed or soaked, as nosocomial bacteria and other external contaminants can be introduced into the wound, leading to severe infection. because of the risk of actually causing infection, probing, flushing, or replacing tissues back into the wound should be performed at the time of formal debridement when the patient is physiologically stable. immediate bactericidal antibiotic therapy with a first-generation cephalosporin should be started immediately to obtain adequate concentrations of antibiotics at the fracture site. the duration of antibiotic therapy should ideally be limited to - days to prevent the risk of superinfection. treatment of open musculoskeletal injury involves three considerations: initial inspection and wound debridement, stabilization and repair, and wound bandaging. emergency care when associated with a fracture, wound is created from the inside out by penetration of bone fragments through the skin or from a low-energy gunshot. simple or comminuted fracture pattern good stability of the two main bone segments treatment and prognosis are good and similar to those of a closed injury if wound is debrided and stabilized within to hours. when associated with a fracture, wound is created from the outside in. major deep injury with considerable soft tissue stripping from bone and muscle damage simple or comminuted fracture pattern prognosis is good if wound is debrided within hours of injury and provided rigid stabilization with a bone plate or external fixator. results from major external force severe damage and necrosis of skin, subcutaneous tissue, muscle, nerve, bone, tendon, and arteries soft tissue damage may vary from crush injury to shearing injury associated with bite wounds or low-speed automobile accidents. requires immediate and delayed sequential debridement and rigid external fixation can require prolonged healing times guarded prognosis initial inspection and wound debridement include the following steps: . after the patient's cardiovascular status has been stabilized and it has been determined that it can withstand anesthesia, place the animal under general anesthesia and remove the temporary splint. . keeping the wound covered, shave the surrounding fur. . remove the covering and then place sterile lubricant jelly over the wound. shave the fur to the edges of the wound margin. . wash away any entrapped fur and the lubricant jelly. . complete an antiseptic scrub of the surrounding skin. . if the wound is a small puncture (e.g., gunshot pellets or bites), probe the wound with a sterile hemostat. do a thorough debridement if tissues deep to the hole are cavitated. if not deep, create a hole for drainage. . flush the wound with a physiologic solution (lactated ringer's solution is preferred). . debride the wound from outward to inward. cut away damaged areas of skin and deeper tissues to open up underlying cavitations and tissue injury. . continuously irrigate with warm physiologic solution (lactated ringer's solution is preferred). the stream must be strong enough to flush debris out of the bottom of the wound. to accomplish this, attach a -gauge needle to a -ml syringe (will deliver psi). excise any obviously devitalized tissue. . do not remove any bone fragments that are firmly attached to soft tissue. do not cut into healthy soft tissue to find bullet or bone fragments, unless the bullet can cause injury to joints or nerve tissue. . do a primary repair of tendons and nerves if the wound is type i and recent (within hours of the initial injury). if the wound is too severe or if there is obvious infection, tag the ends of the tendons and nerves for later repair. it is best to stabilize and repair open fractures as soon as the patient's cardiovascular and respiratory status can tolerate general anesthesia, provided that adequate stabilization is possible. if this is not possible because of the level of experience of the surgeon or the lack of necessary equipment, it is best to perform wound management and place a temporary splint until definitive repair can be performed. wound bandaging is discussed in the section on bandaging techniques. structural injuries to the joints are common and can involve both ligaments and articular cartilage injuries. cartilage does not heal well; therefore, injuries involving articular cartilage can lead to a significant loss of function and degenerative joint disease (osteoarthritis). cartilage injuries that are superficial evoke a short-lived enzymatic and metabolic response that does not stimulate enough cellular growth to repair the defect. superficial lesions remain as defects but do not progress to chondromalacia or osteoarthritis. deep cartilage lacerations that extend to subchondral bone produce an exuberant healing response from the cells of the underlying cartilage. in many cases, this material undergoes degeneration and leads to osteoarthritis. impact injuries to surface cartilage can cause chondrocyte and underlying bone injury. these lesions rapidly progress to osteoarthritis; however, they may be totally or partially reversible. treatment of grade i injuries requires short-term coaptation splints and has a good prognosis. grade ii injuries require surgical treatment with a suture stent and consistent postoperative coaptation splints to heal and maintain good function. healing of grade iii injuries often is a problem, and suture stents or surgical reapproximation may be indicated. failure to immobilize joints that are frequently flexed (elbow and stifle) can result in late complications of ligament repair. ligamentous injuries of joints, particularly the collateral ligaments of the stifle, elbow, and hock, and carpal hyperextension injuries are commonly missed and may require surgical fixation, including arthrodesis (box - ). fractures in immature animals differ from those in adults in that young puppies and kittens have a great ability to remodel bone. remodeling is dependent on the age of the patient and the location of the fracture. the younger the puppy or kitten and the closer the fracture to the epiphysis or growth plate, the greater the potential for remodeling and the development of angular limb deformities. remodeling occurs more effectively in longlimbed breeds of dogs than in short-limbed breeds. fractures through the growth plate of immature animals may potentially cause angular limb deformities, joint dislocations or incongruity, and osteoarthritis. this form of injury is commonly observed in the distal ulnar growth plate and the proximal and distal radial growth plates. high-rise syndrome in cats is seen in cats that fall from a height usually greater than feet. it occurs most frequently in high-rise buildings in urban areas where cats lie on window ledges and suddenly fall out the window. the most common lesions observed in cats that fall from heights are thoracic injuries (rib and sternal fractures, pneumothorax, and pulmonary contusions) and facial and oral trauma (lip avulsions, mandibular symphyseal fractures, fractures of the hard palate, and maxillary fractures). limb and spinal cord fractures and luxations, radius and ulna fractures, abdominal trauma, urinary tract trauma, and diaphragmatic hernias are also common. the injuries sustained are often found in combination, rather than as an isolated injury of one area of the body. follow the mnemonic a crash plan when managing a cat suffering from high-rise syndrome, treating the animal immediately for shock. following cardiovascular and respiratory stabilization, evaluate thoracic and abdominal radiographs, including those of the spine. evaluate the bladder closely, making sure that the cat is able to urinate effectively. examine the hard palate, maxilla, and mandibular symphysis for fractures. palpate the pelvis and carefully manipulate all limbs to examine for fractures or ligamentous injuries. finally, perform a complete neurologic examination. patients that fall less than five stories often have a more guarded prognosis than patients that fall from higher levels. sometimes the owner witnesses the ingestion of a foreign body during play, such as throwing a stick or fetching a ball. cats tend to play with string or thread that becomes caught around the base of the tongue. in many cases, however, ingestion of the foreign object is not witnessed, and diagnosis is made based on clinical signs and physical examination. foreign bodies lodged in the oral cavity often cause irritation and discomfort, including difficulty breathing and difficulty swallowing. often, an animal paws at its mouth in an attempt to dislodge a stick or bones wedged across the roof of the mouth. irritation, inability to close the mouth, and blockage of the orpharynx can result in excessive drooling. the saliva may appear blood-tinged due to concurrent soft tissue trauma (figs - and - ) . obstruction of the glottis by a foreign body (e.g., tennis ball or toy) can result in cyanosis secondary to an obstructed airway and hypoxemia. in many cases, the object is small enough to enter the larynx but too large to be expelled. if a foreign object is lodged in the mouth for more than several days, halitosis and purulent discharge may be present. many animals are anxious at the time of presentation and may require sedation or a light plane of anesthesia to remove the foreign object. the animal may bite personnel and may have bitten the owner during his or her attempt to remove the object from the mouth en route to the hospital. propofol ( mg/kg iv) or a combination of propofol with diazepam ( . - mg/kg iv) is an excellent combination for a light plane of anesthesia. exercise caution when anesthetizing a patient with a ball lodged in the airway, as further compromise of respiratory function may occur and cause worsening of the hypoxemia. before inducing anesthesia, assemble all supplies necessary to remove the object. make sure that rigid towel clamps, sponge forceps, and bone forceps are on hand, because the foreign object is often very slippery with saliva. hemostats and carmalts may slip and not be useful in the removal of the foreign object. place a peripheral intravenous catheter to secure vascular access prior to anesthetic induction. have available the supplies necessary for an emergency tracheostomy, if the foreign object cannot be removed by usual methods. induce a light plane of anesthesia and then grasp the object with the sponge forceps or towel clamps, and extract. monitor the cardiorespiratory status of the animal at all times during the extraction process. if you are unable to remove the object, and if severe respiratory distress, including cyanosis, bradycardia, or ventricular dysrhythmias, develop, perform a tracheostomy distal to the site of obstruction. once the foreign body has been removed, administer supplemental flow-by oxygen until the animal awakens. if laryngeal edema or stridor on inspiration is present, administer a dose of dexamethasone sodium phosphate ( . mg/kg iv, im, sq) to decrease inflammation. the patient should be carefully monitored for hours, because noncardiogenic pulmonary edema can develop secondary to airway obstruction. esophageal foreign bodies pose a serious medical emergency. it is helpful if the owner witnessed ingestion of the object and noted rapid onset of clinical signs. in many cases, however, ingestion is not witnessed, and the diagnosis must be made based on clinical signs, thoracic radiographs, and results of a barium swallow. the most common clinical signs are excessive salivation with drooling, gulping, and regurgitation after eating. many animals will make repeated swallowing motions. some animals exhibit a rigid "sawhorse" stance, with reluctance to move immediately after foreign body ingestion and esophageal entrapment. after completing a physical examination, evaluate cervical and thoracic radiographs to determine the location of the esophageal obstruction. esophageal foreign objects are lodged most commonly at the base of the heart, the carina, or just orad to the lower esophageal sphincter. if the object has been lodged for several days, pleural effusion and pneumomediastinum may be present secondary to esophageal perforation. endoscopy is useful for both diagnosis and removal of the foreign object; however, it is invasive and requires general anesthesia ( fig. - ) . remove foreign objects lodged in the esophagus with a rigid or flexible endoscope after the patient has been placed under general anesthesia. evaluate the integrity of the esophagus both before and after removal of the material because focal perforation or pressure necrosis can be present. necrosis of the mucosa and submucosa of the esophagus often leads to stricture formation or perforation. attempt to retrieve the object with a flexible fiberoptic endoscope if available. rigid tube endoscopy can also be performed. in many cases, smooth objects that cannot be easily grasped can be pushed into the stomach and allowed to dissolve or may be removed by gastrotomy. if the foreign body is firmly lodged in the esophagus and cannot be pulled or pushed into the stomach, or if perforation has already occurred, the prognosis for return to function without strictures is not favorable. in such cases, referral to a surgical specialist is recommended for esophagostomy or esophageal resection. after removal of the object, carefully examine the esophagus and then administer gastroprotectant agents (famotidine, . mg/kg po bid; sucralfate slurry, . - . g/dog) for a minimum of to days. to rest the esophagus, the patient should receive nothing per os (npo) for to hours. if esophageal irritation or erosion is moderate to severe, a percutaneous gastrotomy tube should be placed for feeding until the esophagus heals. perform repeat endoscopy every days to evaluate the healing process and to determine whether stricture formation is occurring. persistent vomiting immediately or soon after eating is often associated with a gastric foreign body. in some cases, the owner knows that the patient has ingested a foreign body of some kind. in other cases, continued vomiting despite lack of response to conservative treatment (npo, antiemetics, gastroprotectant drugs) prompts further diagnostic procedures, including abdominal radiographs and bloodwork. obstruction to gastric outflow and vomiting of hydrochloric acid often cause a hypochloremic metabolic acidosis. radiopaque gastric foreign bodies may be observed on plain films. radiolucent cloth material may require a barium series to delineate the shape and location of the foreign body ( fig. - ) . treatment consists of removal with flexible endoscopy or a simple gastrotomy. most animals with uncomplicated gastric foreign bodies are relatively healthy, but any metabolic and electrolyte abnormalities should be corrected prior to anesthesia and surgery. small intestinal obstruction can be caused by foreign bodies, tumors, intussusception, volvulus, or strangulation within hernias. regardless of the cause, clinical signs of small intestinal obstruction depend on the location and degree of obstruction, and whether the bowel has perforated. clinical signs associated with a high small intestinal obstruction are usually more severe and more rapid in onset compared with partial or complete obstruction of the jejunum or ileum. complete obstructions that allow no fluid or chyme to pass are worse than partial obstructions, which can cause intermittent clinical signs interspersed with periods of normality (table - ). the most common clinical signs associated with a complete small intestinal obstruction are anorexia, vomiting, lethargy, depression, dehydration, and sometimes abdominal pain. early clinical signs may be limited to anorexia and depression, making a diagnosis challenging unless the owner has a suspicion that the animal ingested some kind of foreign object. obstructions cranial to the common bile duct and pancreatic papillae lead to vomiting of gastric contents, namely hydrochloric acid, and a hypochloremic metabolic alkalosis. obstructions caudal to the common bile duct and pancreatic papillae result in loss of other electrolytes and sometimes mixed acid-base disorders. eventually, all animals with small intestinal obstruction vomit and have fluid loss into dilated segments of bowel, leading to dehydration and electrolyte abnormalities. increased luminal pressure causes decreased lymphatic drainage and bowel edema. the bowel wall eventually becomes ischemic and may rupture. linear foreign bodies should be suspected in any vomiting patient, particularly cats. string or thread often is looped around the base of the tongue and can be visualized in many cases by a thorough oral examination. to look properly under the tongue, grasp the top of the animal's head with one hand, and pull the lower jaw open with the index finger of the opposite hand while pushing up the thumb simultaneously on the tongue in between the intermandibular space. thread and string can be observed lying along the ventral aspect of the tongue. in some cases, if a linear foreign body is lodged very caudally, it cannot be visualized without heavy sedation or anesthesia. linear foreign bodies eventually cause bowel obstruction and perforation of the intestines along the mesenteric border. the foreign material (e.g., string, thread, cloth, pantyhose) becomes lodged proximally, and the intestines become plicated as the body attempts to push the material caudally through the intestines ( fig. - ) . continued peristalsis eventually causes a sawing motion of the material and perforation of the mesenteric border of the intestines. once peritonitis occurs, the prognosis is less favorable unless prompt and aggressive treatment is initiated. reevaluate any patient that does not respond to conservative symptomatic therapy, performing a complete blood count, serum biochemical panel (including electrolytes), and abdominal radiographs. intestinal masses may be palpable on physical examination and are often associated with signs of discomfort or pain when palpating over the mass. radiography and abdominal ultrasound are the most useful diagnostic aids. plain radiographs may be diagnostic when the foreign object is radiodense or there is characteristic dilation or plication of bowel loops. as a rule of thumb, the width of a loop of small bowel should be no larger than twice the width of a rib. diagnosis of small intestinal obstruction or ileus can be based on the appearance of stacking loops of dilated bowel. comparison of the width of the bowel with the width of a rib is often performed. with mild dilation, the bowel width is three to four times the rib width; with extensive dilation, five to six times the rib width ( fig. - ) . in cases of linear foreign bodies, c-areas (comma-shaped areas) of gas trapped in the plicated bowel will appear stacked on one another. blunt, wedge-shaped areas of gas or square linear areas of gas adjacent to a distended bowel loop are characteristic of a foreign body lodged in the intestine. contrast radiography is indicated when confirmation of the suspected diagnosis is necessary and ultrasonography is not available. contrast material may outline the object or abruptly stop orad to the obstruction. the definitive treatment of any type of small intestinal foreign body is surgical removal. linear foreign bodies sometimes pass, but they should never be left untreated in a patient that is demonstrating clinical signs of inappetence, vomiting, lethargy, and dehydration. the timing of surgery is critical because the risk of intestinal perforation increases with time. prior to surgery, correct any acid-base and electrolyte abnormalities with intravenous fluid therapy. administer broad-spectrum antibiotics. perform an enterotomy or intestinal resection and anastomosis as soon as possible once the patient's acid-base and electrolyte status have been corrected. clinical signs of a foreign body in the large bowel are usually nonexistent. in most cases, if a foreign object has passed successfully through the small bowel, it will pass through the large bowel without incident unless bowel perforation and peritonitis occur. penetrating foreign bodies such as needles often cause localized or generalized peritonitis, abdominal pain, and fever. hematochezia may be present if the foreign object causes abrasion of the rectal mucosa. symptomatic patients should have abdominal radiographs performed. colonoscopy or exploratory laparotomy should be performed if survey radiographs are suggestive of a large intestinal obstruction or perforation. in most cases, large intestinal foreign bodies will pass without incident. surgery is required to treat perforations, peritonitis, or abscesses. emergency care figure - : after minutes, the barium has stopped moving and has reached a blunt, intraluminal intestinal foreign body. note that barium appears wedge-shaped or square at the site of the foreign body. foreign bodies in the rectum and anus often are the result of ingestion of bones, wood material, needles, and thread, or malicious external insertion. often the material can pass through the entire gastrointestinal tract and then get stuck in the anal ring. clinical signs include hematochezia and dyschezia with straining to defecate. diagnosis is made by visual examination of the item in the anus, or by careful digital palpation after heavy sedation or short-acting general anesthesia. radiography is helpful in locating needles that have penetrated the rectum and lodged in the perirectal or perinatal tissues. treatment consists of careful removal of the needle digitally or surgically. intussusception is the acute invagination of one segment of bowel (the intussusceptum) into another (the intussuscipiens). the proximal segment always invaginates into the distal segment of bowel. intussusception most commonly occurs in puppies and kittens less than year of age but can occur in an animal of any age with hypermotility of the small bowel, gastrointestinal parasites, and severe viral or bacterial enteritis. intussusception occurs primarily in the small bowel in the jejunum, ileum, and ileocolic junction. clinical signs include vomiting, abdominal discomfort, and hemorrhagic diarrhea. usually, hemorrhagic diarrhea is the first noticeable sign, and in puppies, may be due to parvoviral enteritis, with secondary intussusception. usually, the obstruction is partial with mild clinical signs. more serious clinical signs develop as the obstruction becomes more complete. differential diagnoses include hemorrhagic gastroenteritis, parvoviral enteritis, gastrointestinal parasites, intestinal foreign body, bacterial enteritis, and other causes of vomiting and diarrhea. the diagnosis of intussusception is often made based on palpation of a sausage-shaped firm, tubular structure in the abdomen accompanied by clinical signs and abdominal pain. plain radiographs may demonstrate segmental or generalized dilated segments of bowel, depending on the duration of the problem. ultrasonographs of the palpable mass resemble the layers of an onion, with hyperechoic intestinal walls separated by less echogenic edema. treatment consists of correction of the patient's acid-base and electrolyte abnormalities with intravenous fluids and surgical reduction or removal of the intussusception with resection and anastomosis. although enteroplication has been suggested, the technique has fallen out of favor because of the increased risk of later obstruction. the primary cause of intestinal inflammation and hypermotility must be identified and corrected. gastric dilatation can occur with or without volvulus in the dog. gastric dilatationvolvulus (gdv) occurs primarily in large-and giant-breed dogs with deep chests, such as the great dane, labrador retriever, saint bernard, german shepherd dog, gordon and irish setters, standard poodle, bernese mountain dog, and bassett hound. the risk of gdv increases with age; however, it can be seen in dogs as young as months. deep, narrow-chested breeds are more likely to develop gdv than dogs with broader chests. the overall mortality for surgically treated gastric dilatation-volvulus ranges from % to %, with most deaths occurring in patients that required splenectomy and partial gastrectomy. clinical signs of gdv include abdominal distention, unproductive vomiting or retching, lethargy, weakness, sometimes straining to defecate, and collapse. the owner may think that the animal is vomiting productively because of the white foamy froth (saliva) that is not able to pass into the twisted stomach. in some cases, there is a history of the dog's being fed a large meal or consuming a large quantity of water prior to the onset of clinical signs. instruct the owner of any patient with a predisposition for and clinical signs of gdv to transport the animal to the nearest veterinary facility immediately. physical examination often reveals a distended abdomen with a tympanic area on auscultation. in dogs with very deep chests, it may be difficult to appreciate abdominal distention if the stomach is tucked up under the rib cage. depending on the stage of shock, the patient may have sinus tachycardia with bounding pulses, cardiac dysrhythmias with pulse deficits, or bradycardia. the mucous membranes may appear red and injected or pale with a prolonged capillary refill time. the patient may appear anxious and attempt to retch unproductively. if the patient is nonambulatory at the time of presentation, the prognosis is more guarded. the definitive diagnosis of gdv is based on clinical signs, physical examination findings, and radiographic appearance of gas distention of the gastric fundus with dorsocranial displacement of the pylorus and duodenum (the so-called "double-bubble" or "popeye arm" sign) ( fig. - ) . in simple gastric dilatation without volvulus, there is gas distention of the stomach with anatomy appearing normal on radiography. with "food bloat," or gastric distention from overconsumption of food, ingesta is visible in the distended stomach ( fig. - ) . as soon as a patient presents with a possible gdv, place a large-bore intravenous catheter in the cephalic vein(s) and assess the patient's ecg, blood pressure, heart rate, capillary refill time, and respiratory function. obtain blood samples for a complete blood count, serum biochemistry profile, immediate lactate measurement, and coagulation tests before taking any radiographs. rapidly infuse a colloid (hetastarch or oxyglobin, ml/kg iv bolus) along with shock volumes of a crystalloid fluid (up to ml/kg/hour) (see section on shock). monitor perfusion parameters (heart rate, blood pressure, capillary refill time, and ecg) and titrate fluid therapy according to the patient's response. the use of short-acting glucocorticosteroids is controversial. glucocorticosteroids may help stabilize cellular membranes and decrease the mechanisms of ischemia-reperfusion injury, but no detailed studies have proved them to be beneficial versus not using glucocorticosteroids in the patient with gdv. attempt gastric decompression, either with placement of an orogastric tube or by trocharization. to place an orogastric tube, position the distal end of the tube at the level of the patient's last rib ( fig. - ) and place it adjacent to the animal's thorax; then put a piece of tape around the tube where it comes out of the mouth, once it is in place. put a roll of -inch tape in the patient's mouth behind the canine teeth and then secure the roll in place by taping the mouth closed around the roll of tape. lubricate the tube with lubricating jelly and slowly insert the tube through the center of the roll of tape into the stomach. the passing of the tube does not rule out volvulus. in some cases, the front legs of the patient need to be elevated, and the caudal aspect of the patient lowered (front legs standing on a table with back legs on the ground) to allow gravity to pull the stomach down to allow the tube to pass. once the tube has been passed, air within the stomach is relieved, and the stomach can be lavaged. the presence of gastric mucosa or blood in the efflux from the tube makes the prognosis more guarded. if an orogastric tube cannot be passed, clip and aseptically scrub the patient's lateral abdomen and then insert -gauge over-the-needle catheter. "pinging" the animal's side with simultaneous auscultation allows determination of the location that is most tympanic-that is, the proper location for catheter insertion. once intravenous fluids have been started in the animal, take a right lateral abdominal radiograph to document gdv. if no volvulus is present, the owner may elect for more conservative care, and the animal should be monitored in the hospital for a minimum of hours. because some cases of gdv intermittently twist and untwist, the owner should be cautioned that although the stomach is not twisted at that moment, a volvulus can occur at any time. if radiographs demonstrate food bloat, induce emesis (apomorphine, . mg/kg iv) or perform orogastric lavage under general anesthesia. documentation of gastric dilatation-volvulus constitutes a surgical emergency. figure - : example of "food bloat" with severe gastric distention caused by overconsump-following diagnosis of gdv, continue administration of intravenous fluids. serum lactate measurements greater than . mmol/l are associated with an increased risk of gastric necrosis, requirement for partial gastrectomy, and increased mortality. administer fresh frozen plasma ( ml/kg) to patients with thrombocytopenia or prolonged pt, activated partial thromboplastin time (aptt), or activated clotting time (act). cardiac dysrhythmias, particularly ventricular dysrhythmias, are common in cases of gdv and are thought to occur secondary to ischemia and proinflammatory cytokines released during volvulus and reperfusion. lidocaine ( - mg/kg followed by mcg/kg/minute iv cri) can be used to treat cardiac dysrhythmias preemptively that are associated with ischemia-reperfusion injury, or administration can be started when ventricular dysrhythmias are present. correct any electrolyte abnormalities, including hypokalemia and hypomagnesemia. the use of nonsteroidal antiinflammatory drugs (flunixin meglumine, carprofen, ketoprofen) that can potentially decrease renal perfusion and predispose to gastric ulcers is absolutely contraindicated. administer analgesic drugs (fentanyl, µ/kg iv bolus, followed by - µ/kg/hour iv cri; or hydromorphone, . mg/kg iv) before anesthetic induction. after carrying out a balanced anesthesia protocol, the patient should be taken immediately to surgery for gastric derotation and gastropexy. postoperatively, assess the patient's ecg, blood pressure, platelet count, coagulation parameters, and gastric function (see section on rule of twenty). if no resection is required, the animal can be given small amounts of water beginning hours after surgery. depending on the severity of the patient's condition, small amounts of a bland diet can be offered to hours postoperatively. continute supportive care with analgesia and crystalloid fluids until the patient is able to tolerate oral analgesic drugs (tramadol, - mg/kg po q - h). once the patient is ambulatory and able to eat and drink on its own, it can be released from the hospital; instruct the owner to feed the animal multiple small meals throughout the day for the first week. when the intestines twist around the root of the mesentery, a small intestinal or mesenteric volvulus occurs. the problem is most common in the young german shepherd dog, although it has been observed in other large and giant breeds. predisposing factors include pancreatic atrophy, gastrointestinal disease, trauma, and splenectomy. clinical signs of mesenteric volvulus include vomiting, hemorrhagic diarrhea, bowel distention, acute onset of clinical signs of shock, abdominal pain, brick-red mucous membranes (septicemia), and sudden death. diagnosis is based on an index of suspicion and the presence of clinical signs in a predisposed breed. plain radiographs often reveal grossly distended loops of bowel in a palisade gas pattern. in some dogs, multiple, tear-drop-shaped, gas-filled loops appear to rise from a focal point in the abdomen. usually, massive distention of the entire small bowel is observed ( fig. - ) . the presence of pneumoperitoneum or lack of abdominal detail secondary to the presence of abdominal fluid is characteristic of bowel perforation and peritonitis. in a patient with mesenteric volvulus, immediate aggressive action is necessary for the animal to have any chance of survival. treatment consists of massive volumes of iv crystalloid and colloid fluids (see section on iv therapy), broad-spectrum antibiotics (ampicillin, mg/kg iv qid, with enrofloxacin, mg/kg iv once daily), and surgical correction of the bowel. because of the massive release of proinflammatory cytokines, bacterial translocation, and ischemia, treatment for shock is of paramount importance (see sections on rule of twenty and shock). prognosis for any patient with mesenteric volvulus is poor. obstipation (obstructive constipation) is most common in the older cat. in cases of simple constipation, rehydrating the animal with intravenous fluids and stool softeners is often volvulus. this consistutes an immediate surgical emergency, and the prognosis is often poor. this condition is most common in young german shepherd dogs, but can be observed in any breed. sufficient for it to regain the ability to have a bowel movement. obstipation, however, is caused by adynamic ileus of the large bowel that eventually leads to megacolon. affected cats usually are anorectic, lethargic, and extremely dehydrated. treatment consists of rehydration with intravenous crystalloid fluids, correction of electrolyte abnormalities, enemas, and promotility agents such as cisapride ( . mg/kg po q - h). the use of phosphate enemas in cats is absolutely contraindicated because of the risk of causing acute, fatal hyperphosphatemia. in many cases, the patient should be placed under general anesthesia and manual deobstipation is performed with warm water soapy enemas and a gloved finger to relieve and disimpact the rectum. stool softeners such as lactulose and docusate stool sofener (dss) may also be used. predisposing causes of obstipation such as narrowing of the pelvic canal, perineal hernia, and tumors should be ruled out. adenocarcinoma is the most common neoplasm of the gastrointestinal tract that causes partial to complete obstruction. adenocarcinomas tend to be annular and constricting, and they may cause progressive obstruction of the lumen of the small or large bowel. siamese cats tend to have adenocarcinomas in the small intestine, whereas in dogs, the tumor tends to occur in the large intestine. clinical signs of adenocarcinoma are both acute and chronic and consist of anorexia, weight loss, and progressive vomiting that occur over weeks to months. effusion may be present if metastasis to peritoneal surfaces has occurred. diagnosis is based on clinical signs and physical examination findings of a palpable abdominal mass, radiographic evidence of an abdominal mass and small or large intestinal obstruction, or ultrasonographic evidence of an intestinal mass. treatment consists of surgical resection of the affected bowel segment. the prognosis for long-term survival ( - months) is good if the mass is completely resected and if other clinical signs of cachexia or metastasis are observed at the time of diagnosis. median survival is to weeks if metastasis to lymph nodes, liver, or the peritoneum are absent at the time of diagnosis. in dogs, the prognosis is more guarded. leiomyoma and leiomyosarcoma are tumors that can cause partial or complete obstruction of the bowel. clinical signs are often referred to progressive anemia, including weakness, lethargy, inappetence, and melena. hypoglycemia can be observed as a paraneoplastic syndrome, or due to sepsis and peritonitis secondary to bowel perforation. leiomyomas are most commonly observed at the ceco-colic junction or in the cecum. surgical resection and anastomosis is usually curative, and has a favorable prognosis. incarceration of a loop of bowel into congenital or acquired defects in the body wall can cause small bowel obstruction. pregnant females and young animals with congenital hernias are most at risk. rarely, older animals with perineal hernias and animals of any age with traumatic hernias can be affected. clinical signs are consistent with a small intestinal obstruction: anorexia, vomiting, lethargy, abdominal pain, and weakness. diagnosis is often made based on physical examination of a reducible or nonreducible mass in the body wall. hernias whose contents are reducible are usually asymptomatic. treatment consists of supportive care and rehydration, administration of broad-spectrum antibiotics, and surgical correction of the body wall hernia. in some cases, intestinal resection and anastomosis of the affected area is necessary when bowel ischemia occurs. the potential for bowel perforation should be suspected whenever there is any penetrating injury (knife, gunshot wound, bite wound, stick impalement) of the abdomen. injuries that result in bowel ischemia and rupture can also occur secondary to nonpenetrating blunt emergency care trauma or shear forces (e.g., big dog-little dog/cat). perforation of the stomach and small and large intestines can occur with use of nonsteroidal antiinflammatory drugs. diagnosis of bowel perforation first depends on the alertness to the possibility that the bowel may have been perforated or penetrated. as a general rule, all penetrating injuries of the abdomen should be investigated by exploratory laparotomy. diagnostic peritoneal lavage (dpl) can be performed; however, early after penetrating injury of the bowel, dpl may be negative or nondiagnostic until peritonitis develops. whenever any patient with blunt or penetrating abdominal trauma does not respond to initial fluid therapy, or responds and then deteriorates, the index of suspicion for bowel injury should be raised. the findings of pneumoperitoneum on abdominal radiographs or of intracellular bacteria, extracellular bacteria, bile pigment, bowel contents, and cloudy appearance of fluid obtained by abdominocentesis or diagnostic peritoneal lavage fluid (see sections on abdominocentesis and diagnostic peritoneal lavage) warrant immediate surgical exploration. treatment largely consists of stabilizing the patient's cardiovascular and electrolyte status with intravenous fluids, administration of broad-spectrum antibiotics, and definitive surgical exploration and repair of injured structures. prolapse of the rectum is observed most frequently secondary to parasitism and gastrointestinal viral infections in young puppies and kittens with chronic diarrhea. older animals with rectal prolapse often have an underlying problem such as a tumor or mucosal lesion that causes straining and dyschezia. the diagnosis of a rectal prolapse is made based on physical examination findings. the diagnosis of rectal prolapse is sometimes difficult to distinguish from small intestinal intussusception. in rare cases, the intussusception can invaginate through the large bowel, rectum, and anus. the two entities are distinguished from one another by inserting a lubricated thermometer or blunt probe into the cul-de-sac formed by the junction of the prolapsed mucosa and mucocutaneous junction at the anal ring. inability to insert the probe or thermometer indicates that the rectal mucosa is prolapsed. passage of the probe signifies that the prolapsed segment is actually the intussusceptum. treatment can be performed easily if the prolapse is acute and the rectal mucosa is not too irritated or edematous. the presence of severely necrotic tissue warrants surgical intervention. to reduce an acute rectal prolapse, after placing the patient under general anesthesia, lubricate the prolapsed tissue and gently push it back into the rectum, using a lubricated syringe or syringe casing. apply a loose purse-string suture, leaving it in place for a minimum of hours. de-worm the patient and administer stool softeners. if a rectal prolapse cannot be reduced, or if the tissue is nonviable, surgical intervention is warranted. in patients in which viable tissue does not stay reduced with a purse-string suture, a colopexy can be performed during a laparotomy. first, place tension on the colon to reduce the prolapse, and then suture the colon to the peritoneum of the lateral abdominal wall with two to three rows of - or - monofilament suture material. if the prolapsed tissue is nonviable, it must be amputated. place four stay sutures at -degree intervals through the wall of the prolapse at the mucocutaneous junction. resect the prolapse distal to the stay sutures and then reestablish the rectal continuity by suturing the seromuscular layers together in one circumferential line and the mucosal layers together in the other. replace the suture incision into the anal canal. following surgery, de-worm the patient and administer a stool softener and analgesic drugs. avoid using thermometers or other probes in the immediate postoperative period because they may disrupt suture lines. acute gastritis may be associated with a variety of clinical conditions, including oral hemorrhage, ingestion of highly fermentable nondigestable foods or garbage, toxins, foreign bodies, renal or hepatic failure, inflammatory bowel disease, and bacterial and viral infections. diarrhea often accompanies or follows acute gastritis. hemorrhagic gastroenteritis often occurs as a shock-like syndrome with a rapidly rising hematocrit level. clinical signs of gastritis include depression, lethargy, anterior abdominal pain, excessive water consumption, vomiting, and dehydration. differential diagnosis of acute gastritis includes pancreatitis, hepatic or renal failure, gastrointestinal obstruction, and toxicities (box - ). the diagnosis is often a diagnosis of exclusion of other causes (see preceding text). a careful and thorough examination of the vomitus may be helpful in arriving at a diagnosis. a complete blood count, serum biochemistry profile including amylase and lipase, parvovirus test (in young puppies), fecal flotation and cytology, abdominal radiographs (plain and/or contrast studies), and abdominal ultrasound may be warranted to rule out other causes of acute vomiting. while diagnostic tests are being performed, treatment consists of withholding all food and water for a minimum of hours. after calculating the patient's degree of dehydration, administer a balanced crystalloid fluid to normalize acid-base and electrolyte status. control vomiting with antiemetics such as metoclopramide, prochlorperazine, chlorpromazine, dolasetron, and ondansetron (table - ). if vomiting is accompanied by diarrhea, administer broad-spectrum antibiotics (cefazolin, mg/kg iv q h, with metronidazole, mg/kg iv q h; or ampicillin, mg/kg iv q h, with enrofloxacin, mg/kg iv q h) to decrease the risk of bacterial translocation and bacteremia/septicemia. although antacids (famotidine, ranitidine, cimetidine) do not have a direct antiemetic effect, their use can decrease gastric acidity and esophageal irritation during vomiting. if gastritis is secondary to uremia or nonsteroidal antiinflammatory drug use, administer gastroprotectant and antiemetic drugs (ranitidine, mg/kg po q h; sucralfate, . - g/dog po q h; or omeprazole ( . - mg/kg po q h) to decrease acid secretion and coat areas of gastric ulceration (table - ) . once food and water can be tolerated, the patient can be placed on an oral diet and medications, and intravenous fluids can be discontinued. do not use until a gastrointestinal obstruction has been ruled out. hemorrhagic gastroenteritis (hge) is an acute onset of severe hemorrhagic vomiting and diarrhea most commonly observed in young small-breed dogs (e.g., poodles, miniature dachshunds, miniature schnauzers) to years of age. clinical signs develop rapidly and include vomiting and fetid diarrhea with hemorrhage, often strawberry jam-like in appearance. the hematocrit can rise from % to %. often, the animal is extremely hypovolemic but has no apparent signs of abdominal pain. there is no known cause of hge, although clostridium perfringens, escherichia coli, campylobacter, and viral infections have been suggested but not consistently confirmed. other differential diagnoses of of hematemesis and hemorrhagic diarrhea include coronavirus, parvovirus, vascular stasis, sepsis, hepatic cirrhosis with portal hypertension, and other causes of severe shock. immediate treatment consists of placement of a large-bore intravenous catheter and replenishment of intravascular fluid volume with crystalloid fluids (up to ml/kg/hour), while carefully monitoring the patient's hematocrit and total protein. administer broad-spectrum antibiotics (ampicillin, mg/kg iv q h, and enrofloxacin mg/kg iv q h) because of the high risk of bacterial translocation and sepsis. control vomiting with antiemetic drugs. monitor the patient's platelet count and coagulation tests for impending disseminated intravascular coagulation (dic), and administer fresh frozen plasma and heparin, as needed (see section on disseminated intravascular coagulation). when vomiting has ceased for hours, offer the animal small amounts of water, and then a bland diet (e.g., boiled chicken and rice or boiled ground beef and rice mixed with low-fat cottage cheese). pancreatitis occurs most frequently in dogs but can occur in cats as well. in dogs, the onset of pancreatitis is sometimes preceded by ingestion of a fatty meal or the administration of drugs (e.g., potassium bromide or glucocorticoids). glucocorticoids can increase the viscosity of pancreatic secretions and induce ductal proliferation, resulting in narrowing and obstruction of the lumen of the pancreatic duct. pancreatitis can also occur following blunt or penetrating abdominal trauma, high duodenal obstruction causing outflow obstruction of the pancreatic papilla, pancreatic ischemia, duodenal reflux, biliary disease, and hyperadrenocorticism. in cats, acute necrotizing pancreatitis is associated with anorexia, lethargy, hyperglycemia, icterus, and sometimes acute death. chronic pancreatitis is more common in cats and results in intermittent vomiting, anorexia, weight loss, and lethargy. predisposing causes of chronic pancreatitis in cats include pancreatic flukes, viral infection, hepatic lipidosis, drugs, organophosphate toxicity, and toxoplasmosis. clinical signs of acute pancreatitis include sudden severe vomiting, abdominal pain, and lethargy. depending on the severity of pancreatic inflammation, depression, hypotension, and systemic inflammatory response syndrome (sirs) may be present. subacute cases may have minimal clinical signs. severe pancreatic edema can result in vascular changes and ischemia that perpetuates severe inflammation. hypovolemic shock and dic can also decrease pancreatic perfusion. severe pancreatic edema, autolysis, and ischemia lead to pancreatic necrosis. duodenal irritation is manifested as both vomiting and diarrhea. pain may be localized to the right upper abdominal quadrant or may be generalized if peripancreatic saponification occurs. differential diagnosis of pancreatitis is the same as for any other cause of vomiting. complications that occur in patients with severe pancreatitis include dehydration, acidbase and electrolyte abnormalities, hyperlipemia, hypotension, and localized peritonitis. hepatic necrosis, lipidosis, congestion, and abnormal architecture can develop. inflammatory mediators (bradykinin, phospholipase a, elastase, myocardial depressant factor, and bacterial endotoxins) stimulate the inflammatory cascade and can lead to sirs, with severe hypotension, clotting system activation, and dic. electrolyte imbalances and hypovolemia secondary to vomiting all can lead to multiple organ dysfunction syndrome (mods), and ultimately, death. if a patient survives an episode of acute pancreatitis, long-term sequelae can include diabetes mellitus. monitor patients with recurrent pancreatitis for clinical signs of polyuria, polydipsia, polyphagia, hyperglycemia, and glucosuria. the diagnosis of pancreatitis is based on the presence of clinical signs (which may be absent in cats), laboratory findings, and ultrasonographic evidence of pancreatic edema and increased peripancreatic echogenicity. serum biochemistry analyses can sometimes support a diagnosis of pancreatitis; however, serum amylase and lipase are often unreliable indicators of pancreatitis, depending on the chronicity of the process in the individual patient. both serum amylase and lipase are excreted in the urine. impaired renal clearance/ function can cause artifactual elevations of serum amylase and lipase in the absence of pancreatic inflammation. furthermore, serum lipase levels can be elevated as a result of gastrointestinal obstruction (e.g., foreign body). early in the course of the disease, levels can be two to six times normal, but they may decrease to within normal ranges at the time of presentation to the veterinarian. the transient nature of amylase elevation makes this test difficult to interpret, and it is not highly sensitive if a normal value is found. lipase levels also increase later in the course of the disease. amylase and lipase should be tested concurrently with the rest of the biochemistry profile. other changes often observed are elevations in bun and creatinine levels secondary to dehydration and prerenal azotemia, hyperglycemia, and hyperlipemia. hypocalcemia can occur secondary to peripancreatic fat saponification, and its presence warrants a more negative prognosis. a more specific measure is pancreatic lipase immunoreactivity, which becomes elevated in dogs and cats with pancreatitis. this test, combined with ultrasonographic or computed tomography evidence of pancreatitis, is the most sensitive and specific test available for making an accurate diagnosis. however, because the results of this test take time to obtain, animals must be treated in the meantime. abdominal effusion or fluid from diagnostic peritoneal lavage can be compared with serum amylase and lipase activity. abdominal lipase and amylase concentrations in the fluid greater than that in the peripheral blood are characteristic of chemical peritonitis associated with pancreatitis. wbc counts greater than cells/mm , the presence of bacteria, toxic neutrophils, glucose levels less than mg/dl, or lactate levels greater than that of serum are characteristic of septic peritonitis, and immediate exploratory laparotomy is warranted. if a biopsy sample obtained during laparotomy does not demonstrate inflammation, but this does not rule out pancreatitis, because disease can be focal in nature and yet cause severe clinical signs. abdominal radiographs may sometimes reveal a loss of abdominal detail or a ground glass appearance in the right upper quadrant. pancreatic edema and duodenal irritation can displace the gastric axis toward the left, toward the left with dorsomedial displacement of the proximal duodenum (the so-called "backwards " or "shepherd's crook" sign). ultrasonography and ct are