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SUMMARY
Equine rhinopneumonitis (ER) is a collective term for any one of several highly contagious, clinical disease entities of equids that may occur as a result of infection by either of two closely related herpesviruses, equid herpesvirus-1 and -4 (EHV-1 and EHV-4).
Infection by either EHV-1 or EHV-4 is characterised by a primary respiratory tract disease of varying severity that is related to age and immunological status of the infected animal. Infections by EHV-1 in particular are capable of progression beyond the respiratory mucosa to cause the more serious disease manifestations of abortion, perinatal foal death, or neurological dysfunction.
Identification of the agent: The standard method of identification of the herpesviral agents of ER continues to be laboratory isolation of the virus from appropriate clinical or necropsy material, followed by seroconfirmation of its identity. The viruses can be readily isolated in equine cell culture from nasopharyngeal samples taken from horses during the febrile stage of respiratory tract infection, from liver, lung, spleen, or thymus of aborted fetuses and early foal deaths, and from the leukocyte fraction of the blood of animals with acute EHV-1 disease. Positive identification of viral isolates as EHV-1 or EHV-4 can be achieved by immunofluorescence with type-specific monoclonal antibodies.
A rapid presumptive diagnosis of rhinopneumonitis abortion can be achieved by direct immunofluorescent detection of viral antigen in cryostat sections of tissues from aborted fetuses, using conjugated polyclonal antiserum.
Sensitive and reliable methods for EHV-1/4 detection by polymerase chain reaction or immunoperoxidase staining have been developed recently and are useful adjuncts to standard virus cultivation techniques for diagnosis of ER.
Post-mortem demonstration of the characteristic histopathological lesions of EHV-1 in tissues from aborted fetuses, cases or perinatal foal death or in the central nervous system of neurologically affected animals complements the laboratory diagnosis of ER.
Serological tests: Because most horses will possess some level of antibody to EHV-1/4, the demonstration of specific antibody in the serum collected from a single blood sample is not sufficient for a positive diagnosis of recent, active ER. Paired, acute and convalescent sera from animals suspected of being infected with EHV-1 or EHV-4 can be tested for a significant rise in virus-specific antibody titre by either complement fixation (CF), virus neutralisation, or enzyme-linked immunosorbent assay. If a single convalescent serum is the only sample available, the CF test is the most informative serological test to perform.
Requirements for vaccines and diagnostic biologicals: Both live attenuated and inactivated viral vaccines of varying composition are commercially available for use in assisting in the control of ER. While vaccination is helpful in reducing the incidence of abortion in mares, and in ameliorating the severity of clinical signs of respiratory infection in young horses, it should not be considered to be a substitute for strict adherence to the well established tenets of sound management practices known to reduce the risk of rhinopneumonitis. Revaccination at frequent intervals is recommended with each of the products, as the duration of vaccine-induced immunity is relatively short.
Standards for production and licensing of both attenuated and inactivated EHV vaccines are established by appropriate veterinary regulatory agencies in the countries of vaccine manufacture and use. A single set of internationally recognised standards for ER vaccines is not available. In each case, however, vaccine production is based on the system of a detailed outline of production employing a well characterised cell line and a master seed lot of vaccine virus that has been validated with respect to virus identity, safety, virological purity, immunogenicity, and the absence of extraneous microbial agents.
A. INTRODUCTION
Equine rhinopneumonitis (ER) is a historically derived term that describes a constellation of several disease entities of horses that may include respiratory disease, abortion, neonatal foal pneumonitis, or myeloencephalopathy (1, 2, 5, 7). The disease has been recognised for over 60 years as a threat to the international horse industry, and is caused by either of two members of the Herpesviridae family, equid herpesvirus-1 and -4 (EHV-1 and EHV-4). EHV-1 and EHV-4 are closely related alphaherpesviruses of horses with nucleotide sequence identity within individual homologous genes ranging from 55% to 84%, and amino acid sequence identity from 55% to 96% (13, 14). The two herpesviruses are enzootic in all countries in which large populations of horses are maintained as part of the cultural tradition or agricultural economy. There is no recorded evidence that the two herpesviruses of ER pose any health risks to humans working with the agents.
ER is highly contagious among susceptible horses, with viral transmission to cohort animals occurring by inhalation of aerosols of virus-laden respiratory secretions. Extensive use of vaccines has not eliminated EHV infections, and the world-wide annual financial burden from these equine pathogens is immense.
In horses under 3 years of age, clinical ER usually takes the form of an acute, febrile respiratory illness that spreads rapidly through the group of animals. The viruses infect and multiply in epithelial cells of the respiratory mucosa. Signs of infection become apparent 2-8 days after exposure to virus, and are characterised by fever, inappetence, depression, and nasal discharge. The severity of respiratory disease varies with the age of the horse and the level of immunity resulting from previous vaccination or natural exposure. Subclinical infections with EHV-1/4 are common, even in young animals. Although mortality from uncomplicated ER is rare and complete recovery within 1-2 weeks is the normal pattern, the respiratory infection is a frequent and significant cause of interrupted schedules among horses assembled for training, racing, or competitive equestrian events. Fully protective immunity resulting from infection is of short duration, and convalescent animals are susceptible to reinfection by EHV-1/4 after several months. Although reinfections by the two herpesviruses cause less severe or clinically inapparent respiratory disease, the risks of subsequent abortion and/or central nervous system (CNS) disease are not eliminated. The greatest clinical threats to individual breeding, racing, or pleasure horse operations posed by ER are the potential abortigenic and neurological sequelae of EHV-1 respiratory infection.
