key: cord-0005304-1ji2opv0
authors: Kaltenboeck, B.; Hehnen, H. -R.; Vaglenov, A.
title: Bovine Chlamydophila spp. infection: Do we underestimate the impact on fertility?
date: 2005
journal: Vet Res Commun
DOI: 10.1007/s11259-005-0832-4
sha: 491f759d13c08224d276b7da39e22fec35aa10ed
doc_id: 5304
cord_uid: 1ji2opv0

Classical methods for detection of Chlamydophila species, and of antibodies against these agents, have indicated that these bacteria are highly prevalent in cattle and associated with numerous disease conditions. These methods demonstrated acute Chlamydophila-induced diseases such as epizootic bovine abortion, as well as worldwide variable, but generally high, Chlamydophila seroprevalence. However, it was impossible to consistently detect the low levels of these organisms which were suspected to be present in endemic infections. Application of highly sensitive real-time PCR and ELISA methods for detection of Chlamydophila spp. DNA and of antibodies against Chlamydophila spp., respectively, in a series of prospective cohort studies revealed a high prevalence of Chlamydophila spp. genital infections in female calves (61%) and adult heifers (53%). These infections were acquired by extragenital transmission in the first weeks of life, and infection frequency was increased by crowding of the animals. A challenge study demonstrated that infection with C. abortus resulted in decreased fertility of heifers. The experimental use of a C. abortus vaccine provided evidence for immunoprotection against C. abortus-induced suppression of bovine fertility. The results of these investigations suggest that bovine Chlamydophila infection should be viewed more as pervasive, low-level infection of cattle than as rare, severe disease. Such infections proceed without apparent disease or with only subtle expressions of disease, but potentially have a large impact on bovine herd health and fertility.

Classical investigations of diseases induced in cattle by intracellular bacteria of the order Chlamydiales have revealed severe, but rare, disease manifestations such as pneumonia, enteritis, polyarthritis, sporadic encephalomyelitis, and abortion and fertility disorders (Shewen, 1980) . Early investigators realized that these sporadic appearances of acute diseases represented only the`tip of the iceberg' of a ubiquitous distribution of subclinical infections with these agents, and that speci¢c circumstances precipitated the clinical manifestation of disease (Storz, 1971 ).

Today's prevailing view of bovine chlamydial infection is that of a curiosity, signi¢cant in rare incidences of abortion and zoonosis, but not in animal health and production. This view has been created by inadequate sensitivity and inconsistent application of standard diagnostic methods such as complement ¢xation test and culture methods. Investigations using modern ELISA and PCR techniques show a very di¡erent picture of chlamydial infections in cattle. High seroprevalence, approaching 100%, and chlamydial genomic DNA prevalence as high as 50^60% has been associated with clinically inapparent bovine infection with Chlamydophila abortus and Chlamydophila pecorum (Cavirani et al., 2001; DeGraves et al., 2003a; Domeika et al., 1994; Jee et al., in press; Kaltenboeck et al., 1997a,b; Wang et al., 2001) . The ubiquitous presence of two chlamydial species in cattle begs a number of questions, most importantly: what is their impact on herd health and production?

The authors have addressed this question in a series of epidemiological and heifer challenge experiments with C. abortus. These experiments demonstrated that previous infection and established immunity against C. abortus did not protect against decreased fertility following direct uterine challenge with C. abortus or following challenge through contact with herd mates. The results also indicated that pregnancy results after uterine C. abortus or mock inoculation were strongly in£uenced by simultaneous cohort challenge, as well as by pre-challenge immunity against C. abortus, expressed as anti-C. abortus IgM serum antibodies. The experimental use of a C. abortus vaccine provided evidence for immunoprotection against C. abortusinduced suppression of bovine fertility (DeGraves et al., 2002) . The results of these investigations suggest that bovine Chlamydophila spp. infections occur mainly without apparent disease, or only with subtle signs of disease. Nevertheless, these infections may impact on herd health and fertility more profoundly than overt clinical chlamydial disease.