more sensitive in making a diagnosis of pancreatitis. treatment of pancreatitis is largely supportive in nature and is designed to correct hypovolemia and electrolyte imbalances, prevent or reverse shock, maintain vital organ perfusion, alleviate discomfort and pain, and prevent vomiting (see section on rule of twenty). when treating pancreatitis in dogs, all food and water should be restricted. however, food should not be withheld from cats with chronic pancreatitis. give fresh frozen plasma to replenish alpha- -macroglobulins. administer antiemetics such as chlorpromazine (use with caution in a hypovolemic or hypotensive patient), dolasetron, ondansetron, or metoclopramide to prevent or control vomiting. analgesic drugs can be provided in the form of constant rate infusion (fentanyl, - µ/kg/hour iv cri, and lidocaine, - µ/kg/minute iv cri), intrapleural injection (lidocaine, - mg/kg q h), or intermittent parenteral injections (morphine, . - mg/kg sq, im; hydromorphone, . mg/kg im or sq). because the pancreas must be rested, consider using parenteral nutrition. acute hepatic failure may be associated with toxins, adverse reaction to prescription medication, and bacterial or viral infections. the most frequent clinical signs observed in a patient with acute hepatic failure are anorexia, lethargy, vomiting, icterus, bleeding, and cns depression or seizures (associated with hepatic encephalopathy). differential diagnosis and causes of acute hepatic failure are listed in box - . diagnosis of acute hepatic failure is based on clinical signs and biochemical evidence of hepatocellular (ast, alt) and cholestatic (alk phos, t bili, ggt) enzyme elevations. ultrasonography may be helpful in distinguishing the architecture of the liver, but unless a mass or abscess is present, cannot provide a specific diagnosis of the cause of the hepatic damage. management of the patient with acute hepatic failure includes correction of dehydration and acid-base and electrolyte abnormalities, as shown in the following list: • hypoalbuminemia: plasma or concentrated albumin. plasma also is an excellent source of clotting factors that can become depleted. • clotting abnormalities: vitamin k ( . mg/kg sq or po q - h) to • severe anemia: fresh or stored blood • gastric hemorrhage: gastroprotectant drugs (omeprazole, ranitidine, famotidine, cimetidine, sucralfate) • hypoglycemia: dextrose supplementation ( . %- %) • hepatic failure, particularly when hypoglycemia is present: broad-spectrum antibiotics (ampicillin mg/kg iv q h; with enrofloxacin, mg/kg iv q h) • hepatic encephalopathy: lactulose or betadine enemas • cerebral edema: mannitol ( . - . g/kg iv over to minutes) followed by furosemide ( mg/kg iv minutes later). deterioration of clinical signs may signify the development of cerebral edema. applewhite aa, cornell kk, selcer ba: diagnosis and treatment of intussusception in dogs. comp cont educ pract vet ( ) often, systemic hypertension is diagnosed when the animal is seen by the veterinarian because of some other clinical sign, such as acute blindness, retinal detachment, hyphema, epistaxis, and cns signs following intracranial hemorrhage. diagnosis of systemic hypertension is often difficult in the absence of clinical signs and without performing invasive or noninvasive blood pressure monitoring. normal blood pressure (bp) measurements in dogs and cats are listed in table - . hypertension is defined as a consistent elevation in systolic bp > mm hg, consistent diastolic bp > mm hg, and consistent mean arterial blood pressure > mm hg. the effects of systemic hypertension include left ventricular hypertrophy, cerebrovascular accident, renal vascular injury, optic nerve edema, hyphema, retinal vascular tortuosity, retinal hemorrhage, retinal detachment, vomiting, neurologic defects, coma, and excessive bleeding from cut surfaces. emergency care dog - - - cat - - - patients with systemic hypertension should have a thorough diagnostic work-up to determine the underlying cause. although uncommon, hypertensive emergencies can occur with pheochromocytoma, acute renal failure, and acute glomerulonephritis. sodium nitroprusside ( - µ/kg/minute iv cri) or diltiazem ( . - . mg/kg iv given slowly over minutes, followed by µ/kg/minute) can be used to treat systemic hypertension. with the use of sodium nitroprusside or diltiazem, monitor carefully for hypotension. diagnosis is based on consistent elevations in systolic, diastolic, and/or mean arterial bp. because many of the clinical signs associated with systemic hypertension involve hemorrhage into some closed cavity, other causes of hemorrhage, such as vasculitis, thrombocytopenia, thrombocytopathia, and hepatic or renal failure, should be investigated (see section on coagulation disorders). diagnostic testing is based on clinical signs and index of suspicion for an underlying disease and may include a complete blood count; urinalysis; urine protein:creatinine ratio; acth stimulation test; thoracic and abdominal radiographs; thoracic and abdominal ultrasound; tick serology; brain ct or mri; and assays of serum electrolytes, aldosterone concentration, t , endogenous tsh, plasma catecholamine, and growth hormone. management of systemic hypertension involves treatment of the primary underlying disorder, whenever possible. long-term adjunctive management includes sodium restriction in the form of cooked or prescription diets to decrease fluid retention. obese animals should be placed on dietary restrictions and undergo a weight reduction program. thiazide and loop diuretics may be used to decrease sodium retention and circulating blood volume. alpha-and beta-adrenergic blockers may be used, but they are largely ineffective as monotherapeutic agents for treating hypertension. calcium channel blockers and angiotensin-converting enzyme (ace) inhibitors are the mainstay of therapy in the treatment of hypertension in dogs and cats ( diabetic ketoacidosis (dka) is a potentially fatal and terminal consequence of unregulated insulin deficiency and possible glucagon excess. in the absence of insulin, unregulated lipolysis results in the beta-hydroxylation of fatty acids by abnormal hepatic metabolism. as a result, ketoacids-namely, acetoacetic acid, beta-hydroxybutyric acid, and acetoneare produced. early in the course of the disease, patients exhibit clinical signs associated with diabetes mellitus: weight loss, polyuria, polyphagia, and polydipsia. later, as ketoacids stimulate the chemoreceptor trigger zone, vomiting and dehydration occur, with resulting hypovolemia, hypotension, severe depression, abdominal pain, oliguria, and coma. at the time of presentation, often a strong odor of ketones (acetone) is present on the patient's breath. physical examination often reveals dehydration, severe depression or coma, and hypovolemic shock. in extreme cases, the patient exhibits a slow, deep kussmaul respiratory pattern in an attempt to blow off excess co to compensate for the metabolic acidosis. a serum biochemistry profile and complete blood count often reveal prerenal azotemia, severe hyperglycemia (blood glucose > mg/dl), hyperosmolarity (> mosm/kg), lipemia, hypernatremia (sodium > meq/l), elevated hepatocellular and cholestatic enzyme activities, high anion gap, and metabolic acidosis. although a whole body potassium deficit is usually present, the serum potassium may appear artifactually elevated in response to metabolic acidosis. with severe metabolic acidosis, potassium moves extracellularly in exchange for a hydrogen ion. phosphorus too moves intracellularly in response to acidosis, and serum phosphorus is usually decreased. hypophosphatemia > mg/dl can result in intravascular hemolysis. urinalysis often reveals + glucosuria, ketonuria, and a specific gravity of . or greater. the urine of all diabetic animals should be cultured to rule out a urinary tract infection or pyelonephritis. treatment of a patient with dka presents a therapeutic challenge. treatment is aimed at providing adequate insulin to normalize cellular glucose metabolism, correcting acidbase and electrolyte imbalances, rehydration and restoration of perfusion, correcting acidosis, providing carbohydrate sources for utilization during insulin administration, and identifying any precipitating cause of the dka. obtain blood samples for a complete blood count, and serum biochemistry electrolyte profiles. whenever possible, insert a central venous catheter for fluid infusion and procurement of repeat blood samples. calculate the patient's dehydration deficit and maintenance fluid requirements and give appropriate fluid and electrolytes over a period of hours. it is advisable to rehydrate patients with severe hyperosmolarity for a minimum of hours before starting insulin administration. use a balanced electrolyte solution (e.g., plasmalyte-m, normosol-r, lactated ringer's solution) or . % saline solution for maintenance and rehydration. balanced electrolyte solutions contain small amounts of potassium and bicarbonate precursors that aid in the treatment of metabolic acidosis. treat animals with severe metabolic acidosis with an hco − > meq/l or a ph < . with supplemental bicarbonate ( . - . meq/kg). add supplemental dextrose to the patient's fluids as a carbohydrate source during insulin infusion. both insulin and carbohydrates are necessary for the proper metabolism of ketone bodies in patients with dka. the rate and type of fluid and amount of dextrose supplementation will change according to the patient's blood glucose concentration. serum potassium will drop rapidly as the metabolic acidosis is corrected with fluid and insulin administration. measure serum potassium every hours, if possible, and supplement accordingly (see section on fluid therapy for chart of potassium supplementation). if the patient's potassium requirement exceeds meq/l, or if the rate of potassium infusion approaches . meq/ kg/hour in the face of continued hypokalemia, magnesium should be supplemented. magnesium is required as a cofactor for many enzymatic processes and for normal function of the na,k-atpase pump. hypomagnesemia is a common electrolyte disturbance in many forms of critical illness. replenishing magnesium (mgcl , . meq/kg/day iv cri) often helps to correct the refractory hypokalemia observed in patients with dka. patients with hypophosphatemia that approaches . mmol/l should receive potassium phosphate ( . - . mmol/kg/hour iv cri). when providing potassium phosphate supplementation, be aware of the additional potassium added to the patient's fluids, so as to not exceed recommended rates of potassium infusion. to determine the amount of potassium chloride (kcl) to add along with potassium phosphate (kpo ), use the following formula: meq k + derived from kcl = total meq of k + to be administered over hours − meq in which k + is derived from kpo clinical signs of severe hypophosphatemia include muscle weakness, rhabdomyolysis, intravascular hemolysis, and decreased cerebral function that can lead to depression, stupor, seizures, or coma. regular insulin can be administered either im or as a constant rate infusion in the treatment of patients with dka. subcutaneous insulin should not be administered. because of the severe dehydration present in most patients with dka, subcutaneous insulin is poorly absorbed and is not effective until hydration has been restored. in the low-dose intravenous method, place regular insulin ( . units/kg for a cat, and . units/kg for a dog) in ml of . % saline solution. run ml of this mixture through the intravenous line to allow the insulin to adsorb to the plastic tubing. administer the patient's insulin fluid rate according to blood glucose levels ( table - ) . adjust the patient's total fluid volume according to changes in the insulin fluid rate as necessary. in many cases, multiple bags of fluids are necessary because they must be changed when fluctuations in blood glucose concentrations occur in response to therapy. infusion of the insulin mixture should be in a separate intravenous catheter. to replenish hydration, use a second intravenous line for the more rapid infusion of non-insulin-containing fluids. to administer the regular insulin im, first give . unit/kg im and then re-check the patient's blood glucose every hour. additional injections of regular insulin ( . unit/kg other fluid type (ml/hour) > . % nacl - . % nacl + . % dextrose - . % nacl + . % dextrose - . % nacl + . % dextrose < . % nacl + % dextrose im) should be administered based on the patient's response to subsequent injections. once the patient's blood glucose falls to to mg/dl, add . % to % dextrose to the fluids to maintain the blood glucose concentration at to mg/dl. continue intramuscular injection of regular insulin ( . - . unit/kg q - h) until the patient is rehydrated, no longer vomiting, and able to tolerate oral fluids and food without vomiting. even in patients with intramuscular regular insulin therapy, a central venous catheter should be placed for frequent blood sample collection. as the patient begins to respond to therapy, monitor electrolytes, glucose, and acid-base status carefully. hypokalemia, hypophosphatemia, and hypomagnesemia can occur. when the patient's hydration and acid-base status has normalized and the patient is able to tolerate oral food and water, a longer-acting insulin can be administered as for treatment of a patient with uncomplicated diabetes. extreme hyperosmolarity can result in a coma, if uncorrected. in patients with diabetes mellitus, hyperglycemia and hypernatremia secondary to osmotic diuresis and free water loss can lead to severe hyperosmolarity. in dogs, normal serum osmolality is < mosm/l of serum. hyperosmolarity is expected when serum osmolality is > mosm/l. if equipment for determining serum osmolarity is not available, osmolarity can be calculated by the following formula: osm/l = (na + k) + (glucose/ ) + (bun/ . ) patients with severe dehydration, hyperglycemia, hypernatremia, and azotemia may experience cerebral edema without ketonemia. treatment is directed solely at rehydrating the patient and slowly reducing blood glucose levels using a hypotonic solution such as . % nacl + . % dextrose or % dextrose in water (d w). after the initial rehydration period, administer potassium supplementation conservatively. red blood cells and the brain absolutely depend on the oxidation of glucose for energy. hypoglycemia can be caused by various systemic abnormalities that can be related to intestinal malabsorption of nutrients, impaired hepatic glycogenolysis or gluconeogenesis, and inadequate peripheral utilization of glucose. clinical signs of hypoglycemia are extremely variable and can include weakness, tremors, nervousness, polyphagia, ataxia, tachycardia, muscle twitching, incoordination, visual disturbances, and generalized seizures. clinical signs typically occur when serum glucose levels are < mg/dl. the combination of the clinical signs listed previously, documentation of low serum glucose, and alleviation of clinical signs upon glucose administration is known as whipple's triad. whenever a patient presents with hypoglycemia, consider the following important factors: the age of onset, the nature of the hypoglycemic episode (transient, persisent, or recurrent) , and the pattern based on the patient's history . treatment of hypoglycemia is directed at providing glucose supplementation and determining any underlying cause. administer supplemental dextrose ( %- % dextrose, - ml/kg iv; or % dextrose, ml/kg po) as quickly as possible. do not attempt oral glucose supplementation in any patient having a seizure or if the airway cannot be protected. administer intravenous fluids (e.g., normosol-r, lactated ringer's solution, . % saline solution) with . %- % supplemental dextrose until the patient is eating and able to maintain euglycemia without supplementation. in some cases (e.g., insulinoma), eating or administration of supplemental dextrose can promote insulin secretion and exacerbate clinical signs and hypoglycemia. in cases of refractory hypoglycemia secondary to iatrogenic insulin overdose, glucagon ( mg/kg iv bolus, then - ng/kg/minute iv cri) can also be administered along with supplemental dextrose. to make a glucagon infusion of ng/ml, reconstitute ml ( mg/ml) of glucagon according to the manufacturer's instructions and add this amount to ml of . % saline solution. emergency care the diagnosis of eclampsia (puerperal tetany) is often made on the basis of history and clinical signs. clinical signs can become evident when total calcium decreases to < . mg/dl in dogs and < . mg/dl in cats. the disease is often observed in small, excitable dogs, and stress may play a complicating role in the etiology. in most bitches, the disease manifests itself to weeks after parturition. in some cases, however, clinical signs can develop before parturition occurs. hypophosphatemia may accompany hypocalcemia. clinical signs of hypocalcemia include muscle tremors or fasciculations, panting, restlessness, aggression, hypersensitivity, disorientation, muscle cramping, hyperthermia, stiff gait, seizures, tachycardia, a prolonged qt interval on ecg, polydipsia, polyuria, and respiratory arrest. treatment of eclampsia consists of slow, cautious calcium supplementation ( % calcium gluconate, . mg/kg iv over minutes). severe refractory tetanus can be controlled with intravenous diazepam. supportive care includes intravenous fluid administration and cooling (see section on hyperthermia and heat-induced illness). instruct the owner to give the patient oral calcium supplements (e.g., to tablets of tums bid-tid) after discharge from the hospital. also instruct the owner about how to wean the puppies, allowing the bitch to dry up, in order to prevent recurrence. recurrence with subsequent pregnancies is common, particularly in patients that receive calcium supplementation during gestation (table - ) . hypercalcemia can occur from a variety of causes. the gosh darn it mnemonic can be used to remember the various causes of hypercalcemia in small animal patients (box - ) . the gastrointestinal, renal, and nervous systems are most commonly affected, particularly when serum total calcium rises above . mg/dl. clinical signs of severe hypercalcemia include muscle weakness, vomiting, seizures, and coma. ecg abnormalities include prolonged pr interval, rapid qt interval, and ventricular fibrillation. the most serious clinical signs are often seen when hypercalcemia is observed in combination with hyperphosphatemia or hypokalemia. pay special attention to the "calcium × phosphorus product." if this product exceeds , dystrophic calcification can occur, leading to renal failure. renal complications include polyuria, polydipsia, dehydration, and loss of renal tubular concentrating ability. renal blood flow and the glomerular filtration rate (gfr) are impaired when serum total calcium exceeds mg/dl. the extent, location, and number of renal tubular injuries are the main factors in determining whether renal damage secondary to hypercalcemia is reversible or irreversible. emergency therapy of hypercalcemia is warranted when severe renal compromise, cardiac dysfunction, or neurologic abnormalities are present, or if no clinical signs occur but the calcium × phosphorus product exceeds . the treatment of choice is correction of the underlying cause of hypercalcemia, whenever possible. in some cases, the results of diagnostic tests take time, and emergency therapy should be initiated immediately, before a definitive cause of the hypercalcemia is found. emergency management of hypercalcemia consists of reduction of serum calcium levels. administer intravenous fluids ( . % saline solution) to expand extracellular fluid volume and promote calciuresis. to promote diuresis, initial intravenous fluid rates should approach two to three times maintenance levels ( - ml/kg/day). potassium supplementation may be required to prevent iatrogenic hypokalemia. administration of a loop diuretic such as furosemide ( - mg/kg iv) will promote calcium excretion. calcitonin ( iu/kg im q h for cats and iu/kg im q h for dogs) can be administered to decrease serum calcium levels. in severe refractory hypercalcemia secondary to cholecalciferol toxicity, more aggressive calcitonin therapy ( - iu/kg sq q - h) can be attempted. side effects of calcitonin treatment include vomiting and diarrhea. alternatively, bisphosphonates (pamidronate, . - . mg/kg iv) are useful in rapidly reducing serum calcium concentrations. glucocorticosteroids reduce calcium release from the bone, decrease intestinal absorption of calcium, and promote renal calcium excretion. administer glucocorticosteroids only after the underlying cause of hypercalcemia has been determined and appropriate therapy started. because many forms of neoplasia can result in hypercalcemia as a paraneoplastic syndrome, empiric use of glucocorticosteroids can induce multiple drug resistance, making the tumor refractory to the effects of chemotherapeutic agents. hypoadrenocorticism is most commonly observed in young to middle-aged female dogs, but it can occur in animals of any age, gender, and breed. clinical signs, which are referable to deficiency in glucocorticoid (cortisol) and mineralocorticoid (aldosterone) hormones, may develop slowly over time, leading to a waxing and waning course; acute clinical signs occur when > % of the adrenal functional reserve has been destroyed. in such cases, complete adrenocortical collapse can result in an addisonian crisis. lack of aldosterone causes a lack of renal sodium and water retention, and impaired potassium excretion. the most significant clinical signs associated with hypoadrenocorticism are depression, lethargy, weakness, anorexia, shaking, shivering, vomiting, diarrhea, weight loss, abdominal pain, weakness, hypotension, dehydration, and inappropriate bradycardia (box - ) . the diagnosis of hypoadrenocorticism is made based on the patient's clinical signs in combination with electrolyte abnormalities that include hyperkalemia, hyponatremia, and hypochloremia. serum sodium concentration ( - meq/l) is often greatly reduced, and serum potassium is elevated (> . meq/l). a sodium:potassium ratio of < is characteristic of hypoadrenocorticism, although not exactly pathognomonic. electrocardiographic changes associated with hyperkalemia include inappropriate bradycardia, absence of p waves, elevated spiked t waves, and widened qrs complexes. other more variable bloodwork abnormalities include a lack of a stress leukogram, eosinophilia, hypoglycemia, hyperphosphatemia, hypercalcemia, azotemia, and hypocholesterolemia. a definitive diagnosis of hypoadrenocorticism is based on an adrenocorticotropic hormone (acth) stimulation test. in patients with hypoadrenocorticism, baseline cortisol levels are usually low, with a lack of appropriate cortisol release after administration of acth analogue. rarely, animals with "atypical" hypoadrenocorticism lose glucocorticoid secreting ability from the zona fasciculata, but retain mineralocorticoid secretory ability from the zona glomerulosa. atypical addisonian patients have normal serum electrolytes but still have clinical signs of vomiting, diarrhea, weakness, lethargy, inappetence, muscle wasting, and weight loss. the diagnosis is more difficult in such cases because of the presence of normal electrolytes. an acth stimulation test should be considered, particularly in predisposed breeds. treatment of hypoadrenocorticism includes placement of a large-bore intravenous catheter, infusion of intravenous crystalloid fluids ( . % saline solution), and replenishment of glucocorticoid and mineralocorticoid hormones. administer dexamethasone or dexamethasone-sodium phosphate ( . - . mg/kg iv). dexamethasone will not interfere with the acth stimulation test, unlike other longer-acting steroids (e.g., prednisolone, methylprednisolone sodium succinate, triamcinolone). depending on the severity of the patient's condition, consider monitoring using the rule of twenty. administer antiemetics and gastroprotectant drugs to treat nausea, vomiting, and hematemesis. give the patient broad-spectrum antibiotics (ampicillin, mg/kg iv q h) if hematochezia or hemorrhagic diarrhea is present. if severe gastrointestinal blood loss occurs, whole blood, packed red blood cells, or fresh frozen plasma may be required. control hypoglycemia with . %- . % dextrose. use sodium bicarbonate, regular insulin with dextrose, or calcium gluconate to correct severe hyperkalemia with atrial standstill (see section on atrial standstill). chronic therapy for hypoadrenocorticism consists of mineralocorticoid and glucocorticosteroids supplementation for the rest of the animal's life. mineralocorticoid supplementation can be in the form of desoxycorticosterone pivalate (docp) ( . mg/kg im) or fludrocortisone acetate ( . mg/ . - kg body weight daily). fludrocortisone acetate possesses both mineralocorticoid and glucocorticoid activities and can be used as the sole daily treatment of hypoadrenocorticism. (because fludrocortisone is poorly absorbed in some dogs, it may not completely normalize electrolyte abnormalities in these animals.) docp is primarily a mineralocorticoid. give supplemental glucocorticosteroids in the form of prednis(ol)one ( - . mg/kg/day). in dogs, iatrogenic hypoadrenocorticism can be caused by abrupt discontinuation of glucocorticosteroid treatment. long-term glucocorticosteroid supplementation can downregulate the pituitary gland's excretion of endogenous acth and the zona fasciculata's ability to excrete cortisol. however, the zona glomerulosa's ability to secrete aldosterone does not appear to be affected. clinical signs of iatrogenic hypoadrenocorticism include inability to compensate for stress, weakness, lethargy, vomiting, diarrhea, and collapse. treatment of iatrogenic hypoadrenocorticism is the same as for naturally occurring disease. following immediate emergency treatment, the patient should be weaned slowly from exogenous glucocorticosteroid supplementation. severe hyperthyroidism can manifest as a medical emergency as a result of hypermetabolism. clinical signs in affected cats with severe thyrotoxicosis include fever, severe tachycardia (heart rate > bpm), vomiting, hypertension, congestive heart failure with pulmonary edema, and fulminant collapse. clinical signs typically are manifested as an end-stage of chronic debilitation associated with hyperthyroidism and are often preceded by polyphagia, weight loss, cardiac murmur, polyuria/polydipsia (pu/pd), vomiting, and diarrhea. treatment of thyrotoxicosis includes antagonizing the adrenergic activity by administration of a beta-adrenergic blocker (esmolol, ( - µ/kg/minute, or propranolol, . mg/ kg/hour). administration of glucocorticosteroids (dexamethasone, mg/kg) may inhibit the conversion of thyroxine (t ) to the active form triiodothyronine (t ) and decrease peripheral tissue responsiveness to t , effectively blocking its effects. correct hypoglycemia with supplemental dextrose ( . %). use care to avoid overhydration in a patient with cardiac failure or insufficiency. start the patient on methimazole as quickly as possible and consider the use of radioactive iodine therapy. to maintain cerebral perfusion pressure, blood pressure must be normalized. if other concurrent injuries are suspected (e.g., pulmonary contusions), administer synthetic colloid fluids (dextran- , - ml/kg iv, or hetastarch, - ml/kg iv) to normalize blood pressure. although the use of colloids is controversial because of their potential to leak into the calvarium, the benefits of reestablishing cerebral perfusion far outweigh the risks of their use. hypertonic saline ( . % nacl, - ml/kg iv) can also be administered over to minutes to expand intravascular volume. maintain blood glucose within normal reference ranges whenever possible, because hyperglycemia is a negative prognostic indicator in cases of head trauma. if tremors or seizures cause hyperthermia or increased metabolism, active cooling of the patient is warranted (see sections on hyperthermia and heat-induced injury). all patients with head trauma should receive care and monitoring based on the rule of twenty (see section on rule of twenty). examine the patient's level of consciousness, response to various stimuli, pupil size and reactivity to light, physiologic nystagmus, and cranial nerve deficits. in dogs, damage to the midbrain often produces coma and decerebrate rigidity. initial consciousness followed by a unconsciousness or stupor usually involves an injury to the brainstem. brainstem lesions can be caused by compressive skull fractures, extradural or subdural hematomas, or herniation through the foramen magnum from cerebral edema (box - ) . the patient's pupil size and response to light can be used to localize a diagnosis and give a rough prognosis for severity of disease and possibility for return to function. pupils can be normal in size, mydriatic, or miotic. whenever a pupil appears miotic, direct ocular emergency care unconscious with no response to noxious stimuli injury with uveitis or secondary miosis due to brachial plexus injury should be ruled out. the eyes should always be examined to rule out ocular trauma. in a patient with head trauma, a change from dilated to constricted to normal pupil size is suggestive of improvement in clinical function. bilateral mydriatic pupils that are unresponsive to light in an unconscious animal are a grave prognostic sign and usually indicate an irreversible severe midbrain contusion. bilateral miotic pupils with normal nystagmus and ocular movements are associated with diffuse cerebral or diencephalic lesions. miotic pupils that become mydriatic indicate a progressive midbrain lesion with a poor prognosis. unilateral, slowly progressive pupillary abnormalities in the absence of direct ocular injury are characteristic of brainstem compression or herniation caused by progressive brain swelling. asymmetric pupils are seen in patients with rostral brainstem lesions and can change rapidly. unresponsive pupils that are seen in the midposition occur with brainstem lesions that extend into the medulla and are a grave sign. visual deficits are common with intracranial injury. lesions that are less severe and limited to the cerebrum produce contralateral menace deficits with normal pupillary light response. bilateral cerebral edema can cause blindness with a normal response to light if the midbrain is not disturbed. a patient that is severely depressed and recumbent may not respond to menacing gestures, even when visual pathways are intact. ocular, optic tract, optic nerve, or optic chiasm lesions can interfere with vision and the pupillary light response. brainstem contusion and cerebral edema may produce blindness and dilated unresponsive pupils due to disturbance of the oculomotor area. examine all cranial nerves carefully. cranial nerve abnormalities can indicate direct contusion or laceration of the neurons in the brainstem or where they exit the skull. cranial nerves that are initially normal then later lose function indicate a progressively expanding lesion. when specific cranial nerve deficits are present, the prognosis is considered guarded. clinical signs such as rolling to one side, torticollis, head tilt, and abnormal nystagmus are usually associated with petrosal bone or cerebellomedullary lesions that produce vestibular neuron dysfunction. fractures of the petrosal temporal bone often cause hemorrhage and cerebrospinal fluid (csf) leak from the external ear canal. if the lesion is limited to the membranous labyrinth, the loss of balance will be toward the injured side and the quick phase of the nystagmus will be toward the injured side. normal physiologic nystagmus requires that the pathway is between the peripheral vestibular neurons and the pontomedullary vestibular nuclei to the nuclei of the cranial nerves that innervate the extraocular muscles (iii, iv, vi). severe brainstem lesions disrupt this pathway. disruption of the pathway is manifested as an inability to produce normal physiologic nystagmus by moving the patient's head from side to side. in patients with severe central nervous system depression, this reflex may not be observed. next, assess postural changes and motor function abilities. a loss of the normal oculocephalic ("dolls-eye") reflex is an early sign of brainstem hemorrhage and a late sign of brainstem compression and herniation. any intracranial injury may be accompanied by a concurrent cervical spinal cord injury. handle animals with such injuries with extreme care to avoid causing further damage. whenever there is uncertainty whether a spinal cord lesion exists, strap the patient down to a flat surface and obtain radiographs of the spine. at least two orthogonal views may be required to see fractures; however, do not manipulate the patient until radiography has been completed. crosstable views, in which the bucky is turned perpendicular to the patient's spine, with a radiograph plate secured behind the patient, may be required to minimize patient motion. in patients with cerebral lesions, hemiparesis usually resolves within to days. evaluation of cranial nerve function at frequent intervals may reveal an initial injury or a progressively expanding lesion in the brain. signs of vestibular disorientation, marked head tilt, and abnormal nystagmus occur with contusions of the membranous labyrinth and fracture of the petrous temporal bone. hemorrhage and cerebrospinal fluid otorrhea may be visible from the external ear canal. rolling movements indicate an injury to the cerebellar-medullary vestibular system. respiratory dysfunction and abnormal respiratory patterns are sometimes observed with severe head injury. lesions of the diencephalon produce cheyne-stokes respirations, in which the patient takes progressively larger and larger breaths, pauses, then takes progressively smaller and smaller breaths. mesencephalic lesions cause hyperventilation and can result in respiratory alkalosis. medullary lesions result in a choppy, irregular respiratory pattern. clinical signs of respiratory dysfunction in the absence of primary respiratory damage indicate a guarded prognosis. after injury, seizures may be associated with intracranial hemorrhage, trauma, or an expanding intracranial mass lesion. immediately begin medical therapy to control the seizure. administer diazepam ( . mg/kg iv or . - . mg/kg/hour iv cri) to treat seizures. if diazepam is not effective in combination with other treatments to control intracranial edema, consider giving pentobarbital . loading doses of phenobarbital ( - mg/kg iv divided into or doses, given every to minutes) may be beneficial in preventing further seizures. severe refractory seizures or decreased mentation may be associated with cerebral edema and increased intracranial pressure. mannitol, an osmotic diuretic, is effective at reducing cerebral edema ( . - . g/kg iv over to minutes). mannitol also acts as a free radical scavenger that can inhibit the effects of cerebral ischemia-reperfusion injury. mannitol works synergistically with furosemide ( mg/kg iv given minutes after the mannitol infusion). corticosteroids have not been demonstrated to be beneficial in the treatment of head trauma and may induce hyperglycemia. hyperglycemia has been shown to be a negative prognostic indicator in cases of head trauma. also, glucocorticoids can suppress immune system function and impair wound healing. because of the known risks and lack of known benefits of glucocorticosteroids, their use in treatment of head trauma is contraindicated. the prognosis for any patient with severe head trauma is guarded. management of head trauma patients may include intense nursing care for a period of weeks to months, depending on the presence and extent of concurrent injuries. if progressive loss of consciousness occurs, surgery for decompression of compressive skull injuries should be considered. the most common injury associated with head trauma in small animals is a contusion with hemorrhage in the midbrain and pons. subdural or extradural hemorrhage with space-occupying blood clots is uncommon. diagnostic tests of head trauma may include skull radiographs, ct, and mri of the brain. special studies can help detect edema and hemorrhage in the brain and brainstem, and aid in making an accurate diagnosis and prognosis. a cerebrospinal fluid tap is contraindicated in patients with head trauma because of the risk of causing a rapid decrease in intracranial pressure and brainstem herniation. if a compressive skull fracture is present, the patient should be stabilized for surgery to remove the compression. surgery to alleviate increased intracranial pressure is rarely performed in veterinary medicine because of the poor prognosis and results. in some cases, when a lesion can be localized to one area, -to -cm burr holes can be placed through the skull over the affected area of the cerebrum, exposing the underlying brain tissue. blood clots can be removed through the holes. the bone flap may or may not be replaced, depending on the surgeon's preference and the degree of brain swelling. spinal cord injuries may be associated with trauma, disk rupture, fractures, and dislocation of the spinal column. proceed with caution when moving a patient with suspected spinal cord injury. avoid flexion, extension, and torsion of the vertebral column. all animals that are unconscious following a traumatic event should be considered to have cervical or thoracolumbar spinal injury until proved otherwise by radiography, ct, or mri. the animal should be moved onto a flat surface (e.g., board, door, window, picture frame) and taped down to prevent motion and further displacement of vertebrae. sedation with analgesics or tranquilizers may be necessary to keep the animal immobile and to minimize patient motion. whenever possible, avoid the use of narcotics in patients with head trauma because of the risk of increasing intracranial pressure. as in other emergencies, the abcs emergency care should be evaluated, and the patient treated for shock, hemorrhage, and respiratory compromise. once the cardiovascular and respiratory systems have been evaluated and stabilized, a more thorough neurologic examination can be performed. protrusion of an intervertebral disk indicates that the disk is bulging into the vertebral canal as a result of dorsal shifting of the nuclear pulposus disk material. disk extrusion refers to the rupture of the outer disk membrane and extrusion of the nuclear material into the vertebral column. in dogs and cats, there are intervertebral disks that potentially can cause a problem. chondrodystrophic breeds of dogs are predisposed to endochondral ossification and include the dachshund, shih tzu, french bulldog, bassett hound, welsh corgis, american spaniel, beagle, lhasa apso, and pekingese. initial examination of the patient with suspected intervertebral disk disease includes identifying the neuroanatomic location of the lesion based on clinical signs and neurologic deficits and then establishing a prognosis. the neurologic examination should be carried out without excessive manipulation of the animal. the presence of pain, edema, hemorrhage, or a visible deformity may localize an area of vertebral injury. once an area of suspected lesion is localized based on physical examination findings, take radiographs to establish a diagnosis and to institute therapy. in most cases, the animal must receive a short-acting anesthestic for proper radiographic technique and to prevent further injury. lateral and crosstable ventrodorsal (vd) or dorsoventral (dv) radiographs require less manipulation of the animal compared with traditional vd and dv projections. myelography is often required to delineate the location of the herniated disk material. prognosis in spinal cord injury depends on the extent of the injury and the reversibility of the damage. perception of noxious stimuli, or the presence of "deep pain," by the animal when the stimulus is applied caudal to the level of the lesion is a good sign. to apply a noxious stimulus, apply firm pressure to a toe on one of the rear limbs using a thick hemostat or a pair of pliers. flexion or withdrawl of the limb is simply a local spinal reflex, and should not be perceived as a positive response to or patient perception of the noxious stimulus. turning of the head, vocalization, dilation of the pupils, change in respiratory rate or character, or attempts to bite are behaviors that are more consistent with perception of the noxious stimulus. absence of perception of the noxious stimulus ("loss of deep pain") is a very poor prognosis for return to function. focal lesions are usually associated with vertebral fractures and displacement of the vertebral canal. focal lesions in one or more of the spinal cord segments from t to t can cause complete dysfunction of the injured tissue as a result of concussion, contusion, or laceration. the degree of structural damage cannot be determined from the neurologic signs alone. transverse focal lesions result in paraplegia, with intact pelvic limb spinal reflexes and analgesia of the limbs and body caudal to the lesion. clinical signs in patients with spinal injury are summarized in table - . carefully evaluate the cardiovascular and respiratory status of patients with spinal injuries. immediately address specific injuries such as pneumothorax, pulmonary contusions, hypovolemic shock, and open wounds. if there is palpable or radiographic evidence of a vertebral lesion causing compressive injury, surgery is the treatment of choice unless the displacement has compromised most or all of the vertebral canal. displacements through % to % of the vertebral canal are associated with a poor prognosis, particularly if deep pain is absent caudal to the lesion. in the absence of a radiographic lesion and in the presence of continued neurologic deficits, an mri or ct scan or myelography is warranted to localize a potentially correctable lesion. surgical exploration can be considered: with the objectives of providing spinal cord decompression by hemilaminectomy or laminectomy with removal of disk material or blood clots, realign and stabilize the vertebral column, and perform a meningotomy, if necessary. place the patient on a backboard or other rigid surface, taped down for transport and sedated, to be transported to a surgical specialist. the presence of worsening or ascending clinical signs may signify ascending-descending myelomalacia and is characteristic of a very poor prognosis.in acute spinal trauma, the use of glucocorticoids has been the mainstay of therapy; however, controversy exists about whether they actually offer any benefit. traditional glucocorticosteroid therapy is listed in box - . more recently, the use of propylene glycol has proved to be beneficial in the treatment of acute traumatic herniated disk. high-dose glucocorticoids should only be used for the first hours after initial injury. side effects of glucocorticosteroid therapy include gastric and intestinal ulceration. the prophylactic use of gastroprotectant drugs will not prevent gastrointestinal ulcer formation; however, if signs of gastrointestinal ulcer are present, institute gastroprotectant therapy. management of the patient with spinal cord injury includes aggressive nursing care and physical therapy. many patients with spinal cord injury have little to no control over bladder function, which results in chronic dribbling or retention of urine and overdistention of the urinary bladder with overflow incontinence. urinary bladder retention can lead to urinary tract infection, bladder atony, and overflow incontinence. manual expression of the bladder several times a day may be enough to keep the bladder empty. alternatively, place a urinary catheter to maintain patient cleanliness and to keep the bladder decompressed. (see section on urinary catheterization). paralytic ileus and fecal retention are frequent complications of spinal cord injury. to help prevent constipation, provide highly digestable foods and maintain the patient's hydration with oral and intravenous fluids. mild enemas or stool softeners can also be used to treat fecal retention. to prevent decubital ulcer formation, turn the patient every to hours, and use clean, dry, soft padded bedding. apply deep muscle massage and passive range of motion exercises to prevent disuse atrophy of the muscles and dependent edema. the radial nerve innervates the extensor muscles of the elbow, carpus, and digits. the radial nerve also supplies sensory innervation to the distal craniolateral surface of the forearm and the dorsal surface of the forepaw. injuries to the radial nerve at the level of the elbow emergency care cranial to c spastic tetraplegia or tetraparesis hyperreflexive all four limbs severe injury can result in death from respiratory failure. c -t tetraparesis or tetraplegia depressed thoracic limb spinal reflexes (lower motor neuron) hyperreflexive pelvic limbs (upper motor neuron) t -t horner' syndrome (prolapsed nictitans, enophthalmos, and miosis) t -l schiff-sherrington syndrome (extensor rigidity of thoracic limbs, flaccid paralysis with atonia, areflexia, and analgesia of pelvic limbs) result in an inability to extend the carpus and digits. as a result, the animal walks and bears weight on the dorsal surface of the paw. there is also loss of cutaneous sensation, which leads to paw injury. injuries to the radial nerve above the elbow (in the shoulder area) results in an inability to extend the elbow and bear weight on the affected limb. it can take weeks before the full extent of the injury and any return to function are manifested. the animal may need to be placed in a carpal flexion sling or have eventual amputation if distal limb injury or self-mutilation occurs. the sciatic nerve primarily innervates the caudal thigh muscles that flex the stifle and extend the hip. the tibial branch of the sciatic nerve innervates the caudal leg muscles that extend the tarsus and flex the digits. the tibial nerve provides the sole cutaneous sensory innervation to the plantar aspect of the paw and digits. the peroneal branch of the sciatic nerve provides the sole sensory cutaneous innervation to the dorsal surface of the paw ( table - ) . sciatic nerve injury may occur with pelvic fractures, particularly those that involve the body of the ileum at the greater ischiatic notch, or with sacroiliac luxations that contuse the l and l spinal nerves that pass ventral to the sacrum to contribute to the sciatic nerve. with sciatic nerve injury, there is decreased stifle flexion and overflexion of the hock (tibial nerve), and the animal walks on the dorsal surface of the paw (peroneal nerve). clinical signs of tibial or peroneal damage are seen with femur fractures or with inadvertent injection of drugs into the caudal thigh muscles. the femoral nerve innervates the extensor muscles of the stifle. the saphenous branch of the femoral nerve provides the sole cutaneous innervation to an area on the medial distal thigh, the leg, and the paw. the femoral nerve is protected by muscles and is rarely injured in pelvic fractures. clinical signs of femoral nerve injury are inability to support weight on the pelvic limb, absence of a patellar reflex, and analgesia in the area of cutaneous innervation. coma is complete loss of consciousness, with no response to noxious stimuli. in some animals that present in a coma or stuporous state, the immediate cause will be apparent. in other cases, however, a careful and thorough diagnostic work-up must be performed. a coma scale devised to assist in the clinical evaluation of the comatose patient is shown in table - . whenever an animal presents in a comatose state, immediately secure the emergency management of specific conditions c -t nerve roots radial nerve paralysis musculocutaneous nerve inability to flex the elbow axillary or thoracodorsal dropped elbow nerve median and ulnar nerves loss of cutaneous sensation on the caudal surface of the forearm and palmar and lateral surfaces of the paw; inability to flex the carpus and digits c -t nerve roots radial, median, or ulnar nerve injury c -c nerve roots musculocutaneous, suprascapular, and axillary injury c -t horner's syndrome (miosis, enophthalmos, and prolapsed nictitans) airway by placing an endotracheal tube (see section on endotracheal intubation). if necessary, provide respiratory assistance, or at a minimum, supplemental oxygen. control existing hemorrhage and treat shock, if present. take a careful and thorough history from the owner. make careful note of any seizure, trauma, or toxin exposure, and whether prior episodes of coma have ever occurred. perform a careful physical examination, taking note of the patient's temperature, pulse, and respiration. an elevated temperature may suggest the presence of systemic infection, such as pneumonia or hepatitis, or a brain lesion with loss of hypothalamic thermoregulatory control. very high temperatures associated with shock and coma are often observed in animals with heat stroke (see section on heat stroke and heat-induced illness). circulatory collapse or barbiturate overdose can produce coma and hypothermia. abnormal respiratory patterns also may be observed in a comatose patient. hypoventilation may occur with elevated intracranial pressure or barbiturate overdose. rapid respiratory rate may be associated with pneumonia, metabolic acidosis (dka, uremia), or brainstem injury. examine the skin for any bruises or external trauma. examine the mucous membranes and make note of color and capillary refill time. icterus with petechiae or ecchymotic hemorrhage in a comatose patient may be associated with end-stage hepatic failure and hepatic encephalopathy. smell the patient's breath for the odor of ketones that may signify dka or end-stage hepatic failure. motor activity normal gait, normal spinal reflexes hemiparesis, tetraparesis, or decerebrate activity recumbent, intermittent extensor rigidity recumbent, constant extensor rigidity recumbent, constant extensor rigidity with opisthotonus recumbent, hypotonia of muscles, depressed or absent spinal reflexes normal papillary reflexes and oculocephalic reflexes slow pupillary light reflexes and normal to reduced oculocephalic reflexes bilateral unresponsive miosis with normal to reduced oculocephalic reflexes pinpoint pupils with reduced to absent oculocephalic reflexes unilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes bilateral, unresponsive mydriasis with reduced to absent oculocephalic reflexes occasional periods of alertness and responsive to environment depression of delirium, capable of responding to environment but response may be inappropriate semicomatose, responsive to visual stimuli semicomatose, responsive to auditory stimuli semicomatose, responsive only to repeated noxious stimuli comatose, unresponsive to repeated noxious stimuli *neurologic function is assessed for each of the three categories and a grade of to is assigned according to the descriptions for each grade. the total score is the sum of