B. DIAGNOSTIC TECHNIQUES
| 1. | Identification of the agent (3)
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| | Because ER is a highly contagious disease with the potential for occurring as explosive outbreaks with high mortality from abortigenic or neurological sequelae, rapid diagnostic methods are important. Although several rapid and innovative diagnostic techniques based on enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR), immunohistochemical staining with peroxidase, or nucleic acid hybridisation probes have been recently described, their use is often restricted to specialised reference laboratories, and thus the method of choice for diagnosis of ER by diagnostic virology laboratories handling many routine samples continues to be the traditional methodology of cell culture isolation followed by sero-identification of the isolated viruses. Successful laboratory isolation of EHV-1/4 depends on strict adherence to proper methods for both sample collection and laboratory processing.
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| | a) | Collection of samples
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| | | Samples of nasopharyngeal exudate for virus isolation are best obtained from horses during the very early, febrile stages of the respiratory disease, and are collected via the nares by swabbing the nasopharyngeal area with a 5 x 5 cm gauze sponge attached to the end of a 50 cm length of flexible, stainless steel wire encased in latex rubber tubing. A guarded uterine swab devise can also be used. After collection, the swab should be removed from the wire and transported immediately to the virology laboratory in 3 ml of cold (not frozen) fluid transport medium (serum-free MEM [minimal essential medium] with antibiotics). Virus infectivity can be prolonged by the addition of bovine serum albumin or gelatine to 0.1% (w/v).
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| | | Virological examination of fetal tissues from suspect cases of EHV-1 abortion is most successful when performed on aseptically collected samples of liver, lung, thymus, and spleen. The tissue samples should be transported to the laboratory and held at 4°C until inoculated into tissue culture. Samples that cannot be processed within a few hours should be stored at -70°C. In ante-mortem cases of EHV-1 neurological disease, the virus can often be isolated from the leukocyte fraction of the blood of acutely infected horses or, less often, from the nasopharynx of the affected animal or cohort animals. For attempts at virus isolation from blood leukocytes, a 20 ml sample of sterile blood, collected in citrate, EDTA (ethylene diamine tetra-acetic acid), or heparin anticoagulant, should be transported without delay to the laboratory on ice, but not frozen. Although the virus has, on occasion, been isolated from post-mortem cases of EHV-1 neurological disease by culture of samples of brain and spinal cord, such attempts to isolate virus are often unsuccessful.
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| | b) | Virus isolation
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| | | For efficient primary isolation of EHV-4 from horses with respiratory disease, equine-derived cell cultures must be used. Both EHV-1 and EHV-4 may be isolated from nasopharyngeal samples using primary equine fetal kidney cells or cell strains of equine fibroblasts derived from dermal (E-Derm) or lung tissue. EHV-1 can be isolated on other cell types, as will be discussed later. The nasopharyngeal swab and its accompanying 3 ml of transport medium are transferred into the barrel of a sterile 10 ml syringe. Using the syringe plunger, the fluid is squeezed from the swab into a sterile tube. A portion of the expressed fluid is then filtered through a sterile, 0.45 µm membrane syringe filter unit into a second sterile tube. Filtration will decrease bacterial contamination, but may also lower virus titre. Recently prepared cell monolayers in 25 cm2 tissue culture flasks are inoculated with 0.5 ml of the filtered, as well as the nonfiltered, nasopharyngeal swab extract. Multiwell plates may also be used. Virus is allowed to attach by incubating the inoculated monolayers at 37°C on a platform rocker for 1.5-2 hours. Monolayers of uninoculated control cells should be incubated in parallel with sterile transport medium only.
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| | | At the end of the attachment period, the inocula are removed and the monolayers are rinsed twice with phosphate buffered saline (PBS) to remove virus-neutralising antibody that may be present in the nasopharyngeal secretions. After addition of 5 ml of supplemented maintenance medium (MEM containing 2% fetal calf serum [FCS] and twice the standard concentrations of antibiotics [penicillin, streptomycin, gentamicin, and amphotericin B]), the flasks are incubated at 37°C. The use of positive control virus samples to validate the isolation procedure carries the risk that this may lead to eventual contamination of diagnostic specimens. This risk can be minimised by using routine precautions and good laboratory technique, including the use of biosafety cabinets, inoculating positive controls after the diagnostic specimens, decontaminating the surfaces in the hood while the inoculum is asorbing and using a positive control of relatively low titre. Inoculated flasks should be inspected daily by microscopy for the appearance of characteristic herpesvirus cytopathic effect (CPE) (focal rounding, increase in refractility, and detachment of cells). Cultures exhibiting no evidence of viral CPE after 1 week of incubation should be blind-passaged into freshly prepared monolayers of cells, using small aliquots of both media and cells as the inoculum. Further blind passage is usually not productive.