Bacteria of the order Chlamydiales are obligate intracellular parasites that are unable to replicate outside of eukaryotic host cells (Storz, 1971) . They are ubiquitous, infect a wide range of vertebrate and invertebrate hosts, and cause a wide variety of diseases (Schachter, 1999) . It is thought that diseases induced by chlamydial organisms are mainly the result of an immune reaction that is ine¡ective at clearing the organisms, but results in collateral tissue damage (Ward, 1999) . Vertebrate isolates typically have low host and cell type speci¢city, but certain chlamydial strains and species tend to associate with speci¢c hosts and diseases (Shewen, 1980; Storz and Kaltenboeck, 1993) .

Chlamydiae were ¢rst associated with disease of cattle (Bos taurus) when McNutt isolated intracellular organisms from cases of sporadic bovine encephalomyelitis in feedlot cattle (McNutt and Waller, 1940) . After chicken embryo and cell culture methods for chlamydiae became widely used around 1955, a number of studies worldwide documented chlamydiae in many acute diseases of cattle. A prominent example is epizootic bovine abortion, similar to classic ovine abortion caused by C. abortus. This disease was found worldwide in many countries including France (Hidiroglou and Prevost, 1959) , Germany (Schoop et al., 1965; Wehner and Wehr, 1980) , India (Nanda et al., 1992) , Japan (Kawakami et al., 1955; Nabeya et al., 1991) , the United Kingdom (Gri¤ths et al., 1995) , and the USA (Storz et al., 1960) . The causal agent also caused epididymitis and seminal vesiculitis and was excreted in bull semen (Storz et al., 1968) . Chlamydial strains from ruminant abortion were identi¢ed as serotype 1 of ruminant chlamydiae (Schachter et al., 1975) , biotype 1 (Spears and Storz, 1979) , immunotype 1 (Perez-Martinez and Storz, 1985) , or ompA type B577 . Recently, reclassi¢cation as Chlamydophila abortus was proposed (Everett et al., 1999) . C. abortus has also been associated with bovine mastitis (Corner et al., 1968; Kaltenboeck et al., 1997b; RÖnsholt and Basse, 1981; Wehnert et al., 1980) . A second chlamydial agent was associated with clinically severe bovine chlamydial disease manifestations including abortion, sporadic bovine encephalomyelitis, pneumonia, enteritis, polyarthritis, kerato-conjunctivitis, nephritis, or purulent endometritis (Bannister et al., 1962; Dyml, 1965; French and Snowdon, 1960; Ko« lbl and Psota, 1968; Romvary, 1964; White, 1965, Wilson and Thompson, 1968; Wittenbrink et al., 1988) . This chlamydial strain was diagnosed as serotype 2 of ruminant chlamydiae (Schachter et al., 1975) , biotype 2 (Spears and Storz, 1979) , immunotype 2 (Perez-Martinez and Storz, 1985) , ompA type LW613 , or C. pecorum (Fukushi and Hirai, 1992) . Recently, reclassi¢cation as Chlamydophila pecorum was proposed (Everett et al., 1999) .

Numerous studies have con¢rmed the disease potential of C. abortus and C. pecorum by experimentally reproducing acute diseases such as those listed above (Bowen et al., 1978; Jones et al., 1998; Storz et al., 1976; Wittenbrink et al., 1993) . Shewen (1980) summarized the status of our understanding of chlamydial infections in animals: Exceptionally, some animals may experience severe or even fatal disease as a result of chlamydial exposure. A well balanced host-parasite relationship represents the common nature of chlamydial infection. This long-lasting inapparent or`latent' state has been documented in several species: birds, cattle, guinea pigs, sheep and humans. Under circumstances of stress,`carrier' animals may shed the organisms in large numbers or may in fact lapse into clinical disease.'

Despite improvements in diagnostic techniques, most notably the introduction of the PCR, our understanding about the prevalence and pathogenetic signi¢cance of these infections has not substantially changed since Shewen's review in 1980. The major impediment has been the cumbersome nature and insensitivity of diagnostic procedures, particularly of the CFT for determination of seroprevalence of chlamydial infection in cattle (Kaltenboeck et al., 1997a; Perez-Martinez et al., 1986) . Only recently, several investigations reported high prevalence of chlamydial infection or of antibodies against chlamydiae in cattle, and linked these data with increased diseases such as abortion, endometritis, and fertility disorders (Cavirani et al., 2001; Domeika et al., 1994; Wang et al., 2001; Wittenbrink et al., 1988) . One of the main reasons that chlamydial infections of cattle are still not widely recognized is simply that insensitive diagnostic techniques give a misleading indication of the prevalence of these infections.