the three category scores. this scale is designed to assist the clinician in evaluating the neurologic status of the craniocerebral trauma patient. as a guideline and according to clinical impressions, a consistent total score of to represents a grave prognosis, to a poor to guarded prognosis, and to a good prognosis. (modified from the glasgow coma scale used in humans.) from shores a: craniocerebral trauma. in kirk rw, ed: current veterinary therapy x. small animal practice. philadelphia, wb saunders, , p . finally, conduct a complete neurologic evaluation. the presence of asymmetric neurologic signs may suggest an intracranial mass lesion (e.g., hemorrhage, neoplasia, injury). usually, toxicities or metabolic disturbances (e.g., dka, hepatic encephalopathy) cause symmetric clinical signs of neurologic dysfunction, with cerebral signs predominating. in hepatic encephalopathy, pupils are usually normal in size and responsive to light. in toxicities, the pupils are abnormal in size and may be unresponsive to light. obtain a complete blood count, serum biochemistry profile, urinalysis, and specific tests for glucosuria and ketonuria. findings of a drastically elevated blood glucose with glucosuria, ketonuria, and high specific gravity are characteristic of dka. fever and uremic encephalopathy are characterized by severe azotemia with a low urine specific gravity. if barbiturate intoxication is suspected, save urine for later toxin analysis. evaluate urine sediment for calcium oxalate crystalluria that may indicate ethylene glycol toxicity. calculate plasma osmolality (see following section) to check for nonketotic hyperosmolar diabetes mellitus. elevated blood ammonia levels may be associated with hepatic encephalopathy. in uncontrolled diabetes mellitus, hyperosmolarity can result in clinical signs of disorientation, prostration, and coma. plasma osmolarity can be calculated from the formula: mosm/l = (na + k) + (glucose/ ) + (bun/ . ) clinical signs of hyperosmolarity can occur when the plasma osmolarity exceeds mosm/l. treatment of dka or nonketotic hyperosmolar syndrome is aimed at reducing ketoacid production, stimulating carbohydrate utilization, and impeding peripheral release of fatty acids. the treatment of choice is rehydration and provision of supplemental regular insulin and a carbohydrate source (see section on diabetic ketoacidosis). during ketosis, insulin resistance may be present. slow rehydration with . % saline solution or other balanced crystalloid fluids (e.g., normosol-r, plasmalyte-m, lactated ringer's solution), should occur, with the goal of rehydration over to hours. too rapid rehydration can result in cerebral edema and exacerbation of clinical signs. hepatic encephalopathy (he) is characterized by an abnormal mental state associated with severe hepatic insufficiency. the most common cause of he is congenital or acquired c o m a portosystemic shunts. acute hepatic destruction can also be caused by toxins, drugs, or infectious causes. the treatment of he is considered a medical emergency (table - ) . absorption of ammonia and other nitrogenous substances from the gastrointestinal tract is thought to be one of the complicating factors in he. prevent absorption of ammonia and other nitrogenous substances from the gastrointestinal tract by restricting dietary protein to % to % for dogs, and to % to % (on a dry matter basis) for cats. dietary protein should be from a nonanimal plant source (e.g., soybean) whenever possible. caloric requirements are met with lipids and carbohydrates. also prescribe cleansing enemas to rid the colon of residual material, and antibiotic therapy to reduce gastrointestinal tract bacteria. neomycin ( mg/kg q h) can be administered as a retention enema. metronidazole ( . mg/kg po, q - h) or amoxicillin-clavulanate ( . mg po q h) can also be administered. administer lactulose ( . - . ml q h for cats; . - ml q h for dogs) to trap ammonia in the colon to prevent absorption (table - ) . administer lactulose orally to an alert animal, or as a retention enema to a comatose animal. if lactulose is not available, betadine retention enemas will change colonic ph and prevent ammonia absorption. a side effect of lactulose administration (po) is soft to diarrheic stool. a seizure is a transient disturbance of brain function that is sudden in onset, ceases spontaneously, and has a tendency to recur, depending on the cause. most seizures are generalized and result in a loss of consciousness and severe involuntary contraction of the skeletal muscles, resulting in tonic-clonic limb activity and opisthotonus. mastication, salivation, urination, and defecation are common. partial (petit mal) seizures range from limited limb activity, facial muscle twitching, and episodic behavioral abnormalities to brief loss of consciousness. similar clinical signs also can occur with syncopal episodes. conduct a careful cardiac examination in any patient with a history of petit mal seizures. seizures of any form constitute a medical emergency, particularly when they occur in clusters, or as status epilepticus. most seizures are of short duration and may have subsided by the time the animal is presented for treatment. whenever a seizure occurs, however, it is important that the animal does not inadvertently injure itself or a bystander. it is important to evaluate whether the patient has a coexisting disease that can predispose it to seizures, such as hepatic failure, uremia, diabetes mellitus, hypoglycemia, toxin exposure, insulin-secreting tumors, and thiamine deficiency. many toxins are responsible for clinical signs of tremors or seizures (see section on poisons and toxins). treatment of a primary disease entity can help control seizures, in some cases, provided that the underlying cause is investigated and treated. status epilepticus, a state of continuous uncontrolled seizure activity, is a medical emergency. when an animal is in a state of status epilepticus, immediately place a lateral or medial saphenous intravenous catheter and administer diazepam ( . mg/kg iv) to help control the seizure. in most cases, the seizure must be controlled before a diagnostic workup is attempted. whenever possible, however, blood samples should be collected before administration of any anticonvulsant agent because of the risk of incorrect test results. for example, the propylene glycol carrier in diazepam can cause a false-positive ethylene glycol test using an in-house testing kit. whenever possible, check blood glucose levels, particularly in young puppies or kittens, to evaluate and treat hypoglycemia as a cause of seizures. if hypoglycemia exists, administer % dextrose ( g/kg iv). if diazepam partially controls the status epilepticus, administer a constant rate infusion ( . mg/kg/hour in % dextrose in water). diazepam is sensitive to light, and the bag and infusion line must be covered to prevent degradation of the drug. if diazepam fails to control status epilepticus, give pentobarbital ( - mg/kg iv to effect). the animal's airway should be intubated and protected while the patient is kept in the drug-induced coma. protracted cases of seizures may require mannitol and furosemide therapy to treat cerebral edema. administer intravenous fluids (balanced crystalloid at maintenance doses [see section on intravenous fluid therapy]). the patient should be turned every to hours to emergency care prevent atelectasis. insert a urinary catheter for cleanliness, and place the animal on soft dry padded bedding to prevent decubital ulcer formation. depending on the length of time that the patient is rendered unconscious, apply passive range of motion exercises and deep muscle massage to prevent disuse atrophy of the muscles and dependent or disuse edema. monitor the patient's oxygenation and ventilation status by arterial blood gas measurement or pulse oximetry and capnometry (see section on blood gas, pulse oximetry, and capnometry). administer supplemental oxygen to any patient that is hypoxemic secondary to hypoventilation or other causes. severe refractory seizures can result in the development of neurogenic pulmonary edema. lubricate the animal's eyes every hours to prevent drying out and corneal abrasions. depending on the cause of the seizure, administer phenobarbital at a loading dose of to mg/kg iv given in four to five injections, every to minutes; make sure that the patient is rousable in between injections). seizures in cats often are associated with structural brain disease. the occurrence of partial focal seizures is unequivocally associated with a focal cerebral lesion and acquired structural brain disease. an initial high frequency of seizures is also a strong indication that structural brain disease is present. seizure activity in cats may occur as mild generalized seizures or complex partial seizures and may be associated with systemic disorders such as feline infectious peritonitis virus, toxoplasmosis, cryptococcus infection, lymphosarcoma, meningiomas, ischemic encephalopathy, and thiamine deficiency. thiamine deficiency in the cat can be a medical emergency characterized by dilated pupils, ataxic gait, cerebellar tremor, abnormal oculocephalic reflex, and seizures. treatment consists of administration of thiamine ( mg/day) for three days. steffen f, grasmueck s: propofol for treatment of refractory seizures in dogs and a cat with intracranial disorders. j small anim pract ( ) ( ) ( ) ( ) . j am vet med assoc ( ): [ ] [ ] [ ] [ ] [ ] [ ] . an ocular emergency is any serious condition that causes or threatens to cause severe pain, deformity, or loss of vision. treat ocular emergencies immediately, within to several hours after the emergency, whenever possible (box - , - ). to assess the location and degree of ocular injury, perform a complete ocular examination. in some cases, short-acting sedation or general anesthesia in conjunction with topical local anesthetic may be necessary to perform the examination, because of patient discomfort and blepharospasm. the equipment listed in box - may be necessary and may be invaluable in making an accurate diagnosis. to perform a systematic and thorough ocular examination, first obtain a history from the owner. has there been any prior incident of ocular disease? is there any history of trauma or known chemical irritant or exposure? did the owner attempt any irrigation or medical techniques prior to presentation? when was the problem first noticed? has it changed at all since the owner noticed the problem? after a history has been obtained, examine the patient's eyes for discharge, blepharospasm, or photophobia. if any discharge is present, note its color and consistency. do not attempt to force the eyelids open if the patient is in extreme discomfort. administer a short-acting sedative and topical local anesthetic such as . % proparacaine. note the position of the globe within its orbit. if the eye is exophthalmic, strabismus and protrusion of the third eyelid are often visible. exposure keratitis may be present. in cases of retrobulbar or zygomatic salivary gland inflammation, the patient will resist opening the mouth and exhibit signs of discomfort or pain. note any swelling, contusions, abrasions, or lacerations of the eyelids. note whether the lids are able to close completely and cover the cornea. if a laceration of the lid is present, determine the depth of the laceration. palpate the orbit for fractures, swelling, pain, crepitus, and cellulitis. examine the cornea and sclera for penetrating injury or foreign material. the use of lid retractors or small forceps can be very helpful in these cases. if a wound appears to penetrate completely into the globe, look for loss of uveal tissue, lens, or vitreous. do not put any pressure on the globe, because intraocular herniation may result. examine the conjunctiva for hemorrhage, chemosis, lacerations, and foreign bodies. examine the superior and inferior conjunctival cul-de-sacs for foreign material. in such cases, placement of a topical anesthetic and use of a moistened cotton swab is invaluable to sweep the conjunctival fornix to pick up foreign bodies. use a small, fine-tipped forceps to retract the third eyelid away from the globe and examine behind the third eyelid for foreign bodies. next, examine the cornea for opacities, ulcers, foreign bodies, abrasions, or lacerations. place a small amount of fluroescein stain mixed with sterile water or saline on the dorsal sclera. close the eye to disperse the stain over the surface of the cornea, then flush gently with sterile saline irrigation. examine the cornea again for any defects. a linear defect perpendicular to the long axis of the eye should alert the clinician to investigate the conjunctiva for dystechia. record the pupil size, shape, and response to light (both direct and consensual). examine the anterior chamber and note its depth and whether hyphema or aqueous flare are present. is the lens clear and is it in the normal position? lens luxation can cause the lens tissue to touch the cornea and cause acute corneal edema. measure intraocular pressure with a schiotz tonometer or tonopen. finally, dilate the pupil and examine the posterior chamber using a direct or indirect ophthalmoscope to look for intraocular hemorrhage, retinal hemorrhage, retinal detachment, tortuous retinal vessels, optic neuritis, and inflammation. the basic surgical instruments listed in box - may be useful in the treatment of ocular lacerations and other ophthalmic injuries: bite wounds and automobile trauma commonly cause lacerations and abrasions of the lid margins. the lids can be considered to be two-layer structures, with the anterior composed of the skin and orbicularis muscle and the posterior layer composed of the tarsus and conjunctiva. the openings of the meibomian glands in the lid margin form the approximate line separating the lids into anterior and posterior segments. splitting the lid into these two segments facilitates the use of sliding skin flaps to close wound defects, if necessary. clean and thoroughly but gently irrigate the wound with sterile saline solution before attempting any lid laceration repair. use sterile saline solution to irrigate the wound and conjunctiva. a % povidone-iodine scrub can be used on the skin, taking care to avoid getting any scrub material in the soft tissues of the eye. drape the eye with an adhesive ocular drape, if possible, to prevent further wound contamination. trim the ragged wound edges, but be very conservative with tissue debridement. leave as much tissue as possible to insure proper wound contracture with minimal lid deformity. close a small lid wound with a figure-of-eight or two-layered simple interrupted suture of absorbable suture material or nylon in the skin. the lid margins must be absolutely apposed to prevent postoperative lid notching. direct blunt trauma to the eye can cause severe ecchymosis because of the excellent vascular supply of the eyelids. other associated ocular injuries such as orbital hemorrhage, proptosis, and corneal laceration may also occur. trauma, allergic reactions, inflammation of the sebaceous glands (hordeolum), thrombocytopenia, and vitamin k antagonist rodenticide intoxication can all cause ecchymoses of the lids. treat eyelid ecchymoses initially with cool compresses, followed by warm compresses. resorption of blood can occur from to days after the initial insult. ocular allergies respond well to topical application (dexamethasone ophthalmic ointment q - h) and systemic administration of glucocorticosteroids, along with cool compresses. in order to fully assess the conjunctiva for abnormalities, it may be necessary to carefully dissect it away from the underlying sclera. when performing this dissection, do not place undue pressure on the globe because of the risk of herniation of the intraocular contents through a scleral wound. repair large conjunctival lacerations with - absorbable sutures, using an interrupted or continuous pattern. carefully approximate the margins of the conjunctiva to prevent formation of inclusion cysts. when large areas of the conjunctiva have been damaged, advancement flaps may be required to close the defect. subconjunctival hemorrhage is a common sequela of head trauma, and it may also be observed in various coagulopathies. by itself, it is not a serious problem but may signify severe underlying intraocular damage. a complete ocular examination is indicated. other causes of subconjunctival hemorrhage include thrombocytopenia, autoimmune hemolytic anemia, hemophilia, leptospirosis, vitamin k antagonist rodenticide intoxication, severe systemic infection or inflammation, and prolonged labor (dystocia). uncomplicated subconjunctival hemorrhage usually clears on its own within days. if the conjunctiva is exposed because of swelling and hemorrhage, administer a topical protective triple antibiotic ophthalmic ointment every to hours until the conjunctival hemorrhage resolves. toxic, acid, and alkaline chemical injuries to the eye can sometimes occur. the severity of the injury caused by ocular burns depends on the concentration, type, and ph of the chemical and on the duration of exposure. weak acids do not penetrate biologic tissue very well. the hydrogen ion precipitates the protein upon contact and therefore provides some protection to the corneal stroma and intraocular contents. precipitation of corneal proteins produces a ground-glass appearance in the cornea. alkaline solutions and very strong acids penetrate tissues rapidly, causing saponification of the plasma membrane, denaturation of collagen, and vascular thrombosis within the conjunctiva, episclera, and anterior uvea. severe pain, blepharospasm, and photophobia are produced by exposure of free nerve endings in the corneal epithelium and conjunctiva. severe alkaline burns cause an increase in intraocular pressure. intraocular prostaglandins are released, and the intraocular aqueous ph increases, producing changes in the blood-aqueous barrier and secondary uveitis. uveitis with anterior synechia formation, eventual chronic glaucoma, phthisis, secondary cataract, and corneal perforation can occur. healing of the corneal epithelium is usually accomplished by neovascularization and sliding and increased mitosis of the corneal epithelium. severe stromal burns within the cornea heal by degradation and removal of necrotic debris, followed by replacement of the collagen matrix and corneal epithelial cells. the release of collagenase, endopeptidase, and cathepsins from polymorphonuclear cells serves to cause further corneal breakdown. in severe cases, only pmns may be present, and fibroblasts may never invade the corneal stroma. all chemical burns should be washed copiously with any clean aqueous solution available. if any sticky paste or powder is adherent to the conjunctival sac, remove it with moist cotton swabs and irrigation. begin mydriasis and cycloplegia by topical application of % atropine ophthalmic drops or ointment. start antibiotic therapy with triple antibiotic ophthalmic ointment or gentocin ointment every to hours. treat secondary glaucomas with topical carbonic anhydrase inhibitors. to avoid fibrinous adhesions and symblepharon formation, keep the conjunctival cul-de-sacs free of proteinaceous exudate that can form adhesions. analgesics are required for pain. oral nonsteroidal antiinflammatory agents such as carprofen, ketoprofen, meloxicam, or aspirin are recommended. persistent epithelial erosions may require a conjunctival flap left in place for to weeks or placement of a topical collagen shield (contact lens). topical antibiotics, mydriatics, and lubricants (lacrilube or puralube ointment) should also be used. strong acid or alkali burns can result in severe corneal stromal loss. in the past, topical n-acetylcysteine ( % mucomyst) has been recommended. this treatment is very painful. other treatments are also available, such as ethylenediaminetetraacetic acid (edta) ( . m solution) and patient serum to inhibit mammalian collagenase activity. to prepare patient serum, obtain to ml of whole blood from the patient. spin it down in a serum separator tube after a clot forms and then place the serum in a red-topped tube on the patient's cage. (the contents of the tube are viable for days without refrigeration.) apply the serum topically to the affected eye every to hours. avoid using topical steroids because they inhibit fibroblast formation and corneal healing. in severe cases, if conjunctival swelling and chemosis also are present, antiinflammatory doses of oral steroids can be administered short-term. oral steroids and nonsteroidal antiinflammatory drugs should never be administered to the patient concurrently, because of the risk of gastrointestinal ulcer and perforation. corneal abrasions are associated with severe pain, blepharospasm, lacrimation, and photophobia. animals with such intense pain are often difficult to examine until analgesia has been administered. topical use of proparacaine ( . % proparacaine hydrochloride) is usually sufficient to permit relaxation of the eyelids so that the eye can be examined. using a focal source of illumination and an eye loupe, examine the cornea, inferior and superior conjunctival fornixes, and medial aspect of the nictitans for foreign bodies. place a sterile drop of saline on a fluorescein-impregnated strip and touch the superior conjunctiva once to allow the stain to spread onto the surface of the eye. irrigate the eye to remove excess stain and then examine the corneal surface for any areas of stain uptake. if an area of the cornea persistently remains green, there is damage to the corneal epithelium in that area. initial treatment consists of application of a topical mydriatic ( drop of % atropine in affected eye q h) to prevent anterior synechiae and improve cycloplegia. triple antibiotic ointment is the treatment of choice (a / -inch strip in the affected eye q h) until the ulcer heals. in some cases, nonhealing ulcers (e.g., boxer ulcer, indolent ulcer) form in which the epithelial growth does not adhere to the underlying cornea. gently debride the loose edges of the ulcer/erosion with a cotton swab and topical anesthesia. more severe cases in which only minimal healing has occurred after days of treatment require grid keratectomy, in which a -gauge needle is used to gently scratch the surface of the abrasion or ulcer in the form of a grid to promote neovascularization. apply a topical anesthetic before performing the procedure. a collagen contact lens also may be required to promote wound healing. all corneal abrasions should be reevaluated in hours, and then every to days thereafter until they have healed. acute infectious keratitis secondary to bacterial infection is characterized by mucopurulent ocular discharge, rapidly progressing epithelial and corneal stromal loss, inflammatory cellular infiltrates into the corneal stroma, and secondary uveitis, often with hypopyon formation. confirmation of infectious keratitis is based on corneal scrapings and a positive gram stain. initial treatment for bacterial keratitis consists of systemic antibiotics and topical ciprofloxacin ( . % eyedrops or ointment). penetrating injuries through the cornea may result in prolapse of intraocular contents. frequently, pieces of uveal tissue or fibrin effectively but temporarily seal the defect and permit the anterior chamber to re-form. avoid manipulation of these wounds until the animal has been anesthetized, as struggling or excitement can promote loss or dislodgement of the temporary seal and cause the intraocular contents to be extruded. superficial corneal lacerations need not be sutured and can be treated the same as a superficial corneal ulcer or abrasion. if the laceration penetrates more than % the thickness of the cornea, or extends more than to mm, it should be sutured. when placing sutures in the cornea, it is helpful to use magnification. referral to a veterinary ophthalmologist is advised. if a veterinary ophthalmologist is not available, use - or - silk, collagen, or nylon sutures on a micropoint spatula-type needle. use a simple interrupted suture pattern and leave the sutures in place for a minimum of weeks. because many corneal lacerations are jagged and corneal edema forms, most of the wound edges cannot be tightly juxtaposed. in such cases, pull a conjunctival flap across the wound to prevent leakage of aqueous fluid. never suture through the full thickness of the cornea; rather, the suture should pass through the mid-third of the cornea. following closure of the corneal wound, the anterior chamber must be re-formed to prevent anterior synechia formation with secondary glaucoma. taking care to avoid iris injury, use a -or -gauge needle to insert sterile saline at the limbus. any defect in the suture line will be apparent because of leakage of the fluid from the site and should be repaired. incarceration of uveal tissue in corneal wounds is a difficult surgical problem. persistent incarceration of uveal tissue can result in development of a chronic wick in the cornea, a shallow anterior chamber, chronic irritation, edema, vascularization of the cornea, and intraocular infection that can lead to panophthalmitis. referral to a veterinary ophthalmologist is strongly recommended. the most common foreign bodies associated with ocular injuries in small animals are birdshot, bb pellets, and glass. the site of intraocular penetration of the foreign bodies may be obscured by the eyelids. a foreign body entering the eye may penetrate the cornea and fall into the anterior chamber or become lodged in the iris. foreign bodies may occasionally penetrate the lens capsule, producing cataracts. some metallic high-speed foreign bodies may penetrate the cornea, iris, and lens to lodge in the posterior wall of the eye or vitreous chamber. direct visualization of a foreign body is the best means of localization. examination of the eye with an indirect ophthalmoscope or biomicroscope (if available) is invaluable for locating foreign bodies. indirect visualization of the ocular foreign body can also be achieved through radiographic techniques. three separate views should be obtained to determine the plane of location of the foreign object. ct or mri may prove useful, although scatter from the foreign body may make it difficult to directly visualize with these techniques. ocular ultrasound is perhaps the most useful and refined radiographic technique for locating intraocular foreign bodies. before removing any foreign body from the eye, the risk and surgical danger of removing it must be weighed against the risks of leaving it in place. metallic foreign bodies in the anterior chamber are much easier to remove than nonmagnetic ones. attempted removal of foreign objects from the vitreous chamber of the eye has consistently produced poor results. for the best chance of recovery, ocular foreign bodies should be removed by a veterinary ophthalmologist whenever possible. blunt trauma to the globe can result in luxation or subluxation of the lens. the subluxated lens may move anteriorly and make the anterior chamber more shallow. trembling of the iris (iridodonesis) may be noticed when the lens is subluxated. in complete luxation, the lens may fall totally into the anterior chamber and obstruct aqueous outflow, causing secondary glaucoma. alternatively, the lens may be lost into the vitreous cavity. luxation of the lens is almost always associated with rupture of the hyaloid membrane and herniation of the vitreous through the pupillary space. emergency surgery for lens luxation is required if the lens is entirely within the anterior chamber or incarcerated within the pupil, causing a secondary pupillary block glaucoma. acute elevation in intraocular pressure can cause vision loss within hours; thus, lens removal should be accomplished as quickly as possible. referral to a veterinary ophthalmologist is recommended. severe trauma to the globe or a direct blow to the head can result in retinal or vitreous hemorrhage. there may be large areas of subretinal or intraretinal hemorrhage. subretinal hemorrhage assumes a discrete globular form, and the blood appears reddish-blue in color. the retina is detached at the site of hemorrhage. superficial retinal hemorrhage may assume a flame-shaped appearance, and preretinal or vitreous hemorrhage assumes a bright-red amorphous appearance, obliterating the underlying retinal architecture. retinal and vitreous hemorrhage secondary to trauma usually resorbs spontaneously over a -to -week period. unfortunately, vitreous hemorrhage, as it organizes, can produce vitreous traction bands that eventually produce retinal detachment. expulsive choroid hemorrhage can occur at the time of injury and usually leads to retinal detachment, severe visual impairment, and total loss of vision. treatment of vitreal and retinal hemorrhage includes rest and correction of factors that may predispose to intraocular hemorrhage. more complicated cases may require vitrectomy performed by a veterinary ophthalmologist. hyphema refers to blood in the anterior chamber of the eye. the most common traumatic cause of hyphema is an automobile accident. hyphema may also present because of penetrating ocular wounds and coagulopathies. blood within the eye may come from the anterior or posterior uveal tract. trauma to the eye may result in iridodialysis or a tearing of the iris at its root, permitting excessive bleeding from the iris and ciliary body. usually, simple hyphema resolves spontaneously in to days and does not cause vision loss. loss of vision following bleeding into the anterior chamber is associated with secondary ocular injuries such as glaucoma, traumatic iritis, cataract, retinal detachment, endophthalmitis, and corneal scarring. treatment of hyphema must be individualized, but there are severe general principles of treatment. first, stop ongoing hemorrhage and prevent further bleeding whenever possible. this may involve correction of the underlying cause, if a coagulopathy is present. next, aid in the elimination of blood from the anterior chamber, control secondary glaucoma, and treat associated injuries, including traumatic iritis. finally, detect and treat any late complications of glaucoma. in most cases of traumatic hyphema, little can be done to arrest or prevent ongoing hemorrhage. it is best to restrict the animal's activity and prohibit exertion. rebleeding can occur within days, and intraocular pressure must be monitored closely. after to days, the blood in the anterior chamber will change color from a bright red to bluish-black ("eight-ball hemorrhage"). if total hyphema persists and intraocular pressure rises despite therapy, surgical intervention by a veterinary ophthalmologist may be necessary. the primary route of escape of rbcs from the anterior chamber is via the anterior drainage angle. iris absorption and phagocytosis play a minor role in the removal of blood from the anterior chamber. because of the associated traumatic iritis in hyphema, topical administration of a glucocorticoid ( % dexamethasone drops or % prednisolone drops) is advised to control anterior chamber inflammation. a cycloplegic agent ( % atropine) should also be used. the formation of fibrin in the anterior chamber of the eye secondary to hemorrhage can produce adhesions of the iris and secondary glaucoma (see section on glaucoma secondary to hyphema) by blocking the trabecular network. hyphema secondary to retinal detachment (collie ectasia syndrome) and end-stage glaucoma are extremely difficult to treat medically and have a poor prognosis. proptosis of the globe is common secondary to trauma, particularly in brachycephalic breeds. proptosis of the globe in dolichocephalic breeds requires a greater degree of initiating contusion than the brachycephalic breeds because the orbits are so much deeper. therefore, secondary damage to the eye and cns associated with proptosis of the globe may be greater in the collie or greyhound than in the pug. when proptosis occurs, carefully evaluate the cardiovascular system for evidence of hypovolemic or hemorrhagic shock. examine the respiratory and neurologic systems. be sure to establish an airway and treat shock, if present. control hemorrhage and stabilize the cardiovascular system before attempting to replace the globe within its orbit or perform enucleation. during the initial management of the cardiovascular and respiratory systems, the eye should be covered with an ophthalmic grade ointment or sponges soaked in sterile saline to prevent the globe from drying out. proptosis of the globe can be associated with serious intraocular problems including iritis, chorioretinitis, retinal detachment, lens luxation, and avulsion of the optic nerve. stain the surface of the eye with fluorescein to look for topical abrasions or ulcers. carefully examine the sclera, cornea, and conjunctiva for penetrating injuries that may allow aqueous leakage. evaluate the size, location, and response to light of the pupil. a reactive pupil is better than a mydriatic fixed pupil. topical administration of a mydriatic (atropine %) to prevent persistent miosis and synechia formation is indicated, along with topical and oral antibiotics and oral analgesic therapy. reposition the proptosed globe with the patient under general anesthesia. make a lateral canthotomy incision to widen the palpebral fissure. lavage the globe with sterile saline irrigation to remove any external debris. place a copious amount of triple antibiotic ophthalmic ointment on the surface of the eye and then gently press the globe into the orbit using the flat side of a scalpel handle or a moistened sterile surgical sponge. do not probe the retro-orbital space with a needle or attempt to reduce intraocular pressure by paracentesis. when the globe is replaced in the orbit, close the lateral canthotomy incision with simple interrupted sutures. place three non-penetrating mattress sutures in the lid margins but do not draw them together. tighten the lid sutures through small pieces of a red rubber catheter or length of intravenous extension tubing to prevent the sutures from causing lid necrosis. leave the medial canthus of the eye open in order to allow topical treatment. postoperative treatment is directed at preventing further iritis and preventing infection. administer systemic broad-spectrum antibiotics (clavamox, . mg/kg po bid) and analgesic drugs. apply topical triple antibiotic ophthalmic ointment ( / inch in affected eye q - h) and atropine ( % in affected eye q h) to prevent infection, cycloplegia, and anterior synechiae. antiinflammatory doses of systemic steroids can also be added to the treatment if severe periorbital inflammation is present. systemic steroids should never be used in conjunction with nonsteroidal antiinflammatory drugs, because of the risk of gastrointestinal ulceration and perforation. the sutures should remain in place for a minimum of weeks. after this time, remove the sutures and inspect the globe. if proptosis recurs, repeat the treatment. following proptosis, strabismus is common secondary to periorbital muscle injury. even after extensive treatment, vision in the eye may still be lost. nonvisual eyes can remain in place, but phthisis may develop. carbonic anhydrase inhibitors such as acetazolamide and dichlorphenamide decrease aqueous secretion and may effectively reduce intraocular pressure if the trabecular outflow is still functioning at % of its capacity. an eye with a poorly functional trabecular outflow system will respond poorly to therapy with carbonic anhydrase inhibitors. osmotic agents such as mannitol or glycerol may be helpful in controlling glaucoma secondary to hyphema. reduction in vitreous chamber size can make the anterior chamber deeper and may allow increased aqueous outflow. evacuation of blood or blood clots from the anterior chamber is not advisable unless the glaucoma cannot be controlled medically or there is no indication after a prolonged period of time that blood is being resorbed. tissue plasminogen activator (t-pa) has proved to be useful in may be helpful in lysing blood clots and preventing excessive fibrin formation. the t-pa is reconstituted to make a solution of µ/ml, which is then frozen at − °c in . -ml aliquots. the thawed, warmed reconstituted t-pa is injected into the anterior chamber. blind probing of the anterior chamber of the eye and surgical intervention in an attempt to remove blood clots can cause serious complications such as rebleeding, lens luxation, iris damage, and damage to the corneal epithelium, and therefore is not advised. acute glaucoma is a rise in intraocular pressure that is not compatible with normal vision. glaucoma may present as early acute congestive or noncongestive glaucoma, or as end-stage disease. cardinal signs of glaucoma are a sudden onset of pain, photophobia, lacrimation, deep episcleral vascular engorgement, edematous insensitive cornea, shallow anterior chamber depth, dilated unresponsive pupil, loss of visual acuity, and buphthalmia. intraocular pressure usually exceeds mm hg but may be normal or only slightly increased if glaucoma is secondary to anterior uveitis. most forms of clinical glaucoma in dogs are secondary to some other intraocular problem. primary glaucoma is recognized in some breeds, including the bassett hound, cocker spaniel, samoyed, bouvier des flandres, and some terrier breeds either from goniodysgenesis or a predisposition to lens luxation. other common causes of acute glaucoma are anterior uveitis and intumescent lens secondary to rapid cataract development, particularly in dogs with diabetes mellitus. treatment involves investigation of the underlying cause of the sudden rise in intraocular pressure and rapid reduction in intraocular pressure. permanent visual impairment is often associated with chronically buphthalmic globes or the presence of rippling or striae formation on the cornea. referral to a veterinary ophthalmologist is recommended. if the eye is still visual and not buphthalmic, the prognosis is favorable, depending on the cause of the acute glaucoma. treatment to reduce intraocular pressure consists of improving aqueous outflow, reducing intraocular volume with osmotic agents, and reducing aqueous formation (table - ). the use of topical mydriatic agents in acute glaucoma is contraindicated because of the risk of making lens luxation or anterior uveitis worse. referral to a veterinary ophthalmologist for emergency surgery is indicated in cases of iris bombe, intumescent lens, or lens subluxation. administer osmotic agents to reduce the size of the vitreous body and the amount of aqueous. osmotic agents create an osmotic gradient between the intraocular fluids and the emergency management of specific conditions vascular bed, thus allowing osmotic removal of fluid independent of the aqueous inflow and outflow systems. if no other treatments are available, oral glycerol ( %, . ml/kg or . g/kg) can be used to effectively reduce intraocular pressure. an adverse side effect of oral glycerol treatment is protracted vomiting. do not use glycerol in a diabetic patient. mannitol ( - g/kg iv over hour) also effectively reduces intraocular pressure but does not cause vomiting. carbonic anhydrase inhibitors can be used to reduce intraocular volume by reducing aqueous production. oral administration of dichlorphenamide, methazolamide, and acetazolamide ( - mg/kg) is usually not very effective alone in reducing aqueous volume and intraocular pressure and also can cause metabolic acidosis. topical carbonic anhydrase inhibitors appear to be more effective (dorzolamide, trusopt) when used in conjunction with topical beta-blockers (timolol, . % or . % solution q h). the most effective treatment for acute pressure reduction is use of a topical prostaglandin inhibitor (latanaprost). usually just one or two drops effectively reduces intraocular pressure in the emergency stages, until the patient can be referred to a veterinary ophthalmologist the following day. many clinical conditions that are presented as emergencies may be due in part or wholly to the presence of a neoplasm. paraneoplastic signs are summarized in table - . prompt identification of the neoplasia combined with knowledge of treatment, expected response to therapy, and long-term prognosis can aid owners and practitioners in making appropriate treatment decisions. hemorrhage or effusion can occur in any body cavity as a result of the presence of benign or malignant tumors. tumors secrete anticoagulants to allow angiogenesis to grow unchecked. hemorrhage often occurs as a result of rupture of a neoplasm or invasion of a neoplasm into a major vascular structure. effusion may be the result of direct fluid production by the mass or may be due to obstruction of lymphatic or venous flow. hemorrhagic effusions in the abdominal cavity occur most commonly with neoplastic masses of the spleen or liver. the most common causes are hemangiosarcoma and hepatocellular carcinoma. clinical signs associated with acute abdominal hemorrhage, regardless of the cause, are related to hypovolemic shock and decreased perfusion and include pale mucous membranes, tachycardia, anemia, lethargy, and acute collapse. treatment for abdominal hemorrhage includes placement of a large-bore peripheral cephalic catheter and starting one fourth of a shock dose ( ml/kg/hour for dogs, and ml/kg/hour for cats) of intravenous crystalloid fluids, taking care to carefully monitor perfusion parameters of heart rate, capillary refill time, mucous membrane color, and blood pressure. administer intravenous colloids such as dextran- , hetastarch, and oxyglobin ( - ml/kg iv bolus) to restore intravascular volume and normotension. treat severe anemia with whole blood or packed rbcs to improve oxygen-carrying capacity and oxygen delivery (see sections on transfusion medicine and treatment of shock). confirm the presence of hemoabdomen abdominocentesis (see section on abdominocentesis). the presence of nonclotting hemorrhagic effusion is consistent with free blood. packed cell volume of the fluid is usually the same or higher than that of the peripheral blood. an abdominal compression bandage can be placed while further diagnostics are being performed. in cases of acute hemoabdomen, obtain right lateral, left lateral, and ventrodorsal or dorsoventral thoracic radiographs to help rule out obvious metastasis. monitor the patient's ecg and correct dysrhythmias as necessary (see section on cardiac dysrhythmias). surgery is indicated once the patient is stabilized. in some cases, hemorrhage is so severe that the patient should be taken immediately to surgery. when recommending surgery for a hemorrhaging intraabdominal mass, it is important to discuss likely diagnoses and long-term prognosis with the owner. hemangiosarcoma usually involves the spleen or liver or both. the presence of free abdominal hemorrhage is associated with a malignant tumor in % of cases. even when free abdominal hemorrhage is not present, the tumor is malignant in % of cases. approximately % (two thirds) of masses in the spleen are malignant (hemangiosarcoma, lymphoma, mast cell tumor, malignant fibrous histiocytoma, leiomyosarcoma, fibrosarcoma), and approximately one third are benign (hematoma, hemangioma). hepatocellular carcinoma usually affects one liver lobe (usually the left), and surgery is the treatment of choice. with complete surgical excision, median survival in dogs is longer than days. if diffuse disease is observed at the time of surgery, the prognosis is poor. nonhemorrhagic effusions are associated with mesothelioma, lymphoma, carcinomatosis, or any mass that causes vascular or lymphatic obstruction. clinical signs of respiratory distress and abdominal distention with nonhemorrhagic effusions are usually slowly progressive in onset and not as severe as those observed with hemorrhage. treatment is usually aimed at identification of the underlying cause. obtain a fluid sample via thoracocentesis or abdominocentesis. to obtain further cells for cytologic evaluation, aspirate fluid from the thoracic or abdominal mass with ultrasound guidance. cytologic evaluation of the fluid will often elucidate the causative tumor type. an abdominal ultrasound can determine the degree of metastasis. perform therapeutic abdominocentesis or thoracocentesis if the effusion is causing respiratory difficulty. rapid re-accumulation of the fluid potentially can cause hypoproteinemia and hypovolemic shock. mesothelioma is a rare tumor most commonly observed in urban environments. in humans, mesothelioma has been associated with exposure to asbestos. it is sometimes difficult to differentiate between reactive mesothelial cells and malignant mesothelial cells. treatment is aimed at controlling the neoplastic effusion. intracavitary cisplatin has been demonstrated to slow rates of fluid re-accumulation, but is largely a palliative therapy. lymphoma is another tumor type that can cause thoracic or abdominal effusion. cytologic evaluation of the fluid usually reveals abundant lymphoblasts. treatment with multiagent chemotherapy protocols, with or without adjunctive radiation therapy, can prevent tumor remission and stop fluid accumulation. carcinomatosis occurs as a result of diffuse seeding of the abdominal cavity with malignant carcinomas and has a poor prognosis. carcinomatosis may occur de novo or from metastasis of a primary tumor. treatment consists of fluid removal when respiratory difficulty occurs, with or without intracavitary cisplatin as a palliative measure. cisplatin should never be used in cats due to fatal acute pulmonary edema. clinical signs of hemorrhagic thoracic effusion include acute respiratory distress, anemia, hypovolemic or cardiogenic shock, and collapse. hemorrhagic thoracic effusions are rare in association with neoplastic effusions. a notable exception is intrathoracic hemorrhage in young dogs with osteosarcoma of the rib. hemorrhage can result when a primary lung tumor erodes through a vessel. hemangiosarcoma of the lungs or right auricular area can also result in hemorrhagic thoracic effusion. in many cases, hemorrhage may be confined to the pericardial sac with a right auricular mass, causing a globoid cardiac silhouette on thoracic radiographs. treatment consists of pericardiocentesis (see section on pericardial effusion and pericardiocentesis) and placement of a pericardial window, or the mass may be removed if it is in the right auricular appendage and resectable. although surgery can resolve clinical signs of right-sided heart failure, metastatic disease often develops soon afterward. nonhemorrhagic thoracic effusion is more common than hemorrhagic thoracic effusion, and is caused most commonly by mesothelioma, lymphoma, carcinomatosis, and thymoma. clinical signs develop gradually and include respiratory difficulty, cyanosis, and cough. supplemental oxygen should be administered. in many cases, thoracocentesis can be therapeutic and diagnostic. obtain thoracic radiographs both before and after thoracocentesis to determine whether a mass effect is present. following identification of a cause, definitive therapy can be instituted. mesotheliomas are rare and are associated with diffuse serosal disease. they are more common in dogs than in cats. effusions caused by mesotheliomas can affect the pleural or pericardial cavities. treatment is directed at removing effusion fluid and controlling reaccumulation with use of intracavitary platinum compounds, carboplatin, and cisplatin can be used in dogs. (cisplatin and carboplatin should never be used in cats.) chemical or physical pleurodesis may be helpful in controlling reaccumulation of fluid, but it is very painful in small animal patients. thoracic effusion secondary to lymphoma often is associated with an anterior mediastinal mass. t-cell lymphoma is the most common type of mediastinal mass observed in dogs. b-cell lymphoma is associated with a decreased response to chemotherapy and shorter survival times. treatment consists of combination chemotherapy with or without radiation therapy to decrease mass size. carcinomatosis is a diffuse disease of the pleural cavity that often is a result of metastasis from a primary pulmonary carcinoma or mammary adenocarcinoma. treatment is similar to that for mesothelioma and is aimed at controlling the effusion and delaying its recurrence. thymomas have been documented in both dogs and cats. dogs most commonly present with a cough, while cats present with clinical signs of respiratory distress and a restrictive respiratory pattern associated with the presence of pleural effusion. an anterior mediastinal mass is often observed on thoracic radiographs. in some cases, the pleural effusion must be drained via thoracocentesis before a mass is visible. ultrasound-guided aspiration and cytologic evaluation of the mass reveal a malignant epithelial tumor with small lymphocytes and mast cells. prognosis is good if the tumor can be completely excised. treatment consists of surgical removal with or without presurgical radiation therapy to shrink the mass. paraneoplastic syndromes of myasthenia gravis have been documented in dogs with thymomas. if megaesophagus or aspiration pneumonia is present, the prognosis is more guarded because of the high rate of complications. obstructive lesions affecting the urinary tract can be extramural (intra-abdominal, pelvic, or retroperitoneal) or intramural (urethral, bladder, or urethral wall) . transitional cell carcinoma is the most common type of bladder tumor observed in dogs. prostatic adenocarcinoma, or neoplasia of the sublumbar lymph nodes (lymphoma, adenocarcinoma from apocrine gland adenocarcinoma), also can cause urethral obstruction. treatment is aimed at relieving the obstruction and then attempting to identify the cause of the disease. to alleviate the obstruction, pass a urinary catheter whenever possible. perform cystocentesis only as a last resort because of the risk of seeding the peritoneal cavity with tumor cells if transitional cell carcinoma is the cause of the obstruction. institute supportive therapy including intravenous fluids and correction of electrolyte abnormalities. plain radiographs may reveal a mass lesion or may not be helpful without double contrast cystography. abdominal ultrasound is more sensitive in identifying a mass lesion in the urinary bladder. masses in the pelvic urethra are difficult to visualize with ultrasonography. double contrast cystourethrography is preferred. once the patient is stabilized, biopsy or surgery is indicated to identify the cause of the mass and attempt resection. urine tests for transitional cell carcinoma are available for identification of transitional cell carcinoma in the dog. complete surgical excision of transitional cell carcinoma or removal of benign tumors of the urinary bladder yields a favorable prognosis. poorer prognosis is seen with incomplete excision. many transitional cell carcinomas are located in the trigone region of the bladder and cannot be completely excised. the nonsteroidal antiinflammatory drug piroxicam is helpful in alleviating