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| | | A number of cell types may be used for isolation of EHV-1 from the tissues of aborted fetuses or from post-mortem cases of neurological disease (e.g. rabbit kidney [RK-13], baby hamster kidney [BHK-21], Madin-Darby bovine kidney [MDBK], pig kidney [PK-15], etc.), but equine-derived cell cultures are most sensitive and must be used if the infrequent cases of EHV-4 abortion are to be detected. Around 10% (w/v) pooled tissue homogenates of liver, lung, thymus, and spleen (from aborted fetuses) or of CNS tissue (from cases of neurological disease) are used for virus isolation. These are prepared by first mincing small samples of tissue into 1 mm cubes in a sterile Petri dish with dissecting scissors, followed by macerating the tissue cubes further in serum-free culture medium with antibiotics using a mechanical tissue grinder (e.g. Ten-Broeck or Stomacher). After centrifugation at 1200 g for 10 minutes, the supernatant is removed and 0.5 ml is inoculated into duplicate cell monolayers in 25 cm2 flasks. Following incubation of the inoculated cells at 37°C for 1.5-2 hours, the inocula are removed and the monolayers are rinsed twice with PBS. After addition of 5 ml of supplemented maintenance medium, the flasks are incubated at 37°C for up to 1 week or until viral CPE is observed.
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| | | Culture of peripheral blood leukocytes for the presence of EHV-1 is frequently successful in horses during the early stages of the paralytic disease. On receipt by the diagnostic laboratory, the tube of chilled blood, containing citrate or heparin as anticoagulant, is mixed by inversion and allowed to stand for 1 hour at room temperature. The top layer of leukocyte-rich plasma is removed and centrifuged at 640 g for 15 minutes. After decanting the supernatant, the leukocyte pellet is resuspended in the small volume of residual supernatant by brief vortexing of the tube. The resuspended cells are then rinsed twice with 10 ml of sterile PBS by centrifugation (300 g for 10 minutes) and resuspension. After the last centrifugation, the leukocyte pellet is resuspended in 1 ml of MEM containing 2% FCS. Then, 0.5 ml of the rinsed cell suspension is added to duplicate monolayers of equine fibroblast, equine fetal or RK-13 cell monolayers in 25 cm2 flasks containing 8-10 ml freshly added maintenance medium. The flasks are incubated at 37°C for 7 days; the inoculum is not removed. Because CPE may be difficult to detect in the presence of the massive inoculum of leukocytes, each flask of cells is freeze-thawed after 7 days of incubation. Finally, 0.5 ml of the cell-free culture medium from each flask is transferred to freshly made cell monolayers. These are incubated and observed for viral CPE for at least 5-6 days before discarding as negative.
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| | c) | Seroconfirmation of virus identity
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| | | The basis for identification of any herpesvirus isolate recovered from specimens submitted from suspected cases of ER is its immunoreactivity with specific antisera. Specific identification of an isolate as EHV-1 or EHV-4 can be quickly and simply accomplished by immunofluorescent detection of viral antigen in the infected cell culture using type-specific monoclonal antibodies (MAbs), which are available from OIE Reference Laboratories for equine rhinopneumonitis. The test, which is type-specific and accurate, can be performed on a small aliquot of infected cells from the same container inoculated with clinical or post-mortem material. An isolate made in a laboratory that lacks MAbs can be confirmed as EHV1/4 using a virus-specific polyclonal antiserum or by the PCR (see section B.1.f).
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| | | Cell monolayers infected with the isolate are removed by scraping from the flask when at least 75% CPE is evident. The cells are pelleted from the culture medium and resuspended in 0.5 ml of PBS. 50 µl of the cell suspension is placed into two wells of a multiwell microscope slide, air-dried, and fixed for 10 minutes with 100% acetone. Control cell suspensions (uninfected, EHV-1 infected, or EHV-4 infected) are also spotted into each of two wells of the same slide. Control cells may be prepared in advance and stored frozen in small aliquots. A drop of an appropriate dilution of MAb specific for EHV-1 is added to one well of each cell pair, and a drop of MAb specific for EHV-4 is added to each of the other wells. After 30 minutes' incubation at 37°C in a humid chamber, unreacted antibody is removed by two 10-minute washes with PBS. MAbs bound to viral antigen can be detected with goat anti-mouse IgG conjugated with fluorescein isothiocyanate (FITC). A drop of diluted conjugate is added to each well and, after 30 minutes at 37°C, the wells are again washed twice with PBS. Cells are examined with a fluorescence microscope, and positive fluorescence with the antibody of appropriate specificity indicates the virus type.
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| | d) | Virus detection by direct immunofluorescence
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| | | Direct immunofluorescent detection of EHV-1 antigens in samples of post-mortem tissues collected from aborted equine fetuses provides an indispensable method to the veterinary diagnostic laboratory for making a rapid preliminary diagnosis of herpesvirus abortion (9). Side-by-side comparisons of the immunofluorescent and cell culture isolation techniques on more than 100 cases of equine abortion have provided evidence that the diagnostic reliability of direct immunofluorescent staining of fetal tissues obtained at necropsy approaches that of virus isolation attempts from the same tissues. In the United States of America (USA), specific and potent polyclonal antiserum to EHV-1, prepared in swine and conjugated with FITC, is provided to veterinary diagnostic laboratories for this purpose by the National Veterinary Services Laboratories of the United States Department of Agriculture (USDA). The antiserum cross-reacts with EHV-4 and hence is not useful for serotyping. Freshly dissected samples (5 x 5 mm pieces) of fetal tissue (lung, liver, thymus, and spleen) are frozen, sectioned on a cryostat at -20°C, mounted on to microscope slides, and fixed with 100% acetone. After air-drying, the sections are incubated at 37°C in a humid atmosphere for 30 minutes with an appropriate dilution of the conjugated swine antibody to EHV-1. Unreacted antibody is removed by two washes in PBS, and the tissue sections are then covered with aqueous mounting media and a cover-slip, and examined for fluorescent cells indicating the presence of EHV antigen. Each test should include a positive and negative control consisting of sections from known EHV-1 infected and uninfected fetal tissue.