Perez-Martinez and colleagues (1986) demonstrated that the standard CFT for bovine antibodies against chlamydial agents was unable to detect even high antibody levels. Addition of bovine complement was necessary to obtain improved sensitivity because bovine antibodies in essence do not bind guinea pig complement. The lack of sensitivity of the standard CFT is obvious in Figure 1 from research on synthetic antigen ELISAs for chlamydial antigens which compared these assays to the CFT (Kaltenboeck et al., 1997a) . In a random survey of 40 sera from Alabama cattle herds with abortion problems, ELISAs against peptides of the C. abortus major outer membrane protein or against recombinant chlamydial LPS invariably detected very high antibody levels. In fact, immunoglobulin-rich sera from gnotobiotic calves challenged with bovine coronavirus had to be used as negative controls because it was impossible to ¢nd any other Chlamydophila-negative bovine sera. In comparison, the CFT titers of all but one serum sample were negative. The single positive serum had a low titer of 1:10 ( Figure 1 ).

The authors have comparatively examined 317 additional bovine sera with highsensitivity ELISA methods using chemiluminescent substrate and C. abortus and C. pecorum peptide and lysed elementary body antigens, and have failed to detect a single truly anti-Chlamydophila negative serum despite consistently negative reactivity of several control sera from gnotobiotic calves (data not shown). These ELISAs have a wide dynamic range and are highly informative because the resultant luminescence signal is linear over 5 orders of magnitude. The data indicate that signals from all antigens are highly correlated, but agreement between whole organism antigens is better than that between peptide antigens. The serological cross-reactivity between whole organism C. abortus and C. pecorum antigens is 83% (Jee et al., submitted) . In examinations of sera from C. abortus challenged heifers, IgM antibodies provided the best functional correlation between fertility outcome and pre-or post-challenge anti-C. abortus antibodies, or their ratios Jee et al., submitted) .

A major determinant of sensitivity in assays of bovine antibodies against Chlamydophila spp. is the negative control serum. Given the overwhelming seroprevalence of Chlamydophila spp., many sera used as negative controls cannot be truly seronegative. Thus, the vast majority of seroprevalence studies will show variable proportions of false negative data, indicating as negative those chlamydia-positive animals that have serum anti-Chlamydophila antibody levels below the arbitrary cut-o¡ imposed by the control sera. Figure 1 . Chlamydophila spp. ELISA and CFT data obtained from 40 bovine sera from herds with abortion (Kaltenboeck et al., 1997a) . The CFT test used standard Chlamydophila CFT antigen, ELISAs used C. abortus major outer membrane protein (MOMP)-derived peptides from variable domains (VD) 2 or 4, or recombinant chlamydial lipopolysacccharide (LPS). An immunoglobulin-rich serum from a gnotobiotic calf after challenge with bovine coronavirus was used as negative control serum. Animal sera are indicated on the abscissa

The high seroprevalence of Chlamydophila spp. in cattle poses the challenging question why we do not also routinely detect a high prevalence of the agents. The answer might be that standard chicken embryo and cell culture methods for detection are not sensitive enough to detect low amounts of chlamydiae (Huang et al., 2001) . Such low amounts would be expected if chlamydial infections were persistent at the individual host or herd level in an endemic disease situation.

Application of PCR to the direct detection of chlamydial agents is an obvious answer. Following initial reports that PCR detection of chlamydial DNA was possible, speci¢c, and sensitive (Hewinson et al., 1997; Kaltenboeck et al., 1997b) , the limitations of early PCR methods were recognized: (1) nested PCR methods were necessary to maximize sensitivity and speci¢city; and (2) the quantity and stability of the specimen imposes major constraints on the detection limits of the methods (DeGraves et al., 2003b) . Therefore, such PCR methods, while an improvement, are problematic because they lack the robustness needed for routine diagnostic application or large scale research.