clinical signs for a reported -month median survival. in some dogs, cisplatin and carboplatin may delay recurrence of transitional cell carcinoma. tumors of the prostate gland are always malignant and occur with equal frequency in castrated and uncastrated male dogs. diagnosis of prostatic tumors is based on ultrasonographic evidence of a mass effect or prostatomegaly and on transrectal or transabdominal aspiration or biopsy. surgery, chemotherapy, and radiation therapy generally are unrewarding over the long term, although palliative radiation therapy may relieve clinical signs for to months. luminal tumors of the gastrointestinal tract typically cause obstruction, with slowly progressive clinical signs including vomiting, inappetence, and weight loss, or with acute severe protracted vomiting. extraluminal obstructive lesions usually arise from adhesions, or strangulation may occur, resulting in obstruction. perforation of the mass through the gastric or intestinal wall can cause peritonitis. treatment consists of initial stabilization and rehydration, evaluation for evidence of metastasis, and surgical resection of the affected area in cases of adenocarcinoma, leiomyoma, leiomyosarcoma, and obstructive or perforated lymphoma. gastric and intestinal adenocarcinoma are the most common gastrointestinal tumors observed in dogs. affected animals typically have a history of anorexia, weight loss, and vomiting. obtain an abdominal ultrasound before performing any surgery. fine needle aspirates of the mass and adjacent lymph nodes are usually diagnostic and can determine whether there is local metastasis. many tumors are not resectable, and metastasis occurs in approximately % of cases. dogs with smaller tumors that can be resected typically have longer survival times. leiomyosarcomas occur in the intestines of dogs, and carry a more favorable prognosis than adenocarcinoma if the mass can be completely resected. with complete resection, the average survival time is longer than year. the paraneoplastic syndrome of hypoglycemia has been observed with this tumor type. gastrointestinal lymphoma is the most common tumor of the gastrointestinal tract observed in cats. in comparison, it is relatively rare in dogs. unless there is complete obstruction or perforation of the gastrointestinal tract, surgical treatment for gastrointestinal lymphoma is not indicated. rather, multiple chemotherapy drugs are used in combination to achieve remission and resolution of the clinical signs of anorexia, weight loss, and vomiting. treatment responses unfortunately are poor. mast cell tumors of the gastrointestinal tract typically are manifested as gastrointestinal ulceration and hemorrhage in up to % of patients. the gastrointestinal hemorrhage that occurs with mast cell tumors results from increased acid secretion as a result of histamine receptor stimulation. treatment consists of histamine or proton pump inhibition (ranitidine, famotidine, cimetidine, or omeprazole). bowel perforation is a rare complication. many chemotherapy agents exert their effects on rapidly dividing normal and neoplastic cells. normal tissues that are commonly affected include the bone marrow, gastrointestinal tract, skin and hair follicles, and reproductive organs. some drugs have unique organspecific toxicities that must be monitored. knowledge and recognition of the expected type and onset of complications can alleviate their severity by rapid treatment, when complications occur (see table - ) . neutropenia is the most common bone marrow toxicity observed secondary to chemotherapy in small animal patients (table - ) . in most cases, the neutropenia is dose-dependent. the nadir, or lowest neutrophil count, is typically observed to days after chemotherapy treatment. once the nadir occurs, bone marrow recovery is observed, with an increase in circulating neutrophils within to hours (table - ) . treatment of myelosuppression is largely supportive to treat or prevent sepsis. prophylactic antibiotics are recommended in the afebrile patient with a neutrophil count < /µl. acceptable antibiotics include trimethoprim-sulfa and amoxicillin-clavulanate. granulocyte-colony stimulating factor (g-csf) (e.g., neupogen) is a recombinant human product that stimulates the release of neutrophils from the bone marrow, and its use shortens the recovery time following myelosuppressive drug therapy. disadvantages of g-csf include antibody production in response to the drug within weeks of use and its high cost. to prevent ongoing neutropenia, subsequent chemotherapy dosages should be decreased by %, and the interval in between treatments increased. whenever possible, overlap of myelosuppressive drugs should be avoided. acute gastrointestinal toxicity can occur within to hours after administration of cisplatin and actinomycin d. in many cases, pretreatment with the antiemetics metoclopramide, butorphanol, chlorpromazine, dolasetron or ondansetron can prevent chemotherapyinduced nausea and vomiting. vomiting can also occur as a delayed side effect to days after treatment with doxorubicin (adriamycin), actinomycin d, methotrexate, and cytoxan. in delayed reactions, vomiting and diarrhea are caused by damage to intestinal crypt cells. treatment consists of administration of antiemetics, intravenous fluids, and a bland highly digestible diet. doxorubicin also can cause hemorrhagic colitis within to days of administration. treatment includes a bland diet, metronidazole, and tylosin tartrate (tylan powder). emergency care mild to none not observed vincristine (low-dose), l-asparaginase, glucocorticosteroids moderate - days melphalan, cisplatin, mitoxantrone, actinomycin d severe - days doxorubicin, cyclophosphamide, vinblastine paralytic ileus can be observed to days after administration of vincristine. this side effect is more common in humans than animals and can be treated with metoclopramide once a gastrointestinal obstruction has been ruled out. cardiotoxicity doxorubicin (adriamycin) causes a dose-dependent dilative cardiomyopathy when the cumulative dose reaches to mg/m . in many cases, however, clinical signs do not occur until the cumulative dose is mg/m . the myocardial lesions are irreversible. treatment of cardiac dysrhythmias is dependent on the type of dysrhythmia (see section on treatment of dysrhythmias). discontinue doxorubicin and administer diuretics and positive inotropic therapy for dilative cardiomyopathy in order to delay the progression of congestive heart failure (see sections on treatment of congestive heart failure). if abnormalities are shown on electrocardiography performed before beginning therapy, substitute liposome-encapsulated doxorubicin or mitoxantrone substituted in the chemotherapy protocol. cardioprotectant drugs such as vitamin e, selenium, and n-acetyl cysteine have shown some promise in the prevention of doxorubicin-induced cardiotoxicity. cyclophosphamide can cause a sterile hemorrhagic cystitis. damage to the urinary bladder mucosa and vessels is caused by the toxic metabolite acrolein. clinical signs of sterile hemorrhagic cystitis include a history of cyclophosphamide administration, stranguria, hematuria, and pollakiuria. treatment for sterile hemorrhagic cystitis is discontinuation of the drug, treatment of any underlying urinary tract infection with antibiotic therapy based on susceptibility testing, and intravesicle drug administration. in extremely refractory cases, surgical debridement and cauterization of the bladder mucosa may be necessary. prevention of sterile hemorrhagic cystitis includes emptying the bladder frequently and administering the drug in the morning. concurrent administration of prednisone can induce polyuria and polydipsia. if sterile hemorrhagic cystitis occurs, chlorambucil can be substituted as a chemotherapeutic agent. anaphylactic reactions have been observed with the administration of l-asparaginase, adriamycin, etoposide, and paclitaxel. the risk of anaphylaxis increases with repeated administration, although in some animals anaphylaxis will occur on the first exposure to the drug. treatment consists of administration of epinephrine, diphenhydramine, famotidine, and glucocorticosteroids, as with any other life-threatening allergic reaction (see section on treatment of allergic reactions). to decrease the risk of an adverse reaction, give diphenhydramine ( . mg/kg im) to minutes before drug administration. slowing the rate of intravenous infusion also can decrease the chance of an anaphylactic reaction. cisplatin can cause a fatal irreversible pulmonary edema in cats, even at low dosages. -fluorouracil ( -fu) can cause a severe neurotoxicity in cats that results in ataxia and seizures. never use cisplatin or -fu in cats. poisoning cases benefit from a rapid, organized approach. key points in this approach are giving appropriate advice over the telephone, being able to access information sources, and providing appropriate treatment. there are only a few classes of poisons that account for the majority of toxicities reported in dogs and cats. every veterinarian should develop a familiarity with the clinical management of rodenticide and insecticide toxicity and be prepared with antidotes on hand. beyond the most common toxins, the spectrum of possibilities is endless, and the veterinarian must rely on appropriate information resources. it is important to have available a comprehensive source of pharmaceutical and plant identification resources. remarkably, considering the myriad of potentially toxic substances to which an animal can be exposed, relatively few specific antidotes are commonly used in veterinary medicine. because of the lack of specific antidotes, the veterinarian must treat each toxicity with general methods of poison management, applying basic critical care in the treatment of specific clinical signs associated with the poison exposure or toxicity. the adage "treat the patient, not the poison" often comes into play when the exact toxic substance is unknown, or has no specific antidote. before an animal arrives, the staff should be prepared to ask specific questions over the phone, and provide initial advice for clients, particularly if the animal lives some distance from the hospital (box - .) it is important to have access to a database of information on toxic substances. thousands of potentially toxic substances are available on the market today. the american society for the prevention of cruelty to animals (aspca) animal poison control center provides direct access to veterinary toxicologists hours a day, days a year. for additional information, call the nearest veterinary school or emergency center (box - ). also, see section for a table of emergency hotlines. check your local telephone book for a poison control center listing under emergency numbers, usually found on the front cover. although these numbers are for human poisonings, they have access to extensive poison and toxin databases and can potentially provide useful information for veterinarians, particularly regarding antidotal substances suitable for out of the ordinary toxins and human medications. information on the toxic ingredients in thousands of medications, insecticides, pesticides, and other registered commercial products has been confidentially placed by the government in these poison control centers. as new products are marketed, information regarding toxin ingredients is forwarded to the centers. various e-mail discussion lists can serve as an informative resource for practitioners, but access generally requires an initial subscription and may have the disadvantage of delayed *do not keep the client on the telephone for too long. lengthy histories can be performed once the animal is at your hospital and you have started to initiate treatment. † hair dressing products sometimes have hydrogen peroxide as a % w/v; this concentration is not suitable for induction of emesis. is your animal breathing or does it have respiratory difficulty? what is the color of the gums or tongue? is your animal able to walk? is there any vomiting, diarrhea, trembling, or seizures? does it appear lethargic or hyperactive? what is the substance that your animal ingested (was exposed to)? did you witness the ingestion or exposure? how much did the animal consume? how long ago was the exposure? was the substance swallowed, or is it on the animal's skin or eyes? how is the patient acting? how long has the animal been acting that way? or when was the last time you saw your animal act normally? . first aid instructions for the client: induce vomiting at home and save the vomitus. never induce vomiting if the patient is depressed, appears comatose, or is actively seizing. if the animal has ingested a caustic substance (strong alkali or acids) or a petroleum-based product (kerosene or turpentine), never recommend induction of emesis. hydrogen peroxide ( % w/v † ) ml = tsp/ lb of body weight can repeat once if no vomiting occurs after minutes . remind the owner to bring a sample of the toxin and the vomitus in with the patient. . advise the owner to transport the patient as rapidly as possible to the nearest veterinary hospital. response times. they are useful for ideas on standard and long-term therapy, but not emergency stabilization. an exception to this is the veterinary interactive network (vin), which posts message board communications. previous communications from veterinarians who treated a case with the same poison/toxin can be accessed with a subscription. many manufacturers operate an information service about their products. if the product label or name is available, check for a telephone number that may route you to a specialist. there are six essential steps in treating toxicities: . performing a physical examination . stabilizing the patient's vital signs . taking a thorough history . preventing continued absorption of the toxin . administering specific antidotes when available . facilitating clearance or metabolism of the absorbed toxin it is most important to provide symptomatic and supportive care both during and following emergency treatment. immediately on presentation, perform a brief but thorough physical examination. obtain a minimum database as well as serum, urine, or orogastric lavage samples for later toxicologic analyses. it is important at this time to systematically evaluate the patient's physical status, focusing particularly on the toxins most common to a particular geographic location and the organ systems most commonly affected by toxins in veterinary medicinenamely, the neurologic and gastrointestinal tracts. a checklist is useful when performing a complete physical examination (box - ). the minimium database includes a urine sample, packed cell volume, total protein, serum urea, and serum glucose. the information obtained from these simple cage-side tests is useful for determining dehydration, hemoconcentration, azotemia (renal or prerenal), and hypo-or hyperglycemia. when appropriate, obtain samples for serum biochemistry profiles, serum electrolytes, blood gases, serum osmolality, a complete hemogram, and coagulation profiles. samples of serum, urine, and any vomitus or orogastric lavage contents should be collected and saved for later toxicologic analyses as required later. stabilization of vital signs includes four major goals of treatment: maintain respiration, maintain cardiovascular function, control cns excitation, and control body temperature. in any patient with clinical signs of respiratory distress or respiratory dysfunction, supplemental oxygen should be administered via flow-by, oxygen hood, oxygen cage, nasal, nasopharyngeal, or transtracheal oxygen sources. ventilatory assistance may be necessary. irritant or corrosive substances can cause damage to the oropharyngeal mucosa to such an extent that airway obstruction occurs. when necessary, a temporary tracheostomy should be performed. arterial blood gases, pulse oximetry, and capnometry may be required to monitor oxygenation and ventilation. at the time of presentation, immediately place an intravenous catheter for administration of intravenous fluids, inotropes, antiarrhythmics, and antidotes, if necessary. the initial fluid of choice is a balanced crystalloid solution such as normosol-r, plasmalyte-m, or lactated ringer's solution. fluid therapy can later be changed based on the patient's acidbase and electrolyte status. some toxins can cause severe dysrhythmias and hyper-or hypotension. monitor blood pressure and perform ecg and correct any abnormalities according to standard therapy (see sections on hypotension and cardiac dysrhythmias). what is the pupil size? what is the pupil reactivity to light? is the ocular examination normal? what is the sensitivity to light or sound? nose: is it moist, dry, bubbling, or frothy, or caked with dirt? throat: are there any characteristic odors on the breath? are there any traces of foreign material on the tongue or in the crevices of the teeth or gums? are there petechiae or ecchymosis on the gums or bleeding from the gumline? what is the mucous membrane color? is it normal and pink, or dark red (injected), pale, or icteric? what is the capillary refill time? is it fast, normal, or slow? what is the patient's heart rate? are there any pulse deficits or dysrhythmias auscultated? what is the patient's blood pressure? what is the quality of the femoral pulse? is it synchronous with the heart rate, or are there dropped pulses? is the pulse bounding, normal, thready, or not palpable? what is the patient's electrocardiogram? what is the patient's respiratory rate? what is the patient's respiratory character? is it normal, fast, shallow, or labored? what do you hear on thoracic auscultation? do you hear harsh airway sounds or pulmonary crackles? what is the patient's rectal temperature? is there excessive salivation? is there evidence of vomiting or diarrhea? is abdominal palpation painful? do the intestinal loops feel normal, or are they fluid-filled or gas-filled? what is the color and consistency of the feces? is there a palpable urinary bladder? is there urine production? what is the color of the urine? peripheral lymph nodes should be normal in poisonings. some toxins cause hemolysis, methemoglobinemia, heinz body anemia, and coagulopathies. whole blood, fresh frozen plasma, packed rbcs, or hemoglobin-based oxygen carriers should be available and used if necessary. treat methemoglobinemia with a combination of ascorbic acid and n-acetylcysteine. many toxins affect the cns, producing clinical signs of excitation and/or seizures. diazepam is the drug of choice for most but not all seizures and tremors. if an animal has cns excitation secondary to the ingestion of selective norepinephrine reuptake inhibitors, avoid using diazepam, as it can potentially exacerbate clinical signs. muscle relaxants such as guaifenesin or methocarbamol may be required to control muscle spasm and tremors associated with some toxicities. consider animals that are in status epilepticus because of toxin exposure at high risk. such patients may not require the full dose of anesthetics or sedatives for seizure control. give phenobarbital ( - mg/kg iv) or pentobarbital ( - mg/kg iv to effect) for longer-term management of seizures. core body temperature can easily increase or decrease secondary to increased muscle activity or coma. animals may present as hypo-or hyperthermic, depending on the toxin ingested and the stage of toxicity. manage hypothermia with circulating hot water or hot air blankets, or place bubble wrap or saran wrap around the animal's peripheral extremities. manage hyperthermia by placing lukewarm wet towels on the patient until the rectal temperature has decreased to . °c ( °f). (see section on of hyperthermia and heat-induced illness). if sedatives or anesthetics have been used, initial hyperthermia may initially resolve due to hypothalamic loss of thermoregulatory control, cool water bathing should not be performed. when the patient is first presented to the veterinarian, have the owner complete a toxicologic history form (figure - ) while the animal is being initially assessed and vital signs are being stabilized. when initial stabilization of vital signs has been accomplished, the veterinarian can discuss the patient's history with the owner. in urgent situations, the veterinarian should obtain a brief history as an initial procedure (box - ). knowing when the animal was last seen as normal provides a time frame in which the toxic substance was most likely accessed, allowing differential diagnoses to be ranked in some order of probability by rate of onset. in eliciting a history from the owner about the animal's access to poisons, it is important not to take anything for granted. many owners do not realize how poisonous some substances can be, such as insecticide products, garbage, cleaning chemicals, and over-the-counter drugs commonly used by humans. many owners will deny that an animal could have ingested anything that might be toxic, not wanting to believe that the source of the toxin is within their household or property, particularly if recreational drug exposure is suspected. it is useful to phrase questions in a neutral fashion-for example, "is such-and-such present on the premises?" rather than "could the dog have eaten such-and-such?" if recreational drug exposure is suspected, another way to question the owners is to ask whether they have had any guests in their house recently that may have had such-and-such (e.g., marijuana, cocaine, methamphetamine). this approach serves to minimize the suggestion of any bias or preconceptions. when questioning an owner about recent events, it is useful to realize and acknowledge that disruption in the household routine is a distinct factor in the occurrence accidents, including poisonings. examples of such disruptive events include moving from the house, family member is ill or in the hospital, and renovations or recent construction. while these events are occurring, the safeguards followed by a normally careful owner may be disrupted. often, doors or gates may be left open, animals may be outside instead of inside (or vice versa), and inexperienced people may be pet-sitters. once owners are made aware of the importance of assessing such risks, they are often able to provide insight into otherwise baffling circumstances. various methods can be used to remove toxins from the gastrointestinal tract, including emesis, orogastric lavage, cathartics, and enemas. adsorbents, ion exchange resins, or precipitating or chelating agents may be used. removal of a toxic substance from the body surface may be necessary, depending on the toxin.the use of both emesis and orogastric lavage is less and less frequent in human medicine because of the risk of aspiration pneumonia and doubts about their efficacy. currently, management of poisonings in human medicine relies heavily on the use of activated charcoal combined with sorbitol as a cathartic, when appropriate, and supportive critical care. it should be emphasized, however, that the majority of poisonings in humans are due to drug overdoses (illicit or otherwise) (which have a relatively small volume and rapid absorption), for which this treatment is appropriate. furthermore, adoption of the approach rests on the availability of a hospital intensive care infrastructure, which is not always available in veterinary practice. induce emesis if the animal's physiology and neurologic status are stable (i.e., does not have respiratory depression or is not actively seizing, obtunded, unable to swallow or protect its airway). do not administer the same emetic more than twice. if the emetic doesn't work after two doses, give a different emetic or perform orogastric lavage under general anesthesia. emetics are strictly contraindicated for toxicity from petroleum-based products and corrosives because of the risk of aspiration pneumonia and further esophageal damage. emetics may also be of little value if poisons with antiemetic properties have been ingested, such as benzodiazepines, tricyclic antidepressants, and marijuana (table - ) . various emetics traditionally have been recommended for use in veterinary medicine. many have fallen out of favor because of the risk of causing adverse consequences and side effects. apomorphine ( . mg/kg iv or in the conjunctival sac) remains the standard but is less useful in certain situations in which the poison causes cns excitation or stimulation. it is ineffective in cats. other emetics include xylazine and hydrogen peroxide. do not use table salt because of the risk of severe oropharyngeal irritation and hypernatremia. do not use mustard powder or dishwashing liquid detergent because of the risk of severe oropharyngeal, esophageal, and gastric irritation. orogastric lavage is described in detail in the section on emergency procedures gastric lavage is contraindicated in treatment of toxicity from petroleum-based compounds and acid/alkali ingestion. the procedure can be messy but is very effective if performed within to hours of ingestion of the poison. to prevent aspiration, the patient should be placed under general anesthesia. keep the animal's head lowered during the procedure to prevent aspiration of stomach contents into the trachea. it is sometimes helpful to put the animal in both right and left lateral recumbency to allow complete emptying of gastric contents. repeat the procedure until the fluid runs clear from the stomach. in some cases in which solid material has been ingested, this process can take a long time, so be prepared with a large volume of warm water. following successful evacuation and lavage, administer a slurry of activated charcoal through the orogastric tube before removing it. keep the endotracheal tube cuffed and in place until the animal is semi-conscious, is starting the fight the tube, and is visibly able to swallow and protect its airway. • when was the animal last seen as normal? • what clinical signs developed? • how fast did the clinical signs develop? • when was the onset of clinical signs? • what is the animal's activity level? • does the animal have access to any poisonous substances? • this includes known toxins or chemicals, over-the-counter or prescription medications (including the owner's), and recreational drugs. enemas are useful to facilitate the action of cathartics and in cases in which the poison is a solid material (e.g., compost, snail bait, garbage) (box - ). it is best to use just lukewarm water. commercially available phosphate enema solutions can cause severe electrolyte disturbances (hyperphosphatemia, hyponatremia, hypocalcemia, and hypomagnesemia) and acid-base abnormalities (metabolic acidosis); therefore, they are absolutely contraindicated in small animal patients. use nonsterile nonspermicidal water-soluble lubricants (k-y jelly) old intravenous fluid bag enema bag -to -ml syringe fluid warm water, with or without hand or liquid dish soap the fluid volume required depends on the size of the animal and the state of its lower gastrointestinal tract. as with orogastric lavage, continue the procedure until the water runs clear. if difficulty is encountered emptying the lower gastrointestinal tract, repeat the enema in or hours, rather than be overzealous on the first attempt. cathartics are useful for hastening gastrointestinal elimination of toxins, and they are particularly useful for elimination of most solid toxicants (e.g., compost, garbage, snail baits). cathartics can be used in conjunction with activated charcoal. do not use magnesium-based cathartics in patients with cns depression, because hypermagnesemia can worsen this disorder and also cause cardiac rhythm disturbances (table - ) . activated charcoal ( - ml/kg) is the safest and to date the most effective adsorbent for the treatment of ingested toxins. activated charcoal can be administered after emesis or orogastric lavage or can be administered as the sole treatment. various preparations are available on the market, including dry powder, compressed tablets, granules, liquid suspensions, and concentrated paste preparations. commercially available products are relatively inexpensive and should be used whenever possible for ease of administration. vegetableorigin activated charcoal is the most efficient adsorbent and binds compounds with weak, nonionic bonds. some preparations are combined with sorbitol to provide simultaneous administration of an adsorbent and a cathartic; this combination has been shown to be most efficacious. repeated administration of activated charcoal every to hours has been shown to be beneficial in the management of a toxin that undergoes enterohepatic recirculation. administration of an oily cathartic or mixing the activated charcoal with food only serves to reduce the absorptive surface of the activated charcoal and therefore is not recommended. in general, substances that are very soluble and are rapidly absorbed are not well adsorbed by activated charcoal, including alkalis, nitrates, mineral acids, ethanol, methanol, ferrous sulfate, ammonia, and cyanide. kaolin and bentonite are clays that have been used as adsorbents. both are usually less effective than activated charcoal. however, they are reported to be better adsorbents than activated charcoal for the herbicide paraquat. ion exchange resins can ionically bind certain drugs or toxins. cholestyramine is one such resin, commonly used in human medicine to bind intestinal bile acids and thereby decrease cholesterol absorption. its application in toxicology extends to the absorption of fat-soluble toxins such as organochlorine and certain acidic compounds such as digitalis. ion exchange resins also have been used to delay or reduce the absorption of phenylbutazone, warfarin, chlorothiazide, tetracycline, phenobarbital, and thyroid preparations. precipitating, chelating, and diluting agents precipitating, chelating, and diluting agents are used primarily in the management of heavy metal intoxications, such as alkaloids or oxalates. they work by binding preferentially to the metal ion and creating a more soluble complex that is amenable to renal excretion. those chelating agents in common usage are calcium edta, deferoxamine, and d-penicillamine. calcium edta and deferoxamine should both be on hand in the veterinary hospital because they are necessary to treat zinc and iron toxicity, respectively, both of which have a short window of opportunity for therapeutic intervention. d-penicillamine has a wide application for a number of metal toxicities but tends to be used for long-term chronic therapy because it can be administered orally. various agents used for nonspecific dilution of toxins, including milk of magnesia and egg whites, although old-fashioned, still have wide application in many cases in which low-grade irritants have been ingested. bathing the animal is an important aspect of treatment for topical exposures to toxins such as insecticidal products, petroleum-based products, and aromatic oils. bathing an animal is not an innocuous procedure. to avoid hypothermia and shock, use warm water at all times. actively dry the animal to further minimize the risk of hypothermia. when bathing the animal, use rubber gloves and a plastic apron to avoid exposure to noxious agents. in most cases, a mild dishwashing soap is appropriate. medicated or antibacterial shampoos are less appropriate in this situation. for petroleum-based products in particular, dawn dishwashing liquid that "cuts the grease" works well to remove the oils. if dawn is not available, mechanics' hand cleaners or coconut oil-based soaps can be used instead. as a general principle, best results are obtained by barely wetting the patient's fur until the detergent is worked well into the fur, keeping the amount of water to a minimum until ready for the rinse. oil-based paint is best removed by clipping rather than by attempting removal with solvents, because solvents are also toxic. to remove powder products, brush and vacuum the animal before bathing it to eliminate further toxic exposure. with caustic alkaline or acidic products, the primary treatment is to dilute and flush the skin with warm water; do not attempt neutralization. neutralization can cause an exothermic reaction that causes further damage to the underlying tissues. eliminating poison from the eyes for ocular exposures, irrigate the eyes for a minimum of to minutes with warm (body temperature) tap water or warmed . % sterile saline solution. the use of neutralizing substances is not recommended because of the risk of causing further ocular damage. following adequate irrigation, treat chemical burns of the eyes with lubricating ointments and possibly a temporary tarsorrhaphy. atropine may be indicated as a cycloplegic agent. systemic nonsteroidal antiinflammatory drugs can be used to control patient discomfort. daily follow-up examinations are required because epithelial damage may be delayed, especially with alkali burns, and it is difficult to predict the final extent of ocular damage. topical glucocorticosteroids are contraindicated if the corneal epithelium is not intact. if severe conjunctival swelling is present with a corneal ulcer, parenteral glucocorticosteroids can be administered to help alleviate inflammation, but nonsteroidal antiinflammatory drugs should not be used simultaneously due to the risk of gastrointestinal ulceration or perforation. whenever possible, administer specific antidotes to negate the effects of the toxin and prevent conversion of the substance to the toxic metabolite. three categories of agents are used in the management of poisonings. the first category is specific antidotes. unfortunately, few specific antidotes are available for use in veterinary medicine. some "classic" toxins and antidotes are now considered to be rare, such as curare and physostigmine, thallium and prussian blue, and fluoride and calcium borogluconate. these and a few others have been omitted from the table. the second, broader category of antidotes includes those drugs used in the symptomatic management of clinical signs, which are part of our routine veterinary stock. drugs such as atropine, sedatives, steroids, antiarrhythmics, and beta-blockers fall into this category. the third category comprises nonspecific decontaminants such as activated charcoal, cathartics, and emetics. these were discussed previously. many patients benefit from efforts to enhance clearance or metabolism of the absorbed toxins. some specific therapies have been developed for this purpose, including -methylpyrazole for ethylene glycol toxicity and specific antibodies such as digibind (digoxin immune fab [ovine]) for digitalis toxicity. other strategies are aimed at promoting renal excretion. renal excretion strategies include diuresis, ion trapping, and peritoneal dialysis or hemodialysis (see section on peritoneal dialysis). diuresis and ion trapping are applicable to a large number of toxins and are discussed here in more detail. other toxins respond to urine acidification and urine alkalinization. enhancing renal excretion of substances is most useful for those organic substances that are present in significant concentrations in the plasma. substances that are non-ionic and lipid-soluble, such as certain herbicides, are likely to be less affected by attempts to promote rapid renal elimination. before starting diuresis or ion trapping, intravenous fluid therapy should be adequate as determined by normal central venous pressure, urine output, and mean arterial blood pressure. if any of these values are less than normal, use other measures to ensure adequate renal perfusion, including but not limited to a constant rate infusion of dopamine. simple fluid diuresis can influence the excretion of certain substances. the use of mannitol as an osmotic diuretic may reduce the passive reabsorption of some toxic substances in the proximal renal convoluted tubule by reducing water reabsorption. dextrose ( %) can be used as an osmotic diuretic. furosemide can be used to promote diuresis, but again, there is no substitute for intravenous fluid therapy. the use of mannitol, dextrose, and furosemide is contraindicated in hypotensive or hypovolemic patients. take care to avoid causing dehydration with any diuretic; central venous pressure monitoring is strongly recommended. ion trapping is based on the principle that ionized substances do not cross renal tubular membranes easily, and are not well reabsorbed. if the urinary ph can be changed so that the toxin's chemical equilibrium shifts to its ionized form, then that toxin can be "trapped" in the urine and excreted. alkaline urine favors the ionization of acidic compounds, and acidic urine favors the ionization of alkaline compounds. those toxins that are amenable to ion trapping are mostly weak acids and weak bases. ammonium chloride can be used to promote urinary acidification. contraindications to the use of ammonium chloride include a preexisting metabolic acidosis, hepatic or renal insufficiency, and hemolysis or rhabdomyolysis leading to hemoglobinuria or myoglobinuria. signs of ammonia intoxication include cns depression and coma. when performing urine acidification, frequently check the serum potassium concentration and urine ph. urine alkalinization can be performed with use of sodium bicarbonate. contraindications to the use of sodium bicarbonate include metabolic alkalosis (particularly with concurrent use of furosemide), hypocalcemia, and hypokalemia. as with urine acidification, monitor the serum potassium concentration and urine ph frequently. the major steps in management of poisonings discussed here must be accompanied by application of the fundamentals of critical care. respiratory and cardiovascular support have been discussed previously. renal and gastrointestinal function and analgesia are particularly important in the management of the poisoning patient. maintenance of renal perfusion is a priority in the poisoning patient. fluid, electrolyte, and acid-base balance must be controlled and be accurate. poisoning patients are at particularly high risk for renal damage and acute renal failure, whether by primary toxic insult to the renal parenchyma or by acute or prolonged renal hypoperfusion. for this reason, a protocol that aims at preventing oliguria and ensuing renal failure is one of the therapeutic strategies that should be routinely employed. this protocol is described in box - . gastrointestinal protectant drugs may be indicated for the management of those poisons that are gastrointestinal irritants or ulcerogenic. commonly used gastroprotectant drugs include cimetidine, ranitidine, famotidine, omeprazole, sucralfate, and misoprostol. antiemetics may be used to suppress intractable vomiting. metoclopramide is commonly used, and it is the drug of choice for centrally mediated nausea. antiemetics that work by different mechanisms can be used in combination as necessary. examples are dopamine -receptor antagonists such as prochlorperazine, -hydroxytryptamine antagonists such as ondansetron and dolasetron, and h- receptor antagonists such as diphenhydramine and meclizine. analgesics are more appropriate to treat poisonings than once thought. common effects of poisons including severe gastroenteritis and topical burns or ulcerations may warrant the use of analgesics. longer-acting analgesics such as morphine, hydromorphone, and buprenorphine are particularly useful. nutritional support may be necessary in the form of enteral or parenteral feeding in patients that have esophageal or gastric damage or that need to be sedated for long periods of time. endoscopy may be useful in assessing the degree of esophageal and gastric damage, particularly after ingestion of caustic substances. introduction: acetaminophen (paracetamol) is the active ingredient in tylenol and many over-thecounter cold products. acetaminophen is converted to n-acetyl-p-benzoquinonimine in the liver, a toxic substance that can cause oxidative injury of red blood cells and hepatocytes. clinical signs of acetaminophen toxicity include respiratory distress from lack of oxygen-carrying capacity, cyanosis, methemoglobinemia (chocolate-brown appearance of the blood and mucous membranes), lethargy, vomiting, and facial and paw swelling (cats). the toxic dose of acetaminophen is > mg/kg for dogs, and mg/kg for cats. treatment of acetaminophen toxicity includes induction of emesis or orogastric lavage if the substance has been ingested within minutes. activated charcoal should also be administered. in cases of severe anemia, give supplemental oxygen along with a packed rbc transfusion. administer intravenous fluids to maintain renal and hepatic perfusion. n-acetylcysteine, vitamin c, and cimetidine are the treatments of choice for methemoglobinemia in patients with acetaminophen toxicity. introduction: hydrochloric, nitric, and phosphoric acids cause chemical burns through contact with the skin and/or eyes. localized superficial coagulative necrosis occurs upon contact. usually, the patient's skin is painful to the touch or the animal may lick or chew at an irritated area that is not visible under the haircoat. if the chemical is swallowed, do not induce emesis or perform orogastric lavage, because of the risk of worsening esophageal irritation. rinse the patient's skin and eyes with warm water or warm saline for a minimum of / hour. use analgesics and treat corneal ulcers (see section on corneal ulcers) as required. do not attempt chemical neutralization, because of the risk of causing an exothermic reaction and worsening tissue injury. aflatoxin (aspergillus flavus) is found in moldy feed grains. clinical signs of toxicity occur after ingestion and include vomiting, diarrhea, and acute hepatitis; abortion may occur in pregnant bitches. treatment of suspected aflatoxin ingestion consists of gastric decontamination, administration of activated charcoal, intravenous fluids, and hepatic supportive care (s-adenosyl methionine [same], milk thistle). drinking (ethanol), rubbing (isopropyl), and methyl (methanol) alcohols can be harmful if ingested ( . to . g/kg po). all cause disruption of neuronal membrane structure, impaired motor coordination, cns excitation followed by depression, and stupor that can lead to cardiac and respiratory arrest, depending on the amount ingested. affected animals may appear excited and then ataxic and lethargic. contact or inhalant injury can occur, causing dermal irritation and cutaneous hyperemia. methanol also can cause hepatotoxicity. and diarrhea result from muscarinic overload. nicotinic overload produces muscle tremors. toxicity can result in seizures, coma, and death. and cause severe irritation and corrosion of the mucous membranes and skin. some compounds also can cause clinical signs similar to those observed with anticholinesterase compounds, including muscle tremors, seizures, paralysis, and coma. methemoglobinemia can occur. signs of ethylene glycol intoxication and renal impairment or failure, a negative test for the presence of calcium oxalate crystalluria means that there is no more ethylene glycol in the patient's serum because it has all been metabolized. cats are very sensitive to the toxic effects of ethylene glycol. in many cases, cat may have ingested a toxic dose, but because the sensitivity of the assay is low, test results will be negative. lack of treatment can result in death. there are three phases of ethylene glycol intoxication. in the first to hours after ingestion (stage i), the patient may appear lethargic, disoriented, and ataxic. in stage ii ( to hours following ingestion), the patient improves and appears clinically normal. in stage iii ( to hours following ingestion), the patient demonstrates clinical signs of renal failure (polyuria and polydipsia) that progress to uremic renal failure (vomiting, lethargy, oral ulceration). finally, seizures, coma, and death occur. crosses, old english sheepdogs, and some terriers. clinical signs of ivermectin toxicity include vomiting, ataxia, hypersalivation, agitation, tremors, hyperactivity, hyperthermia, hypoventilation, coma, seizures, signs of circulatory shock, bradycardia, and death. clinical signs often occur within to hours after ingestion or iatrogenic overdose. blood ivermectin levels can be measured, but diagnosis is often made based on clinical signs and knowledge of exposure in predisposed breeds. there is no known antidote. the clinical course can be prolonged for weeks to months before recovery occurs. to treat known exposure, induce emesis or perform orogastric lavage if the substance was ingested was within hour of presentation and the patient is not symptomatic. administer activated charcoal. control seizures with phenobarbital, pentobarbital, or propofol administered as intermittent boluses or as a constant rate infusion. diazepam, which potentially can worsen central nervous stimulation, is contraindicated. administer intravenous fluids to maintain perfusion and hydration, and treat hyperthermia. supportive care may be necessary, including supplemental oxygen (or mechanical ventilation, if necessary), frequent turning of the patient and passive range-of-motion exercises, placement of a urinary catheter to maintain patient cleanliness and monitor urine output, lubrication of the eyes, and parenteral nutrition (see section on rule of twenty). specific antidotes used to treat ivermectin toxicity include physostigmine and picrotoxin. physostigmine therapy was beneficial in some patients for a short period; picrotoxin caused severe violent seizures and therefore should be avoided. introduction d-limonene and linalool are components of citrus oil extracts used in some flea control products. the toxic dose is unknown, but cats appear to be very sensitive to exposure. clinical signs of toxicity include hypersalivation, muscle tremors, ataxia, and hypothermia. treatment of d-limonene and linalool exposure includes treatment of hypothermia, administration of activated charcoal to prevent further absorption, and careful, thorough bathing to prevent further dermal exposure. lead is ubiquitous, and is found in some paints, car batteries, fishing equipment/ sinkers, and plumbing materials. lead can be toxic at doses of mg/kg. if more than than - mg/kg of lead is ingested, death can occur. lead causes toxicity by inhibiting sulfur-containing enzymes, leading to increased rbc fragility, and cns damage. clinical signs of hyperexcitability, dementia, vocalization, seizures, and lower motor neuron polyneuropathy can occur. affected animals may appear blind, or vomiting, anorexia, and constipation or diarrhea may occur. if lead toxicity is suspected, blood and urine lead levels can be measured. treatment of lead toxicity is supportive and is directed at treatment of clinical signs. control seizures with diazepam or phenobarbital. if cerebral edema is present, administer mannitol ( . - . g/kg iv), followed by furosemide ( mg/kg iv minutes after mannitol). sodium or magnesium sulfate should be administered as a cathartic. initiate chelation therapy with dimercaprol, penicillamine, or calcium edta. if a lead object is identified in the gastrointestinal tract on radiographs, remove the object using endoscopy or exploratory laparotomy. hyperthermia, that occurs within - minutes of ingestion. diarrhea and convulsions can develop. if hyperthermia is severe, renal failure secondary to myoglobinuria and disseminated intravascular coagulation can result. delayed hepatic failure has been described days after initial recovery. if metaldehyde toxicosis is suspected, analysis of urine, serum, and stomach contents is warranted. to treat metaldehyde toxicity, procure and maintain a patent airway and control cns excitation and muscle tremors. if an animal has just ingested the metaldehyde and is not symptomatic, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by administration of one dose of activated charcoal. administer intravenous fluids to control hyperthermia, prevent dehydration, and correct acid-base and electrolyte abnormalities. methocarbamol is the treatment of choice to control muscle tremors. diazepam can be used to control seizures if they occur. introduction mushroom ingestion most commonly causes activation of the autonomic nervous system, resulting in tremors, agitation, restlessness, hyperexcitability, and seizures. in some cases slud (salivation, lacrimation, urination, and defecation) is seen. some mushrooms (amanita spp.) also can cause hepatocellular toxicity. clinical signs include vomiting, anorexia, lethargy, and progressive icterus. hemoglobinuria and pigment damage of the renal tubular epithelium. heinz bodies may be observed on cytologic evaluation of the peripheral blood smear. paint in a sorbitol or glycerol carrier. when large quantities of these osmotically active sugars are ingested, osmotic shifts of fluid cause a sudden onset of neurologic or gastrointestinal signs, including ataxia, seizures, and osmotic diarrhea caused by massive fluid shifts into the gastrointestinal tract. the loss of water in excess of solute can result in hypernatremia, a free water deficit, and increased serum osmolality. following orogastric lavage, treatment of ingestion includes administering warm water enemas to help speed the movement of the paintballs through the gastrointestinal tract. do not administer activated charcoal (usually in a propylene glycol carrier), because the compound's cathartic action will pull more fluid into the gastrointestinal tract. baseline electrolytes should be obtained and then carefully monitored. if severe hypernatremia develops, administer hypotonic solutions such as . % nacl + . % dextrose or % dextrose in water after calculating the patient's free water deficit. because of the large volume of fluid loss, intravenous fluid rates may seem excessive but are necessary to normalize acid-base, electrolyte, and hydration status. in most cases, these patients can survive if the problem is recognized promptly and corrected with careful electrolyte monitoring, aggressive decontamination strategies, and intravenous fluid support. introduction paraquat, a dipyridyl compound, is the active ingredient in some herbicides. the ld of paraquat is - mg/kg. paraquat initially causes cns excitation. it also causes production of oxygen-derived free radical species in the lungs, that can lead to the development of acute respiratory distress syndrome. initial clinical signs include vomiting, diarrhea, and seizures. within to days, clinical signs associated with severe respiratory distress and acute respiratory distress syndrome (ards) can develop, leading to death. chronic effects include pulmonary fibrosis, if the patient survives the initial toxicity period. the prognosis for paraquat toxicity is generally unfavorable. to treat paraquat ingestion, remove the toxin from the gastrointestinal tract as rapidly as possible after ingestion. there are no known antidotes. if the compound was ingested within the past hour and the animal is able to protect its airway, induce emesis. otherwise, perform orogastric lavage. activated charcoal is not as