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| | e) | Virus detection by immunoperoxidase staining
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| | | Enzyme immunohistochemical (IH) staining methods (e.g. immunoperoxidase) have been developed recently as procedures for detecting EHV-1 antigen in paraffin-embedded tissues of aborted equine fetuses or neurologically affected horses (12, 19). Such ancillary IH techniques for antigen detection may facilitate identification of the virus in archival tissue samples or in clinical cases in which traditional laboratory methods for EHV-1 detection have been unsuccessful. Immunoenzymatic staining for EHV-1 is particularly useful for the simultaneous evaluation of morphological lesions and the identification of the infectious agent. Adequate controls must be included with each immunoperoxidase test run for evaluation of both the method specificity and antibody specificity.
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| | f) | Virus detection by polymerase chain reaction
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| | | The PCR can be used for rapid amplification and diagnostic detection of nucleic acids of EHV-1 and -4 in clinical specimens, paraffin-embedded archival tissue, or inoculated cell cultures (4, 10, 11, 16, 17). A variety of type-specific PCR primers have been designed to distinguish between the presence of EHV-1 and EHV-4. The correlation between PCR and virus isolation techniques for diagnosis of EHV-1 or EHV-4 is high (16). Diagnosis of ER by PCR is rapid, sensitive, and does not depend on the presence of infectious virus in the clinical sample. It now forms an integral part of a range of diagnostic tests currently available for ER, each with its own advantages and limitations.
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| | | For diagnosis of active infection by EHV, PCR methods are most reliable with samples from aborted fetuses or from nasopharyngeal swabs of foals and yearlings; they are most useful in explosive epizootics of abortion or respiratory tract disease in which a rapid identification of the virus is critical for guiding management strategies. The interpretation of the amplification by PCR of genomic fragments of EHV-1 or EHV-4 in tissues (Iymph nodes, peripheral blood leukocytes, or CNS) from adult horses is complicated by the high prevalence of latent EHV-1 and EHV-4 DNA in circulating Iymphocytes and the trigeminal ganglia of such animals (18).
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| | | A simple multiplex PCR assay for simultaneous detection of both EHV-1 and EHV-4 has been described (17). A more sensitive protocol for semi-nested PCR detection of EHV-1 or EHV-4 in clinical or pathological specimens (nasal secretions, blood leukocytes, fetal tissues, etc.) is as follows (16):
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| | | i) | Prepare template DNA from test specimens: Following sample homogenisation and cell (and virion) lysis in the presence of a chaotropic salt, nucleic acids bind selectively to silica or cationic resin substrates. Substrate-bound nucleic acids are purified in a series of rapid wash steps followed by recovery with low-salt elution. The reagents for performing such steps for rapid nucleic acid isolation are available in kit format from a number of commercial sources (e.g. High Pure PCR Template Preparation Kit, Roche Molecular Biochemicals, Indianapolis, USA; QIAamp DNA Kit, Qiagen, Valencia, USA).
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| | | ii) | Semi-nested primer sequences specific for EHV-1 (16):
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| | | | EHV1-gH-F = 5'-AAG-AGG-AGC-ACG-TGT-TGG-AT-3'
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| | | | EHV1-gH-R = 5'-TTG-AAG-GAC-GAA-TAG-GAC-GC-3'
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| | | | EHV1-gH-RN = 5'-AGT-AGG-TCA-GGC-CGA-TGC-TT-3'
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| | | iii) | Semi-nested primer sequences specific for EHV-4 (16):
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| | | | EHV4-gB-F = 5'-CTG-CTG-TCA-TTA-TGC-AGG-GA-3'
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| | | | EHV4-gB-R = 5'-CGT-CTT -CTC-GAA-GAC-GGG-TA-3'
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| | | | EHV4-gB-RN = 5'-CGC-TAG-TGT-CAT-CAT-CGT-CG-3'
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| | | iv) | PCR conditions for first stage amplification: Specimen template DNA (5 µl) is added to a PCR mixture (total volume of 25 µl) containing 1 x PCR buffer (50 mM KCl, 10 mM Tris/HCl, pH 9.0, 0.1% Triton X-100), 200 µM of each deoxynucleotide triphosphate (dNTP), 2.5 mM MgCl2, 2.0 µM of each outer primer (EHV1-gH-F and EHV1-gH-R for EHV-1 detection and, in a separate reaction mixture, EHV4-gB-F and EHV4-gB-R for EHV-4 detection) and 0.5 u Taq DNA polymerase. Cycling parameters are: initial denaturation at 95°C for 5 minutes; 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute; with a final extension at 72°C for 5 minutes. Separate reaction mixtures containing either known viral DNA or no DNA (water) should be prepared and amplified as positive and negative controls.