The advent of single-tube real-time PCR platforms has removed most limitations to routine diagnostic use of PCR methodology, and has added the bene¢t of quanti¢cation and di¡erentiation of the nucleic acid targets (DeGraves et al., 2003a) . This research group has adapted the LightCycler 1 real-time PCR as a high-throughput platform with extreme sensitivity for routine detection and quanti¢cation of even single DNA targets by modifying assay chemistry (Huang et al., 2001) and nucleic acid extraction, and adopting specimen collection and preservation with guanidinium thiocyanate bu¡er before freezing (DeGraves et al., 2003b) .

Application of these technologies has already demonstrated the surprisingly high prevalence of 53% vaginal infection of virgin heifers with C. abortus and C. pecorum (Table I, DeGraves et al., 2003a) . These data also clearly demonstrate that these genital infections are transmitted by non-venereal means, and that typically very low amounts of Chlamydophila genomes are present. They also show substantial di¡erences between sensitivities of di¡erent PCR methods, as evident in the overall 22% prevalence found by Chlamydophila ompA PCR and the 53% prevalence of Chlamydophila spp. infection found by 23S rRNA PCR in the same extracted specimens. The lower sensitivity of the ompA PCR is related to the longer ampli¢cation fragment as compared the 23S rRNA PCR (287 bp vs. 168 bp) and the higher degree of degeneracy of the ompA primers (DeGraves et al., 2003b) .

Upon investigation of the acquisition of these infections by female calves in serial sampling beginning at birth, it became evident that the calves acquired natural infection with C. abortus and C. pecorum at overall 61% prevalence primarily in the ¢rst 2 months after birth, without showing signs of clinical disease (Table II, Jee et al., in press ). The percentage of Chlamydophila-positive calves and the level of infection showed a highly signi¢cant quadratic, but not linear, increase as the number of calves per weekly group was increased . Thus, crowding greatly increased the prevalence and intensity of Chlamydophila spp. vaginal infections in calves.

The authors conclude that sample collection and preservation, nucleic acid extraction, as well as the type of PCR profoundly in£uence Chlamydophila spp. detection in bovine specimens. Results of epidemiological studies may thus range from low single digit to above 50% prevalence for bovine vaginal Chlamydophila spp. infection. For reliable and comparable data it will be imperative to standardize the overall methodology of PCR testing. The high seroprevalence of chlamydiae in cattle, con¢rmed by chlamydial genomic DNA prevalence data, poses the challenge of determining the health consequences, if any, of these endemic infections. Economic losses caused by late-term C. abortus infection and subsequent epizootic bovine abortion are readily apparent. However, infection may result in unrecognized economic losses as the consequence of subclinical infertility (Bowen et al., 1978) . Bovine infertility associated with C. abortus and C. pecorum infection has been addressed, but not extensively characterized (Jones et al., 1998; Storz et al., 1968 Storz et al., , 1976 Wehner and Wehr, 1980; Wittenbrink et al., 1988 Wittenbrink et al., , 1993 . It is recognized that bulls can shed C. abortus in semen; that breeding bulls which shed C. abortus in semen have decreased fertility; that semen spiked with C. abortus is associated with reduced fertility; and that uterine challenge with C. abortus can cause metritis. As described above, cattle typically experience a ¢rst infection with Chlamydophila spp. at an early age without any clinical signs of disease, and respond by establishing immune responses as evidenced by sustained high antibody levels.

However, it is unclear if infection is cleared from the tissues (Papp et al., 1993 (Papp et al., , 1994 , and what the consequences of persistent or repeated infections are.

To approach this question, the authors investigated the in£uence of subclinical uterine and systemic infection with C. abortus on the fertility of virgin heifers . A herd of 30 Holstein heifers, all highly positive for serum antibodies against C. abortus, was estrus-induced by injection with dinoprost tromethamine. Twenty heifers responding with estrus in the ¢rst challenge round were inseminated with the semen of a bull with high fertility, and simultaneously received an intracervical challenge of 0, 10 4 , 10 5 , 10 6 , or 10 8 inclusion forming units (IFU) of C. abortus type strain B577 (Storz, 1966) via the cervix. Two weeks later, the remaining 10 heifers were injected again, and similarly bred and inoculated in the second challenge round. Pregnancy was determined after 42 days by rectal palpation. Second-round heifers were considered cohort-challenged at the time of insemination by contact with C. abortus-shedding ¢rst-round animals. Pregnancy results indicated that the highest challenge dose abolished fertility, demonstrating that previous infection and established immunity against C. abortus did not protect against chlamydial disease . The data also indicated that pregnancy results at lower uterine challenge were strongly in£uenced by concomitant cohort challenge, as well as by the level of pre-challenge immunity against C. abortus, expressed as pre-challenge anti-C. abortus IgM serum antibodies. Logistic regression modeling predicted fertility of heifers challenged with C. abortus (Figure 2 ). This study demonstrated that subclinical chlamydial infections may potentially have a profound negative e¡ect on bovine fertility, and that these losses may be reduced with appropriate herd management practices.