effective as clay or bentonite adsorbents for removing this particular toxin. early in the course of paraquat toxicity, oxygen therapy is contraindicated because of the risk of producing oxygen-derived free radical species. later, oxygen therapy, including mechanical ventilation, is necessary if ards develops. experimentally, free radical scavengers (n-acetyl cysteine, vitamin c, vitamin e, same) have been shown to be useful in preventing damage caused by oxygen-derived free radical species. hemoperfusion may be useful in eliminating the toxin, if it is performed early in the course of toxicity. pennyroyal oil is an herbal flea control compound that contains menthofuran as its toxic compound. menthofuran is hepatotoxic and may cause gastrointestinal hemorrhage and coagulopathies. to treat toxicity, administer a cathartic and activated charcoal and antiemetic and gastroprotectant drugs, and thoroughly bathe the animal to prevent further dermal exposure. petroleum distillates: see fuels phenobarbital: see barbiturates phenylcyclidine (angel dust) introduction phenylcyclidine (angel dust) is an illicit recreational drug that causes both cns depression and excitation, decreased cardiac output, and hypotension. to treat phenylcyclidine toxicity, place an intravenous catheter, and administer intravenous fluids and antiarrhythmic drugs to maintain organ perfusion. administer supplemental oxygen, and administer diazepam to control seizures. urine alkalinization can help eliminate the compound. phenylephrine is an α-adrenergic agonist in many over-the-counter decongestant preparations. clinical signs of intoxication include mydriasis, tachypnea, agitation, hyperactivity, and abnormal flybiting and staring behavior. tachycardia, bradycardia, hypertension, hyperthermia, and seizures can occur. to treat phenylephrine toxicity, place an intravenous catheter and give intravenous fluids to maintain hydration, promote diuresis, and treat hyperthermia. administer prazosin or sodium nitroprusside to treat hypertension, antiarrhythmic drugs as necessary, and diazepam to control seizures. phenylpropanolamine has both αand β-adrenergic agonist effects, and is used primarily in the treatment of urinary incontinence in dogs. the drug was taken off of the market for use in humans because of the risk of stroke. clinical signs of phenylpropanolamine intoxication include hyperactivity, hyperthermia, mydriasis, tachyarrhythmias or bradycardia, hypertension, agitation, and seizures. to treat toxicity, administer prazosin or nitroprusside to control hypertension, a betablocker (esmolol, propranolol, atenolol) to control tachyarrhythmias, diazepam to control seizures, and intravenous fluids to maintain hydration and promote diuresis. urine acidification may aid in facilitating excretion. if bradycardia occurs, do not use atropine. pseudoephedrine is an αand β-adrenergic agonist that is a component of many over-thecounter decongestants and is used in the manufacture of crystal methamphetamine. clinical signs of toxicity include severe restlessness, tremors, mydriasis, agitation, hyperthermia, tachyarrhythmias or bradycardia, hypertension, and seizures. to treat toxicity, administer activated charcoal, intravenous fluids to promote diuresis and treat hyperthermia, chlorpromazine to combat α-adrenergic effects, a beta-blocker (propranolol, esmolol, atenolol) to treat β-adrenergic effects, and cyproheptadine (per rectum) to combat serotoninergic effects. piperazine is a gaba agonist, and causes cervical and truncal ataxia, tremors, seizures, coma, and death. salt used for thawing ice commonly contains calcium chloride, a compound that has a moderate toxic potential. calcium chloride produces strong local irritation and can cause gastroenteritis and gastrointestinal ulcers if ingested. respiratory emergencies consist of any problem that impairs delivery of oxygen to the level of the alveoli or diffusion of oxygen across the alveolar capillary membrane into the pulmonary capillary network. decreased respiratory rate or tidal volume can result in hypoxia and buildup of carbon dioxide, or hypercarbia, leading to respiratory acidosis. conditions most frequently encountered result in airflow obstruction, prevention of normal lung expansion, interference with pulmonary gas exchange (ventilation-perfusion mismatch), and alterations of pulmonary circulation. evaluation of the patient with respiratory distress is often challenging, because the most minimal stress can cause rapid deterioration, or even death in critical cases. careful observation of the patient from a distance often allows the clinician to determine the severity of respiratory distress and localize the lesion based on the patient's respiratory pattern and effort. animals in respiratory distress often have a rapid respiratory rate (> breaths per minute). as respiratory distress progresses, the patient may appear anxious and start openmouth breathing. the animal often develops an orthopneic posture, characterized by neck extension, open-mouthed breathing, and elbows abducted or pulled away from the body. cyanosis of the mucous membranes often indicates extreme decompensation. clinical signs of respiratory distress can develop acutely, or from decompensation of a more chronic problem that was preceded by a cough, noisy respirations, or exercise intolerance. localization of the cause of respiratory distress is essential to successful case management. in any patient with clinical signs of respiratory distress, the differential diagnosis should include primary pulmonary parenchymal disease, airway disease, thoracic cage disorders, congestive heart failure, dyshemoglobinemias (carbon monoxide, methemoglobin), and anemia. careful observation of the patient's respiratory pattern can aid in making a diagnosis of upper airway disease/obstruction, primary pulmonary parenchymal disease, pleural space disease, and abnormalities of the thoracic cage. it is often helpful to rest a hand on the patient and breathe along with the patient's effort, to confirm the periods of inhalation and exhalation. the pharynx, larynx, and extrathoracic trachea comprise the upper airway. obstructive lesions are associated with a marked inspiratory wheeze or stridor and slow deep inspiratory effort. auscultation of the larynx and trachea may reveal more subtle obstructions of normal air flow. stridor can usually be auscultated without the use of a stethoscope. lung sounds are usually normal. the neck should be carefully palpated for a mass lesion, tracheal collapse, and subcutaneous emphysema. subcutaneous emphysema suggests tracheal damage or collapse secondary to severe trauma. in some cases, there is a history of voice, or bark, change secondary to laryngeal dysfunction. differential diagnosis is usually based on the patient's signalment, history, and index of suspicion of a particular disease process. differential diagnoses of upper airway obstruction are listed in box - . diseases of the pleural space often are associated with a restrictive respiratory pattern. inspiratory efforts are short, rapid, and shallow, and there is often a marked abdominal push. the pattern has been referred to as a choppy "dysynchronous" respiratory pattern. depending on the disease present, lung sounds may be muffled ventrally and enhanced dorsally. percussion of the thorax reveals decreased resonance if fluid is present. increased resonance is present with pneumothorax. decreased compressibility of the anterior thorax may be present with an anterior mediastinal mass lesion, particularly in cats and ferrets. a pneumothorax or diaphragmatic hernia is commonly associated with evidence of trauma, with or without rib fractures. respiratory distress due to hemothorax may be exacerbated by anemia. differential diagnoses for patients with evidence of pleural cavity disease include pneumothorax, diaphragmatic hernia, neoplasia, and various types of pleural effusion. primary pulmonary parenchymal disease can involve the intrathoracic airways, alveoli, interstitial space, and pulmonary vasculature. a rapid, shallow, restrictive respiratory pattern may be observed with a marked push on exhalation, particularly with obstructive airway disease such as chronic bronchitis (asthma) in cats. crackles or wheezes are heard on thoracic auscultation. differential diagnoses for pulmonary parenchymal disease include cardiogenic and noncardiogenic pulmonary edema, pneumonia, feline bronchitis (asthma), pulmonary contusion, aspiration pneumonitis, pulmonary thromboembolism, neoplasia, infection (bacterial, fungal, protozoal, viral) , and/or chronic bronchitis. other abnormal respiratory patterns may be evident, and warrant further consideration. tachypnea present in the absence of other signs of respiratory distress can be a normal response to nonrespiratory problems, including pain, hyperthermia, and stress. a restrictive respiratory pattern with minimal thoracic excursions can be associated with diseases of neuromuscular function, including ascending polyradiculoneuritis, botulism, and tick paralysis. if adequate ventilation cannot be maintained by the patient, mechanical ventilation may be indicated. kussmaul respiration manifests as very slow, very deep respirations when a metabolic acidosis is present. this type of respiratory pattern typically is observed in patients with severe diabetic ketoacidosis and renal failure in a compensatory attempt to blow off carbon dioxide. cheyne-stokes respiration is usually observed with a defect in the central respiratory control center. the classic pattern of cheyne-stokes respiration is normal or hyperventilation followed by a period of apnea or hypoventilation. in cases of lower cervical cord damage or damage to the central respiratory control center in the cns, the diaphragm alone may assume most of the ventilatory movement. with diaphragmatic fatigue, severe hypoventilation and resultant hypoxemia may require mechanical ventilation. immediate management of any patient in respiratory distress is to minimize stress at all costs. relatively benign procedures such as radiography or intravenous catheter placement can be fatal in patients with severe respiratory compromise. stabilization should always precede further diagnostic evaluation. in some cases, sedation may be required before performing any diagnostics, to prevent further stress. all patients should receive some form of supplemental oxygen, either by mask, cage, or flow-by techniques. in cases in which a severe pneumothorax or pleural effusion is suspected, perform therapeutic and diagnostic thoracocentesis bilaterally to allow lung re-expansion and alleviate respiratory distress, whenever possible. if thoracocentesis alone is not effective at maintaining lung re-expansion, place a thoracostomy tube (particularly in cases of tension pneumothorax). if hypovolemic/ hemorrhagic shock is present, initiate treatment while stabilizing the respiratory system (see section on shock). if an animal is suspected of having an upper airway obstruction, reestablish airflow. in cases of laryngeal paralysis, tracheal collapse, and brachycephalic airway syndrome, sedation is often very useful in alleviating the distress of airway obstruction. in cases of laryngeal collapse, however, sedation may make the condition worse. if laryngeal edema is severe, administer a dose of short-acting glucocorticosteroids (dexamethasone sodium phosphate) to decrease laryngeal inflammation and edema. if a foreign body is lodged in the pharynx, perform the heimlich maneuver by thrusting bluntly several times on the patient's sternum. objects such as balls or bones may be small enough to enter the larynx but too large to be expelled, and will require rapid-acting general anesthesia to facilitate dislodgement and removal. if the obstruction cannot be removed, bypassing the obstruction with an endotracheal tube or temporary tracheostomy should be considered. in an emergency, a temporary transtracheal oxygen catheter can quickly be placed in the following manner. connect a -or -gauge needle to a length of intravenous extension tubing and a -ml syringe. place the male connector of the syringe into the female portion of the extension tubing. cut off the syringe plunger and connect the resulting blunt end to a length of flexible tubing attached to a humidified oxygen source. run the oxygen at l/minute to provide adequate oxygenation until a tracheostomy can be performed. (see sections on oxygen supplementation and tracheostomy). once the animal's condition has been stabilized, specific diagnostic tests, including arterial blood gas analyses, thoracic radiographs, and/or transtracheal wash, can be performed, depending on the patient's condition and needs. specific therapies for management of upper airway obstruction, pleural space disease, and pulmonary disease are discussed next. upper airway obstruction can occur as a result of intraluminal or extraluminal mass lesions or foreign bodies in the oropharynx (abscess, neoplasia), laryngeal paralysis, trauma, and anatomic abnormalities. clinical signs of an upper airway obstruction are associated with an animal's extreme efforts to inhale air past the obstruction. marked negative pressure occurs in the extrathoracic airways and can cause worsening of clinical signs. mucosal edema and inflammation further worsen the obstruction. therapy for upper airway obstruction is aimed at breaking the cycle of anxiety and respiratory distress. administer the anxiolytic tranquilizer acepromazine ( . - . mg/kg iv, im, sq) to decrease patient anxiety. many animals develop hyperthermia from increased respiratory effort and extreme anxiety. implement cooling measures in the form of cool intravenous fluids and wet towels soaked in tepid water placed over the animal (see section on hyperthermia). administer supplemental oxygen in a manner that is least stressful for the animal. short-acting glucocorticosteroids can also be administered (dexamethasone sodium phosphate, . mg/kg iv, sq, im) to decrease edema and inflammation. if the airway obstruction is severe and there is no response to initial measures to alleviate anxiety and decrease inflammation, establish control of ventilation by placement of an endotracheal tube (see section on endotracheal intubation), tracheal oxygen catheter, or temporary tracheostomy. to obtain airway control, administer a rapid-acting anesthetic (propofol, - mg/kg iv to effect), and intubate with a temporary tracheostomy. an intratracheal oxygen catheter can be placed with sedation and/or a local anesthetic (see technique for transtracheal wash). laryngeal paralysis is a congenital or acquired condition that occurs primarily in largebreed dogs secondary to denervation of the arytenoid cartilages by the recurrent laryngeal nerve. congenital laryngeal paralysis occurs in the bouvier des flandres, siberian husky, and bull terrier. acquired laryngeal paralysis occurs in labrador retrievers, saint bernards, and irish setters. acquired laryngeal paralysis can be idiopathic, acquired secondary to trauma to the recurrent laryngeal nerve, or can be a component of systemic neuromuscular disease. although rare, this condition also occurs in cats. with dysfunction of the recurrent laryngeal nerve, the intrinsic laryngeal muscles atrophy and degenerate. as a result, the vocal folds and arytenoid cartilage move in a paramedian position within the airway and fail to abduct during inhalation, causing airway obstruction. laryngeal paralysis can be partial or complete, unilateral or bilateral. in many cases, a change in bark is noted prior to the development of clinical signs of respiratory distress or exercise intolerance. when a patient presents with severe inspiratory stridor (with or without hyperthermia) initiate stabilization with anxiolytic tranquilizers, supplemental oxygen, and cooling measures. once the patient's condition has been stabilized, definitive measures to accurately document and assess the patient's airway should be considered. place the patient under very heavy sedation with short-acting barbiturates or propofol ( - mg/kg iv) and observe the arytenoid cartilages closely in all phases of respiration. administer just enough drug to allow careful examination without getting bitten. if the arytenoid cartilages do not abduct during inhalation, administer dopram (doxapram hydrochloride, - mg/kg iv) to stimulate respiration. absent or paradoxical laryngeal motion (closed during inspiration and open during exhalation) is characteristic of laryngeal paralysis. correction of the defect involves documentation and treatment of any underlying disorder and surgical repair of the area to open the airway. partial laryngectomy, arytenoid lateralization ("tie-back" surgery), or removal of the vocal folds has been used with some success. aspiration pneumonitis is common following these procedures. brachycephalic airway syndrome is associated with a series of anatomic abnormalities that collectively increase resistance to airflow. affected animals typically have stenotic nares, an elongated soft palate, and a hypoplastic trachea. components of the syndrome can occur alone or in combination. in severe cases, laryngeal saccular edema and eversion, and eventual pharyngeal collapse, can occur secondary to the severe increase in intrathoracic airway pressure required to overcome the resistance of the upper airways. specific airway anomalies can be identified with general anesthesia and laryngoscopy. severe respiratory distress should be treated as discussed previously. treatment requires surgical correction of the anatomic abnormalities. in animals with laryngeal collapse, surgical correction may not be possible, and a permanent tracheostomy may be required. because an elongated soft palate and stenotic nares can be identified before the onset of clinical signs, surgical correction to improve airflow when the animal is young may decrease the negative intra-thoracic pressure necessary to move air past these obstructions. the chronic consequences of everted laryngeal saccules and laryngeal collapse potentially can be prevented. tracheal collapse is common in middle-aged and older toy and small-breed dogs. the owner typically reports a chronic cough that is readily induced by excitement or palpation of the trachea. the cough often sounds like a "goose honk." diagnostic confirmation is obtained by lateral radiography or fluoroscopy of the cervical and thoracic trachea during all phases of respiration. acute decompensation is uncommon but does occur, particularly with excitement, exercise, and increased environmental temperatures or ambient humidity. therapy of the patient with acute respiratory distress secondary to tracheal collapse includes sedation, administration of supplemental oxygen, and provision of cooling measures to treat hyperthermia. cough suppressants (hydrocodone bitartrate-homatropine methylbromide, . mg/kg po q - h, or butorphanol, . mg/kg po q - h) are useful. tracheal collapse is a dynamic process that usually involves both the upper and lower airways. because of this, bypassing the obstruction is often difficult. tracheal stents have been emergency care used with limited success in combination with treatment of chronic lower airway disease. crush or bite injuries to the neck can result in fractures or avulsion of the laryngeal or tracheal cartilages. bypassing the obstructed area may be necessary until the patient is stable and can undergo surgical correction of the injury. if there is avulsion of the cranial trachea, it may be difficult to intubate the patient. a long, rigid urinary catheter can be inserted past the area of avulsion into the distal segment, and an endotracheal tube passed over the rigid catheter, to establish a secure airway. neck injury can also result in damage to the recurrent laryngeal nerve and laryngeal paralysis. foreign bodies can lodge in the nasal cavity, pharynx, larynx, and distal trachea. signs of foreign bodies in the nares include acute sneezing and pawing at or rubbing the muzzle on the ground. if the object is not removed, sneezing continues and a chronic nasal discharge develops. respiratory distress is uncommon, but the foreign body is severely irritating. pharyngeal and tracheal foreign bodies can cause severe obstruction to airflow and respiratory distress. diagnosis of a foreign body is based on the patient history, physical examination findings, and thoracic or cervical radiographs. smaller foreign bodies lodged in the distal airways may not be apparent radiographically but can cause pulmonary atelectasis. foreign bodies of the nose or pharynx can often be removed with an alligator forceps with the patient under anesthesia. if removal is not possible with a forceps, flushing the nasal cavity from cranial to caudal (pack the back of the mouth with gauze to prevent aspiration) can sometimes dislodge the foreign material into the gauze packing. rhinoscopy may be necessary. if an endoscope is not available, an otoscope can be used. foreign objects lodged in the trachea can be small and function like a ball valve during inhalation and exhalation, causing episodic hypoxia and collapse. when attempting to remove these objects, suspend the patient with its head down. remove the object with an alligator forceps, using a laryngoscope to aid in visualization. foreign bodies lodged in the trachea or bronchi require removal with endoscopic assistance. nasopharyngeal polyps (in cats, tumors, obstructive laryngitis, granulomas, abscesses, and cysts) can cause upper airway obstruction. clinical signs are usually gradual in onset. the lesions can be identified through careful laryngoscopic examination performed with the patient under general anesthesia. the nasopharynx above the soft palpate should always be included in the examination. pedunculated masses and cysts are excised at the time of evaluation. biopsy of diffusely infiltrative masses is indicated for histologic examination and prognosis. it is impossible to distinguish obstructive laryngitis from neoplasia based on gross appearance alone. whenever possible, material should be collected from abscesses and granulomas for cytologic evaluation and bacterial culture. extraluminal masses impinge on and slowly compress the upper airways, resulting in slow progression of clinical signs. masses are usually identified by palpation of the neck. enlarged mandibular lymph nodes, thyroid tumors, and other neoplasms may be present. diagnosis is usually based on a combination of radiography and ultrasonography. ct and/or mri are helpful in identifying the full extent and invasiveness of the lesion. definitive diagnosis is made with a fine-needle aspirate or biopsy. many thyroid tumors bleed excessively. the inside of each side of the hemithorax is covered in parietal pleura. the lung lobes are covered in visceral pleura. the two surfaces are in close contact with each other, and are contiguous at the hilum under normal circumstances. pneumothorax refers to free air within the pleural space, accumulating in between the parietal and visceral pleura. the term pleural effusion refers to fluid accumulation in that area but does not reflect the amount or type of fluid present. the mediastinal reflections of the pleura typically are thin in dogs and cats, and usually, but not always, connect. bilateral involvement of pneumothorax or pleural effusion is common. both pneumothorax and pleural effusion compromise the lungs' ability to expand and result in hypoxia and respiratory distress. pneumothorax can be classified as open versus closed, simple versus complicated, and tension. an open pneumothorax communicates with the external environment through a rent in the thoracic wall. a closed pneumothorax results from tears in the visceral pleura but does not communicate with the outside. a tension pneumothorax occurs as a result of a tear in the lung or chest wall that creates a flap valve, such that air is allowed to leave the lung and accumulate in the pleural space during inhalation, and closes to seal off exit of air from the pleural space during exhalation. tension pneumothorax can cause rapid decline in cardiopulmonary status and death if not recognized and treated immediately. a simple pneumothorax is one that can be controlled with a simple thoracocentesis. complicated pneumothorax involves repeated accumulation of air, requiring placement of a thoracic drainage catheter. in many cases, pneumothorax develops as a result of trauma. spontaneous pneumothorax occurs with rupture of cavitary lesions of the lung that may be congenital or acquired as a result of prior trauma, heartworm disease, airway disease (emphysema), paragonimiasis, neoplasia, or lung abscess. pneumothorax also rarely occurs as a result of esophageal tears or esophageal foreign bodies. rapid circulatory and respiratory compromise following traumatic pneumothorax can develop as a result of open or tension pneumothorax, rib fractures, airway obstruction, pulmonary contusions, hemothorax, cardiac dysrhythmias, cardiac tamponade, and hypovolemic shock. any patient that is rapidly decompensating after a traumatic episode must be quickly assessed, and emergency therapy initiated (see section on immediate management of trauma, a crash plan). diagnosis of pneumothorax is usually made based on a history of trauma, a rapid, shallow, restrictive respiratory pattern, and muffled heart and lung sounds on thoracic auscultation. the clinical signs and history alone should prompt the clinician to perform a bilateral diagnostic and therapeutic thoracocentesis before taking thoracic radiographs (see section on thoracocentesis). the stress of handling the patient for radiography can be deadly in severe cases of pneumothorax. although the mediastinum on both sides of the thorax connects, it is necessary to perform thoracocentesis on both sides to ensure maximal removal of free air in the pleural space and allow maximal lung expansion. if negative pressure cannot be obtained, or if the patient rapidly reaccumulates air, place a thoracostomy tube connected to continuous suction. (see section on thoracostomy tube placement). treat all penetrating wounds to the thorax as open sucking chest wounds unless proved otherwise. to "close" an open sucking chest wound, clip the fur around the wound as quickly as possible, and place sterile lubricant jelly or antimicrobial ointment circumferentially around the wound. cut a sterile glove to provide a covering. place the covering over the wound, making sure to cover all of the sterile lubricant, thus creating a seal to close the wound temporarily from the external environment. evaluate the patient's thorax via thoracocentesis while placing a thoracostomy tube. once the patient is stable, the open chest wound can be surgically explored, lavaged, and definitively corrected. all animals with open chest wounds should receive antibiotics (first-generation cephalosporin) to prevent infection. following stabilization, radiographs can be taken and evaluated. pneumothorax is confirmed by evidence of elevation of the cardiac silhouette above the sternum, increased density of the pulmonary parenchymal tissue, free air in between the parietal and visceral pleura (making the outline of the lungs visible), and absence of pulmonary vascular structures in the periphery. parenchymal lesions within the lungs are best identified after as much air as possible has been removed from the thorax. obtain left and right lateral and ventrodorsal or dorsoventral views. a standing lateral view may reveal air-or fluid-filled cavitary masses. if underlying pulmonary disease is suspected as a cause of spontaneous pneumothorax, a transtracheal wash, fecal flotation, and heartworm test may be indicated. treatment of pneumothorax includes immediate bilateral thoracocentesis, covering of any open chest wounds, administration of supplemental oxygen, and placement of a thoracostomy tube if negative pressure cannot be obtained or if air rapidly reaccumulates. serial radiography, ct, or mri should be performed in dogs with spontaneous pneumothorax, because the condition can be associated with generalized pulmonary parenchymal disease. strict cage rest is required until air stops accumulating and the thoracostomy tube can be removed. the patient's chest tube should be aspirated every hours after discontinuing continuous suction. if no air reaccumulates after hours, the chest tube can be removed. exercise restriction is indicated for a minimum of week. if bullae or mass lesions are present, exploratory thoracotomy should be considered as a diagnostic and potentially therapeutic option for long-term management in prevention of recurrence. pleural fluid cytologic analysis is indicated for all patients with pleural effusion before administration of antibiotics. the general term pleural effusion means a collection of fluid in the space between the parietal and visceral pleura but does not indicate what kind or how much fluid is present. clinical signs associated with pleural effusion depend on how much fluid is present, and how rapidly the fluid has accumulated. clinical signs associated with pleural effusion include respiratory distress, reluctance to lie down, labored breathing with an abdominal component on exhalation, cough, and lethargy. auscultation of the thorax may reveal muffled heart and lung sounds ventrally and increased lung sounds dorsally, although pockets of fluid may be present, depending on the chronicity of the effusion. percussion of the thorax may reveal decreased resonance. in stable patients, the presence of pleural effusion can be confirmed radiographically. radiographic confirmation of the pleural effusion should include right and left lateral and dorsoventral or ventrodorsal views. a handling or standing lateral view should be obtained if an anterior mediastinal mass is suspected. the standing lateral view will allow the fluid to collect in the costophrenic recess. in patients with respiratory distress, muffled heart and lung sounds, and suspicion of pleural effusion, thoracocentesis should be performed immediately. thoracocentesis can be both therapeutic and diagnostic. radiography is contraindicated because the procedure can cause undue stress and exacerbation of clinical signs in an unstable patient. pleural effusion can cause severe respiratory distress, and can be the result of a number of factors that must be considered when implementing an appropriate treatment plan. pathology of the pleura is almost always a secondary process except for primary bacterial pleuritis and pleural mesotheliomas. causes of pleural effusion in the cat and dog include pyothorax, feline infectious peritonitis, congestive heart failure, chylothorax, heartworm disease, hemothorax, hypoalbuminemia, lung lobe torsions, neoplasia, diaphragmatic hernia, and pancreatitis (box - ). in stable animals, diagnosis of pleural effusion can be made based • imbalance of transpleural or hydrostatic or protein osmotic forces • change in membrane permeability • decrease in rate of fluid reabsorption • combination of foregoing mechanisms on thoracic radiography or ultrasound. thoracic radiographs can show whether the pleural effusion is unilateral or bilateral. effusions in dogs and cats are usually bilateral. the lung parenchyma and the cardiac silhouette cannot be fully evaluated until most of the fluid has been evacuated from the pleural cavity. following thoracocentesis, radiography should be performed with left and right lateral and ventrodorsal or dorsoventral views. in cases of suspected heart failure, echocardiography also is necessary. pleural fluid cytologic analysis is indicated for all patients with pleural effusion. collect specimens before administering antibiotics, whenever possible, because treatment with antibiotics can make a septic condition (pyothorax) appear nonseptic. the remainder of the diagnostic workup and treatment is based on the type of fluid present (table - ). the fluid may be a transudate, nonseptic exudate, septic exudate, chylous, hemorrhagic, or neoplastic. ultrasonographic evaluation of the thorax can be helpful in identifying intrathoracic masses, diaphragmatic hernias, lung lobe torsions, and cardiac abnormalities. unlike radiography, ultrasonography is facilitated by the presence of fluid in the pleural space. pyothorax refers to a septic effusion of the pleural cavity. the infection is generally the result of a combination of aerobic and anaerobic bacteria. rarely, fungal organisms are present. the source of the underlying organisms is rarely identified, particularly in cats, but can be caused by penetrating wounds through the chest wall, esophagus, migrating foreign bodies (especially grass awns), or primary lung infections. the most common organisms associated with pyothorax in the cat are pasteurella, bacteroides, and fusobacterium. fever is often present in addition to clinical signs of pleural effusion. septic shock is ununcommon. diagnosis of pyothorax is made based on cytologic analysis and the demonstration of intracellular and extracellular bacteria, toxin neutrophils and macrophages, and sometimes the presence of sulfur granules. gram stains of the fluid can assist in the initial identification of some organisms. bacterial cultures are indicated for bacteria identification and antibiotic susceptibility testing. administration of antibiotics before cytologic evaluation can cause a septic effusion to appear nonseptic. emergency treatment for pyothorax involves placement of an intravenous catheter, intravenous fluids to treat hypovolemic shock, and broad-spectrum antibiotics (ampicillin, mg/kg iv q h, and enrofloxacin, mg/kg iv q h). chloramphenicol also is an appropriate antibiotic to use for penetration into pockets of fluid. administration of a beta-lactam antibiotic (ampicillin or amoxicillin) with a beta-lactamase inhibitor (amoxicillin clavulanate or ampicillin sulbactam) is helpful in achieving better coverage of bacteroides spp. treatment of pyothorax differs in the cat and dog. in the cat, placement of one or two thoracic drainage catheters is recommended to allow continuous drainage of the intrathoracic abscess. inadequate drainage can result in treatment failure. fluid should be evaluated and the pleural cavity lavaged with ml/kg of warmed . % saline or lactated ringer's solution every hours. approximately % of the infused volume should be recovered after each lavage. in dogs, or in cats with refractory pyothorax, perform an exploratory thoracotomy to remove any nidus of infection. rarely a foreign body is visible that can be removed at the time of surgery, but this finding is rare. antibiotics are indicated for a minimum of to weeks after removal of the thoracostomy tube. early diagnosis and aggressive treatment result in a good prognosis in the majority of patients with pyothorax. in cats, clinical signs of ptyalism and hypothermia at the time of presentation worsen the prognosis. chylothorax refers to the abnormal accumulation of chyle (lymphatic fluid) in the pleural cavity. the cisterna chili is the dilated collection pool of lymphatic ducts in the abdomen that accumulate chyle prior to entry into the thoracic duct located within the thoracic cavity. the thoracic duct enters the thorax at the aortic hiatus. numerous tributaries or collateral ducts exist. the functions of the lymphatic vessels collectively serve to deliver triglycerides and fat-soluble vitamins into the peripheral vascular circulation. damage of the thoracic duct or lymphatic system or obstruction to lymphatic flow can result in the development of chylous effusion in the pleural or peritoneal space. it is difficult to identify chylous effusions based on their milky appearance alone. to identify a chylous effusion versus a pseudochylous effusion, the triglyceride and cholesterol levels of the fluid must be compared with those of peripheral blood. chylous effusions have a higher triglyceride and lower cholesterol levels than peripheral blood. pseudochylous effusions have a higher cholesterol and lower triglyceride levels than peripheral blood. disease processes that can result in chylous effusions are listed in the box - . clinical signs associated with chylous effusion are typical of any pleural effusion and of the disease process that caused the effusion. weight loss may be evident, depending on the chronicity of the process. the diagnosis is made based on thoracocentesis, cytology, and biochemical evaluation of the fluid (i.e., triglyceride and cholesterol levels). the fluid often appears milky or bloodtinged but can be clear if the patient has significant anorexia. typical cytologic characteristics are listed in table - . lymphangiography can be used to confirm trauma to the thoracic duct, but this is usually not necessary unless surgical ligation is going to be attempted. the diagnostic evaluation must also attempt to identify an underlying cause. therapy for chylothorax is difficult and primarily involves documentation and treatment of the underlying cause. if an underlying cause is not found, treatment is largely supportive and consists of intermittent thoracocentesis to drain the fluid as it accumulates and causes respiratory dysfunction, nutritional support, and maintenance of fluid balance. a variety of surgical techniques, including ligation of the thoracic duct, pleural-peritoneal shunts, and pleurodesis, have been attempted but have had limited success. most recently, the combination of thoracic duct ligation with subtotal pericardectomy has been shown to improve surgical success rates in the treatment of chylothorax. rutin, a bioflavinoid, has been used with limited success in the treatment of idiopathic chylothorax in cats. prognosis in many cases of chylothorax is guarded. extensive hemorrhage into the pleural cavity can cause fulminant respiratory distress due to sudden hypovolemia and anemia and interference with lung expansion. hemothorax typically is associated with trauma, systemic coagulopathy, lung lobe torsions, and erosive lesions within the thorax (usually neoplasia). diagnosis of hemothorax involves obtaining a fluid sample via thoracocentesis. hemorrhagic effusion must be differentiated from systemic blood inadvertently collected during the thoracocentesis procedure. unless the hemorrhage is peracute, fluid in cases of hemothorax is rapidly defibrinated and will not clot, has a packed cell volume less than that of venous blood, contains rbcs and macrophages. hemorrhagic effusions also usually contain a disproportionately higher number of white blood cells compared with peripheral blood. hemothorax commonly is the sole clinical sign observed in animals with vitamin k antagonist rodenticide intoxication and systemic coagulopathy. whenever an animal presents with signs of a hemorrhagic pleural effusion, perform coagulation testing immediately to determine whether a coagulopathy exists. the prothrombin time test is fast and can be performed as a cage-side test (see section on coagulopathy). therapy for hemorrhagic pleural effusions should address the blood and fluid loss. administer intravenous crystalloid fluids and rbc products (see section on transfusion therapy). when necessary, administer coagulation factors in the form of fresh whole blood or fresh frozen plasma, along with vitamin k ( mg/kg sq in multiple sites with a -gauge needle). if severe respiratory distress is present, evacuate the blood within the pleural space via thoracocentesis until clinical signs of respiratory distress resolve. fluid that remains aids in the recovery of the patient, because rbcs and proteins eventually will be reabsorbed. autotransfusion can be performed to salvage blood and reinfuse it into the anemic patient. in cases of neoplastic or traumatic uncontrollable hemorrhagic effusions, surgical exploration of the thorax is warranted. diaphragmatic hernia, or a rent in the diaphragm, can result in the protrusion of abdominal organs into the thoracic cavity and impair pulmonary expansion. organs that are commonly herniated into the thorax include the liver, stomach, and small intestines. diaphragmatic hernia usually is secondary to trauma but can occur as a congenital anomaly. in cases of trauma, rib fractures, pulmonary contusions, traumatic myocarditis, hemothorax, and shock are also often present concurrently with diaphragmatic hernia. respiratory distress can be caused by any one or a combination of the above lesions. animals with prior or chronic diaphragmatic hernias may have minimal clinical signs despite the presence of abdominal organs within the thorax. clinical signs of acute or severe diaphragmatic hernia include respiratory distress, cyanosis, and shock. a diagnosis of diaphragmatic hernia is made based on the patient's history (traumatic event), clinical signs, and radiographs. in some cases, ultrasonography or contrast peritoneography is necessary to confirm the diagnosis. contrast radiographs may show the presence of the stomach or intestines within the thorax following oral administration of barium. never administer barium directly into the peritoneal cavity or in cases of suspected gastrointestinal rupture. treatment of a patient with a diaphragmatic hernia includes cardiovascular and respiratory system stabilization before attempting surgical repair of the diaphragm. if the stomach is within the thorax, or if the patient's respiratory distress cannot be alleviated with medical management alone, immediate surgery is necessary. if the respiratory distress is minimal and the stomach is not located within the thorax, surgery can be postponed until the patient is a more stable anesthetic candidate. at the time of surgery, the abdominal organs are replaced into the abdominal cavity, and the rent in the diaphragm is closed. air must be evacuated from the thorax following closure of the diaphragm. if chronic diaphragmatic hernia is repaired, the complication of reexpansion pulmonary edema can occur. cardiac injury is a common complication secondary to blunt thoracic trauma. in most cases, cardiac injury is manifested as arrhythmias, including multiple premature ventricular contractions, ventricular tachycardia, st segment depression or elevation secondary to myocardial hypoxemia, and atrial fibrillation (see section on cardiac emergencies). myocardial infarction and cardiac failure can occur. careful and repeated assessments of the patient's blood pressure and ecg tracing should be a part of any diagnostic work-up for a patient that has sustained blunt thoracic trauma. rib fractures are associated with localized pain and painful respiratory movements. radiographs are helpful to confirm the diagnosis. careful palpation may reveal crepitus and instability of the fractured ribs. common problems associated with rib fractures emergency care include pulmonary contusions, pericardial laceration, traumatic myocarditis, diaphragmatic hernia, and splenic laceration or rupture. a flail segment results from rib fractures of more than three adjacent ribs that produce a "floating segment" of the chest wall. the flail segment moves paradoxically with respiration-that is, it moves inward during inhalation and outward during exhalation. respiratory distress is associated with the pain caused by the fractures and the presence of traumatic underlying pulmonary pathology. therapy for rib fractures and flail chest includes administration of supplemental oxygen, treatment of pneumothorax or diaphragmatic hernia, and administration of systemic and local anesthesia to alleviate the discomfort associated with the fractures. although controversial, positioning the patient with the flail segment up may reduce pain and improve ventilation. avoid the use of chest wraps, which do nothing to stabilize the flail segment and can further impair respiratory excursions. following administration of a systemic analgesic, administer a local anesthetic at the dorsocaudal and ventrocaudal segment of each fractured rib, and in one rib in front of and behind the flail segment. often, pulmonary function will improve once the pain associated with rib fractures has been adequately treated. in rare cases in which the flail segment involves five or more ribs, surgical stabilization may be necessary. single rib fractures or smaller flail segments are allowed to heal on their own. feline bronchitis has a variety of names (bronchial asthma, asthma, acute bronchitis, allergic bronchitis, chronic asthmatic bronchitis, feline lower airway disease) and refers to the acute onset of respiratory distress secondary to narrowing of the bronchi. cats may present with an acute onset of severe restrictive respiratory pattern associated with lower airway obstruction. acute bronchitis in cats typically has an inflammatory component in the lower airways, resulting in acute bronchoconstriction, excessive mucus production, and inflammatory exudates. in cats with chronic bronchitis, there may be damage of the bronchial epithelium and fibrosis of the airways. these patients often have a history if intermittent exacerbation of clinical signs, intermittent cough, and periods of normality throughout the year. because there appears to be an allergic or inflammatory component in feline bronchitis, clinical signs can be acutely exacerbated by stress and the presence of aerosolized particles such as perfume, smoke, and carpet powders. causes of feline bronchitis include heartworm disease, parasitic infestation (lungworms), and (rarely) bacterial infection. on presentation, the patient should be placed in an oxygen cage and allowed to rest while being observed from a distance. postpone performing stressful diagnostic procedures until the patient's respiratory status has been stabilized. after careful thoracic auscultation, administer a short-acting bronchodilator (terbutaline, . mg/kg sq or im) along with a glucocorticosteroid (dexamethasone sodium phosphate mg/kg im, sq, iv) to alleviate immediate bronchospasm and airway inflammation. clinical signs of feline bronchitis are characterized by a short, rapid respiratory pattern with prolonged expiration with an abdominal push. wheezes may be heard on thoracic auscultation. in some cases, no abnormalities are found on auscultation, but become acutely worse when the patient is stimulated to cough by tracheal palpation. radiographs may reveal a hyperinflated lung field with bronchial markings and caudal displacement of the diaphragm. in some cases, consolidation of the right middle lung lobe is present. a complete blood count and serum biochemistry profile can be performed, but results usually are unrewarding. in endemic areas, a heartworm test is warranted. fecal examination by flotation and the baermann technique is helpful in ruling out lungworms and other parasites. bronchoalveolar lavage or transtracheal wash is useful for cytologic and bacterial examination. long-term management of feline bronchitis includes isolation from environmental exposure to potential allergens (litter dust, perfumes, smoke, incense, carpet powders) and treatment of bronchoconstriction and inflammation with a combination of oral and inhaled glucocorticosteroids and bronchodilators (table - ). antibiotic therapy is contraindicated unless a pure culture of a pathogen is documented. oral therapy with steroids and bronchodilators should be used for a minimum of weeks after an acute exacerbation and then gradually decreased to the lowest dose possible to alleviate clinical signs. metered dose inhalers are now available (aerokat.com) for administration of inhaled bronchodilators and steroids. fluticasone (flovent, mcg/puff ) can be administered initially every hours for week and then decreased to once daily, in most cases. inhaled glucocorticosteroids are not absorbed systemically, and therefore patients do not develop the adverse side effects sometimes documented with oral glucocorticosteroid administration. because it takes time for glucocorticosteroids to reach peak effects in the lungs, administration of inhaled glucocorticosteroids should overlap with oral prednisolone administration for to days. treatment of pulmonary contusions is supportive. administer supplemental oxygen in a manner that is least stressful for the animal. arterial blood gas analysis or pulse oximetry can determine the degree of hypoxemia and monitor the response to therapy. intravenous fluids should be administered with caution to avoid exacerbating pulmonary hemorrhage or fluid accumulation in the alveoli. treat other conditions associated with the traumatic event. possible complications of pulmonary contusions are rare but include bacterial infection, abscessation, lung lobe consolidation, and the development of cavitary lesions. the routine use of antibiotics or steroids in cases of pulmonary contusions is contraindicated unless external wounds are present. empiric antibiotic use without evidence of external injury or known infection can potentially increase the risk of a resistant bacterial infection. steroids have been shown to decrease pulmonary alveolar macrophage function and impair wound healing and are contraindicated. aspiration pneumonia can occur in animals as a result of abnormal laryngeal or pharyngeal protective mechanisms or can be secondary to vomiting during states of altered mentation, including anesthesia, recovery from anesthesia, and sleep. megaesophagus, systemic polyneuropathy, myasthenia gravis, and localized oropharyngeal defects such as cleft palate can increase the risk of developing aspiration pneumonitis. iatrogenic causes of aspiration pneumonia include improper placement of nasogastric feeding tubes, overly aggressive force-feeding, and oral administration of drugs. aspiration of contents into the airways can cause mechanical airway obstruction, bronchoconstriction, chemical damage to the alveoli, and infection. severe inflammation and airway edema are common. pulmonary hemorrhage and necrosis can occur. diagnosis of aspiration pneumonia is based on clinical signs of pulmonary parenchymal disease, a history consistent with vomiting or other predisposing causes, and thoracic radiographs demonstrating a bronchointerstitial to alveolar pulmonary infiltrate. the most common site is the right middle lung lobe, although the pneumonia can occur anywhere, depending on the position of the patient at the time of aspiration. a transtracheal wash or bronchoalveolar lavage is useful for bacterial culture and susceptibility testing. treatment of aspiration pneumonia includes antibiotic therapy for the infection, administration of supplemental oxygen, and loosening the debris in the airways. administer intravenous fluids to maintain hydration. nebulization with sterile saline and chest physiotherapy (coupage) should be performed at least every hours. antibiotics to consider in the treatment of aspiration pneumonia include ampicillin/enrofloxacin, amoxicillinclavulanate, ampicillin-sulbactam, trimethoprim sulfa, and chloramphenicol. the use of glucocorticosteroids is absolutely contraindicated. continue antibiotic therapy for a minimum of weeks after the resolution of radiographic signs of pneumonia. pulmonary edema arises from the accumulation of fluid in