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| | | v) | PCR conditions for second stage (semi-nested) amplification: One-half µl of the first amplification product is added to a fresh PCR mixture (total volume of 25 µl) containing 1 x PCR buffer, 200 µM of each dNTP, 2.5 mM MgCl2, 2.0 µM of each primer (EHV1-gH-F and EHV1-gH-RN for EHV-1 detection and, in a separate reaction mixture, EHV4-gB-F and EHV4-gB-RN for EHV-4 detection) and 0.5 u Taq DNA polymerase. Cycling parameters are: initial denaturation at 95°C for 5 minutes; 35 cycles of 95°C for 30 seconds, 60°C for 30 seconds, and 72°C for 1 minute; with a final extension at 72°C for 5 minutes.
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| | | vi) | Gel analysis of amplified products: 5 µl of each final amplified product, including controls, is electrophoresed on a 2.0% agarose gel in Tris/acetate running buffer, along with a 100 base pairs (bp) DNA ladder. The gel is stained with ethidium bromide and viewed by UV transillumination for amplified products of either 287 bp for EHV-1 or 323 bp for EHV-4.
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| | g) | Histopathology
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| | | Histopathological examination of sections of formalin-fixed, paraffin-embedded tissues from aborted fetuses or from neurologically affected horses is an essential part of the laboratory diagnosis of these two clinical manifestations of ER. In aborted fetuses, typical herpetic intranuclear inclusion bodies present within bronchiolar epithelium or in cells at the periphery of areas of hepatic necrosis are pathognomonic lesions for EHV-1. The characteristic, but not pathognomonic, microscopic lesion associated with EHV-1 neuropathy is a degenerative thrombotic vasculitis of small blood vessels in the brain or spinal cord (perivascular cuffing and infiltration by inflammatory cells, endothelial proliferation and necrosis, and thrombus formation).
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| 2. | Serological tests
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| | Because of the ubiquity of the viral agents of ER and the high seroprevalence among horses in most parts of the world, the demonstration of a negative antibody titre to EHV-1/4 by serological testing of horses designated for export is not part of present veterinary regulations that seek to prevent international spread of infectious diseases of horses. Serological testing can, however, be a useful adjunct procedure for assisting in the diagnosis of ER in horses. Serodiagnosis of ER is based on the demonstration of significant increases in antibody titres in paired sera taken during the acute and convalescent stages of the disease. The results of tests performed on sera from a single collection date are, in most cases, impossible to interpret with any degree of confidence. The initial (acute phase) serum sample should be taken as soon as possible after the onset of clinical signs, and the second (convalescent phase) serum sample should be taken 3-4 weeks later. Samples of cerebrospinai fluid may also be of value in serodiagnosis of EHV-1 neurological disease. 'Acute phase' sera from mares after abortion or from horses with EHV-1 neurological disease may already contain maximal titres of EHV-1 antibody, with no increase in titres detectable in sera collected at later dates. In such cases, serological testing of paired serum samples from clinically unaffected cohort members of the herd for rising antibody titres against EHV-1/4 may provide information useful for retrospective diagnosis of ER within the herd. Finally, the serological detection of antibodies to EHV-1 in heart or umbilical cord blood or other fluids of equine fetuses can be of diagnostic value in rare cases of virologically negative fetuses aborted as a result of EHV-1 infection.
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| | Serum antibody levels to EHV-1/4 may be determined by ELISA (8), virus neutralisation (VN) (15), or complement fixation (CF) tests (15). Because CF antibody titres to EHV-1/4 become negative within a few months following recovery from infection, the CF test is most useful in diagnosing recent infection by the herpesviruses if a convalescent serum is the only sample available for testing. The CF test performed on sera collected from horses with the paralytic form of EHV-1 infection is also useful for making the rapid diagnosis necessary for initiating appropriate measures for control of the spread of this contagious disease. Because there are no internationally recognised reagents or standardised techniques for performing any of the serological tests for detection of EHV-1/4 antibody, antibody titre determinations on the same serum may differ from one laboratory to another. Furthermore, all of the serological tests mentioned detect antibodies that are cross-reactive between EHV-1 and EHV-4. Nonetheless, the demonstration, by any of the tests, of a four-fold or greater rise in antibody titre to EHV-1 or EHV-4 during the course of a clinical illness provides serological confirmation of recent infection with one of the viruses. The ELISA and CF test have the advantage that they are quicker to provide results and do not require cell culture facilities. Recently, a type-specific ELISA that can distinguish between antibodies to EHV-1 and EHV-4 was developed and was made commercially available (6). The microneutralisation test is a widely used and sensitive serological assay for detecting EHV-1/4 antibody and will thus be described here.
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| | a) | Virus neutralisation test
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| | | This serological test is most commonly performed in flat-bottom 96-well microtitre plates (tissue culture grade) using a constant dose of virus and doubling dilutions of equine test sera. At least two replicate wells for each serum dilution are required. Serum-free MEM is used throughout as a diluent. Virus stocks of known titre are diluted just before use to contain 100 TCID50 (50% tissue culture infective dose) in 25 µl. Monolayers of E-Derm or RK-13 cells are monodispersed with EDTA/trypsin and resuspended at a concentration of 5 x 105/ml. Note that RK-13 cells can be used with EHV-1 but do not give clear CPE with EHV-4. Antibody positive and negative control equine sera and controls for cell viability, virus infectivity, and test serum cytotoxicity, must be included in each assay. End-point VN titres of antibody are calculated by determining the reciprocal of the highest serum dilution that protects 100% of the cell monolayer from virus destruction in both of the replicate wells.
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| | | A suitable test procedure is as follows:
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| | | i) | Inactivate test and control sera for 30 minutes in a water bath at 56°C.
