key: cord-0294202-bgk4vofq
authors: Long, Maureen T.
title: Appendix B Laboratories Molecular Testing for Infectious Diseases
date: 2014-12-31
journal: nan
DOI: 10.1016/b978-1-4557-0891-8.00070-1
sha: 9dca036b1130284139b9e19aece16a4db39f4b78
doc_id: 294202
cord_uid: bgk4vofq

Unknown

Since the 1990s, a rapid explosion in molecular-based diagnostics has occurred in veterinary medicine. Contemporaneous with this expansion, training in diagnostic medicine is declining because of loss of state and federal support of veterinary training and lack of interest in veterinary students, who primarily track into clinical programs. 1, 2 Most molecular diagnostic tests presently emphasize the polymerase chain reaction (PCR) format. The most current application is a real-time format; most companies have now developed formats that can perform a 40-cycle PCR test in 35 to 45 minutes in a high-throughput format once nucleic acids are extracted. 3 With robotics, samples can be processed and results obtained within a very short window of time. Regardless of technologic advances that apply, the same basic concepts apply when submitting samples for diagnostic testing that are based on either high-throughput or new technologies. 4, 5 Understanding Older and Newer Formats for Detection of Infection Table 1 lists the basic definitions of PCR methods. A basic understanding of the different PCR test formats is needed for continuing education of practitioners and diplomates. 1 Although it is not necessary for the practitioner to be a molecular biologist, Table 1 provides a short review of the definitions of molecular testing.

The main advantage of genomic strategies for testing is that a live organism is not needed for diagnosis. Although diagnosis based on molecular techniques does not require a live organism, DNA and RNA are subject to the same microbiologic, biochemical, and physical factors as live organisms for degradation. 6 In some situations, genomic DNA may be robust in its survival for forensic purposes; however, overgrowth and chemical contamination of a small microbiologic sample could result in false-negative reactions for any of the aforementioned molecular techniques. A sample collected using the correct media that is solely dedicated for PCR testing should be taken. For blood, ethylenediamine tetraacetic acid (EDTA) or purple top tubes are best. Use of EDTA as an anticoagulant for testing of virus in plasma or buffy coat is applicable for most genomic techniques and viral culture techniques. However, if one wishes to culture plasma (e.g., that of a foal) for bacteria, EDTA is bacteriostatic, and a blood culture bottle must also be collected. Anticoagulants such as heparin can inhibit PCR reactions especially when a kit extraction method is used, which is likely to be the typical formats in most diagnostic laboratories.

For detection of nasal or respiratory pathogens, wooden swabs contain formalin, which can inhibit PCR (fixed tissue methods are performed on paraffin, and formalin has been removed in the paraffin process). Plastic, polypropylene sticks with Dacron or rayon swabs, not those with calcium alginate, are essential for nasal swabbing. 7 Cotton allows bacteria and virus to become embedded in fibers, and frequently the extraction method calls for direct extraction off the swab. This step inactivates most viruses and bacteria because of the detergent used in the first lysis step. Finally, use of a transport medium is best for viruses because this inhibits overgrowth of bacteria that may inactivate and break down nucleic acids needed for the successful detection of viruses. The proteins in the viral medium also assist in keeping the virus in a biologically active state, so this is preferable for viral culture also.

Most testing has been validated on specimens that have been collected under the best conditions without inhibitors and contamination stored appropriately. Specimens requiring storage before shipping to a laboratory should not be stored, and the most optimal time for testing by PCR is less than 3 days in a sample stored consistently at 4°C. If one cannot ship a horse sample within the allotted time, the sample should be allowed to sit in a refrigerator for 20 minutes and then the plasma drawn off and placed in a new tube without any anticoagulants. For horses, the sample should not be centrifuged. The white blood cells of horses settle in this time and do not form a buffy coat that is adhered to red blood cells (RBCs). This allows collection of as few RBCs as possible. 8 This is essential because as RBCs lyse in older samples, heavy protein contamination occurs, and these particular proteins (iron) are toxic to many PCR reactions. Many laboratories historically have indicated shipment at either 4°C or not to exceed 75°C. The latter is not appropriate for blood or nasal specimens because of the potential for hemolysis or bacterial overgrowth, respectively. All samples should be shipped on ice packs (no wet ice) overnight for arrival within 24 hours. One should not ship expecting successful Saturday delivery, and it is our experience that samples become lost more frequently with weekend deliveries.