the pulmonary interstitial alveolar spaces, and airways. ventilation-perfusion abnormalities result in hypoxia. pulmonary edema can be caused by increased pulmonary vasculature hydrostatic pressure, decreased pulmonary oncotic pressure, obstruction of lymphatic drainage, or increased capillary permeability. multiple factors can occur simultaneously. the most common cause of edema is increased pulmonary hydrostatic pressure resulting from left-sided congestive heart failure. decreased plasma oncotic pressure with albumin < . g/dl can also result in accumulation of fluid in the pulmonary parenchyma. overzealous intravenous crystalloid fluid administration can result in dilution of serum oncotic pressure and vascular overload. obstruction of lymphatic drainage is usually caused by neoplasia. other causes of pulmonary edema include pulmonary thromboembolic disease, severe upper airway obstruction (noncardiogenic pulmonary edema), seizures, and head trauma. increased capillary permeability is associated with a variety of diseases that cause severe inflammation (systemic inflammatory response syndrome). the resultant pulmonary edema contains a high amount of protein and is known as acute respiratory distress syndrome (ards). ards can be associated with pulmonary or extrapulmonary causes, including direct lung injury from trauma, aspiration pneumonia, sepsis, pancreatitis, smoke inhalation, oxygen toxicity, electrocution, and immune-mediated hemolytic anemia with disseminated intravascular coagulation. diagnosis of pulmonary edema is made based on clinical signs of respiratory distress and the presence of crackles on thoracic auscultation. in severe cases, cyanosis and fulminant blood-tinged frothy edema fluid may be present in the mouth and nostrils. immediate management includes administration of furosemide ( - mg/kg iv, im) and supplemental oxygen. sedation with low-dose morphine sulfate ( . - . mg/kg iv) is helpful in dilating the splanchnic capacitance vasculature and relieving anxiety for the patient. if fluid overload is suspected secondary to intravenous fluid administration, fluids should be discontinued. severely hypoalbuminemic patients should receive concentrated human albumin ( ml/kg of a % solution) or fresh frozen plasma. furosemide as a constant rate infusion ( . - . mg/kg/hour) also can dilate the pulmonary vasculature and decrease fluid accumulation in cases of ards. following initial stabilization of the patient, thoracic radiographs and an echocardiogram should be assessed to determine cardiac side, pulmonary vascular size, and cardiac contractility. further diagnostic testing may be required to determine other underlying causes of pulmonary edema. heart failure is managed with vasodilators, diuretics, oxygen, and sometimes positive inotropes. treatment ultimately consists of administration of supplemental oxygen, minimal stress and patient handling, and judicious use of diuretics. in cases of cardiogenic pulmonary edema, administer furosemide ( - mg/kg iv, im) every to minutes until the patient loses % of its body weight. positive inotropic and antiarrhythmic therapy may be necessary to improve cardiac contractility and control dysrhythmias. the clinician should determine whether the cause of the pulmonary edema is secondary to congestive heart failure with pulmonary vascular overload, volume overload, hypoalbuminemia, or increased permeability (ards). pulmonary edema secondary to ards typically is refractory to supplemental oxygen and diuretic therapy. in many cases, mechanical ventilation should be considered. a diagnosis of pulmonary thromboembolism (pte) is difficult to make and is based on clinical signs of respiratory distress consistent with pte, lack of other causes of hypoxemia, a high index of suspicion in susceptible animals, the presence of a condition associated with pte, and radiographic findings. virchow's triad consists of vascular endothelial injury, sluggish blood flow with increased vascular stasis, and a hypercoagulable state as predisposing factors for thromboembolic disease. clinical conditions that predispose an animal to pte include hyperadrenocorticism, disseminated intravascular coagulation (dic), catheterization of blood vessels, bacterial endocarditis, protein-losing nephropathy or enteropathy, hyperviscosity syndromes, heat-induced illness, pancreatitis, diabetes mellitus, inflammatory bowel disease, and immune-mediated hemolytic anemia. definitive diagnosis requires angiography or a lung perfusion scan. clinical signs associated with pte include an acute onset of tachypnea, tachycardia, orthopnea, and cyanosis. if the embolism is large, the patient may respond poorly to supplemental oxygen administration. pulmonary hypertension can cause a split second heart sound on cardiac auscultation. in some cases, a normal thoracic radiograph is present in the face of severe respiratory distress. this is a classic finding in cases of pte. potential radiographic abnormalities include dilated, tortuous, or blunted pulmonary arteries; wedge-shaped opacities in the lungs distal to an obstructed artery; and interstitial to alveolar infiltrates. the right heart may be enlarged. echocardiography can show right heart enlargement, tricuspid regurgitation, pulmonary hypertension, and evidence of underlying cardiac disease, possibly with clots in the atria. measurement of antithrombin (at) and d-dimer levels can be useful in the identification of hypercoagulable states, including dic. treatment of any patient with at deficiency or dic includes replenishment of at and clotting factors in the form of fresh frozen plasma. treatment of pte includes therapy for cardiovascular shock, oxygen supplementation, and thrombolytic therapy (see section on thromboembolic therapy). for short-term treatment, administer heparin (heparin sodium, - units/kg sq once, followed by units/kg q h of unfractionated heparin; or fractionated heparin). thrombolytic therapy may include tissue plasminogen activator, streptokinase, or urokinase. long-term therapy with low molecular weight heparin or warfarin may be required to prevent further thromboembolic events. ideally, management should include treatment and elimination of the underlying disease. smoke inhalation commonly occurs when an animal is trapped in a burning building. the most severe respiratory complications of smoke inhalation are seen in animals that are close enough to the flames to also sustain burn injuries (see section on burn injury). at the scene, many animals are unconscious from the effects of hypoxia, hypercapnia, carbon monoxide intoxication, and hydrogen cyanide gases that accumulate in a fire. carbon monoxide produces hypoxia by avidly binding to and displacing oxygen binding to hemoglobin, resulting in severe impairment of oxygen-carrying capacity. the percentage of carboxyhemoglobin in peripheral blood depends on the amount or carbon monoxide in inhaled gases and the length of time of exposure. clinical signs of carbon monoxide intoxication include cyanosis, nausea, vomiting, collapse, respiratory failure, loss of consciousness, and death. smoke inhalation of superheated particles also causes damage to the upper airways and respiratory tree. the larynx can become severely edematous and obstruct inspiration. emergency endotracheal intubation, tracheal oxygen, or tracheostomy tube may be required in the initial resuscitation of the patient, depending on the extent of airway edema. inhalation of noxious gases and particles can cause damage to the terminal respiratory bronchioles. specific noxious gases that can cause alveolar damage include combustible particles from plastic, rubber, and other synthetic products. pulmonary edema, bacterial infection, and ards can result. in any case of smoke inhalation, the first and foremost treatment is to get the animal away from the source of the flames and smoke and administer supplemental oxygen at the scene. at the time of presentation, carefully examine the animal's eyes, mouth, and oropharynx suction soot and debris from the mouth and upper airways. evaluate the patient's respiratory rate, rhythm, and pulmonary sounds. arterial blood gases should be analyzed with co-oximetry to evaluate the pao and carboxyhemoglobin concentrations. evaluation of sao by pulse oximetry is not accurate in cases of smoke inhalation, as the pao may appear normal, even when large quantities of carboxyhemoglobin are present. radiographs are helpful in determining the extent of pulmonary involvement, although radiographic signs may lag behind the appearance of clinical respiratory abnormalities by to hours. bronchoscopy and bronchoalveolar lavage provide a more thorough and accurate evaluation of the respiratory tree; however, these procedures should be performed only in patients whose cardiovascular and respiratory status is stable. management of the patient with smoke inhalation includes maintaining a patent airway, administration of supplemental oxygen, correction of hypoxemia and acid-base abnormalities, preventing infection, and treating thermal burns (see section on burn injury). if severe laryngeal edema is present, a temporary tracheostomy may be necessary to allow adequate oxygenation and ventilation. glucocorticosteroids should not be empirically used in the treatment of smoke inhalation, because of the risk of decreasing pulmonary alveolar macrophage function and increasing the potential for infection. in cases of severe laryngeal edema, however, glucocorticosteroids may be necessary to decrease edema and inflammation. the use of empiric antibiotics is contraindicated unless clinical signs of deterioration and bacterial pneumonia develop. epistaxis can be caused by facial trauma, a foreign body, bacterial or fungal rhinitis, neoplasia, coagulopathies, and systemic hypertension. acute, severe bilateral hemorrhage without wounds have been classified in several ways according their degree of tissue integrity, etiologic force, degree of contamination and duration, and degree of contamination and infection (table - ) . there are also unique causes of wounds such as burns, psychogenic dermatoses, frostbite, decubital ulcers, and snake bite. the animal should be transported to the nearest veterinary facility for definitive care. the wound should be covered or packed with dry gauze or clean linen to protect the wound, and to prevent further hemorrhage and contamination. if an open fracture is present, the limb should be splinted without placing the exposed bone back into the wound. replacing the exposed bone fragment back through the skin wound can cause further damage to underlying soft tissue structures and increase the degree of contamination of deeper tissues. if a spinal fracture is suspected, the patient should be transported on a stable flat surface to prevent further spinal mobilization and neurologic injury. at the time of presentation, first refer to the abcs of trauma, taking care to evaluate and stabilize the patient's cardiovascular and respiratory status. after a complete physical examination and history, ancillary diagnostic techniques can be performed if the patient is hemodynamically stable (see section on triage, assessment, and treatment of emergencies). initially, every patient with superficial wound should receive some degree of analgesia and an injection of a first-generation cephalosporin, preferably within hours of the injury. evaluate the wound after the patient's cardiovascular and respiratory status have been stabilized. always cover an open wound before taking an animal to the hospital to prevent a nosocomial infection. evaluate limb wounds for neural, vascular, and orthopedic abnormalities. carefully examine the structures deep to the superficial wounds. when there has been a delay in assessment of the wound, obtain samples for culture and antimicrobial susceptibility testing. if the wound is older and obviously infected, a gram stain can help guide appropriate antimicrobial therapy pending results of culture and susceptibility testing. place a support bandage saturated with a water-soluble antibiotic ointment or nonirritating antimicrobial solution (e.g., . % chlorhexidine, if bone or joint tissue is not exposed) around the wound. in addition to a first-generation cephalosporin, other appropriate antibiotic choices include amoxicillin-clavulanate, trimethoprim-sulfadiazine, amoxicillin, and ampicillin. if gram-negative flora are present, administer enrofloxacin. administer the antibiotics of choice for a minimum of days unless a change of antibiotic therapy is indicated. at the time of wound cleansing or definitive wound repair, the patient should be placed under general anesthesia with endotracheal intubation, unless the procedure will be brief (i.e., less than minutes). in such cases, a short-acting anesthetic combination open lacerations or skin loss closed crushing injuries and contusions etiologic force abrasion loss of epidermis and portions of dermis, usually caused by shearing between two compressive surfaces avulsion tearing of tissue from its attachment because of forces similar to those causing abrasion but of a greater magnitude incision wound created by a sharp object; wound edges are smooth and there is minimal trauma in the surrounding tissues laceration irregular wound caused by tearing of tissue with variable damage to the superficial and underlying tissue puncture penetrating wound caused by a missile or sharp object; superficial damage may be minimal; damage to deeper structures may be considerable; contamination by fur and bacteria with subsequent infection is common class i - hours with minimal contamination class ii - hours with significant contamination class iii > hours with gross contamination (analgesia + propofol, analgesia + ketamine/diazepam) can be administered to effect. heavy sedation with infiltration of a local anesthetic may also be appropriate for very small wounds, depending on the location of the wound and temperament of the patient. protect the wound by packing it with sterile gauze sponges soaked in sterile saline, or with watersoluble lubricating gel such as k-y jelly. clip the fur surrounding the wound, moving from the inner edge of the wound outward, to help prevent wound contamination with fur or other debris. scrub the wound and surrounding skin with an antimicrobial soap and solution such as dilute chlorhexidine until the area is free of all gross debris. gross debris within the wound itself can be flushed using a -ml syringe filled with sterile saline or lactated ringer's solution and an -gauge needle. pressure-lavage systems are also available for use, if desired. grossly contaminated wounds can be rinsed first with warm tap water to eliminate gross contamination, and then prepared as just described. debride the wound, removing skin and other soft tissue that is not obviously viable. obviously viable and questionable tissue should remain, and the wound left open for frequent reassessment on a daily basis. remove any dark or white segments of skin. questionable skin edges may or not regain viability and should be left in place for hours, so the wound can fully reveal itself. excise grossly contaminated areas of fat and underlying fascia. blood vessels that are actively bleeding should be ligated to control hemorrhage, if collateral circulation is present. if nerve bundles are ligated cleanly in a clean wound, the nerve edges should be reapposed and anastomosed. if gross contamination is present, however, definitive neurologic repair should be delayed until healthy tissue is present. excise contaminated muscle until healthy bleeding tissue is present. anastamoe tendon lacerations if the wound is clean and not grossly contaminated. if gross contamination is present, the tendon can be temporarily anastomosed and a splint placed on the limb until definitive repair of healthy tissue is possible. thoroughly lavage open wounds to a joint with sterile saline or lactated ringer's solution. infusion of chlorhexidine or povidone-iodine solution into the joint can cause a decrease in cartilage repair and is contraindicated. smooth sharp edges and remove any obvious fragments. whenever possible, the joint capsule and ligaments should be partially or completely closed. after removing bullets and metal fragments, the subcutaneous tissue and skin should be left open to heal by second intention, or should be partially closed with a drain. the joint should then be immobilized. injuries and exposed bone should be carefully lavaged, taking care to remove any gross debris without pushing the debris further into the bone and wound. the bone should be covered with a moist dressing and stabilized until definitive fracture repair can be made. this type of injury typically is seen with shearing injuries of the distal extremities caused by interaction with slow-moving vehicles. perform wet-to-dry or enzymatic debridement until a healthy granulation bed is present. if large areas of contamination are present (e.g., necrotizing fasciitis), en bloc debridement may be necessary. en bloc debridement consists of complete excision of badly infected wounds without entering the wound cavity, to prevent systemic infection. this technique should be used only if there is sufficient skin and soft tissue to allow later closure and it can be performed without damaging any major nerves, tendons, or blood vessels. open wounds often are managed by second intention healing, delayed primary closure, or secondary closure. see section on wound management and bandaging for a more complete discussion on the use of various bandaging materials in the treatment of open wounds. if an animal is presented very shortly after a wound has occurred and there is minimal contamination and trauma, the wound can be closed after induction of anesthesia and careful preparation of the wound and surrounding tissues. close any dead space under the skin with absorbable suture material in an interrupted suture pattern. avoid incising major blood vessels or nerves. close the subcutaneous tissues with absorbable suture material in an interrupted or continuous suture pattern. take care that there is not too much tension on the wound, or else surgical dehiscence will occur with patient movement. close the skin with nonabsorbable suture or surgical staples ( - to - ) . if there is any doubt at the time of repair about tissue status or inability to close all dead space, place a passive drain (penrose drain) so that the proximal end of the drain is anchored in the proximal aspect of the wound with a suture(s). leave the ends long so that the suture can be accurately identified at the time of drain removal. pass the suture through the skin, through the drain, and out the other side of the skin. place the rest of the drain into the wound and then secure it at the most ventral portion of the wound or exit hole in the most dependent area of the body, to allow drainage and prevent seroma formation. close the subcutaneous tissue over the drain before skin closure. during wound closure, be sure to not incorporate the subcutaneous or skin sutures into the drain, or it will not be possible to remove the drain without reopening the wound. bandage the area to prevent contamination. the drain can be removed once drainage is minimal (usually to days). active drains can be constructed or purchased; their use is indicated in wounds that are free of material that can plug the drain. to construct a small suction drain, remove the female portion or catheter hub at the end of a butterfly catheter. fenestrate the tubing so that there are multiple side holes, taking care to avoid making the holes larger than % of the circumference of the tubing. place the tubing into the wound via a small stab incision distal to the wound. use a purse-string suture around the tubing to facilitate a tight seal and prevent the tubing from exiting the wound. following wound closure, insert the butterfly needle into a -to -ml evacuated blood collection tube to allow fluid to drain into the tube. incorporate the tube into the bandage, and replace it when it becomes full. alternatively, the butterfly portion of the system can be removed and the tube fenestrated as described previously. place the tube into the wound and suture it in place to create a tight seal. secure the catheter hub to a syringe in which the plunger has been drawn back slightly to create suction. insert a metal pin or -to -gauge needle through the plunger at the top of the barrel to hold it at the desired level. incorporate the suction apparatus into the bandage and replace it when it becomes full. delayed primary closure should be considered when there is heavy contamination, purulent exudate, residual necrotic debris, skin tension, edema and erythema, and lymphangitis. delayed primary closure usually is made to days after the initial wound infliction and open wound management has been performed. once healthy tissue is observed, the skin edges should be debrided and the wound closed as with primary closure. secondary wound closure should be considered when infection and tissue trauma necessitate open wound management for more than days. secondary wound closure is performed after the development of a healthy granulation bed. this technique also is useful when a wound has dehisced and has formed granulation tissue. if the wound edges can be manipulated into apposition and if epithelialization has not begun, the wound can be cleansed and the wound edges apposed and sutured. this is known as early secondary closure. late secondary closure should be performed whenever there is a considerable amount of granulation tissue, the edges of the wound cannot be manipulated into position, and epithelialization has already started. in such cases, the wound should be cleaned, and the skin edges debrided to remove the epithelium. the remaining wound edges are then sutured over the granulation tissue ( shock is defined as a state of inadequate circulating volume and inability to meet cellular oxygen demands. there are three types of shock: hypovolemic, cardiogenic, and septic. early recognition of the type of shock present is crucial in the successful clinical management of shock syndrome. tissue oxygen delivery is based on cardiac output and arterial oxygen concentration. knowledge of the components of normal oxygen delivery is essential to the treatment of shock in the critical patient. improper handling of animal during further tissue and neurologic damage may occur transport (e.g., improper limb or spine immobilization). inadequate assessment of animal's animal's condition may worsen or animal may general condition or wounded tissues succumb; tissue injuries may be overlooked. inadequate wound protection during further wound contamination may occur at assessment, resuscitation, or veterinary facility. stabilization procedures inadequate wound protection while further wound contamination with fur and preparing the surrounding area debris may occur. insufficient wound lavage wound infection may occur. hydrogen peroxide wound lavage lavage offers little bactericidal activity and contributes to irritation of tissues and delayed healing. lavage has short residual activity and absorption with large wound. overly aggressive initial layered debridement may result in the removal of viable debridement tissue. en bloc debridement debridement results in removal of large amounts of tissue and a large defect for closure. use of drains potential exists for bacteria to ascend along the drain, for drain removal by the animal or breakage of the drain, and for possible tissue emphysema with air being sucked under the skin with patient movement. tube-type drains drains may cause postoperative discomfort; fenestrations may become occluded to stop intraluminal drainage. deeply placed sutures in the presence drain may be incorporated into the repair and of a drain prevent drain removal. active drains high negative pressure may cause tissue injury; highly productive wounds may necessitate changing the evacuated blood tubes several times a day with constructed drains. oxygen delivery (do ) = cardiac output (q) × arterial oxygen content (cao ) where q = heart rate × stroke volume. stroke volume is affected by preload, afterload, and cardiac contractility. where hb = hemoglobin concentration, sao = oxygen saturation, and pao = arterial partial pressure of oxygen in mm hg. thus, factors that can adversely affect oxygen delivery include inadequate preload or loss of circulating volume, severe peripheral vasoconstriction and increased afterload, depressed cardiac contractility, tachycardia and decreased diastolic filling, cardiac dysrhythmias, inadequate circulating hemoglobin, and inadequate oxygen saturation of hemoglobin. during septic shock, enzymatic dysfunction and decreased cellular uptake and utilization of oxygen also contribute to anaerobic glycolysis. an inadequate circulating volume may develop secondary to maldistribution of available blood volume (traumatic, septic, and cardiogenic origin) or as a result of absolute hypovolemia (whole blood or loss of extracellular fluid). normally, the animal compensates by ( ) splenic and vascular constriction to translocated blood from venous capacitance vessels to central arterial circulation, ( ) arteriolar constriction to help maintain diastolic blood pressure and tissue perfusion, and ( ) an increase in heart rate to help maintain cardiac output. arteriolar vasoconstrictions support perfusion to the brain and heart at the expense of other visceral organs. if vasoconstriction is severe enough to interfere with delivery of adequate tissue oxygen for a sufficient period of time, the animal may die. hypovolemic shock can result from acute hemorrhage or from severe fluid loss from vomiting, diarrhea, or third spacing of fluids. early in shock, baroreceptors in the carotid body and aortic arch sense a decrease in wall stretch from a decrease in circulating fluid volume. tonic inhibition of sympathetic tone via vagal stimulation is diminished, and heart rate and contractility increase and peripheral vessels constrict to compensate for the decrease in cardiac output. the compensatory mechanisms protect and support blood supply to the brain and heart at the expense of peripheral organ perfusion. this is called early compensatory shock. early compensatory shock is characterized by tachycardia, normal to fast capillary refill time, tachypnea, and normothermia. as shock progresses, the body loses its ability to compensate for ongoing fluid losses. early decompensatory shock is characterized by tachycardia, tachypnea, delayed capillary refill time, normotension to hypotension, and a fall in body temperature. end-stage decompensatory shock is characterized by bradycardia, markedly prolonged capillary refill time, hypothermia, and hypotension. aggressive treatment is necessary for any hope of a favorable outcome. septic shock should be considered in any patient with a known infection, recent instrumentation that could potentially introduce infection (indwelling intravenous or urinary catheter, surgery or penetrating injury), disorders or medical therapy that can compromise immune function (diabetes mellitus, immunodeficiency virus, parvovirus or feline panleukopenia virus infection, stress, malnutrition, glucocorticoids, chemotherapy). the presence of bacteria, viruses or rickettsiae, protozoa, or fungal organisms in the blood constitutes septicemia. septic shock is characterized by the presence of sepsis and refractory hypotension that is unresponsive to standard aggressive fluid therapy and inotropic or pressor support. septic shock and other causes of inflammation can lead to systemic inflammatory response syndrome (sirs). in animals, the presence of two or more of the criteria in table - in the presence of suspected inflammation or sepsis constitutes sirs (table - ). clinical signs associated with sepsis may be vague and nonspecific, including weakness, lethargy, vomiting, and diarrhea. cough and pulmonary crackles may be associated with pneumonia. decreased lung sounds may be associated with pyothorax. abdominal pain and fluid may be associated with septic peritonitis. vaginal discharge may or may not be present in patients with pyometra. diagnostic tests should include a white blood cell count, serum biochemical profile, coagulation tests, thoracic and abdominal radiographs, and urinalysis. the white blood cell count in a septic patient that is appropriately responding to the infection will be elevated with a left-shifted neutrophilia and leukocytosis. a degenerative left shift, in which leukopenia with elevated band neutrophils suggests an overwhelming infection. biochemical analyses may demonstrate hypoglycemia and nonspecific hepatocellular and cholestatic enzyme elevations. in the most severe cases, metabolic (lactic) acidosis, coagulopathies, and end-organ failure, including anuria and ards, may be present. cardiogenic shock occurs as a result of cardiac output inadequate to meet cellular oxygen demands. cardiogenic shock is associated with primary cardiomyopathies, cardiac dysrhythmias, pericardial fluid, and pericardial fibrosis. abnormalities seen on physical examination often are similar to those seen in other categories of shock, but they can also include cardiac murmurs, dysrhythmias, pulmonary rales, bloody frothy pulmonary edema fluid from the nares or mouth, orthopnea, and cyanosis. it is important to distinguish the primary cause of shock before implementing treatment (table - ) , whenever possible, because treatment for a suspected ruptured hemangiosarcoma differs markedly from the treatment for end-stage dilatative cardiomyopathy. the patient's clinical signs may be similar and include a peritoneal fluid wave, but the treatment for hypovolemia can dramatically worsen the congestive heart failure secondary to dilatative cardiomyopathy. when a patient presents with some form of shock, immediate vascular access is of paramount importance. place a large-bore peripheral or central venous catheter for the infusion of crystalloid or colloid fluids, blood component therapy, and drugs. monitor the patient's cardiopulmonary status (by ecg), blood pressure, oxygen saturation (as determined by pulse oximetry or arterial blood gas analyses), hematocrit, bun, and glucose. ancillary diagnostics, including thoracic and abdominal radiography, urinalysis, serum biochemistry profile, coagulation tests, complete blood count, abdominal ultrasound, and echocardiography, should be performed as determined by the individual patient's needs and the type of shock. the following list, called the "rule of twenty," is a guideline for case management of the shock patient. consideration of each aspect of the rule of twenty on a daily basis ensures temperature < °f or > . °f < °f or > . °f heart rate > beats/minute in dogs < or > beats/minute in cats respiratory rate > breaths/minute or paco > breaths/minute or paco < mm hg < mm hg white blood cell > , cells/µl , cells/µl count or < cells/µl o r < cells/ml or > % bands or > % bands that major organ systems are not overlooked. the list also provides a means to integrate and relate changes in different organ systems functions with one another.* the treatment of hypovolemic and septic shock requires the placement of large-bore intravenous catheters in peripheral and central veins. if vascular access cannot be obtained percutaneously or by cutdown methods, intraosseous catheterization should be considered. once vascular access is achieved, rapidly administer large volumes of crystalloid or colloid fluids. as a rule of thumb, administer / of a calculated shock dose of fluids-that is, / × ( ml/kg/hour) in dogs and / × ( ml/kg/hour) in cats) of a balanced crystalloid fluid ( normosol-r, plasmalyte-m, lactated ringer's solution, or . % sterile saline). reassess the patient's perfusion parameters (heart rate, capillary refill time, blood pressure, urine output) on a continual basis to direct further fluid therapy. synthetic colloid fluids (hetastarch, dextran , or oxyglobin) can also be administered in the initial resuscitation from shock. a guideline is to administer to ml/kg of hetastarch or dextran as a bolus over to minutes and then reassess perfusion parameters. hypertonic saline ( . % nacl, ml/kg) can be used in cases of hemorrhagic shock to temporarily restore intravascular fluid volume by drawing fluid from the interstitial space. because this type of fluid resuscitation is short-lived, hypertonic saline should always be used with another crystalloid or colloid fluid, and it should not be used in patients with interstitial dehydration. if hemorrhagic shock is present, the goal should be to return a patient's blood pressure to normal (not supraphysiologic) levels (i.e., systolic pressure - mm hg, diastolic pressure > mm hg, and mean arterial pressure ≥ mm hg) to avoid iatrogenically causing clots to fall off and hemorrhage to re-start. in critically ill patients, fluid loss can be measured in the form of urine, vomit, diarrhea, body cavity effusions, and wound exudates. additionally, insensible losses (those that cannot be readily measured from sweat, panting, and cellular metabolism) constitute ml/kg/ day. measurement of fluid "ins and outs" in conjunction with the patient's central venous pressure, hematocrit, albumin, and colloid oncotic pressure can help guide fluid therapy (see also section on fluid therapy). maintenance of normotension is necessary for adequate oxygen delivery to meet cellular energy demands. blood pressure can be measured using direct arterial catheterization, or through indirect means such as doppler plesthymography or oscillometric methods. the systolic pressure should remain at or greater than - mm hg at all times. the diastolic pressure is very important, too, as it constitutes two thirds of the mean arterial pressure; it must be greater than mm hg for coronary artery perfusion. the mean arterial pressure should be greater than mm hg for adequate tissue perfusion. if fluid resuscitation and pain management are not adequate in restoring blood pressure to normal, vasoactive drugs including positive inotropes and pressors should be considered (table - ). in cases of cardiogenic shock, vasodilator drugs (table - ) can be used to decrease vascular resistance and afterload. low-dose morphine ( . mg/kg, iv, im) dilates splanchnic vessels and helps reduce pulmonary edema. furosemide ( mg/kg/hour) also can dilate pulmonary vasculature and potentially reduce edema fluid formation in cases of ards. cardiac output is a function of both heart rate and stroke volume. stroke volume or (the amount of blood that the ventricle pumps in minute) is affected by preload, afterload, and contractility. during hypovolemic shock, there is a fall in cardiac preload due to a decrease in circulating blood volume. during septic and cardiogenic shock, there is a decrease in contractility secondary to inherent defects of the myocardium or due to the negative inotropic effects of inflammatory cytokines such as tnf-alpha, myocardial depressant factor, il- , and il- released during sepsis and systemic inflammation. afterload also may be increased because of the compensatory mechanisms and neurohumoral activation of the renin-angiotensin-aldosterone axis in hypovolemic or cardiogenic shock. as heart rate increases to compensate for a decline in cardiac output, myocardial oxygen demand increases and diastolic filling time becomes shorter. because the coronary arteries are perfused during diastole, coronary perfusion can be impaired, and myocardial lactic acidosis can develop, causing a further decline in contractility. in addition to lactic acidosis, acid-base and electrolyte abnormalities, inflammatory cytokines, direct bruising of the myocardium from trauma, and areas of ischemia can further predispose the patient to ventricular or atrial dysrhythmias. cardiac dysrhythmias should be controlled whenever possible. treatment of bradycardia should be directed at treating the underlying cause. administer anticholinergic drugs such as atropine ( . mg/kg im) or glycopyrrolate ( . mg/kg im) as necessary. in cases of third-degree or complete atrioventricular (av) block, administer a pure betaagonist such as isoproterenol ( . - . µg/kg/minute iv cri, or . mg in ml of % dextrose in water iv slowly). perform passive rewarming if the patient is hypothermic. receptor activity dosage (iv) dopamine da , da , α +++ , - µg/kg/minute (blood pressure support)* β +++ - µg/kg/minute (renal afferent diuresis) dobutamine α + , β +++ - µg/kg/minute* (blood pressure support, positive inotrope) norepinephrine α +++ , β + . - . mg/kg/minute; . - . mg/kg phenylephrine α +++ , β . - . mg/kg epinephrine α +++ , β +++ . - . mg/kg, . - . mg/kg/minute +++, strong receptor activity; , no receptor activity; +, weak receptor activity. *monitor for tachyarrhythmias at higher doses. correct any underlying electrolyte abnormalities such as hyperkalemia and hypo-and hypermagnesemia. treat ventricular dysrhythmias such as multifocal premature ventricular contractions (pvcs), sustained ventricular tachycardia > beats per minute, and r on t phenomenon (the t wave of the preceding beat occurs superimposed on the qrs complex of the next beat, and there is no return to isoelectric shelf), or if runs of ventricular tachycardia cause a drop in blood pressure. intravenous lidocaine and procainamide are the first drugs of choice for ventricular dysrhythmias. supraventricular tachycardia can impair cardiac output by impairing diastolic filling time. control supraventricular dysrhythmias with calcium channel blockers, beta-adrenergic blockers, or quinidine (table - ) . (disorientation); is minute; minutes) light sensitive and must be covered in foil and not kept for longer than hours albumin can decrease as a result of loss from the gastrointestinal tract, urinary system, and wound exudates, or into body cavity effusions. albumin synthesis can decrease during various forms of shock due to a preferential increase in hepatic acute phase protein synthesis. serum albumin contributes % of the colloid oncotic pressure of blood, in addition to its important roles as a free radical scavenger at sites of inflammation and as a drug and hormone carrier. albumin levels < . g/dl have been associated with an increase in morbidity and mortality in human and veterinary patients. administer fresh frozen plasma ( ml/kg) or concentrated human albumin ( ml/kg of % solution) to maintain serum albumin ≥ . g/dl. additional oncotic support can be in the form of synthetic colloids, as indicated. colloid oncotic pressure within the intravascular and interstitial spaces contributes to fluid flux. oncotic pressure can be measured with a colloid osmometer. normal oncotic pressure is mm hg. in cases of sepsis and sirs, increased vascular permeability increases the tendency for leakage of fluids into the interstitial spaces. colloids that can be administered until the source of albumin loss resolves include the synthetic colloids hetastarch and dextran ( - ml/kg/day), synthetic hemoglobin-based oxygen carriers (oxyglobin, - ml/kg/day), concentrated human albumin ( % albumin, ml/kg), and plasma ( ml/kg). oxygenation and ventilation can be evaluated by arterial blood gas analysis or by the noninvasive means of pulse oximetry and capnometry (see sections on pulse oximetry and capnometry). oxygen delivery can be impaired in cases of hypovolemic shock because of hemorrhage and anemia, and thus a decrease in functional capacity to carry oxygen, and is not to be used for more than weeks due to idiosyncratic blindness. in cases of cardiogenic shock as a result of impaired ability to saturate hemoglobin due to pulmonary edema in the lungs, or decrease in cardiac output. in septic shock, decreases in cardiac output due to inflammatory cytokines and a decrease in cellular oxygen extraction can lead to lactic acidosis. increased cellular metabolism and decreases in respiratory function can lead to respiratory acidosis as co increases. administer supplemental oxygen as flow-by, nasal or nasopharyngeal catheter, oxygen hood, or oxygen cage. supplemental oxygen should be humidified, and delivered at - ml/kg/minute. if oxygenation and ventilation are so impaired that the pao remains < mm hg with the patient on supplemental oxygen, a paco > mm hg, or severe respiratory fatigue, develops, and mechanical ventilation should be considered. glucose is a necessary fuel source for red blood cells and neuronal tissues, and serum glucose should be maintained within normal reference ranges. glucose supplementation can be administered as . - % solutions in crystalloid fluids, or in parenteral and enteral nutrition products. arterial and venous ph can be measured by performing blood gas analyses. decrease in tissue perfusion, impaired oxygen delivery, and decreased oxygen extraction in the various forms of shock can lead to anaerobic metabolism and metabolic acidosis. in most cases, improving tissue perfusion and oxygen delivery with crystalloid and colloid fluids, supplemental oxygen, and inotropic drugs will help normalize metabolic acidosis. serial measurements of serum lactate (normal, < . mmol/l) can be used as a guide to evaluate the tissue response to fluid resuscitative efforts. serum electrolytes often become severely deranged in shock states. serum potassium, magnesium, sodium, chloride, and total and ionized calcium should be maintained within normal reference ranges. if metabolic acidosis is severe, sodium bicarbonate can be administered by calculating the formula base deficit × . × body weight in kg = meq bicarbonate to administer because iatrogenic metabolic alkalosis can occur, a conservative approach is to administer / of the calculated dose and then recheck the patient's ph and bicarbonate levels. if the base excess is unknown, sodium bicarbonate can be administered in incremental doses of meq/kg until the ph is above . . complications associated with bicarbonate therapy include iatrogenic hypocalcemia, metabolic alkalosis, paradoxical cerebrospinal fluid acidosis, hypotension, restlessness, and death. massive trauma, neoplasia, sepsis, and systemic inflammation can all lead to coagulation abnormalities, including disseminated intravascular coagulation (dic). cage-side coagulation monitors are available for daily measurement of prothrombin time (pt), activated partial thromboplastin time (aptt), and platelet counts. fibrin degradation products (fibrin split products) become elevated in dic, trauma, hepatic disease, and surgery. coagulation proteins (clotting factors) and antithrombin often are lost with other proteins in hypoproteinemia or are consumed when microclots are formed and then dissolved. antithrombin levels can be measured by commercial laboratories. antithrombin and clotting factors can be replenished in the form of fresh frozen plasma transfusions. a more sensitive and specific test for dic is the detection of d-dimers, which can be measured by commercial laboratories. treatment for dic involves treatment and resolution of the underlying disease and administration of antithrombin and clotting factors in the form of fresh frozen plasma ( ml/kg) and heparin (unfractionated, - units/kg sq tid; fractionated [lovenox], mg/kg sq bid). monitor the patient for changes in mental status, including stupor, coma, decreased ability to swallow and protect the airway, and seizures. elevation of the patient's head can help to protect the airway and decrease the risk of increased intracranial pressure. serum glucose should be maintained within normal levels to prevent hypoglycemia-induced seizures. one of the major components of oxygen delivery is the binding to hemoglobin. packed cell volume must be kept above - % for adequate cellular oxygen delivery. acid-base status can adversely affect oxygen offloading at the tissue level if metabolic or respiratory alkalosis is present. oxygen-carrying capacity and hemoglobin levels can be increased with administration of rbc component therapy or with hemoglobin-based oxygen carriers. monitoring of renal function includes daily measurement of bun, creatinine, and urine output. normal urine output in a hydrated euvolemic patient is - ml/kg/hour. fluid ins and outs should be measured in cases of suspected oliguria or anuria. in patients with oliguria or anuria, furosemide can be administered as a bolus ( - mg/kg) or by constant rate infusion (cri)( . - mg/kg/hour). mannitol should also be administered ( . - g/kg over to minutes). dopamine ( - µg/kg/minute cri) can be administered to dilate renal afferent vessels and improve urine output. the patient's white blood cell count may be elevated, normal, or decreased, depending on the type of shock. the decision to administer antibiotics should be made on a daily basis. superficial or deep staphylococcus or streptococcus infection usually can be treated with a first-generation cephalosporin (cefazolin, mg/kg iv tid). if a known source of infection is present, administer a broad-spectrum antibiotic (cefoxitin, mg/kg iv tid; ampicillin, mg/kg qid, or enrofloxacin, - mg/kg once daily) pending results of culture and susceptibility testing. if broader anaerobic coverage is required, metronidazole ( mg/kg iv tid) should be considered. gentamicin ( - mg/kg iv once daily) is a good choice for gram-negative sepsis, provided that the patient is well hydrated and has normal renal function. ideally, patients receiving any aminoglycoside antibiotic should have a daily urinalysis to check for renal tubular casts that signify renal damage. in dogs, the gut is the shock organ. impaired gastrointestinal motility and vomiting should aggressively be treated with antiemetics and promotility drugs (dolasetron, . mg/kg iv once daily, and metoclopramide, - mg/kg/day iv cri). metoclopramide is contraindicated in cases of suspected gastrointestinal obstruction. histamine-receptor blockers such as famotidine ( . mg/kg bid iv) and ranitidine ( . to mg/kg iv bid, tid) or proton-pump inhibitors (omeprazole, . - mg/kg po once daily) can be administered for esophagitis. administer sucralfate ( . - g po tid) to treat gastric ulceration. if the gastrointestinal barrier function is diminished due to poor perfusion, infection, or inflammation, administer broad-spectrum antibiotics such as ampicillin ( mg/kg iv qid) to prevent gastrointestinal bacterial translocation. the course of drug therapy should be reviewd daily and the patient should be monitored for potential drug interactions. for example, metoclopramide and dopamine, working at the same receptor, can effectively negate the effects of each other. cimetidine, a cytochrome p enzyme inhibitor, can decrease the metabolism of some drugs. drugs that are avidly protein-bound may have an increase in unbound fraction with concurrent hypoalbuminemia or when hypoalbuminemia is present. decreased renal function may impair the renal clearance of some drugs, requiring increased dosing interval or decreased dose. nutrition is of utmost importance in any critically ill patient. patients with septic shock may become hypermetabolic and require supraphysiologic nutrient caloric requirements, while others may actually become hypometabolic. enteral nutrition is preferred, whenever possible, because enterocytes undergo atrophy without luminal nutrient stimulation. a variety of enteral feeding tubes can be placed, depending on what portion of the gut is functional, to provide enteral nutrition in an inappetent patient. loss of gastrointestinal mucosal barrier function may predispose the patients to the development of bacterial translocation and may contribute to sepsis. if enteral nutrition is impossible because of protracted vomiting or gastrointestinal resection, glucose, lipid, and amino acid products are available that can be administered parenterally to meet nutrient needs until the gastrointestinal tract is functioning and the patient can be transitioned to enteral nutrition. assessment of pain in animals in shock can be challenging. pain can result in the release of catecholamines and glucocounterregulatory hormones that can impair nutrient assimilation and lead to negative nitrogen balance, impaired wound healing, and immunocompromise. in any animal determined to be in pain, analgesic drugs should be administered to control pain and discomfort at all times. opioids are cardiovascularly friendly, and their effects can easily be reversed with naloxone if adverse effects such as hypotension and hypoventilation occur. if the patient is nonambulatory, rotate the animal from side to side every to hours to prevent lung atelectasis. passive range-of-motion exercises and deep muscle massage should be performed to increase tissue perfusion, decrease dependent edema, and prevent disuse atrophy. animals should be kept completely dry on soft, padded bedding to prevent the development of decubital ulcers. all bandages, wound sites, and catheter sites should be checked daily for the presence of swelling, erythema, and pain. soiled bandages should be changed to prevent strike-through and contamination of the underlying catheter or wound. hospitalization can be a stressful experience for patient and client alike. allowing brief visits and walks outside in the fresh air can improve a patient's temperament and decrease stress. the preemptive use of analgesic drugs on a regular schedule (not prn) should be used to prevent pain before it occurs. pain decreases the patient's ability to sleep. lack of sleep can promote further stress and impaired wound healing. the use of glucocorticosteroids and antiprostaglandins in shock therapy remains a topic of wide controversy. although the use of these agents potentially may stabilize membranes, decrease the absorption of endotoxin, and decrease prostaglandin release, the routine use of glucocorticosteroids and antiprostaglandins can decrease renal perfusion and gastrointestinal blood flow, promoting gastrointestinal ulceration and impaired renal function. the administration of supraphysiologic levels of glucocorticosteroids in patients in any type of shock can increase sodium and water retention, depress cellular immune function, and impair wound healing. in clinical studies of small animal patients, the routine use of glucocorticosteroids and antiprostaglandins has not demonstrated definite improved survival. the risks of therapy do outweigh the anecdotal reported benefits, and therefore the empiric use of glucocorticosteroids and antiprostaglandins in any shock patient is urinary tract emergencies azotemia azotemia occurs when % or more of the nephrons are nonfunctional. the magnitude of the azotemia alone cannot be used to determine whether the azotemia is prerenal, renal, or postrenal in origin, or whether the disease process is acute or chronic, reversible or irreversible, progressive or nonprogressive. before beginning treatment for azotemia, the location or cause of the azotemia must be identified. take a thorough history and then perform a physical examination. obtain blood and urine samples before initiating fluid therapy, for accurate assessment of the location of the azotemia. for example, an azotemic