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| | | ii) | Add 25 µl of serum-free MEM to all wells of the microtitre assay plates.
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| | | iii) | Pipette 25 µl of each test serum into duplicate wells of both rows A and B of the plate. The first row serves as the serum toxicity control and the second row as the first dilution of the test. Make doubling dilutions of each serum starting with row B and proceeding to the bottom of the plate by sequential mixing and transfer of 25 µl to each subsequent row of wells. Six sera can be assayed in each plate.
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| | | iv) | Add 25 µl of the appropriately diluted EHV-1 or EHV-4 virus stock to each well (100 TCID50/well) except those of row A, which are the serum control wells for monitoring serum toxicity for the indicator cells. Note that the final serum dilutions, after addition of virus, run from 1/4 to1/256.
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| | | v) | A separate control plate should include titration of both a negative and positive horse serum of known titre, cell control (no virus), virus control (no serum), and a virus titration to calculate the actual amount of virus used in the test.
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| | | vi) | Incubate the plates for 1 hour at 37°C in 5% CO2 atmosphere.
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| | | vii) | Add 50 µl of the prepared E-Derm or RK-13 cell suspension (5 x 105 cells/ml) in MEM/10% FCS to each well.
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| | | viii) | Incubate the plates for 4-5 days at 37°C in an atmosphere of 5% CO2 in air.
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| | | ix) | Examine the plates microscopically for CPE and record the results on a worksheet. Alternatively, the cell monolayers can be scored for CPE after fixing and staining as follows: after removal of the culture fluid, immerse the plates for 15 minutes in a solution containing 2 mg/ml crystal violet, 10% formalin, 45% methanol, and 45% water. Then, rinse the plates vigorously under a stream of running tap water.
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| | | x) | Wells containing intact cell monolayers stain blue, while monolayers destroyed by virus do not stain. Verify that the cell control, positive serum control, and serum cytotoxicity control wells stain blue, that the virus control and negative serum control wells are not stained, and that the actual amount of virus added to each well is between 101.5 and 102.5 TCID50. Wells are scored as positive for neutralisation of virus if 100% of the cell monolayer remains intact. The highest dilution of serum resulting in complete neutralisation of virus (no CPE) in both duplicate wells is the end-point titre for that serum.
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| | | xi) | Calculate the neutralisation titre for each test serum, and compare acute and convalescent phase serum titres from each animal for a four-fold or greater increase.
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
Both live attenuated and inactivated vaccines are available as licensed, commercially prepared products for use as prophylactic aids in reducing the burden of disease in horses caused by EHV-1/4 infection. Clinical experience has demonstrated that none of the vaccine preparations should be relied on to provide an absolute degree of protection from ER. Multiple doses, repeated annually, of each of the currently marketed ER vaccines are recommended by their respective manufacturers, with vaccination schedules that vary with the particular vaccine.
Guidelines for the production of veterinary vaccines are given in Chapter I.1.7. Principles of veterinary vaccine production. The guidelines given here and in Chapter I.1.7 are intended to be general in nature and may be supplemented by national and regional requirements.
At least sixteen vaccine products for ER, each containing different permutations of EHV-1, EHV-4, and the two subtypes of equine influenza virus, are currently marketed by five veterinary biologicals manufacturers.
The clinical indications stated on the product label for use of the several available vaccines for ER are either herpesvirus-associated respiratory disease, abortion, or both. Only four vaccine products have met the regulatory requirements for claiming efficacy in providing protection from herpesvirus abortion as a result of successful vaccination and challenge experiments in pregnant mares. None of the vaccine products has been tested for its ability to prevent the occurrence of neurological disease sometimes associated with EHV-1 infection.
| 1. | Seed management
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| | a) | Characteristics and culture
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| | | The master seed virus (MSV) for ER vaccines must be prepared from strains of EHV-1 and/or EHV-4 that have been positively and unequivocally identified by both serological and genetic tests. Seed virus must be propagated in a cell line approved for equine vaccine production by the appropriate regulatory agency. A complete record of original source, passage history, medium used for propagation, etc., shall be kept for the master seed preparations of both the virus(es) and cell stock(s) intended for use in vaccine production. Permanently stored stocks of both MSV and master cell stock (MCS) used for vaccine production must be demonstrated to be pure, safe and, in the case of MSV, also immunogenic. Generally, the fifth passage from the MSV and the twentieth passage from the MCS are the highest allowed for vaccine production. Results of all quality control tests on master seeds must be recorded and made a part of the licensee's permanent records.
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| | b) | Validation as a vaccine
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| | | i) | Purity
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| | | | Tests for master seed purity include prescribed procedures that demonstrate the virus and cell seed stocks to be free from bacteria, fungi, mycoplasmas, and extraneous viruses. Special tests must be performed to confirm the absence of equine arteritis virus, equine infectious anaemia virus, equine influenza virus, equine herpesvirus-2, -3, and -5, equine rhinovirus, the alphaviruses of equine encephalomyelitis, bovine viral diarrhoea virus (BVDV - common contaminant of bovine serum), and porcine parvovirus (PPV - potential contaminant of porcine trypsin). The purity check should also include the exclusion of the presence of EHV-1 from EHV-4 MSV and vice versa.