The risk factors for reduction of bovine fertility identi¢ed in the study may provide insight for the development of viable prophylactic and therapeutic measures. Transmission frequency of Chlamydophila spp may be decreased by reducing group size and population density. Vaccination may also serve as a component of a prophylactic or Figure 2 . Cohort challenge and pre-challenge anti-C. abortus serum IgM levels strongly modulate fertility after uterine challenge with C. abortus. Fertility of challenged heifers (percent animals pregnant) is signi¢cantly predicted in logistic regression models by uterine C. abortus inoculum dose and cohort challenge by C. abortus (A), or by uterine inoculum dose and concentration of IgM against C. abortus (B). The solid line represents fertility in dependence on uterine inoculum under conditions of cohort challenge to C. abortus (A) or under condition of below-median (low) levels of anti-C. abortus IgM (B). The dashed line represents fertility without cohort challenge (A) or with above-median (high) levels of anti-C. abortus IgM (B). These logistic regression models of fertility of heifers with established immunity against C. abortus indicate that (A) with cohort challenge a uterine infection of 10 5.38 IFU of C. abortus is necessary to reduce fertility of heifers from 100% to 50%, as compared to the 8.5-fold higher dose of 10 6.31 IFU required for the same reduction without cohort challenge; and (B) at low pre-challenge anti-C. abortus IgM levels 10 6.01 intrauterine IFU of C. abortus reduce fertility of heifers from 100% to 50%, as compared to the 17-fold higher dose of 10 7.24 IFU required for the same reduction at high pre-challenge anti-C. abortus IgM levels. therapeutic program, by protecting individual animals from disease and by reducing Chlamydophila transmission within the herd.

Several reasons make a vaccine against bovine chlamydial infections desirable: (1) it is likely that vaccination can create a di¡erential in immunity to C. abortus that would aid in evaluating the impact of subclinical chlamydial infection on bovine fertility in controlled trials; (2) prophylaxis or therapy of chlamydial infections by vaccination is highly preferable in production animals as compared to regimens using antibiotics; (3) successful vaccination might substantially improve bovine herd fertility.

Conventional approaches to searching for vaccines against Chlamydia-induced disease have led to whole organism or subunit vaccines with less than optimal responses, including inconsistent protection in animal models (Rank, 1999) and exacerbation of disease in human trachoma ¢eld trials (Ward, 1999) . A leading candidate for a vaccine, the major outer membrane protein (MOMP), has been tested as a genetic vaccine in several host species with variable and inconclusive results (Pal et al., 1999; Tan et al., 1990) . The authors chose expression library immunization (ELI) for identi¢cation of protective vaccine candidate genes (Barry et al., 1995) . ELI is an unbiased whole-genome approach in which pools of expression plasmids containing inserts from the C. abortus genome were tested in a mouse pneumonia model for the ability to confer protection against challenge infection. Pools of protective plasmids were fractionated into smaller pools, until single protective gene candidates were tested (Stemke-Hale et al., in press) . Five gene fragments conferred protection at levels as good or better than the live-vaccine control of mice that had received a low-level respiratory C. abortus inoculation one month prior to the high-dose challenge inoculation. Four of these fragments (dnaX 2 , gatA, gatC, pbp3) encoded peptides of cytosolic proteins; only one (pmp5) encoded a peptide of an outer membrane protein, traditionally considered the best vaccine candidates. The dnaX 2 fragment, encoding a portion of the C. abortus DNA polymerase, completely protected mice from C. abortus disease, better than the best protection achieved with live vaccination by low-level intranasal C. abortus inoculation. (Stemke-Hale et al., in press).