animal with a history of vomiting and diarrhea that appears clinically dehydrated on physical examination, normally should have a concentrated urine specific gravity (> . ) reflecting the attempt to conserve fluid. if this level is found, the azotemia is much less likely to be renal in origin, and the azotemia will likely resolve after rehydration. if, however, the urine specific gravity is isosthenuric or hyposthenuric ( . - . ) in the presence of azotemia and dehydration, primary intrinsic renal insufficiency is likely present. if the azotemia resolves with fluid therapy, the patient has prerenal and primary renal disease. if the azotemia does not resolve after rehydration, the patient has prerenal and primary renal failure. dogs with hypoadrenocorticism can have both prerenal and primary renal disease secondary to the lack of mineralocorticoid (aldosterone) influence on the renal collecting duct and renal interstitial medullary gradient. medullary washout can occur, causing isosthenuric urine in the presence of dehydration from vomiting and diarrhea. the patient often has azotemia due to fluid loss (dehydration and urinary loss) and gastric or intestinal hemorrhage (elevated bun). the prerenal component will resolve with treatment with glucocorticoids and crystalloid fluids, but the renal component may take several weeks to resolve, until the medullary concentration gradient is reestablished with the treatment and influence of mineralocorticoids. drugs such as corticosteroids and diuretics can influence renal tubular uptake and excretion of fluid, and cause a prerenal azotemia and isosthenuric urine in the absence of primary renal disease. treatment of azotemia includes calculation of the patient's dehydration estimate and maintenance fluid volumes, and administering that volume over the course of hours. identify and treat underlying causes of prerenal azotemia (shock, vomiting, diarrhea). monitor urine output closely. once a patient is euvolemic, oliguria is defined as urine output < - ml/kg/hour. urine output should return to normal in patients with prerenal azotemia as rehydration occurs. if a patient remains oliguric after rehydration, consider the possibility of oliguric acute intrinsic renal failure, and administer additional fluid therapy based on the patient's urine output, body weight, central venous pressure, and response to other medical therapies. prerenal azotemia is caused by conditions that decrease renal perfusion, including hypovolemic shock, severe dehydration, hypoadrenocorticism, congestive heart failure, cardiac tamponade, cardiac dysrhythmias, and hypotension. once renal perfusion is restored, the kidneys can resume normal function. glomerular filtration rate decreases when the mean arterial blood pressure falls to less than mm hg in a patient with normal renal autoregulation. renal autoregulation can be impaired in some diseases. passive reabsorption of urea from the renal tubules can occur during states of low tubular flow (dehydration, hypotension) even if glomerular filtration is not decreased. if renal hypoperfusion is not quickly restored, the condition can progress from prerenal disease to acute intrinsic renal failure. prerenal and renal azotemia can coexist in animals with primary renal disease, as a result of vomiting and ongoing polyuria in the absence of any oral fluid intake. the treatment of prerenal azotemia consists of rehydration, antiemetic therapy, and treatment of the underlying cause of vomiting, diarrhea, or third spacing of fluids. acute intrinsic renal failure is characterized by an abrupt decline in renal function to the extent that azotemia and an inability to regulate solute and fluid balance. patients with acute intrinsic renal failure may be oliguric or polyuric, depending on the cause and state of renal failure. in small animals, the most common causes of acute intrinsic renal failure are renal ischemia and toxins. there are three phases of acute intrinsic renal failure: induction, maintenance, and recovery. during the induction phase, some insult (ischemia or toxin) to the kidneys occurs, leading to a defective concentrating mechanism, decreased renal clearance of nitrogenous waste (azotemia), and polyuria or oliguria. if treatment is initiated during the induction phase, progression to the maintenance phase potentially can be stopped. as the induction phase progresses, there is worsening of the urine-concentrating ability and azotemia. renal tubular epithelial cells and renal tubular casts can be seen on examination of the urine sediment. glucosuria may be present. the maintenance phase of acute intrinsic renal failure occurs after a critical amount of irreversible nephron injury. correction of the azotemia and removal of the cause of the problem do not result in return to normal function. in patients with oliguria, the extent of nephron damage is greater than that observed in patients with polyuria. the maintenance phase may last for several weeks to months. recovery of renal function may or may not occur, depending on the extent of injury. the most serious complications (overhydration and hyperkalemia) are observed in patients with oliguria. the recovery phase occurs with sufficient healing of damaged nephrons. azotemia may resolve, but concentrating defects may remain. if the patient was oliguric in the maintenance phase, a marked diuresis develops during the recovery phase that may be accompanied by fluid and electrolyte losses. this phase may last for weeks to months. treatment of acute intrinsic renal failure consists of determining the cause and ruling out obstruction or uroabdomen whenever possible. a careful history can sometimes determine whether there has been exposure to nephrotoxic drugs, chemicals, or food items. if ingestion or exposure to a toxic drug, chemical, or food occurred recently (within to hours), induce emesis with apomorphine ( . mg/kg iv). next, administer activated charcoal either orally or via stomach tube, to prevent further absorption of the toxin. obtain blood and urine samples for toxicologic analysis (e.g., ethylene glycol) and to determine whether azotemia or abnormalities in the urine sediment exist. (see section on ethylene glycol, grapes and raisins, and nonsteroidal antiinflammatory drugs). obtain a complete blood count, biochemical profile, and urinalysis to determine the presence of signs of chronic renal failure, including polyuria, polydipsia, and nonregenerative anemia. radiographs and abdominal ultrasound can help in determining the chronicity of renal failure. normal renal size is . - . times the length of l in dogs and . - . times the length of l in cats. monitor the patient's body weight at least twice a day to avoid overhydration. also monitor urine output; normal output is - ml/kg/hour. in cases of polyuric renal failure, massive fluid and electrolyte losses can occur. place a urinary catheter for patient cleanliness and to facilitate urine quantitation. measure fluid ins and outs (see section on fluid therapy). after the patient has been rehydrated, the amount of fluids administered should equal maintenance and insensible needs plus the volume of urine produced each day. if a urinary catheter cannot be placed or maintained, serial body weight measurements and central venous pressure should be used to monitor the patient's fluid balance and prevent overhydration. if the patient is oliguric (urine output < - ml/kg/hour), pharmacologic intervention is necessary to increase urine output. first, administer furosemide ( - mg/kg or . mg/kg/hour iv cri). repeat bolus doses of furosemide if there is no response to initial treatment. if necessary, administer low-dose dopamine ( - µg/kg/minute iv cri) to increase renal afferent dilatation and renal perfusion. dopamine and furosemide may be synergistic if administered together. if dopamine and furosemide therapy is ineffective, administer mannitol ( . - . g/kg iv) once only. if polyuria is present, management is simplified because of the decreased risk of overhydration. if oliguria cannot be reversed, monitor the central venous pessure, body weight, and respiratory rate and effort, auscultate for crackles, and examine the patient carefully for signs of chemosis and the presence of serous nasal discharge. correct hyperkalemia with sodium bicarbonate ( . - . meq/kg iv) or with insulin ( . units/kg) plus dextrose ( g/unit of insulin iv, followed by . % dextrose iv cri). treat severe metabolic acidosis (ph < . or hco − < meq/l) with sodium bicarbonate. if anuria develops or oliguria is irreversible despite this therapy, begin peritoneal dialysis. obtain a renal biopsy to establish a diagnosis and prognosis (see section on renal biopsy). administer gastroprotectant drugs and antiemetics to control nausea and vomiting. if possible, avoid the use of nephrotoxic drugs and general anesthesia. initiate nutritional support in the form of an enteral feeding tube or parenteral nutrition as early as possible. once the patient enters the recovery phase, diuresis may occur that can lead to dehydration and electrolyte imbalances (hyponatremia, hypokalemia). dehydration and electrolyte imbalances can be treated with parenteral fluid and electrolyte supplementation. postrenal azotemia is primarily caused by urethral obstruction or leakage from the urinary tract into the abdomen (uroabdomen). complete urinary tract obstruction and uroabdomen are both ultimately fatal within to days if left untreated. in dogs, the most common causes of urethral obstruction are urinary (urethral) calculi or tumors of the urinary bladder or urethra. in male cats, feline urologic syndrome (fus) is the most common cause of urethral obstruction, although there has been an increased incidence of urethral calculi observed in recent years. a ruptured urinary bladder is the most common cause of uroabdomen and is usually secondary to blunt trauma. clinical signs of urinary tract obstruction include dysuria, hematuria, inability to urinate or initiate an adequate stream of urine, and a distended painful urinary bladder. late in the course of obstructive disease, clinical signs referable to uremia and azotemia (vomiting, oral ulcers, hematemesis, dehydration, lethargy, and anorexia) occur. the initial goal of treatment of urinary tract obstruction is to relieve the obstruction. in male dogs, a lubricated catheter can be inserted past the area of obstruction with the animal under heavy sedation or general anesthesia (see section on urohydropulsion). depending on the chronicity of the obstruction, serum electrolytes should be measured;an ecg should be obtained before administering any anesthetic drugs, because of the cardiotoxic effects of hyperkalemia (see section on atrial standstill). correct fluid, electrolyte, and acid-base abnormalities. if a urinary catheter cannot be placed, perform cystocentesis only as a last resort, because of the risk of urinary bladder rupture. definitive treatment includes identification and treatment of the underlying cause (tumor versus urinary calculi). in most cases, surgical intervention is necessary. if an unresectable tumor is present, a low-profile permanent cystostomy tube can be placed, if the owner desires. administration of piroxicam (feldene, . mg/kg po q - h) with or without chemotherapy may shrink the tumor mass and delay the progression of clinical signs. a complete discussion of this disorder is beyond the scope of this text (see additional reading for other sources of information). feline lower urinary tract disease can cause urethral obstruction, particularly in male cats. clinical signs include stranguria, dribbling of small amounts of urine, lethargy, inappetence, and vomiting. often, owners call with the primary complaint of constipation, because the cat is making frequent trips to the litterbox and straining. cases with a duration of obstruction < hours are considered uncomplicated; those with a duration > hours are complicated. treatment of urethral obstruction includes stabilizing and normalizing the patient's electrolyte status, induction of sedation or general anesthesia, and relieving the obstruction. obtain blood samples for analysis of electrolyte abnormalities. treat hyperkalemia (k + > . meq/l) with sodium bicarbonate ( . - . meq/kg iv), regular insulin ( . unit/ kg iv) plus dextrose ( g//unit of insulin iv), followed by . % dextrose iv cri to prevent hypoglycemia; or calcium gluconate ( . ml/kg % iv slowly). administer non-potassiumcontaining intravenous fluids in . % saline solution. obtain an ecg to detect atrial standstill (see section on atrial standstill). in some cases, a urethral plug is visible at the tip of the penis. the urethral plug can sometimes be manually extracted or massaged from the penis, and the obstruction temporarily relieved. in such cases, it is still necessary to pass a urethral catheter to flush sediment from the urethra and urinary bladder. unless a patient is obtunded, administer an anesthetic such as ketamine, atropine, or propofol ( - mg/kg iv) with diazepam iv for patient comfort and muscle relaxation. once the patient is under anesthesia or heavily sedated, urinary catheterization should be performed. in some cases, it will be difficult to advance the catheter. lubricate a closedended tomcat catheter and pass the tip into the distal urethra. fill a -ml syringe with sterile saline and sterile lubricant and connect the syringe to the hub of the catheter. pulse the fluid into the catheter as you gently move the catheter tip back and forth against the urethral obstruction. when the catheter has been passed into the urinary bladder, obtain a urine sample for urinalysis. drain the bladder and flush with sterile saline solution until the urine efflux appears clear. remove the tomcat catheter and insert a - fr red rubber tube or argyle infant feeding catheter into the urethra for urine collection and quantitation. secure the urinary catheter to prepuce with a butterfly strip of -inch adhesive tape secured around the catheter and then sutured to either side of the prepuce. the catheter should be connected to a closed urinary collection system for cleanliness and to reduce the risk of ascending bacterial infection. an elizabethan collar should be placed at all times to prevent the patient from damaging or removing the catheter. when the urethral obstruction has been relieved and the catheter placed, continue intravenous fluid diuresis to alleviate postrenal azotemia. monitor the urine for bacteria and other sediment. in some cases, postobstructive diuresis can be severe. carefully monitor fluid ins and outs, along with body weight, to maintain adequate hydration and perfusion. remove the urinary catheter can be removed after to hours. palpate the bladder frequently to make sure that the patient is voiding normally and to detect the recurrence of obstruction. in patients with severe penile or urethral trauma or edema, administer a short-acting steroid (dexamethasone sodium phosphate, . mg/kg iv, im, sq). at the time of initial diagnosis and again at the time of discharge, the clients need to be instructed about the long-term management of feline lower urinary tract disease at home, and informed of the risks and consequences of recurrence. uroabdomen can occur from trauma or leakage from the kidneys, ureter, or urinary bladder. clinical signs of uroabdomen (azotemia, uremia, hyperkalemia) can also occur secondary to third spacing of urine and leakage into muscular tissue from a ruptured urethra. in most cases, urinary bladder trauma and rupture are secondary to blunt trauma. abdominocentesis should be performed in any animal with suspected blunt abdominal trauma, and any fluid obtained should be analyzed for creatinine or potassium and compared with the patient's serum levels. an abdominal effusion that has a low packed cell volume and a potassium or creatinine level greater than that of the patient's serum is consistent with the diagnosis of uroabdomen. uroabdomen is not a surgical emergency. however, medical management consists of placement of a temporary abdominal drainage catheter into the abdomen, to facilitate removal of urine from the peritoneal cavity. to place the catheter, position the patient in dorsal or lateral recumbency, shave the ventral abdomen, as for any exploratory laparotomy. aseptically scrub the clipped area, and instill a local anesthestic (lidocaine, - mg/kg) caudal and to the right of the umbilicus, through the skin, subcutaneous tissues, and rectus emergency care clinical differentiation of acute necrotizing from chronic nonsuppurative pancreatitis in cats: cases acute pancreatitis in dogs mesenteric volvulus in the dog: a retrospective study of cases incidence and prognostic value of low plasma ionized calcium concentration in cats with pancreatitis: cases ( - ) review of feline pancreatitis. part : clinical signs, diagnosis and treatment gastric dilatation-volvulus syndrome in dogs diagnostic approach to acute pancreatitis pathophysiology of organ failure in severe acute pancreatitis in dogs washabau rj: gastrointestinal motility disorders and gastrointestinal prokinetic therapy watson pt: exocrine pancreatic insufficiency as an end-stage of pancreatitis in dogs clinical signs, underlying cause, and outcome in cats with seizures: cases fibrocartilaginous embolism in dogs: clinical findings and factors influencing the recovery rate kirk's current veterinary therapy xiii intervertebral disc extrusion in six cats medical management of acute spinal cord disease risk factors for recurrence of clinical signs associated with thoracolumbar intervertebral disk herniation in dogs: cases intervertebral disk disease in cats long-term functional outcome of dogs with severe injuries of the thoracolumbar spinal cord: cases canine status epilepticus: a retrospective study of cases risk factors for development of status epilepticus in dogs with idiopathic epilepsy and effects of status epilepticus on outcome and survival time: cases ( - ) skills laboratory part i: performing a neurologic examination skills laboratory part ii: interpreting the results of the neurologic examination accuracy of localization of cervical intervertebral disk extrusion or protrusion using survey radiography in dogs medical and surgical management of the glaucoma patient the feline glaucomas: cases ( - ) the canine glaucomas traumatic ocular protrusion in dogs and cats: cases traumatic glaucoma in a dog ocular and orbital porcupine quills in the dog: a review and case series hyphema: pathophysiologic considerations. comp cont educ pract vet van der woerdt a: the treatment of acute glaucoma in dogs and cats administer crystalloid intravenous fluids at maintenance rates using a balanced electrolyte solution perform urinary catheterization and collection to monitor urine output monitor serum urea nitrogen and creatinine every hours treat oliguria, defined as a drop in urine output to less than ml/kg/hour ml/kg) bolus start dopamine at to µg/kg/minute if no response to crystalloid/colloid bolus occurs within minutes consider mannitol ( . to g/kg iv) administration if no response to dopamine occurs within minutes consider furosemide ( to mg/kg iv, or . to mg/kg/hour iv cri) if no response to dopamine or mannitol occurs in to minutes if no response to furosemide, peritoneal dialysis or hemodialysis is indicated immediately, particularly if anuria is present administered with caution, because of the risk of exacerbating increased capillary permeability and causing pulmonary edema. animal patients. chlorphenoxy derivatives exert their toxic effects by an unknown mechanism, and cause clinical signs of gastroenteritis and muscle rigidity severe anemia should be treated with packed rbcs or hemoglobin-based oxygen carriers handbook of small animal toxicology and poisonings macadamia nut toxicosis in dogs the recognition and treatment of the intermediate syndrome of organophosphate poisoning in a dog acute renal failure in four dogs after raisin or grape ingestion pleural effusion in cats pulmonary function, ventilator management, and outcome of dogs with thoracic trauma and pulmonary contusions: cases ( - ) acute lung injury and acute respiratory distress syndrome smoke exposure in cats: cases ( - ) smoke exposure in dogs: cases ( - ) thoracic duct ligation and pericardectomy for treatment of idiopathic chylothorax use of intraluminal nitinol stents in the treatment of tracheal collapse in a dog clinical approach to epistaxis the veterinary icu book. teton newmedia radiographic diagnosis of diaphragmatic hernia: review of cases in dogs and cats tracheal collapse: diagnosis and medical and surgical management acute respiratory distress syndrome brachycephalic syndrome in dogs outcome and postoperative complications in dogs undergoing surgical treatment of laryngeal paralysis: cases ( - ) full recovery following delayed neurologic signs after smoke inhalation in a dog aspiration pneumonitis the veterinary icu book. teton newmedia allergic airway disease canine pleural and mediastinal effusion, a retrospective study of cases suggested strategies for ventilatory management in veterinary patients with acute respiratory distress syndrome laryngeal and tracheal disorders the veterinary icu book. teton newmedia medical and surgical treatment of pyothorax in dogs: cases traumatic diaphragmatic hernia in cats: cases canine pyothorax: clinical presentation, diagnosis, and treatment canine pyothorax: pleural anatomy and pathophysiology treatment of chronic pleural effusion with pleuroperitoneal shunt in dogs: cases ( - ) effects of doxapram hydrochloride on laryngeal function of normal dogs and dogs with naturally occurring laryngeal paralysis an overview of positive pressure ventilation risk factors, prognostic indicators, and outcome of pyothorax in cats: cases ( - ) use of percutaneous arterial embolization for the treatment of intractable epistaxis in dogs systemic inflammatory response syndrome, sepsis, and multiple organ dysfunction cardiogenic shock and cardiac arrest hemostatic changes in dogs with naturally occurring sepsis multiple organ dysfunction syndrome in humans and dogs increased lactate concentrations in ill and injured dogs the role of albumin in health and disease pathophysiologic characteristics of hypovolemic shock usefulness of systemic inflammatory response syndrome criteria as an index for prognosis judgement current principles and application of d-dimer analysis in small animal practice choosing fluids in traumatic hypovolemic shock: the role of crystalloids, colloids and hypertonic saline colloid and crystalloid resuscitation thromboembolic disease: predispositions and management marks sl: systemic arterial thromboembolism retrospective study of streptokinase administration in cats with arterial thromboembolism feline arterial thromboembolism: an update arterial thromboembolism in cats: acute crises in cases ( - ) and long-term management with low-dose aspirin in cases cut multiple holes in the side of a - fr red rubber tube or thoracic drainage catheter, using care not to make the cut wider than % of the circumference of the tube. insert the catheter into the abdominal cavity in a dorsal caudal direction. make sure that all incisions within the abdomen. secure the tube by placing a pursestring suture around the tube entrance site in the abdominal musculature with absorbable suture material. close the dead space in the subcutaneous tissues with absorbable suture. close the skin around the tube with another purse-string suture secured using a finger-trap technique. connect the tube to a closed urinary collection system and bandage the catheter to the abdomen. the tube can remain in place until the patient retrospective evaluation of acute renal failure in dogs uroabdomen in dogs and cats drug-induced nephrotoxicity: recognition and prevention peritoneal dialysis in emergency and critical care acute renal failure caused by lily ingestion in six cats early diagnosis of renal disease and renal failure acute renal failure in four dogs after raisin or grape ingestion disorders of the feline lower urinary tract the use of a low-profile cystostomy tube to relieve urethral obstruction in a dog renal biopsy: methods and interpretation feline idiopathic cystitis: current understanding of pathophysiology and management today's problem when did you first notice that something was wrong with your pet? when was the last time you noticed your pet act normally? what was the first abnormal sign noticed? what other conditions have developed and what are they? how soon did other signs develop? have the signs become better or worse since you first saw them? what is the name of the product? do you have the container with you today? is it a liquid concentrate, dilute spray, or solid? how long ago do you think that your pet was exposed to the poison? where do you think it happened? do you have any over-the-counter or prescription medications that your animal may have had access to? did you give any medications to your animal? is there any possibility of recreational drug exposure?your pet's recent activity did your pet eat this morning or last night? what is he/she normally fed? is there a chance that your pet may have gotten into the garbage? have you fed table scraps or anything new recently? if so, what? has your pet been off your property in the last - hours? does your pet run loose unattended? has your pet had any antiflea/tick medication within the last week?your pet's environment is your animal kept inside or outside of the house? is your pet kept in a fenced-in yard or allowed to run loose unattended? does your pet have access to neighboring properties (even for a short time)? where has your pet been in the last hours? has your pet traveled outside of your immediate geographic location? if so, when? has your pet been to rural areas in the last week? has there been any gardening work recently? does your pet have access to a compost pile? any fertilizers or weed killer used in the last week? any construction work or renovation recently? any mouse or rat poison in your house, yard, or garage? any cleaning products used inside or outside the house within the last hours? if so, which? have you changed your radiator fluid or does a car leak antifreeze? induce and maintain a patent airway and stabilize the patient's cardiovascular and respiratory status. control cns excitation with diazepam, if necessary, and control the patient's body temperature (both hypo-and hyperthermia) . induce vomiting if the patient is alert and can protect its airway; otherwise, perform orogastric lavage with the patient under general anesthesia with a cuffed endotracheal tube in place. alcohols do not bind well with activated charcoal. treat dermal exposure by bathing the area with warm water. introduction: if ingested, sodium or potassium hydroxide can cause severe contact dermatitis or irritation of the gastrointestinal tract. esophageal burns and full-thickness coagulative necrosis can occur. if an animal ingests a caustic alkali substance, feed the animal four egg whites mixed with quart of warmed water. perform endoscopy within hours to evaluate the extent of injury and to place a feeding tube, in severe cases. do not induce emesis , and do not perform orogastric lavage, because of the risk of worsening esophageal irritation. in cases of contact exposure to the skin or eyes, rinse the exposed area with warm water baths for at least minutes. administer gastroprotectant, antiemetic, and analgesic drugs as necessary. avoid neutralization, which can cause a hyperthermic reaction and worsen injury to the skin and gastrointestinal tract. amitraz is the active ingredient in ascaricides and anti-tick and anti-mite products such as mitaban and taktic. the toxic dose is to mg/kg. amitraz exerts its toxic effects by causing α-adrenergic stimulation, and causes clinical signs similar to those observed with administration of xylazine: bradycardia, cns depression, ataxia, hypotension, hyperglycemia, hypothermia, cyanotic mucous membranes, polyuria, mydriasis, and emesis. a coma can develop. treatment of amitraz intoxication includes cardiovascular support with intravenous crystalloid fluids and induction of emesis in asymptomatic animals. if clinical signs are present, orogastric lavage may be required. many toxic compounds are impregnated in a collar form. if the patient has ingested a collar and does not vomit it, it should be removed using endoscopy or gastrotomy. administer activated charcoal to prevent or delay absorption of the toxic compound. yohimbine or atepamizole, both α-adrenergic antagonists, are the treatment(s) of choice to reverse the clinical signs of toxicity. avoid the use of atropine, because it can potentially increase the viscosity of respiratory secretions and cause gastrointestinal ileus, thus promoting increased absorption of the toxic compound. ammonium hydroxide, or cleaning ammonia, can be caustic at high concentrations (see alkalis/caustics) and cause severe injury to the respiratory system if inhaled. pulmonary edema or pneumonia can occur, resulting in respiratory distress. ingestion of ammonia can cause severe irritation to the gastrointestinal tract and cause vomiting and esophageal injury. if ammonia is ingested, administer a dilute solution of egg white.administer gastroprotectant, antiemetic, and analgesic drugs as necessary. if pneumonia or pulmonary edema occurs secondary to aspiration of ammonia into the airways and alveolar spaces, treatment is largely supportive with supplemental oxygen administration, antibiotics, fluid therapy, and mechanical ventilation as necessary. diuretics may or may not be useful in the treatment of pulmonary edema secondary to ammonia inhalation. amphetamines cause cns excitation due to neurosynaptic stimulation, resulting in hypersensitivity to noise and motion, agitation, tremors, vomiting, diarrhea, and seizures. clinical signs of amphetamine toxicity include muscle tremors, tachyarrhythmias, mydriasis, ptyalism, and hyperthermia. amphetamines are rapidly absorbed from the gastrointestinal tract. treatment includes administration of intravenous fluids to maintain hydration and renal perfusion and correction of hyperthermia. administer sedative drugs such as chlorpromazine to control agitation and tremors, and diazepam to control seizures. urinary acidification can promote excretion and prevent reabsorption from the urinary bladder. in severe cases, treat cerebral edema with a combination of mannitol followed by furosemide to control increased intracranial pressure.antifreeze: see ethylene glycol antihistamines introduction antihistamines (loratadine, diphenhydramine, doxylamine, clemastine, meclizine, dimenhydrinate, chlorpheniramine, cyclizine, terfenadine, hydroxyzine) are available as over-thecounter and prescription allergy and anti-motion sickness products. clinical signs of antihistamine toxicity include restlessness, nausea, vomiting, agitation, seizures, hyperthermia, and tachyarrhythmias. treatment of antihistamine intoxication is largely symptomatic and supportive, as there is no known antidote. if ingestion is recent (within to hours) and the patient is not actively seizing and can protect its airway, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. monitor the patient's heart rate, rhythm, and blood pressure. treat cardiac arrhythmias, if present, with appropriate therapies (see section on cardiac dysrhythmias). administer cooling measures and intravenous fluids to treat hyperthermia. a constant rate infusion of guaifenasin can be used to control muscle tremors. introduction α-naphthylthiourea (antu) is manufactured as a white or blue-gray powder. the toxic dose in dogs is - mg/kg, and in cats is - mg/kg. younger dogs appear to be more resistant to its toxic effects. antu usually causes profound emesis and increased capillary permeability that eventually leads to pulmonary edema. treatment of antu toxicity includes respiratory support. mechanical ventilation may be required in severe cases of pulmonary edema. if an animal does not vomit, orogastric lavage should be performed. administer gastrointestinal protectant, antiemetic, and analgesic drugs. cardiovascular support in the form of intravenous crystalloids should be arsenic introduction inorganic arsenic (arsenic trioxide, sodium arsenite, sodium arsenate) is the active ingredient in many herbicides, defoliants, and insecticides, including ant killers. the toxic dose of sodium arsenate is - mg/kg; that of sodium arsenite is - mg/kg. sodium arsenite is less toxic, although cats are very susceptible. arsenic compounds interfere with cellular respiration by combining with sulfhydryl enzymes. clinical signs of toxicity include severe gastroenteritis, muscle weakness, capillary damage, hypotension, renal failure, seizures, and death. in many cases, clinical signs are acute in onset. treatment of arsenic toxicity involves procuring and maintaining a patent airway. administer intravenous crystalloid fluids to correct hypotension and hypovolemia, and normalize acidbase and electrolyte balance. if no clinical signs are present and if the compound was ingested within hours, induce emesis. if clinical signs are present, perform orogastric lavage followed by administration of activated charcoal. if dermal exposure has occurred, throughly bathe the animal to prevent further absorption. dimercaprol (bal, - mg/kg im q h) can be administered as a chelating agent. n-acetylcysteine (mucomyst) (for cats, - mg/kg po iv, then mg/kg po iv q h for days; for dogs, mg/kg po or iv, then mg/kg po iv q h for days) has been shown to decrease arsenic toxicity in rats. aspirin causes inhibition of the production of prostaglandins, a high anion gap metabolic acidosis, gastrointestinal ulceration, hypophosphatemia, and decreased platelet aggregation when ingested in high quantities (> mg/kg/ hours in dogs; > mg/kg/ hours in cats). clinical signs of aspirin toxicity include tachypnea, vomiting, anorexia, lethargy, hematemesis, and melena. treatment of aspirin toxicity is largely supportive. if the ingestion was recent (within the last hour), induce emesis or perform orogastric lavage followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration and correct acid-base abnormalities. administer synthetic prostaglandin analogues (misoprostol), gastroprotectant drugs, and antiemetics. alkalinization of the urine can enhance excretion. introduction baclofen is a gaba agonist centrally acting muscle relaxant. clinical signs of toxicity include vomiting, ataxia, vocalization, disorientation, seizures, hypoventilation, coma, and apnea. clinical signs can occur at doses as low as . mg/kg. treatment of baclofen ingestion includes induction of emesis if the animal is asymptomatic. otherwise, perform orogastric lavage. emesis or orogastric lavage should be followed by administration of activated charcoal. perform intravenous crystalloid fluid diuresis to promote elimination of the toxin, maintain renal perfusion, and normalize body temperature. supplemental oxygen or mechanical ventilation may be required for hypoventilation or apnea. if seizures occur, avoid the use of diazepam, which is a gaba agonist and can potentially worsen clinical signs. control seizures with intravenous introduction β-adrenergic agonists, including terbutaline, albuterol (salbutamol), and metaproterenol, are commonly used in inhaled form for the treatment of asthma. animals commonly are exposed to the compounds after chewing on their owners' inhalers. clinical signs of β-adrenergic stimulation include tachycardia, muscle tremors, and agitation. severe hypokalemia can occur. treatment of β-adrenergic agonist intoxication includes treatment with beta-blockers (propranolol, esmolol, atenolol), intravenous fluids, and intravenous potassium supplementation. diazepam or acepromazine may be administered for sedation and muscle relaxation. introduction barbiturates such as phenobarbital are gaba agonists and induce cns depression. clinical signs of barbiturate overdose or toxicity include weakness, lethargy, hypotension, hypoventilation, stupor, coma, and death. treatment of barbiturate toxicity includes maintenance and support of the cardiovascular and respiratory systems. if clinical signs are absent and the patient can protect its airway, induce emesis followed by repeated doses of activated charcoal. perform orogastric lavage if emesis is contraindicated. administer supplemental oxygen if hypoventilation occurs. some animals may require mechanical ventilation. administer intravenous fluids to control perfusion and blood pressure. positive inotropic drugs may be required if dosedependent decrease in cardiac output and blood pressure occurs. alkalinization of the urine and peritoneal dialysis can be performed to enhance excretion and elimination. hemodialysis should be considered in severe cases, if available. automotive and dry cell batteries contain sulfuric acid that can be irritating on contact with the eyes, skin, and gastrointestinal tract. button batteries, which contain sodium or potassium hydroxide, cause contact irritation if chewed. to treat exposure, rinse the eyes and skin with copious amounts of warm tap water or sterile saline solution for a minimum of minutes. if ingestion occurred, administer gastroprotectant and antiemetic drugs. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of aspiration pneumonia and worsening esophageal irritation. no attempt should be made at performing neutralization because of the risk of causing an exothermic reaction and worsening tissue damage. administer analgesics to control discomfort. benzoyl peroxide is the active ingredient in many over-the-counter acne preparations. ingestion can result in production of hydrogen peroxide, gastroenteritis, and gastric dilatation. topical exposure can cause dermal irritation and blistering. if an animal has ingested benzoyl peroxide, do not induce emesis, because of the risk of worsening esophageal irritation. instead, perform orogastric lavage. administer gastroprotectant and antiemetic medications and closely observe the patient observed for signs of gastric dilatation.bismuth subsalicylate (pepto-bismol): see aspirin bleach, chlorine (sodium hypochlorite) introduction sodium hypochlorite is available in dilute ( %- %) or concentrated ( % industrial strength or swimming pool) solutions for a variety of purposes. sodium hypochlorite can cause severe contact irritation and tissue destruction, depending on the concentration. affected animals may have a bleached haircoat. treatment of exposure includes dilution with copious amounts of warm water or saline baths and ocular lavage. induction of emesis and orogastric lavage is absolutely contraindicated because of the risk of causing further esophageal irritation. to treat ingestion, give the animal milk or large amounts of water, in combination with gastroprotectant and antiemetic drugs, to dilute the contents in the stomach. administration of sodium bicarbonate or milk of magnesia is no longer recommended. nonchlorine bleaches (sodium peroxide or sodium perborate) have a moderate toxic potential if ingested. sodium peroxide can cause gastric distention. sodium perborate can cause severe gastric irritation, with vomiting and diarrhea; renal damage and cns excitation followed by depression can occur, depending on the amount ingested. to treat dermal or ocular exposure, rinse the skin or eyes with copious amounts of warm tap water or sterile saline for a minimum of minutes; treat ocular injuries as necessary, if corneal burns have occurred. if the bleach has been ingested, do induce emesis and perform orogastric lavage. administer milk of magnesia ( - ml/kg). boric acid is the active ingredient in many ant and roach killers. the toxic ingredient (in amounts of - g/kg) can cause clinical signs in dogs by an unknown mechanism. clinical signs include vomiting (blue-green vomitus), blue-green stools, renal damage, and cns excitation and depression. treatment of boric acid or borate ingestion includes gastric decontamination with induction of emesis or orogastric lavage, followed by administration of a cathartic to hasten elimination. activated charcoal is not useful to treat ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. clostridium botulinum endospores can be found in carrion, food, garbage, and the environment. ingestion of endospores and c. botulinum endotoxin rarely can cause generalized neuromuscular blockade of spinal and cranial nerves, resulting in miosis, anisocoria, lower motor neuron weakness, and paralysis. respiratory paralysis, megaesophagus, and aspiration pneumonia can occur. clinical signs usually develop within days of ingestion. differential diagnosis includes acute polyradiculoneuritis (coonhound paralysis), bromethalin intoxication, and tick paralysis. treatment of botulism is largely supportive; although an antitoxin exists, it often is of no benefit. treatment may include administration of intravenous fluids, frequent turning of the patient and passive range-of-motion exercises to prevent disuse muscle atrophy, and supplemental oxygen administration or mechanical ventilation. administer amoxicillin, ampicillin, or metronidazole. recovery may be prolonged, up to to weeks in some cases. bromethalin is the active ingredient in some brands of mouse and rat poisons. it usually is packaged as . % bromethalin in green or tan pellets, and packaged in - . g place packs. the toxic dose for dogs is . g/kg, and for cats g/kg. bromethalin causes toxicity by uncoupling of oxidative phosphorylation. an acute syndrome of vomiting, tremors, extensor rigidity, and seizures occurs within hours of ingestion of high doses. delayed clinical signs occur within to days of ingestion of a lower dose and include posterior paresis progressing to ascending paralysis, cns depression, and coma. treatment of known bromethalin ingestion includes induction of emesis or orogastric lavage, and repeated doses of activated charcoal every to hours for days, because bromethalin undergoes enterohepatic recirculation. supportive care includes intravenous fluids, anticonvulsants, muscle relaxants (methocarbamol up to mg/kg/day iv to effect), frequent turning of the patient, and passive range-of-motion exercises. supplemental oxygen and /or mechanical ventilation may be required in patients with coma and severe hypoventilation. administer mannitol ( . - g/kg) in conjunction with furosemide ( mg/kg iv) if cerebral edema is suspected. the majority of caffeine toxicities occur in dogs that ingest coffee beans. caffeine causes phosphodiesterase inhibition, and can cause cardiac tachyarrhythmias, cns stimulation (hyperexcitability and seizures), diuresis, gastric ulcers, vomiting, and diarrhea. muscle tremors and seizures can occur, resulting in severe hyperthermia. treatment of caffeine toxicity is largely symptomatic and supportive, as there is no known antidote. if clinical signs are not apparent and the patient is able to protect its airway, induce emesis. alternatively, orogastric lavage can be performed, followed by administration of activated charcoal. administer diazepam to control seizures. administer betaadrenergic blockers (e.g., esmolol, propranolol, atenolol) to control tachyarrhythmias. give intravenous fluids to maintain hydration and correct hyperthermia. the patient should be walked frequently or have a urinary catheter placed to prevent reabsorption of the toxin from the urinary bladder. carbamate compounds are found in agricultural and home insecticide products. examples of carbamates include carbofuran, aldicarb, propoxur, carbaryl, and methiocarb. the toxic dose of each compound varies. carbamate compounds function by causing acetylcholinesterase inhibition. toxic amounts cause cns excitation, muscarinic acetylcholine overload, and slud (salivation, lacrimation, urination, and defecation). miosis, vomiting, treatment of carbamate intoxication includes maintaining an airway and, if necessary, artificial ventilation. administer intravenous crystalloid fluids to control the patient's hydration, blood pressure, and temperature. cooling measures may be warranted. induce emesis if the substance was ingested within minutes and the animal is asymptomatic. give repeated doses of activated charcoal if the animal can swallow and protect its airway. control seizures with diazepam ( . mg/kg iv). bathe the patient thoroughly. atropine ( . mg/kg iv) is useful in controlling some of the muscarinic signs associated with the toxicity. pralidoxime hydrochloride ( -pam) is not useful in cases of carbamate intoxication. control muscle tremors with methocarbamol (up to mg/kg iv) or guaifenesin. in humans, ingestion or inhalation of - ml of carbon tetrachloride can be fatal. clinical signs of carbon tetrachloride toxicity include vomiting and diarrhea, then progressive respiratory and central nervous system depression. ventricular dysrhythmias and hepatorenal damage ensue. the prognosis is grave. treatment of carbon tetrachloride inhalation includes procurement and maintenance of a patent airway with supplemental oxygen, and cardiovascular support. to treat ingestion, administer activated charcoal, and give intravenous fluids to maintain hydration and support renal function. chlorinated hydrocarbons include ddt, methoxychlor, lindane, dieldrin, aldrin, chlordane, chlordecone, perthane, toxaphene, heptachlor, mirex, and endosulfan. the toxic dose of each compound varies. chlorinated hydrocarbons exert their toxic effects by an unknown mechanism, and can be absorbed through the skin and the gastrointestinal tract. clinical signs are similar to those observed in organophosphate toxicity: cns excitation, seizures, slud, (salivation, lacrimation, urination, defecation), excessive bronchial secretions, vomiting, diarrhea, muscle tremors, and respiratory paralysis. secondary toxicity from toxic metabolites can cause renal and hepatic failure. chronic exposure may cause anorexia, vomiting, weight loss, tremors, seizures, and hepatic failure. the clinical course can be prolonged in small animal patients. treatment of chlorinated hydrocarbon toxicity is largely supportive in nature, as there is no known antidote. procure and maintain the patient's airway. normalize the body temperature to prevent hyperthermia. if the substance was just ingested and the patient is not demonstrating any clinical signs, induce emesis. if the patient is symptomatic, perform orogastric lavage followed by activated charcoal administration. bathe the patient thoroughly in cases of topical exposure. administer intravenous crystalloid fluids to maintain hydration. these compounds do not appear to be amenable to fluid diuresis. introduction: chlorphenoxy derivatives are found in , -d, , , -t, mcpa, mcpp, and silvex. the ld of , -d is mg/kg; however, the toxic dose appears to be much lower in small treatment treatment of chlorphenoxy derivative toxicity is largely supportive in nature, as there is no known antidote. secure the patient's airway and administer supplemental oxygen, as necessary. control cns excitation with diazepam ( . mg/kg iv). intravenous crystalloid fluid diuresis and urinary alkalinization can promote elimination. administer gastroprotectant and antiemetic drugs, as needed. the toxic effects of chocolate are related to theobromine. various types of chocolate have different concentrations of theobromine and thus can cause clinical signs of toxicity with ingestion of varying amounts of chocolate, depending on the type. the toxic dose of theobromine is - mg/kg in dogs. milk chocolate contains mg/oz ( mg/ g) of chocolate, and has a low toxic potential. semisweet chocolate contains mg/oz ( mg/ g), and baking chocolate contains mg/oz ( mg/ g). semisweet and baking chocolate, being the most concentrated, have a moderate to severe toxic potential, even in large dogs.clinical signs of theobromine intoxication are associated with phosphodiesterase inhibition and include cns stimulation (tremors, anxiety, seizures), myocardial stimulation (tachycardia and tachyarrhythmias), diuresis, and (at very high doses) gastrointestinal ulceration. with treatment, the condition of most dogs returns to normal within to hours (t / = . hours in dogs). potential side effects include gastroenteritis and pancreatitis due to the fat content of the chocolate. treatment of chocolate toxicity includes obtaining and maintaining a protected airway (if necessary), intravenous fluid diuresis, induction of emesis or orogastric lavage followed by administration of repeated doses of activated charcoal, and placement of a urinary catheter to prevent reabsorption of the toxin from the urinary bladder. cholecalciferol rodenticide ingestion can lead to increased intestinal and renal reabsorption of calcium, causing an increase in serum calcium and dystrophic mineralization of the kidneys and liver at - mg/kg. clinical signs include lethargy, anorexia, vomiting, constipation, and renal pain within to days of ingestion. seizures, muscle twitching, and central nervous system depression may be observed at very high doses. as renal failure progresses, polyuria, polydipsia, vomiting/hematemesis, uremic oral ulcers, and melena may be observed. if the compound was ingested recently (within to hours) induce emesis or perform orogastric lavage, followed by administration of activated charcoal. check the patient's serum calcium once daily for three days following ingestion. if clinical signs of toxicity or hypercalcemia are present, decrease serum calcium with loop diuretics (furosemide, - mg/kg po or iv q h) and glucocorticosteroids (prednisone or prednisolone, - mg/kg po bid) to promote renal calcium excretion. in severe cases, salmon calcitonin ( - iu/kg sc q - h in dogs) or bisphosphonate compounds may be required. correct acid-base abnormalities with intravenous crystalloid fluid diuresis and sodium bicarbonate, if necessary. (see section on hypercalcemia.) denture cleaners contain sodium perborate as the active compound. sodium perborate can cause severe direct irritation of the mucous membranes and may also act as a cns depressant. clinical signs are similar to those seen if bleach or boric acid compound is ingested, namely vomiting, diarrhea, cns excitation then depression, and renal failure. treatment for ingestion of denture cleaner includes gastric decontamination along with induction of emesis or orogastric lavage and administration of a cathartic to hasten elimination. activated charcoal is not useful for treatment of ingestion of this toxin. administer intravenous fluid therapy to maintain renal perfusion. administer gastroprotectant and antiemetic drugs, as necessary. deodorants are usually composed of aluminum chloride and aluminum chlorohydrate. both have a moderate potential for toxicity. ingestion of deodorant compounds can cause oral irritation or necrosis, gastroenteritis, and nephrosis. treatment of deodorant ingestion includes orogastric lavage, and administration of antiemetic and gastroprotectant drugs. introduction anionic detergents include sulfonated or phosphorylated forms of benzene. dishwashing liquid is an example of an anionic