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| | | ii) | Safety
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| | | | Samples of each lot of MSV to be used for preparation of live attenuated ER vaccines must be tested for safety in horses determined to be susceptible to the virulent wild-type virus, including pregnant mares in the last 4 months of gestation. Vaccine safety must be demonstrated in a 'safety field trial' in horses of various ages from three different geographical areas. The safety trial should be conducted by independent veterinarians using a prelicensing batch of vaccine. EHV-1 vaccines making a claim for efficacy in controlling abortion must be tested for safety in a significant number of late gestation pregnant mares, using the vaccination schedule that will be recommended by the manufacturer for the final vaccine product.
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| | | iii) | Immunogenicity
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| | | | Tests for immunogenicity of the EHV-1/4 MSV stocks should be performed in horses on an experimental test vaccine prepared from the highest passage level of the MSV allowed for use in vaccine production. The prescribed test for MSV immunogenicity consists of vaccination of horses with low antibody titres to EHV-1/4, with doses of the test vaccine that will be recommended on the final product label. Second serum samples should be obtained and tested for significant increases in neutralising antibody titre against the virus, 21 days after the final dose.
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| | | iv) | Efficacy
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| | | | An important part of the validation process is the capacity of a prelicensing lot of the ER vaccine to provide a significant level of clinical protection in horses from the particular disease manifestation of EHV-1/4 infection for which the vaccine is offered, when used under the conditions recommended by the manufacturer's product label. Serological data are not acceptable for establishing the efficacy of vaccines for ER. Efficacy studies must be designed to ensure appropriate randomisation of test animals to treatment groups, blinding of the recording of clinical observations, and the use of sufficient numbers of animals to permit statistical evaluation for effectiveness in prevention or reduction of the specified clinical disease. The studies should be performed on fully formulated experimental vaccine products (a) produced in accordance with, (b) at or below the minimum antigenic potency specified in, and, (c) produced with the highest passage of MSV and MCS allowed by the approved 'Outline of Production' (see Section C.2.). Vaccine efficacy is demonstrated by vaccinating a minimum of 20 EHV-1/4-susceptible horses possessing low serum neutralising antibody titres, followed by challenge of the vaccinates and ten nonvaccinated control horses with virulent virus. A significant difference in the clinical signs of ER must be demonstrated between vaccinates and nonvaccinated control horses. The vaccination and challenge study must be performed on an identical number of pregnant mares and scored for abortion if the vaccine product will make a label usage claim 'for prevention of' or 'as an aid in the prevention of' abortion caused by EHV-1.
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| 2. | Method of manufacture
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| | A detailed protocol of the methods of manufacture to be followed in the preparation of vaccines for ER must be compiled, approved, and filed as an Outline of Production with the appropriate licensing agency. Specifics of the methods of manufacture for ER vaccines will differ with the type (live or inactivated) and composition (EHV-1 only, EHV-1 and EHV-4, EHV-4 and equine influenza viruses, etc.) of each individual product, and also with the manufacturer.
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| 3. | In-process control
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| | Cells, virus, culture medium, and medium supplements of animal origin that are used for the preparation of production lots of vaccine must be derived from bulk stocks that have passed the prescribed tests for bacterial, fungal, and mycoplasma sterility; nontumorigenicity; and absence of extraneous viral agents.
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| 4. | Batch control
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| | Each bulk production lot of ER vaccine must pass tests for sterility, safety, and immunogenic potency.
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| | a) | Sterility
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| | | Samples taken from each batch of completed vaccine are tested for bacteria, fungi, and mycoplasma contamination. Procedures to establish that the vaccine is free from extraneous viruses are also required; such tests should include inoculation of cell cultures that allow detection of the common equine viruses, as well as techniques for the detection of BVDV and PPV in ingredients of animal origin used in the production of the batch of vaccine.
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| | b) | Safety
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| | | Tests to assure safety of each production batch of ER vaccine must demonstrate complete inactivation of virus (for inactivated vaccines) as well as a level of residual virus-killing agent that does not exceed the maximal allowable limit (e.g. 0.2% for formaldehyde). Safety testing in laboratory animals is also required.
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| | c) | Potency
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| | | Batch control of antigenic potency for EHV-1 vaccines may be tested by measuring the ability of dilutions of the vaccine to protect hamsters from challenge with a lethal dose of hamster-adapted EHV-1 virus. Although potency testing on production batches of ER vaccine may also be performed by vaccination of susceptible horses followed by either viral challenge or assay for seroconversion, the recent availability of virus type-specific MAbs has permitted development of less costly and more rapid in-vitro immunoassays for antigenic potency. The basis for such in-vitro assays for ER vaccine potency is the determination, by use of the specific MAb, of the presence of at least the minimal amount of viral antigen within each batch of vaccine that correlates with the required level of protection (or seroconversion rate) in a standard animal test for potency.
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| | d) | Duration of immunity
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| | | Tests to establish the duration of immunity to EHV-1/4 achieved by immunisation with each batch of vaccine are not required. The results of many reported observations indicate that vaccination-induced immunity to EHV-1/4 is not more than a few months in duration; these observations are reflected in the frequency of revaccination recommended on ER vaccine product labels.
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| | e) | Stability
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| | | At least three production batches of vaccine should be tested for shelf life before reaching a conclusion on the vaccine's stability. When stored at 4°C, inactivated vaccine products generally maintain their original antigenic potency for at least 1 year. Lyophilised preparations of the live virus vaccine are also stable during storage for 1 year at 4°C. Following reconstitution, live virus vaccine is unstable and cannot be stored without loss of potency.