There is no federal body or international body that consistently provides methodologic standardization for infectious diseases. Several organizations exist to which laboratories may belong or adhere to and can be accredited for quality assurance and control standards; however, these organizations, owing to their mission and funding constraints, usually focus on either Generally, the regulated equine diseases do not have PCR as a trade test; however, PCR methodologies and their recommended reagents are listed in the OIE for several equine tests. As of this writing, the OIE is in the process of adopting PCR for screening of horses for AHS. With adoption of this format as a gold standard, this could revolutionize the South African laboratory maintenance as a form of quality assurance or a subset of diseases for a given set of syndromes. There is a relative lack of standardization in both human and veterinary molecular diagnostic testing, and it is estimated that in herpes simplex virus testing alone, there is a 7.9% false-positive rate and a 14% false-negative rate when proficiency testing is examined. 9

The World Organization for Animal Health (OIE), based in France, is an intergovernmental agency whose main mission is to track contagious animal diseases. 8 The Web site of this organization, http://www.oie.int/eng/en_index.htm, contains some of the most up-to-date information regarding world disease outbreaks in animals. Within the last several years, the focus has included veterinary pathogens with public health significance, animal welfare, food safety, and ethical use of antimicrobials. In terms of infectious diseases, this body has generated an important document, "Terrestrial Animal Health Code." This is a reference document and historically has been used by governmental veterinary officers, epidemiologists, and import/export services. However, any practitioner that is involved with the international shipment of animals should be familiar with these principles for disease control. Since the 1960s, this body has strived to develop standards, guidelines, and recommendations that contribute to preventing the transfer of infectious agents 

The American Association of Veterinary Laboratory Diagnosticians (AAVLD) is a nonprofit organization that works to communicate through the diagnostic laboratory system information regarding the diagnosis of animal diseases (http://www.aavld.org/ mc/page.do). A main tenet of their mission is to assist laboratories in establishing uniform diagnostic techniques and improvement of existing techniques. Only public supported laboratories can be part of this system, and the laboratories must be integrated. Table 2 lists all of the North American accredited laboratories and the tests available by PCR. The AAVLD laboratories must provide in their inspections evidence for quality assurance and quality control of their tests but do not standardize individual techniques. Because these are public institutions, in-state testing is usually subsidized and therefore much less expensive than a private laboratory. By the same token, overnight service may not be available through these laboratories.

The United States Department of Agriculture Animal Health inspection service provides proficiency testing and several laboratories below to the National Animal Health Laboratory Network. This is a state and federal partnership to protect mainly against FADs. Currently, there is no certification for any equine disease except for VSV. Public and private laboratories can be approved to run regulated tests by APHIS (http:// www.aphis.usda.gov/animal_health/lab_info_services/ approved_labs.shtml), which for horses includes contagious equine metritis, equine infectious anemia, equine viral arteritis, and VSV.

There is no accreditation that an outside laboratory can seek that provides protocol-based guidelines for laboratories to follow except for proficiency in regulated tests. One private laboratory has dry card testing available, whereby blood and other fluid samples are spotted on the card and tested. This type of testing is appropriate only for infections that are highly represented in fluid samples and for which there is a high amount of agent in the sample. 10 This would include blood, cerebrospinal fluid (CSF), and joint fluid, which usually is not where equine infectious agents are in abundance during clinical stages of disease. Many of these laboratories trademark their testing protocols, and understandably the primers and targets are kept as trade secrets; thus, further standardization and independent validation are not likely except through proficiency laboratory testing. Table 3 lists private molecular testing laboratories.

Given the problems with inappropriate sampling and handling and lack of standards for these new techniques, what can be said regarding interpretation of test results? There is no question that molecular medicine has dramatically revolutionized infectious disease diagnosis, treatment, and especially biosecurity. In the long run, molecular-based assays are more efficient and allow for minimal exposure of laboratory personnel and veterinarians to many infectious agents. 11 Ultimately, nothing will take the place of isolation of an infectious agent as confirmation of active infection from a properly collected and handled sample, but the efficiency and accuracy with good sampling and laboratory standardization make testing strategy of PCR diagnostics the most common in the future.