detergent that can be toxic at doses of - g/kg. anionic detergents cause significant mucosal damage and edema, gastrointestinal irritation, cns depression, seizures, and possible hemolysis. ocular exposure can cause corneal ulcers and edema. treatment of anionic detergent exposure is largely symptomatic, as there is no known antidote. to treat topical toxicity, flush the patient's eyes and skin with warmed tap water or . % saline solution for a minimum of minutes, taking care to avoid hypothermia. to treat ingestion, feed the patient milk and large amounts of water to dilute the toxin. do not induce emesis, because of the risk of worsening esophageal irritation. to dilute the toxin, perform orogastric lavage, followed by administration of activated charcoal. closely monitor the patient's respiratory status, because oropharyngeal edema can be severe. if necessary, perform endotracheal intubation in cases of airway obstruction. monitor the patient for signs of intravascular hemolysis. administer intravenous crystalloid fluids to maintain hydration until the patient is able to tolerate oral fluids. cationic detergents and disinfectants include quaternary ammonia compounds, isopropyl alcohol, and isopropanol. quaternary ammonia compounds have a serious toxic potential treatment treatment of cationic detergent exposure includes careful bathing and ocular rinsing of the patient for a minimum of minutes, taking care to avoid hypotension. secure the patient's airway and monitor the patient's respiratory status. administer supplemental oxygen, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation. give milk or large amounts of water orally, as tolerated by the patient, to dilute the toxin. nonionic detergents include alkyl and aryl polyether sulfates, alcohols, and sulfonates; alkyl phenol; polyethylene glycol; and phenol compounds. phenols are particularly toxic in cats and puppies. clinical signs of exposure include severe gastroenteritis and topical irritation. some compounds can be metabolized to glycolic and oxalic acid, causing renal damage similar to that observed with ethylene glycol toxicity. topical and ocular exposure should be treated with careful bathing or ocular irrigation for at least minutes. administer activated charcoal to prevent absorption of the compound. as tolerated, give dilute milk or straight tap water orally to dilute the compound. administer antiemetic and gastroprotectant drugs to control vomiting and decrease gastrointestinal irritation. administer intravenous crystalloid fluids to maintain hydration and decrease the potential for renal tubular damage. monitor the patient's acid-base and electrolyte status and correct any abnormalities with appropriate intravenous fluid therapy. introduction diclone (phigone) is a dipyridyl compound that is a cns depressant. the ld in rats is - mg/kg. dichlone reacts with thiol enzymes to cause methemoglobinemia and hepatorenal damage. to treat dichlone ingestion, induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. procure and maintain a patent airway. perform intravenous fluid diuresis to maintain renal perfusion. n-acetylcysteine may be useful in the treatment of methemoglobinemia. diethyltoluamide (deet) is the active ingredient in many insect repellants (e.g., off, cutters, hartz blockade). the mechanism of action of deet is not fully understood, but it acts as a lipophilic neurotoxin within to minutes of exposure. cats appear to be particularly sensitive to deet. a lethal dermal dose is . g/kg; if ingested, the lethal dose is much less. the toxic dose of dermal exposure in dogs is g/kg. clinical signs of toxicity include aimless gazing, hypersalivation, chewing motions, and muscle tremors that progress to seizures. recumbency and death can occur within minutes of exposure at high doses. treatment of deet toxicity is largely supportive, as there are no known antidotes. procure and maintain a patent airway and perform mechanical ventilation, if necessary. place an intravenous catheter and administer intravenous crystalloid fluids to control hydration and treat hypotension, as necessary. treat seizures with diazepam ( . mg/kg iv) or phenobarbital. because of the rapid onset of clinical signs, induction of emesis is contraindicated. perform orogastric lavage if the compound was ingested within the last hours. administer multiple repeated doses of activated charcoal. cooling measures should be implemented to control hyperthermia. if dermal exposure has occurred, bathe the patient thoroughly to avoid further exposure and absorption. diquat is a dipyridyl compound that is the active ingredient in some herbicide compounds. the ld of diquat is - mg/kg. like paraquat, diquat induces its toxic effects by causing the production of oxygen-derived free radical species. clinical signs of diquat intoxication include anorexia, vomiting, diarrhea, and acute renal failure. massive dehydration and electrolyte imbalances can occur as a result of fluid loss into the gastrointestinal tract. treatment of diquat intoxication is similar to that for paraquat ingestion. if the animal had ingested diquat within hour of presentation, induce emesis. in clinical cases, orogastric lavage may be required. both emesis and orogastric lavage should be followed by administration of kaolin or bentonite as an adsorbent, rather than activated charcoal. place an intravenous catheter and administer crystalloid fluids to restore volume status and maintain renal perfusion. monitor urine output. if oliguria or anuria occurs, treatment with mannitol, furosemide, and dopamine may be considered. ecstasy ( , -methylenedioxymethylamphetamine; mdma) is a recreational drug used by humans. ecstasy causes release of serotonin. clinical signs of intoxication are related to the serotonin syndrome (excitation, hyperthermia, tremors, and hypertension), and seizures may be observed. a urine drug screening test can be used to detect the presence of mdma. treatment of ecstasy intoxication is largely supportive, as there is no known antidote. administer intravenous fluids to maintain hydration, correct acid-base status, and treat hyperthermia. serotonin antagonist drugs (cyproheptadine) can be dissolved and administered per rectum to alleviate clinical signs. intravenous propranolol has additional antiserotonin effects. administer diazepam ( . - mg/kg iv) to control seizures. if cerebral edema is suspected, administer mannitol, followed by furosemide. ethylene glycol is most commonly found in antifreeze solutions but is also in some paints, photography developer solutions, and windshield wiper fluid. ethylene glycol in itself is only minimally toxic. however, when it is metabolized to glycolate, glyoxal, glyoxylate, and oxalate, the metabolites cause an increased anion gap metabolic acidosis and precipitation of calcium oxalate crystals in the renal tubules, renal failure, and (ultimately) death.the toxic dose in dogs is . ml/kg, and in cats is . ml/kg. the toxin is absorbed quite readily from the gastrointestinal tract and can be detected in the patient's serum within an hour of ingestion. colorimetric tests that can be performed in most veterinary hospitals can detect larger quantities of ethylene glycol in the patient's serum. in a dog with clinical treatment begin treatment of known ethylene glycol ingestion immediately. induce emesis or perform orogastric lavage and adminiser repeated doses of activated charcoal. place an intravenous catheter and perform crystalloid fluid diuresis with a known antidote. the treatment of choice for dogs is administration of -methylpyrrazole ( -mp), which directly inhibits alcohol dehydrogenase, thus preventing the conversion of ethylene glycol to its toxic metabolites. the dose for dogs is mg/kg initially, followed by mg/kg at and hours and mg/kg at hours. -mp has been used experimentally at . times the recommended dose for dogs. in cats, treatment with -mp is effective if it is administered within the first hours of ingestion.cats will demonstrate signs of sedation and hypothermia with this treatment. if -mp is not available, administer ethanol ( mg/kg iv loading dose, followed by mg/ kg/hour), or as a % solution (for dogs, . ml/kg iv q h for five treatments, then q h for five more treatments; for cats, ml/kg q h for four treatments). grain alcohol ( proof) contains approximately mg/ml of ethanol. antiemetics and gastroprotective agents should be considered. urinary alkalinization and peritoneal dialysis may enhance the elimination of ethylene glycol and its metabolites. many fertilizers are on the market, and may be composed of urea or ammonium salts, phosphates, nitrates, potash, and metal salts. fertilizers have a moderate toxic potential, depending on the type and amount ingested. clinical signs of fertilizer ingestion include vomiting, diarrhea, metabolic acidosis, and diuresis. nitrates or nitrites can cause formation of methemoglobin and chocolate-brown blood. electrolyte disturbances include hyperkalemia, hyperphosphatemia, hyperammonemia, and hyperosmolality. treatment of fertilizer ingestion includes cardiovascular support, and administration of milk or a mixture of egg whites and water, followed by induction of emesis or orogastric lavage. correct electrolyte abnormalities as they occur (see section on hyperkalemia). administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to control hydration and maintain blood pressure. n-acetylcysteine may be useful if methemoglobinemia is present. fipronil is the active ingredient in frontline, a flea control product. fipronil exerts its effects by gaba antagonism and can cause cns excitation. treatment of fiprinol toxicity includes treatment of cns excitation, treatment of hyperthermia by cooling measures, and administration of activated charcoal. fire extinguisher fluid contains chlorobromomethane or methyl bromide, both of which have a serious toxic potential. dermal or ocular irritation can occur. if ingested, the compounds can be converted to methanol, and cause high anion gap metabolic acidosis, cns excitation and depression, aspiration pneumonitis, and hepatorenal damage. to treat ocular or dermal exposure to fire extinguisher fluids, flush the eyes or skin with warmed tap water or . % saline solution for a minimum of minutes. do not induce emesis or perform orogastric lavage to treat ingestion, because of the risk of causing severe aspiration pneumonitis. gastroprotectant and antiemetic drugs may be used, if indicated. administer intravenous fluids to maintain hydration and renal perfusion. supplemental oxygen or mechanical ventilation may be required in severe cases of aspiration pneumonitis. fireplace colors contain salts of heavy metals-namely, copper rubidium, cesium, lead, arsenic, antimony, barium, selenium, and zinc, all of which have moderate toxic potential, depending on the amount ingested and the size of the patient. clinical signs are largely associated with gastrointestinal irritation (vomiting, diarrhea, anorexia). zinc toxicity can cause intravascular hemolysis and hepatorenal damage. to treat ingestion of fireplace colors, administer cathartics and activated charcoal and gastroprotectant and antiemetic drugs. place an intravenous catheter for intravenous crystalloid fluid administration to maintain hydration and renal perfusion. specific chelating agents may be useful in hastening elimination of the heavy metals. fireworks contain oxidizing agents (nitrates and chlorates) and metals (mercury, copper, strontium, barium, and phosphorus). ingestion of fireworks can cause hemorrhagic gastroenteritis and methemoglobinemia. to treat firework ingestion, induce emesis or perform orogastric lavage and administer activated charcoal. administer specific chelating drugs if the amount and type of metal are known, and administer gastroprotectant and antiemetic drugs. if methemoglobinemia occurs, administer n-acetylcysteine; a blood transfusion may be necessary. introduction fuels such as barbecue lighter fluid, gasoline, kerosene, and oils (mineral, fuel, lubricating) are petroleum distillate products that have a low toxic potential if ingested but can cause severe aspiration pneumonitis if as little as ml is inhaled into the tracheobronchial tree. cns depression, mucosal damage, hepatorenal insufficiency, seizures, and corneal irritation can occur. if fuels are ingested, administer gastroprotectant and antiemetics drugs. do not induce emesis or perform orogastric lavage, because of the risk of aspiration pneumonia. to treat topical exposure, rinse the skin and eyes copiously with warm tap water or . % saline solution. administer antiemetic and gastroprotectant drugs, as necessary. administer intravenous fluids to maintain hydration and treat acid-base and electrolyte abnormalities. children's glue contains polyvinyl acetate, which has a very low toxic potential. if inhaled, the compound can cause pneumonitis. treatment of polyvinyl acetate should be performed as clinical signs of pneumonitis (increased respiratory effort, cough, lethargy, respiratory distress) occur. introduction superglue contains methyl- -cyanoacrylate, a compound that can cause severe dermal irritation on contact. do not induce emesis. do not bathe the animal, and do not apply other compounds (acetone, turpentine) in an attempt to remove the glue from the skin. the fur can be shaved, using care to avoid damaging the underlying skin. the affected area should be allowed to exfoliate naturally. glyophosate is a herbicide found in roundup and kleenup. if applied properly, the product has a very low toxic potential. clinical signs of toxicity include dermal and gastric irritation, including dermal erythema, anorexia, and vomiting. cns depression can occur. treatment includes thorough bathing in cases of dermal exposure, and induction of emesis or orogastric lavage followed by administration of activated charcoal. administer antiemetic and gastroprotectant drugs as necessary. administer intravenous crystalloid fluids to prevent dehydration secondary to vomiting. even small amounts of grapes and raisins can be toxic to dogs. the mechanism of toxicity remains unknown. clinical signs occur within hours of ingestion of raisins or grapes, and include vomiting, anorexia, lethargy, and diarrhea (often with visible raisins or grapes in the fecal matter). within hours, dogs demonstrate signs of acute renal failure (polyuria, polydipsia, vomiting) that can progress to anuria. to treat known ingestion of raisins or grapes, induce emesis or perform orogastric lavage, followed by repeated doses of activated charcoal. if clinical signs of vomiting and diarrhea are present, administer intravenous fluids and monitor urine output. aggressive intravenous fluid therapy, in conjunction with maintenance of renal perfusion, is necessary. in cases of anuric renal failure, dopamine, furosemide, and mannitol can be useful in increasing urine output. peritoneal or hemodialysis may be necessary in cases of severe oliguric or anuric renal failure. calcium channel blockers such as amlodipine and diltiazem can be used to treat systemic hypertension. supportive care includes treatment of hyperkalemia, and administration of gastroprotectant and antiemetic drugs and (if the animal is eating) phosphate binders. aromatic hydrocarbons include phenols, cresols, toluene, and naphthalene. all have a moderate toxic potential if ingested. toxicities associated with ingestion of aromatic hydrocarbons include cns depression, hepatorenal damage, muscle tremors, pneumonia, methemoglobinemia, and intravascular hemolysis. if an aromatic hydrocarbon is ingested, do not induce emesis, because of the risk of aspiration pneumonia. a dilute milk solution or water can be administered to dilute the compound. perform orogastric lavage. carefully monitor the patient's respiratory and cardiovascular status. administer supplemental oxygen if aspiration pneumonia is present. to treat topical exposure, thoroughly rinse the eyes and skin with copious amounts of warm tap water or . % saline solution. imidacloprid is the compound used in the flea product advantage. clinical signs of toxicity are related to nicotinic cholinergic stimulation, causing neuromuscular excitation followed by collapse. the compound may induce respiratory paralysis. to treat imidacloprid toxicity, procure and maintain a patent airway with supplemental oxygen administration. control cns excitation with diazepam, phenobarbital, or propofol. administer enemas to hasten gastrointestinal elimination, and administer activated charcoal. bathe the animal thoroughly to prevent further dermal absorption. closely monitor the patient's oxygenation and ventilation status. if severe hypoventilation or respiratory paralysis occurs, initiate mechanical ventilation. iron and iron salts can cause severe gastroenteritis, myocardial toxicity, and hepatic damage if high enough doses are ingested. lawn fertilizers are a common source of iron salts. treatment of ingestion of iron and iron salts includes cardiovascular support in the form of intravenous fluids and antiarrhythmic drugs, as needed. induce emesis or perform orogastric lavage for gastric decontamination. a cathartic can be administered to promote elimination from the gastrointestinal tract. antiemetic and gastroprotectant drugs should be administered to prevent nausea and vomiting. in some cases, radiographs can aid in making a diagnosis of whether the compound was actually ingested. iron toxicity can be treated with the chelating agent deferoxamine. ivermectin is a gaba agonist that is used in commercial heartworm prevention and antihelminthic compounds and can be toxic in predisposed breeds, including collies, collie loperamide is an opioid derivative that is used to treat diarrhea. clinical signs of loperamide intoxication include constipation, ataxia, nausea, and sedation. induce emesis or perform orogastric lavage, followed by administration of activated charcoal and a cathartic. naloxone may be beneficial in the temporary reversal of ataxia and sedation. ingestion of macadamia nuts can cause clinical signs of vomiting, ataxia, and ascending paralysis in dogs. the toxic principle in macadamia nuts is unknown. there is no known antidote. treatment consists of supportive care, including administration of intravenous fluids and antiemetics and placement of a urinary catheter for patient cleanliness. clinical signs resolve in most cases within hours. marijuana is a hallucinogen that can cause cns depression, ataxia, mydriasis, increased sensitivity to motion or sound, salivation, and tremors. along with these findings, a classic clinical sign is the sudden onset of dribbling urine. urine can be tested with drug test kits for tetrahydrocannabinoid (thc), the toxic compound in marijuana. there is no known antidote for marijuana toxicity; therefore, treatment is largely symptomatic. place an intravenous catheter and administer intravenous fluids to support hydration. administer atropine if severe bradycardia exists. induction of emesis can be attempted but because of the antiemetic effects of thc, is usually unsuccessful. orogastric lavage can be performed, followed by repeated doses of activated charcoal. clinical signs usually resolve within to hours. introduction "strike anywhere" matches, safety matches, and the striking surface of matchbook covers contain iron phosphorus or potassium chlorate. both compounds have a low toxic potential but can cause clinical signs of gastroenteritis and methemoglobinemia if large quantities are ingested. treatment of match and matchbook ingestion includes gastric decontamination with induction of emesis or orogastric lavage and administration of activated charcoal and a cathartic. if methemoglobinemia occurs, administer n-acetylcysteine, intravenous fluids, and supplemental oxygen. metaldehyde is the active ingredient in most brands of snail bait. the exact mechanism of toxicity is unknown but may involve inhibition of gaba channels. clinical signs associated with metaldehyde toxicity include severe muscle tremors, cns excitation, and treatment treatment of mushroom toxicity is largely supportive. if the mushroom was ingested within the last hours, induce emesis or perform orogastric lavage and then administer activated charcoal. symptomatic treatment includes intravenous fluids to promote diuresis and treat hyperthermia and skeletal muscle relaxants to control tremors and seizures (methocarbamol, diazepam). if amanita ingestion is suspected, administer hepatoprotectant agents including milk thistle. mycotoxins from penicillium spp. are found in moldy foods, cream cheese, and nuts. clinical signs of intoxication include tremors, agitation, hyperesthesia, and seizures. if tremorigenic mycotoxin toxicity is suspected, a sample of the patient's serum and gastric contents or vomitus can be submitted to the michigan state university veterinary toxicology laboratory for tremorigen assay. there is no known antidote. perform orogastric lavage, followed by administration of activated charcoal. control tremors and seizures with methocarbamol, diazepam, phenobarbital, or pentobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. in cases in which cerebral edema is suspected secondary to severe refractory seizures, administer intravenous mannitol and furosemide. naphthalene is the active ingredient in mothballs and has a high toxic potential. clinical signs associated with naphthalene toxicity include vomiting, methemoglobinemia, cns stimulation, seizures, and hepatic toxicity. a complete blood count often reveals heinz bodies and anemia. do not induce emesis if naphthalene ingestion is suspected. if the ingestion was within hour of presentation, perform orogastric lavage. control seizures with diazepam or phenobarbital. administer intravenous fluids to control hyperthermia and maintain hydration. n-acetylcysteine can play a role in the treatment of methemoglobinemia. a packed rbc transfusion may be necessary if anemia is severe. observe the patient for clinical signs associated with hepatitis. nicotine toxicity occurs in animals as the result of ingestion of cigarettes, nicotine-containing gum, and some insecticides. nicotine stimulates autonomic ganglia at low doses, and blocks autonomic ganglia and the neuromuscular junction at high doses. absorption after ingestion is rapid. clinical signs include hyperexcitability and slud (salivation, lacrimation, urination, and defecation). muscle tremors, respiratory muscle fatigue or hypoventilation, tachyarrhythmias, seizures, coma, and death can occur. if the patient presents within hour of ingestion and has no clinical signs, induce emesis, followed by administration of repeated doses of activated charcoal. in patients with clinical signs of toxicity, perform orogastric lavage. administer intravenous fluids to maintain hydration and promote diuresis, and treat hyperthermia. administer atropine to treat cholinergic symptoms. urinary acidification can promote nicotine excretion. nonsteroidal antiinflammatory drugs (nsaids) include ibuprofen, ketoprofen, carprofen, diclofenac, naproxen, celecoxib, valdecoxib, rofecoxib, and deracoxib. nsaids cause inhibition of prostaglandin synthesis, leading to gastrointestinal ulceration, renal failure and hepatotoxicity. ibuprofen toxicity has been associated with seizures in dogs, cats, and ferrets. the toxic dose varies with the specific compound ingested. to treat nsaid toxicity, induce emesis or perform orogastric lavage, followed by administration of multiple repeated doses of activated charcoal. place an intravenous catheter for crystalloid fluid diuresis to maintain renal perfusion. administer the synthetic prostaglandin analogue misoprostol to help maintain gastric and renal perfusion. control seizures, if present, with intravenous diazepam. administer gastroprotectant and antiemetic drugs to control vomiting and gastrointestinal hemorrhage. continue intravenous fluid diuresis for a minimum of hours, with frequent monitoring of the patient's bun and creatinine. when the bun and creatinine levels are normal or have plateaued for hours, slowly decrease fluid diuresis % per day until maintenance levels are restored. onions, garlic, and chives contain sulfoxide compounds that can cause oxidative damage of rbcs, leading to heinz body anemia, methemoglobinemia, and intravascular hemolysis. clinical signs of toxicity include weakness, lethargy, tachypnea, tachycardia, and pale mucous membranes. vomiting and diarrhea can occur. intravascular hemolysis can cause treatment treatment of onion, chive, and garlic toxicity includes administration of intravenous fluid diuresis, and induction of emesis or orogastric lavage, followed by administration of activated charcoal and a cathartic. in cases of severe anemia, packed rbc transfusion or administration of a hemoglobin-based oxygen carrier should be considered. opiate drugs include heroin, morphine, oxymorphone, fentanyl, meperidine, and codeine. opiate compounds bind to specific opioid receptors throughout the body and produce clinical signs of miosis or mydriasis (cats), and cns excitation, followed by ataxia and cns depression, leading to stupor and coma. hypoventilation, bradycardia, hypoxia, and cyanosis can occur. to treat known overdose or ingestion of an opiate compound, induce emesis (in asymptomatic animals) or perform orogastric lavage, followed by administration of activated charcoal. administer intravenous fluids and supplemental oxygen to support the cardiovascular and respiratory systems. mechanical ventilation may be necessary until hypoventilation resolves. administer repeated doses of naloxone as a specific antidote to reverse clinical signs of narcosis and hypoventilation. if seizures are present (meperidine toxicity), administer diazepam. organophosphate compounds traditionally are used in flea control products and insecticides. common examples of organophosphates include chlorpyrifos, coumaphos, diazinon, dichlorvos, and malathion. the toxic dose varies, depending on the particular compound and individual animal sensitivity. organophosphate toxicity causes acetylcholinesterase inhibition, resulting in clinical signs of cns stimulation, including tremors and seizures. muscarinic acetylcholine overload causes the classic slud signs of salivation, lacrimation, urination, and defecation. miosis, excessive bronchial secretions, muscle tremors, and respiratory paralysis can occur. an intermediate syndrome of generalized weakness, hypoventilation, and eventual paralysis with ventral cervical ventroflexion that may require mechanical ventilation has been described. if organophosphate toxicity is suspected, whole-blood acetylcholinesterase activity can be measured and will be low. treatment of toxicity includes careful and thorough bathing in cases of dermal exposure and, if the substance was ingested, gastric decontamination with induction of emesis or orogastric lavage, followed by administration of activated charcoal, and administration of the antidote pralidoxime hydrochloride . atropine can help control the muscarinic clinical signs. supportive care in the form of cooling measures, intravenous crystalloid fluids, and supplemental oxygen or mechanical ventilation may be required, depending on the severity of clinical signs. introduction ingestion of large amounts of paintballs can cause neurologic signs, electrolyte abnormalities, and occasionally death. paintballs are gelatin capsules that contain multiple colors of if ingestion was recent and if no clinical signs of toxicity are present, induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal. there is no known antidote. treatment includes supportive care in the form of intravenous fluids and administration of phenobarbital or methocarbamol to control seizures and tremors. diazepam, a gaba agonist, is contraindicated, because it can potentially worsen clinical signs. urine acidification may hasten elimination. clinical signs can last from to days. pyrethrin and pyrethroid compounds are extracted from chrysanthemums, and include allethrin, decamethrin, tralomethrin, fenpropanthrin, pallethrin, sumethrin, permethrin, tetramethrin, cyfluthrin, and resemethrin. the oral toxicity is fairly low; however, the compounds can be significantly harmful if inhaled or applied to the skin. pyrethrin and pyrethroid compounds cause depolarization and blockade of nerve membrane potentials, causing clinical signs of tremors, seizures, respiratory distress, and paralysis. contact dermatitis can occur. to distinguish between pyrethrin/pyrethroid toxicity and organophosphate toxicity, acetylcholinesterase levels should be obtained; they will be normal if pyrethrins are the cause of the animal's clinical signs. treatment of toxicity is supportive, as there is no known antidote. carefully bathe the animal in lukewarm water to prevent further oral and dermal exposure. both hyperthermia and hypothermia can worsen clinical signs. administer activated charcoal to decrease enterohepatic recirculation. atropine may control clinical signs of excessive salivation. to control muscle tremors, administer methocarbamol to effect. administer diazepam or phenobarbital to control seizures, as necessary. rotenone is used as a common garden and delousing insecticide. fish and birds are very susceptible to rotenone toxicity. rotenone inhibits mitochondrial electron transport. clinical signs of tissue irritation and hypoglycemia can occur after topical or oral exposure. if the compound is inhaled, cns depression and seizures can occur. to treat toxicity, perform orogastric lavage, followed by administration of a cathartic and activated charcoal. bathe the animal carefully to prevent further dermal exposure and further ingestion. administer diazepam or phenobarbital to control seizures. the prognosis generally is guarded. treatment of ingestion includes dilution with milk, water, or egg whites. perform orogastric lavage, followed by administration of activated charcoal. administer intravenous crystalloid fluids to maintain hydration. administer antiemetic and gastroprotectant drugs to treat gastroenteritis and vomiting.shampoos, nonmedicated: see detergents, nonionic shampoos, selenium sulfide introduction selenium sulfide shampoos (e.g., selsun blue) have a low toxic potential, and primarily cause gastroenteritis. treatment of ingestion includes dilution with water, milk, or egg whites and administration of activated charcoal. carefully and thoroughly rinse the skin and eyes to prevent further exposure. administer antiemetic and gastroprotectant drugs in cases of severe gastroenteritis. zinc-based (zinc pyridinethione) anti-dandruff shampoos have a serious toxic potential if ingested or if ocular exposure occurs. gastrointestinal irritation, retinal detachment, progressive blindness, and exudative chorioretinitis can occur. treatment of ingestion includes gastric decontamination. induce emesis or perform orogastric lavage, followed by administration of a cathartic and activated charcoal.to treat ocular exposure, thoroughly rinse the patient's eyes for a minimum of minutes. carefully monitor the animal for clinical signs of blindness. implement intravenous fluid to maintain hydration and renal perfusion in cases of severe gastroenteritis. silver polish contains the alkali substance sodium carbonate and cyanide salts, and has a serious toxic potential. ingestion results in rapid onset of vomiting and possibly cyanide toxicity. to treat ingestion, monitor and maintain the patient's respiration and cardiovascular status and administer intravenous crystalloid fluids. induce emesis, followed by administration of activated charcoal. administer sodium nitrite or sodium thiosulfate iv for cyanide toxicity. bath soap (bar soap) usually has low toxic potential and causes mild gastroenteritis with vomiting if ingested. to treat ingestion, include dilution with water, administration of intravenous fluids to maintain hydration, and administration of antiemetic and gastroprotectant drugs to treat gastroenteritis. sodium fluoroacetate is a colorless, odorless, tasteless compound that causes uncoupling of oxidative phosphorylation. the toxic dose in dogs and cats is . - . mg/kg. clinical signs of toxicity include cns excitation, seizures, and coma secondary to cerebral edema. the prognosis is guarded. to treat toxicity, procure and maintain a patent airway, monitor and stabilize the cardiovascular status, and control hyperthermia. perform orogastric lavage, followed by administration of activated charcoal. if clinical signs are not present at the time of presentation, induce emesis. administer intravenous fluids and supplemental oxygen, as necessary. strattera (atomoxetine hydrochloride) is a selective norepinephrine reuptake inhibitor used in the treatment of attention deficit hyperactivity disorder (adhd) in humans. peak serum concentrations occur in dogs within to hours of ingestion, with a peak half-life at to hours following ingestion. clinical signs of toxicity include cardiac tachyarrhythmias, hypertension, disorientation, agitation, trembling, tremors, and hyperthermia. treatment of intoxication is largely symptomatic and supportive in nature. first, induce emesis if the patient is conscious and has an intact gag reflex. orogastric lavage can also be performed. administer one dose of activated charcoal to prevent further absorption of the compound from the gastrointestinal tract. identify cardiac dysrhythmias and treat accordingly. control hypertension with sodium nitroprusside or diltiazem as a constant rate infusion. administer acepromazine or chlorpromazine to control agitation. do not use diazepam, because it can potentially worsen clinical signs. administer intravenous fluids to maintain hydration and promote diuresis. strychnine is the active ingredient in pesticides used to control rodents and other vermin. the toxic dose in dogs is . mg/kg, and in cats is mg/kg. strychnine antagonizes spinal inhibitory neurotransmitters and causes severe muscle tremors, muscle rigidity, and seizures. clinical signs are stimulated or exacerbated by noise, touch, light, and sound. mydriasis, hyperthermia, and respiratory paralysis can occur. if strychnine toxicity is suspected, gastric contents should be collected and saved for analysis. if the animal is asymptomatic at the time of presentation, induce emesis. if clinical signs are present, perform orogastric lavage. both emesis and orogastric lavage should be followed by the administration of activated charcoal. administer intravenous crystalloid fluids to support the cardiovascular system, aid in cooling measures, and improve renal diuresis. treat cns stimulation with methocarbamol, diazepam, or phenobarbital. the animal should have cotton packed in its ears to prevent noise stimulation, and should be placed in a quiet, dark room. treatment of ingestion includes dilution with milk of magnesia or water, administration of antiemetic and gastroprotectant drugs, and administration of intravenous crystalloid fluids to maintain hydration. do not induce emesis, because of the risk of causing further esophageal irritation.sunscreen: see zinc and zinc oxide suntan lotion: see shampoos, zinc-based, and alcohols tar: see fuels tea tree oil (melaleuca oil) introduction tea tree (melaleuca) oil is an herbal-origin flea-control product. the toxic principles in tea tree oil are monoterpenes, which produce clinical signs of neuromuscular weakness, and ataxia. treatment of tea tree oil toxicity includes administration of cathartics and activated charcoal to prevent further absorption. carefully bathe the animal to prevent further dermal exposure. tetanus spores from clostridium tetani organisms are ubiquitous in the soil and feces, particularly in barnyards. cases have been reported in dogs after tooth eruption and after abdominal surgeries performed with cold sterilization packs. anaerobic wound infections can contain tetanus spores. the neurotoxin from c. tetani inhibits spinal inhibitory neurons, causing motor neuron excitation. extensor muscle rigidity ("sawhorse stance"), erect ears, and risus sardonicus (a sardonic grin) are characteristic features of tetanus. administer tetanus antitoxin if toxin has not already been bound in the cns. to eliminate the source of the toxin (e.g., abscess), open and debride all wounds. intravenous administration of ampicillin or penicillin g is the treatment of choice for tetanus. supportive care in the form of skeletal muscle relaxants, intravenous fluids and parenteral nutrition, and nursing care to prevent decubitus ulcer formation is required. in extreme cases, mechanical ventilation may be necessary. triazene compounds include atrazine, prometone, and monuron (telvar). the toxic mechanism of triazene compounds is unknown. clinical signs of toxicity include salivation, ataxia, hyporeflexia, contact dermatitis, hepatorenal damage, muscle spasms, respiratory difficulty, and death. treatment of triazene exposure includes cardiovascular and renal support in the form of intravenous crystalloid fluids, inotropic drugs, and antiarrhythmic agents, as necessary. if the exposure is recent, induce emesis. perform orogastric lavage in animals that cannot protect the airway. emesis and orogastric lavage should be followed by the administration of activated charcoal and a cathartic. carefully bathe the patient to prevent further dermal absorption. a variety of tricyclic antidepressants are available for use in both humans and animals, including amitriptyline, amoxapine, desipramine, doxepine, fluoxetine (prozac), fluvoxamine (luvox), imipramine, nortriptyline, paroxetine (paxil), protriptyline, sertraline (zoloft), and trimipramine. selective serotonin reuptake inhibitors (ssris) are rapidly absorbed from the digestive tract, with peak serum concentrations occurring to hours after ingestion. the elimination half-life for each drug differs in dogs, but typically last to hours. ssris inhibit the reuptake of serotonin, causing serotonin to accumulate in the brain. this can cause "serotonin syndrome," characterized by trembling, seizures, hyperthermia, ptyalism or hypersalivation, cramping or abdominal pain, vomiting, and diarrhea. other clinical signs of ssri intoxication include depression, tremors, bradycardia, tachyarrhythmias, and anorexia. any animal that has ingested an ssri should be promptly treated and carefully observed for at least hours for side effects. the treatment of suspected ssri intoxication involves gastric decontamination if the patient is not depressed and has an intact gag reflex. perform orogastric lavage and administer activated charcoal to prevent further toxin absorption and hasten elimination from the gastrointestinal tract. treat other clinical signs symptomatically. administer intravenous diazepam to control seizures. treat tachyarrhythmias according to type. administer methocarbamol to control muscle tremors. cyproheptadine ( mg/kg), a serotonin antagonist, can be dissolved in water and administered per rectum. vitamin k antagonist rodenticides, which are commonly found in pelleted or block form, inhibit the activation of the vitamin k-dependent coagulation factors ii, vii, ix, and x. clinical signs of hemorrhage occur within to days of exposure. hemorrhage can occur anywhere in the body, and can be manifested as petechiation of the skin or mucous membranes, hemorrhagic sclera, epistaxis, pulmonary parenchymal or pleural hemorrhage, gastrointestinal hemorrhage, pericardial hemorrhage, hematuria, retroperitoneal hemorrhage, hemarthrosis, and central nervous system hemorrhage. clinical signs include respiratory distress, cough, bleeding from the gums or into the eyes, ataxia, paresis, paralysis, seizures, hematuria, joint swelling, lameness, lethargy, weakness, inappetence, and collapse.diagnosis is made based on clinical signs and a prolonged activated clotting time, or prothrombin time. the pivka (proteins induced by vitamin k antagonism) test may be helpful but usually cannot be performed in-house. slight thrombocytopenia may be present secondary to hemorrhage; however, blood levels usually do not reach the critical level of < , platelets/µl to cause clinical signs of hemorrhage. in some cases, severe stressinduced hyperglycemia and glucosuria may be present but resolves within hours. if the rodenticide was ingested within the last hours, induce emesis. alternatively, orogastric lavage can be performed in an uncooperative patient. both emesis and orogastric lavage should be followed by administration of activated charcoal. the stomach contents can be submitted for analysis. following successful treatment, administer oral vitamin k for days after the exposure; or a check prothrombin time days after gastric decontamination. if the prothrombin time is prolonged, administer fresh frozen plasma and vitamin k.if the prothrombin time is normal, gastric decontamination was successful, and no further treatment is necessary.if an animal presents with clinical signs of intoxication, administer activated clotting factors in the form of fresh frozen plasma ( ml/kg), and vitamin k ( mg/kg sq in multiple sites with a -gauge needle). packed rbcs or fresh whole blood may be required if the patient is also anemic. supportive care in the form of supplemental oxygen may be necessary in cases of pulmonary or pleural hemorrhage. following initial therapy and discharge, the patient should receive vitamin k ( . mg/kg po q - h for days), and prothrombin time should be checked days after the last vitamin k capsule is administered. in some cases, depending on the type of anticoagulant ingested, an additional weeks of vitamin k therapy may be required. xylitol is a sugar alcohol that, when ingested by humans, does not cause a significant increase in blood glucose, and therefore does not stimulate insulin release from the human pancreas. in dogs, however, xylitol causes a massive rapid and dose-dependent release of insulin from pancreatic beta-cells. following insulin release, clinically significant hypoglycemia can develop, followed by signs of vomiting, weakness, ataxia, mental depression, hypokalemia, hypoglycemic seizures, and coma. clinical signs associated with xylitol ingestion can be seen within minutes of ingestion and can last for more than hours, even with aggressive treatment. known xylitol ingestion should be treated as for other toxin ingestion. if no neurologic abnormalities exist at the time the patient is seen, induce emesis, followed by administration of activated charcoal. it remains unknown at this time whether activated charcoal actually delays or prevents the absorption of xylitol from the canine gastrointestinal tract. if clinical signs have already developed, perform orogastric lavage and gastric decontamination. blood glucose concentrations should be analyzed and maintained with supplemental dextrose as a constant rate infusion ( . %- %) until normoglycemia can be maintained with multiple frequent small meals. hypokalemia may develop because it is driven intracellularly by the actions of insulin. treat hypokalemia with supplemental potassium chloride by infusion, not to exceed . meq/kg/hour. pennies minted in the u.s. after contain large amounts of zinc rather than copper. other sources of zinc include zinc oxide ointment and hardware such as that found in metal bird cages. zinc toxicity causes intravascular hemolysis, anemia, gastroenteritis, and renal failure. if zinc toxicity is suspected, take an abdominal radiograph to document the presence of the metal in the stomach or intestines. (if zinc-containing ointment was ingested, this will not be visible on radiographs.) induce emesis or perform orogastric lavage, depending on the size of the object ingested. often, small objects such as pennies can be retrieved using endoscopy or surgical gastrotomy/enterotomy. always take an additional radiograph after the removal procedure to ensure that all objects have been successfully removed. administer intravenous fluids to maintain renal perfusion and promote fluid diuresis. administer gastroprotectant and antiemetic drugs. chelation therapy with succimer, calcium edta, dimercaprol, or penicillamine may be necessary. do not administer pulmonary contusions are a common sequela of blunt traumatic injury. a contusion basically is a bruise characterized by edema, hemorrhage, and vascular injury. contusions may be present at the time of presentation or can develop over the first hours after injury. a diagnosis of pulmonary contusion can be made based on auscultation of pulmonary crackles, presence of respiratory distress, and the presence of patchy interstitial to alveolar infiltrates on thoracic radiographs. radiographic signs can lag behind the development of clinical signs of respiratory distress and hypoxemia by hours. in most cases, cage rest is sufficient to temporarily diminish blood loss. sedation (acepromazine, . - . mg/kg iv, im, sq) may be helpful in alleviating anxiety and decreasing blood pressure. the hypotensive effects of acepromazine are potentially harmful if severe blood loss has occurred. if evidence of hypovolemia is present (see section on hypovolemic shock), intravenous fluid resuscitation should be administered. rapid assessment of clotting ability, with a platelet count estimate and clotting profile (act or aptt and pt), should be performed. if epistaxis secondary to vitamin k antagonist rodenticide intoxication is suspected, administer vitamin k and fresh frozen plasma or fresh whole blood.persistent hemorrhage from a nasal disorder can be treated with dilute epinephrine ( : , ) into the nasal cavity with the nose pointed toward the ceiling to promote vasoconstriction. if this fails, the animal can be anesthetized, and the nasal cavity packed with gauze, and the caudal oropharynx and external nares covered with umbilical tape to control hemorrhage. a rhinoscopy should be performed to determine the cause of ongoing hemorrhage. continued excessive hemorrhage can be controlled with ligation of the carotid artery on the side of the hemorrhage, or with percutaneous arterial embolization. systemic thromboembolism is most commonly recognized in cats with cardiomyopathies (hypertrophic, restrictive, unclassified, and dilatative) but can also occur in dogs with hyperadrenocorticism, disseminated intravascular coagulation (dic), systemic inflammatory response syndrome (sirs), protein-losing enteropathy and nephropathy, and tumors affecting the aorta and vena cava. thrombosis occurs through a complex series of mechanisms when the components of virchow's triad (hypercoaguable state, sluggish blood flow, and vascular endothelial injury or damage) are present. in cats, blood flow through a severely stretched left atrium is a predisposing factor to the development of clots and thromboembolism.the most common site of embolism is the aortic bifurcation, or "saddle thrombus." other, less common locations of thromboembolism include the forelimbs, kidneys, gastrointestinal tract, and cerebrum. diagnosis usually is made based on clinical signs of cool extremities, the presence of a cardiac murmur or gallop rhythm, auscultation of pulmonary crackles resulting from pulmonary edema, acute pain or paralysis of one or more peripheral extremities, respiratory distress, and pain and lack of a palpable pulse in affected limbs. the affected nailbeds and paw pads are cyanotic, and nails do not bleed when cut with a nail clipper.client education is one of the most important aspects of emergency management of the patient with thromboembolic disease. concurrent congestive heart failure (chf) occurs in % to % of cats with arterial thromboembolism. more than % of cats are euthanized during the initial thromboembolic event because of the poor long-term prognosis and the high risk of recurrence within days to months after the initial event, even with aggressive therapy. although the long-term prognosis varies from months to years after initial diagnosis and treatment, in the majority of cats thromboembolic disease recurs within months. rectal temperature hypothermia and bradycardia on presentation are negative prognostic indicators.immediate treatment of a patient with chf and thromboembolic disease involves management of the chf with furosemide, oxygen, and vasodilators (nitroglycerine paste, morphine, nitroprusside). additional management includes analgesia (butorphanol, . - . mg/kg iv, im) and prevention of further clot formation. aspirin ( mg/kg po q h) is beneficial bcause of its antiplatelet effects. heparin works in conjunction with antithrombin to prevent further clot formation ( - units/kg iv, followed by - units/kg sq q h in cats, and - units/kg sq q h in dogs). acepromazine can cause peripheral vasodilation and decreased afterload but also can promote hypotension in a patient with concurrent chf. acepromazine ( . - . mg/kg sq) should be used with extreme caution, if at all.thrombolytic therapy can also be attempted, but in most cases is not without risk, and may be cost-prohibitive for many clients. streptokinase ( , units iv over minutes and then , units/hour iv cri for hours) was administered with some success in cats; however, many died of hyperkalemia or other complications during the infusion. tissue plasminogen activator ( . - mg /kg/hour iv cri, up to mg/kg total dose, to effect) has been used with some success but is cost-prohibitive for most clients. side effects of thrombolytic therapy include hyperkalemia with reperfusion and hemorrhage.in cats, the primary cause of arterial thromboembolism is cardiomyopathy. once an animal is determined to be stable enough for diagnostic procedures, lateral and dv thoracic radiographs and an echocardiogram should be performed. ultrasound of the distal aorta and renal arteries should also be performed to determine the location of the clot and help establish the prognosis.other diagnostic procedures to evaluate the presence and cause of thromboembolism include a complete blood count, serum biochemistry profile, urinalysis (to rule out proteinlosing nephropathy), urine protein:creatinine ratio, antithrombin levels, acth stimulation test (to rule out hyperadrenocorticism), heartworm antigen test (in dogs), thyroid profile (to rule out hyperthyroidism in cats, and hypothyroidism in dogs), thoracic radiographs, arterial blood gas analyses, coagulation tests, and coombs' test. selective and nonselective angiography can also be performed to determine the exact location of the thrombus.long-term management of thromboembolism involves management of the underlying disease process and preventing further clot formation. begin therapy with heparin until the aptt becomes prolonged . times; then administer warfarin ( . - . mg/kg/day). monitoring therapy based on prothrombin time and the international normalized ratio (inr, . - . ) is recommended. low-dose aspirin ( - mg/kg q h) also has been recommended. physical therapy with warm water bathing, deep muscle massage, and passive range-of-motion exercises should be performed until the patient regains motor function. future therapy may involve the use of platelet receptor antagonists to prevent platelet activation and adhesion.