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| 5. | Tests on the final product
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| | Before release for labelling, packaging, and commercial distribution, randomly selected filled vials of the final vaccine product must be tested by prescribed methods for freedom from contamination and safety in laboratory test animals.
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| | a) | Safety
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| | | See Section C.4.b.
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| | b) | Potency
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| | | See Section C.4.c.
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REFERENCES
| 1. | Allen G.P. & Bryans J.T. (1986). Molecular epidemiology, pathogenesis and prophylaxis of equine herpesvirus-1 infections. In: Progress in Veterinary Microbiology and Immunology, Vol. 2, Pandey R., ed. Karger, Basel, Switzerland & New York, USA, 78-144.
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| 2. | Allen G.P., Kydd J.H., Slater J.D. & Smith K.C. (1999). Recent advances in understanding the pathogenesis, epidemiology, and immunological control of equid herpesvirus-1 (EHV-1) abortion. Equine Infect. Dis., 8, 129-146.
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| 3. | Allen G.P., Kydd J.H., Slater J.D. & Smith K.C. (2004). Equid herpesvirus-1 (EHV-1) and -4 (EHV-4) infections. In: Infectious Diseases of Livestock, Coetzer J.A.W., Thomson G.R. & Tustin R.C., eds. Oxford University Press, Cape Town, South Africa (in press).
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| 4. | Borchers K. & Slater J. (1993). A nested PCR for the detection and differentiation of EHV-1 and EHV-4. J. Virol. Methods, 45, 331-336.
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| 5. | Bryans J.T. & Allen G.P. (1988). Herpesviral diseases of the horse. In: Herpesvirus Diseases of Animals, Wittman G., ed. Kluwer, Boston, USA, 176-229.
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| 6. | Crabb B.S., MacPherson C.M., Reubel G.H., Browning G.F., Studdert M.J. & Drummer H.E. (1995). A type-specific serological test to distinguish antibodies to equine herpesviruses 4 and 1. Arch. Virol., 140, 245-258.
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| 7. | Crabb B.S. & Studdert M.J. (1995). Equine herpesviruses 4 (equine rhinopneumonitis virus) and 1 (equine abortion virus). Adv. Virus Res., 45, 153-190.
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| 8. | Dutta S.K., Talbot N.C. & Myrup A.C. (1983). Detection of equine herpesvirus-1 antigen and the specific antibody by enzyme-linked immunosorbent assay. Am. J. Vet. Res., 44, 1930-1934.
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| 9. | Gunn H.M. (1992). A direct fluorescent antibody technique to diagnose abortion caused by equine herpesvirus. Irish Vet. J., 44, 37-40.
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| 10. | Lawrence G.L., Gilkerson J., Love D.N., Sabine M. & Whalley J.M. (1994). Rapid, single-step differentiation of equid herpesvirus 1 and 4 from clinical material using the polymerase chain reaction and virus-specific primers. J. Virol. Methods, 47, 59-72.
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| 11. | O'Keefe J.S., Julian A., Moriarty K., Murray A. & Wilks C.R. (1994). A comparison of the polymerase chain reaction with standard laboratory methods for the detection of EHV-1 and EHV-4 in archival tissue samples. N.Z. Vet. J., 42, 93-96.
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| 12. | Schultheiss P.C., Collins J.K. & Carman J. (1993). Use of an immunoperoxidase technique to detect equine herpesvirus-1 antigen in formalin-fixed paraffin-embedded equine fetal tissues. J. Vet. Diagn. Invest., 5, 12-15.
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| 13. | Telford E.A.R., Watson M.S., McBride K. & Davison A.J. (1992). The DNA sequence of equine herpesvirus-1. Virology, 189, 304-316.
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| 14. | Telford E.A.R., Watson M.S., Perry J., Cullinane A.A. & Davison A.J. (1998). The DNA sequence of equine herpesvirus 4. J. Gen. Virol., 79, 1197-1203.
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| 15. | Thomson G.R., Mumford J.A., Campbell J., Griffiths L. & Clapham P. (1976). Serological detection of equid herpesvirus 1 infections of the respiratory tract. Equine Vet. J., 8, 58-65.
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| 16. | Varrasso A., Dynon K., Ficorilli N., Hartley C.A., Studdert M.J. & Drummer H.E. (2001). Identification of equine herpesviruses 1 and 4 by polymerase chain reaction. Aust. Vet. J., 79, 563-569.
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| 17. | Wagner W.N., Bogdan J., Haines D., Townsend H.G.G. & Misra V. (1992). Detection of equine herpesvirus and differentiation of equine herpesvirus type 1 from type 4 by the polymerase chain reaction. Can. J. Microbiol., 38, 1193-1196.
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| 18. | Welch H.M., Bridges C.G., Lyon A.M., Griffiths L. & Edington N. (1992). Latent equid herpesviruses 1 and 4: detection and distinction using the polymerase chain reaction and cocultivation from lymphoid tissues. J. Gen. Virol., 73, 261-268.
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| 19. | Whitwell K.E., Gower S.M. & Smith K.C. (1992). An immunoperosidase method applied to the diagnosis of equine herpesvirus abortion, using conventional and rapid microwave techniques. Equine Vet. J., 24, 10-12.
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NB: There are OIE Reference Laboratories for Equine rhinopneumonitis (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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