The protective genes and their corresponding proteins were used in a preliminary vaccine trial to evaluate protection against C. abortus (DeGraves et al., 2002) . The ¢ve most protective genes were selected for vaccination of virgin heifers in a C. abortus cohort challenge model. This vaccine was composed of a pool of plasmids that contained the full ORFs of the genes, recoded for mammalian codon usage. A corresponding recombinant protein vaccine against C. abortus was also developed. This vaccine was Alum-Quil A based and contained a¤nity-puri¢ed full-length proteins expressed in E. coli, using the same genes that were incorporated into the genetic vaccine.

A natural Holstein heifer infection model was used in which intracervically inoculated^but not bred^heifers were placed in contact with herd mates that were bred 2 weeks later and were indirectly C. abortus challenged by contact exposure. The Holstein herd had a 53% vaginal Chlamydophila spp. PCR pre-challenge prevalence, without signi¢cant di¡erence between vaccinated and non-vaccinated heifers. The experimental herd mates were used to evaluate the genetic and recombinant protein vaccines, compared to mock vaccinated controls. Twenty-seven heifers were synchronized for estrus and then inoculated with an intrauterine dose of C. abortus. When the challenged heifers were at maximum shedding of C. abortus as determined by real-time PCR of vaginal swabs, estrus was synchronized in experimental heifers (n = 24). Experimental heifers were then monitored for estrus with a computerized estrus detection system and bred by arti¢cial insemination. Pregnancy was determined by rectal palpation at 42 days post breeding. The results shown in Table III suggest protection by both genetic and protein vaccines from C. abortus-induced infertility. The odds ratio for improvement of fertility with vaccination against C. abortus is 4.5 (p = 0.12).

The *32% increase in fertility of vaccinated heifers as compared to the 50% ¢rstservice conception rate of control heifers would represent a substantial improvement in bovine herd health. Thus, immunization with a subunit C. abortus vaccine may hold promise to improve bovine herd fertility. It is important to note that the vaccines did not create a fresh adaptive immune response to C. abortus in naive heifers, but rather enhanced and/or modulated an existing response in animals with Chlamydophila spp. infection. Thus, the vaccines were used as therapeutic, not prophylactic vaccines, and may be more appropriately termed`antigen-speci¢c immune modulators'.

The results presented of Chlamydophila prevalence studies, and of challenge experiments with and without vaccination indicate that (1) C. abortus and C. pecorum are endemic at low levels in cattle and infect virtually 100% of the animals; (2) Chlamydophila spp. infections are acquired early in life by non-sexual transmission;

(3) such infections typically do not result in clinical disease; (4) C. abortus (and possibly C. pecorum) infections at the time of breeding have the potential to substantially reduce fertility in cattle; and (5) enhanced immunity mediated by recent natural infection or by vaccination may protect cattle from suppression of fertility by C. abortus (and possibly C. pecorum). These conclusions suggest that subclinical Chlamydophila spp. infections have the potential to cause substantial production losses in bovine herds due to reduced fertility. A renewed focus towards quantitative prospective studies of subtle disease manifestations is warranted to improve our understanding of the impact of Chlamydophila spp. infections on bovine herd health. These infections may also negatively a¡ect frequencies and intensities of bovine herd health problems such as mastitis, and respiratory and neonatal diseases. Rational application of standardized, highly sensitive PCR and serological methodology will be required for conclusive results. Furthermore, combination C. abortus^C. pecorum vaccines may aid in prevention of losses by these infections, but also serve together with antibiotics as`perturbation' tools in prospective intervention studies on the impact of Chlamydophila spp. infections on bovine herd health.

Sporadic bovine encephalomyelitis in Canada

Protection against mycoplasma infection using expression-library immunization

Mechanisms of infertility in genital tract infections due to Chlamydia psittaci transmitted through contaminated semen

Association between Chlamydia psittaci seropositivity and abortion in Italian dairy cows

A histological study on the e¡ects of enzootic abortion of ewes virus in the lactating bovine mammary gland

Quantitative detection of Chlamydia psittaci and C. pecorum by high-sensitivity real-time PCR reveals high prevalence of vaginal infection in cattle

High-sensitivity quantitative PCR platform

Re-infection with Chlamydophila abortus by uterine and indirect cohort routes reduces fertility in cattle pre-exposed to Chlamydophila