Given that sampling, handling, and all quality assurance for a particular laboratory are reliable, one must understand what a positive or a negative genomic-based test means. For instance, a positive PCR test result means that nucleic acids that belong to the genome of that particular pathogen were detected in the sample. This agent may or may not be live, infectious, or capable of replication in that sample. At least three different scenarios may be occurring in regard to the sample tested: (1) the pathogen is present and directly is causing the clinical signs observed, (2) the pathogen is present but is not responsible for the clinical signs observed, or (3) the pathogen is not present but the reaction mixture is binding to some other target in the assay. By the same token, a negative sample has failed to detect the nucleic acids of the infectious agent. A negative result can reflect at least three different scenarios in regard to the sample tested: (1) the pathogen was absent at the time of testing, (2) the pathogen is present but not detectable within the limits of sensitivity, and (3) there was some type of inhibition of the positive reaction. The mere results cannot be interpreted without understanding the context in which testing was performed in the first place unless this is used for regulatory purposes. In the absence of case criteria, the results by themselves are not confirmatory for disease causation. This is especially true for negative tests-hence repeated sampling is recommended should the case criteria create a high degree of suspicion for that disease. In the end, a comprehensive investigation using multiple samples employing different detection formats may be the only way to confirm disease causation in an outbreak or new emergence of disease in a group of animals. There is no "magic bullet" when it comes to testing.

Vesicular diseases are highly infectious agents that usually have very high morbidity with low mortality. However, their intensive infectiousness and painful nature when involved in outbreaks in hoofed livestock result in economic catastrophe. Investigatory laboratories around the world have developed several PCR assays that detect simultaneously (called a multiplex reaction) several vesicular viruses. 12 These assays will likely revolutionize early detection of outbreak spread but so far have several issues. Both conventional and real-time formats are available, and the OIE investigatory laboratories are working on some of these assays. The initial assays focused on detection of foot and mouth disease virus (FMDV) and SVD. No incorporation was made for VSV, a disease affecting cattle and horses of economic importance with disease activity in the United States. The VSV PCR is still run as a separate assay format. In addition, many subtypes of FMDV exist, and not all of the techniques available incorporate primers and probes that detect all subtypes. The most comprehensive assay in the literature is a "conventional" PCR format, and this was validated by testing "spiked" samples and experimentally inoculated swine. 13 Limited multiplex real-time PCR formats have been developed, but these were validated on a limited number of samples. Development of these tests is crucial for rapid disease surveillance. In experimental inoculation, virus detection (SVD, VSV, and FMDV) was possible even with multiplex conventional reverse transcriptase PCR in either blood or serum by the first and second 

PCR protocols are described in the literature for many diarrheal pathogens. Specifically, diagnostics for horses include Salmonella enterica, Clostridium perfringens A toxins, Lawsonia intracellularis, rotavirus, and several miscellaneous pathogens (see Table 2 ). Regarding Salmonella PCR in the horse, its excellent sensitivity is most useful for identification of subclinical shedders and environmental contamination during an outbreak. 14 Standard microbial culture methods are still required to obtain the isolates and confirm actual presence of the organism. A clinically ill horse that tests positive by PCR but negative by repeated culture should be interpreted with caution. Given the ultrasensitivity of this technique and the ability for bacterial elements to mobilize between fecal bacteria, only validated PCR techniques for Salmonella should be used. For horses, there is little standardization between the few laboratories that use PCR for detection of Salmonella.

There are many different PCR techniques for detection of equine herpesviruses (EHV). EHV 1 through 4 as a group all are detectable by conventional PCR. [28] [29] [30] [31] [32] [33] Real-time methods have been described for EHV-1 and EHV-4, and several simultaneous detection assays (multiplex) are in the literature. 28, 30, [34] [35] [36] [37] [38] Assays that target DNA of the polymerase gene of EHV appear to be most sensitive. 34 Use of RNA targets to examine latency has been described, which may allow study of pathogenesis. 39, 40 Location of virus and amount for latent infections in biopsy specimens of pharyngeal tissues with conventional nested PCR (double round of PCR essentially) currently defines latency. [41] [42] [43] In vaccinated horses, viral shedding of EHV-1 (non-neurotropic) and EHV-4 is extremely short and must be performed when horses are febrile if early identification of emergence is to be obtained.

One of the most frustrating areas of diagnostic medicine is diagnosis of infectious encephalitis. [44] [45] [46] [47] Although a conventional nested PCR test has been described and shown to be more sensitive for detection of WNV in horse tissues, this technique is fraught with greater probability of false-positive results. Also, use of the real-time format is more amenable to automation (hence, more rapid results). Although one article compared nested PCR with the real-time format, many of the samples in the literature are not controlled for sampling site of tissue. In our laboratory with experimental inoculation and in studies where field specimens were evaluated, the highest viral load for WNV was seen in thalamus and pons/medulla. Should these sections of brain be consistently evaluated, real-time PCR is likely reliable in horse brain. There is no question that the use of the CDC primers for detection of eastern equine encephalitis virus is sensitive and reliable. Horses have high viral load in thalamus, pons, and medulla. The same samples used for diagnostic testing for WNV also can be used for eastern equine encephalitis and western equine encephalitis testing. In arbovirus testing, plasma and serum are not appropriate for testing in horses with neurologic disease.

With neurotropic EHV-1, in our experience, there are high amounts of nasal shedding of virus and a high viral load in the hindbrain of neurologically affected horses. Several real-time PCR techniques have been developed and validated by OIE laboratories and investigators. 34, 42 Currently, the recommended protocol is to screen for EHV-1 using a generalized target for EHV-1 glycoprotein B gene. This gene is highly conserved and can be used to differentiate EHV-1 from EHV-4. In cases in which EHV-1 presence is questionable, the OIE nested protocol is considered the PCR gold standard. When EHV-1 is identified, a special PCR protocol (a single nucleotide polymorphism assay) is run for the differentiation of the strain associated with neurologic disease from the other EHV-1 not usually associated with central nervous system disease. The specific viral mutation (ORF 30) accounts for only approximately 80% to 85% of Detection and typing of C. perfringens in the human field have advanced beyond PCR to microarray to elucidate the complexities involved in differentiation of possible clostridial food-borne and water-borne poisoning. In horses, much attention has been given to the beta-2 C. perfringens toxin as an important cause of diarrhea in adult horses and foals; however, only in the pig has active transcription of beta-2 toxins in the positive strains been documented, and only in the pig has correlation been strong to disease. [15] [16] [17] [18] [19] [20] One study has provided a wider epidemiologic correlation in horses. At the molecular level, although the toxin is present in C. perfringens type A isolates garnered from equine clinical cases, the expression of this toxin is extremely low compared with the pig. 15 Welldesigned case-control and molecular epidemiology studies are paramount for further analysis of this toxin in horses. The most commonly used technique is a conventional PCR protocol that detects the presence of the toxin genes (not activity or expression). 16 Many AAVLD laboratories now offer this technique, and it has largely supplanted biologic assays, which use rodents. This is usually performed on C. perfringens isolates rather than directly on fecal samples, although this is likely the more practical approach. Interpretation is very important because C. perfringens is a common component of fecal flora.

Molecular techniques have greatly altered the efficiency of diagnosis of equine respiratory pathogens. Nowhere is this more apparent than for the diagnosis of Streptococcus equi subsp. equi. Because S. equi subsp. equi is in many cases the notifiable pathogen and one that control is directly correlated to biocontainment practices, differentiation and early identification of S. equi subsp. equi in an outbreak of respiratory disease is crucial. Historically, S. equi subsp. equi is differentiated from S. equi subsp. zooepidemicus on the basis of sugar fermentation. Conventional PCR was first performed with the 16S ribosomal gene and sequencing or in terms of the more easily differentiated superoxide dismutase A gene. 21 In addition, other genes, such as the SePE-I gene (pyrogenic mitogen), have been characterized and found present in S. subsp. equi but not S. subsp. zooepidemicus. 21 Both of these genes have also been characterized using a realtime format. 22 In this format, real-time PCR was able to detect and identify correctly all cultivatable S. equi subsp. equi isolates. In addition, six additional samples meeting the case criteria were positive for S. equi subsp. equi, two of which were identified as S. equi subsp. equi and four of which were identified as S. equi subsp. zooepidemicus. This technique did not identify two S. equi subsp. zooepidemicus isolates. Sequencing demonstrated that the target gene had molecular differences not previously described for S. equi subsp. zooepidemicus. These results compare with previously reported results for conventional PCR. Isolation and identification of S. equi subsp. equi-positive horses can be greatly enhanced by multiple sampling. Three consecutively obtained nasal swabs increase sensitivity of detection to 85%, which is equal to a single guttural pouch flushing.