Vaccine identi¢ed by in vivo genomic screening enhances fertility in cattle during environmental challenge with Chlamydia

Comparison of polymerase chain reaction, direct immuno£uorescence, cell culture and enzyme immunoassay for the detection of Chlamydia psittaci in bull semen

Isolation of a virus of the psittacosis-lymphogranuloma group from cattle with infectious kerato-conjunctivitis

Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and ¢ve new species, and standards for the identi¢cation of organisms

The occurrence of sporadic bovine encephalomyelitis in Australia

Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants

Epizootic bovine abortion in a dairy herd: characterization of a Chlamydia psittaci isolate and antibody response

Detection of Chlamydia psittaci DNA in avian clinical samples by polymerase chain reaction

Quelques observations sur la sterilite¨des bovins occasionne¨e par un virus du groupe psittacose

Quantitative detection of Chlamydia spp. by £uorescent PCRs in the LightCycler

High prevalence of natural Chlamydophila spp. infection in calves

Correlation between anti-Chlamydophila antibodies and fertility in cattle challenged with Chlamydophila abortus

Experimental infections of the genital tract of cattle with Chlamydia psittaci and Chlamydia pecorum

Use of synthetic antigens improves detection by enzyme-linked immunosorbent assay of antibodies against abortigenic Chlamydia psittaci in ruminants

Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species

Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR

Studies on the disease of cattle caused by a psittacosis-lymphogranuloma group virus (Miyagawanella). VII. Isolation of virus, identi¢ed as a member of the psittacosis-lymphogranuloma group of viruses, from feces of cattle

Miyagawanellen-Isolierungen bei Polyarthritis, Pneumonie, Encephalomyelitis and interstitieller Herdnephritis (Fleckniere) der KÌlber. Wiener TierÌrztliche Monatsschrift

Sporadic bovine encephalomyelitis. The Cornell Veterinarian

Abortion in Japanese cows caused by Chlamydia psittaci

Diagnosis of bovine chlamydial abortion in cattle

Vaccination of mice with DNA plasmids coding for the Chlamydia trachomatis major outer membrane protein elicits an immune response but fails to protect against a genital challenge

Chlamydia psittaci infection and associated infertility in sheep

Abortion and subsequent excretion of chlamydiae from the reproductive tract of sheep during estrus

Antigenic diversity of Chlamydia psittaci of mammalian origin determined by microimmuno£uorescence

Bovine chlamydial abortion: serodiagnosis by modi¢ed complement-¢xation and indirect inclusion £uorescence tests and enzyme-linked immunosorbent assay

Models of immunity

Respiratory disease caused in suckling calves by a PLV virus

Bovine mastitis induced by a common intestinal Chlamydia psittaci strain. A pathogenetic and serological investigation

Infection and disease epidemiology

Serotyping of Chlamydia: II. isolates of bovine origin

Zur Isolierung von Miyagawanellen aus abortierten Rinderfeten

Chlamydial infection in animals: a review

Biotyping of Chlamydia psittaci based on inclusion morphology and response to diethylaminoethyl-dextran and cycloheximide

Screening the whole genome of a pathogen in vivo for protective antigens

Psittacosis-lymphogranuloma infection of sheep. Antigenic structures and interrelations of PL agents associated with polyarthritis, enzootic abortion, intrauterine and latent intestinal infections

Isolation of a psittacosis agent (Chlamydia) from semen and epididymis of bulls with seminal vesiculitis syndrome

Urogenital infection and seminal excretion after inoculation of bulls and rams with chlamydiae

Disease diversity of chlamydial infections

The isolation of a viral agent from epizootic bovine abortion

Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein

Prevalence of Chlamydophila abortus infection in domesticated ruminants in Taiwan

Mechanisms of Chlamydia-induced disease

Genital infections and abortions in cattle caused by chlamydiae

Investigations on the contribution of chlamydiae to a mastitis episode

Experimental production of pneumonia in calves by infection with an organism of the psittacosis-lymphogranuloma group

Chlamydia pneumonia of calves

Investigations into the incidence of Chlamydia psittaci in the genital tract and feces of female cattle

Endometritis in cattle experimentally induced by Chlamydia psittaci

The authors wish to thank for the advice and support of numerous individuals over more than a decade of research on bovine chlamydial infections.