Influenza testing has remarkable efficiency for detection of influenza A in human patients. With vaccination in horses, the window of positive testing is restricted mainly to the period of clinical signs, although even vaccinated horses shed virus during an outbreak. 23 Because most outbreaks in horses are currently caused by equine-2 H3N8 influenza strains, the specificity of most viral testing is unquestioned. Virus isolation is considered the gold standard, but real-time reverse transcriptase PCR and antigen test kits are supplanting this very specialized culturing because it must be done in egg cultures (a real art). 24, 25 Real-time PCR was more sensitive than five antigen detection kits and viral isolation. 26, 27 In addition, viral detection using real-time was correlated with quantization by tissue culture techniques. PCR techniques have been developed for detection of Pneumocystis jiroveci in human immunodeficiency virus infection. Because Pneumocystis organisms are considered host-species specific, these techniques must be validated for equine infections. Likewise, invasive Aspergillus infections are another area of interest for molecular detection formats. These techniques would likely be most useful for tissue invasion rather than detection of primary respiratory infection because Aspergillus can be a transtracheal wash contaminant. Candida infection occurs in the blood of equine neonates. These infections can be extremely hard to diagnose. Other pathogenic yeasts for which PCR techniques are highly applicable include Cryptococcus neoformans, Coccidioides immitis, Histoplasma, and Blastomyces dermititidis. The differentiation of Pythium insidiosum infection from mucormycotic fungi in horses is extremely useful because the former is highly resistant to treatment, whereas the latter can be removed by surgical excision/debulking and respond to antifungal therapies.

Faster, more discriminating identification of equine pathogens is possible through development of molecular assays. However, standardization of molecular techniques between laboratories, validation with appropriate sampling, and use of appropriate controls for quality assurance is necessary for expert and quality results in this rapidly expanding service for stakeholders. Ultimately, it is up to the equine practitioner to have a basic understanding of the methods and interpret the disease in the face of appropriate case criteria.

The complete reference list is available online at www. equineinfectiousdiseases.com equine herpes myeloencephalitis cases. Regardless, field investigation, outbreak details, and recent molecular studies have indicated that horses affected with equine herpes myeloencephalitis shed high amounts of virus early in the course of disease, and early quarantine and detection of nasal shedding within the exposed population is paramount to control. Automated, rapid molecular assays are essential for containment of outbreaks.

One of the most exciting areas of diagnostic investigation with PCR techniques is for detection and differentiation of cyathostome infection in horses. 48 There are approximately 43 cyathostomin species. Not only will PCR allow differentiation of these species, but it is also being developed to detect egg and L3 and L4 stages of infection in horse feces. 49,50 PCR protocols also have been used to detect helminth resistance. A PCR technique for detection of Anoplocephala has been described. 20, 51 This technique is sensitive and will likely aid in detection of another equine parasite that is notoriously difficult to identify. Furthermore, this technique will likely contribute to our understanding of the relationship between acute abdominal disease in horses and infection. Habronema infection can be extremely difficult to confirm in premortem biopsy sections, especially in the Southeast United States where diagnosis is complicated by fungal infection. [51] [52] [53] An extremely sensitive technique has been developed for detection in feces. This technique needs to be validated for peripheral tissue sections.

Molecular tests that detect pathogen fungi are also a much needed area for diagnosis of infection in the equine. Several

Survey of the largeanimal diplomates of the American College of Veterinary Internal Medicine regarding knowledge and clinical use of polymerase chain reaction: implications for veterinary education

Leutenegger CM: Real-time polymerase chain reaction: a novel molecular diagnostic tool for equine infectious diseases

Detection of the C282Y and H63D polymorphisms associated with hereditary hemochromatosis using the ABI 7500 fast real time PCR platform

The molecular diagnosis of porcine viral diseases: a review

Aspects of kit validation for tests used for the diagnosis and surveillance of livestock diseases: producer and end-user responsibilities

Experiences of an OIE Collaborating Centre in molecular diagnosis of transboundary animal diseases: a review

Impact of nasopharyngeal swab types on detection of Bordetella pertussis by PCR and culture

Validation of molecular-diagnostic techniques in the parasitological laboratory

An international external quality assessment of nucleic acid amplification of herpes simplex virus

Diagnosis of congenital CMV infection via dried blood spots

Multiplex assay based on single-nucleotide polymorphisms for rapid identification of Brucella isolates at the species level

Rapid and differential diagnosis of foot-and-mouth disease, swine vesicular disease, and vesicular stomatitis by a new multiplex RT-PCR assay

Detection of three porcine vesicular viruses using multiplex real-time primerprobe energy transfer

Evaluation of a PCR to detect Salmonella in fecal samples of horses admitted to a veterinary teaching hospital

Regulated expression of the beta2-toxin gene (cpb2) in Clostridium perfringens type a isolates from horses with gastrointestinal diseases

Multiplex PCR assay for detection of Clostridium perfringens in feces and intestinal contents of pigs and in swine feed

Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin

Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype

Enterotoxigenic Clostridium perfringens type A necrotic enteritis in a foal

Successful treatment and polymerase chain reaction (PCR) confirmation of Tyzzer's disease in a foal and clinical and pathologic characteristics of 6 additional foals

Determination of species-specific sequences of superoxide dismutase A encoding gene sodA and chaperonin 60 encoding gene cpn60 for identification and phylogenetic analysis of Streptococcus phocae

Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Immune responses to commercial equine vaccines against equine herpesvirus-1, equine influenza virus, eastern equine encephalomyelitis, and tetanus

Comparison of sensitivities of virus isolation, antigen detection, and nucleic acid amplification for detection of equine influenza virus

A rapid and highly sensitive method for diagnosis of equine influenza by antigen detection using immuno-PCR

Evaluation of antigen detection kits for diagnosis of equine influenza

Real-time reverse transcription PCR for detection and quantitative analysis of equine influenza virus

Detection of EHV-1 and EHV-4 DNA in unweaned Thoroughbred foals from vaccinated mares on a large stud farm

Detection of equine herpesvirus 3 in equine skin lesions by polymerase chain reaction

Utility of a multiplex PCR assay for detecting herpesvirus DNA in clinical samples

Identification of equine herpesvirus 3 (equine coital exanthema virus), equine gammaherpesviruses 2 and 5, equine adenoviruses 1 and 2, equine arteritis virus and equine rhinitis A virus by polymerase chain reaction

Detection of equine herpesvirus type 2 (EHV-2) in horses with keratoconjunctivitis

Development of a differential multiplex PCR assay for equine herpesvirus 1 and 4 as a diagnostic tool

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

Detection of viruses in nasal swab samples from horses with acute, febrile, respiratory disease using virus isolation, polymerase chain reaction and serology

Detection and quantification of equine herpesvirus-1 viremia and nasal shedding by real-time polymerase chain reaction

Equine herpesvirus-4 kinetics in peripheral blood leukocytes and nasopharyngeal secretions in foals using quantitative realtime TaqMan PCR

Genomic diversity among equine herpesvirus-4 field isolates

Characterization of viral loads, strain and state of equine herpesvirus-1 using real-time PCR in horses following natural exposure at a racetrack in California

Prevalence of equine herpesvirus-1 infection among Thoroughbreds residing on a farm on which the virus was endemic

Prevalence of latent, neuropathogenic equine herpesvirus-1 in the Thoroughbred broodmare population of central Kentucky

Development of a real-time polymerase chain reaction assay for rapid diagnosis of neuropathogenic strains of equine herpesvirus-1

Antemortem detection of latent infection with neuropathogenic strains of equine herpesvirus-1 in horses

Nucleic acid amplification assays for detection of La Crosse virus RNA

Detection of North American eastern and western equine encephalitis viruses by nucleic acid amplification assays

Molecular amplification assays for the detection of flaviviruses

Nucleic acid sequence-based amplification assays for rapid detection of West Nile and St. Louis encephalitis viruses

Comparative sequence analysis of the intergenic spacer region of cyathostome species

TaqMan minor groove binder real-time PCR analysis of beta-tubulin codon 200 polymorphism in small strongyles (Cyathostomin) indicates that the TAC allele is only moderately selected in benzimidazole-resistant populations

New method for simultaneous species-specific identification of equine strongyles (nematoda, strongylida) by reverse line blot hybridization

A comparison of coprological, serological and molecular methods for the diagnosis of horse infection with Anoplocephala perfoliata (Cestoda, Cyclophyllidea)

Molecular crosssectional survey of gastric habronemosis in horses

Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces