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SUMMARY
Equine infectious anaemia (EIA) is caused by a lentivirus. It can be diagnosed on the basis of clinical signs, pathological lesions and serology. Infected horses can remain viraemic carriers for life and will yield a positive serological result. Antibody response usually persists and antibody-positive animals, older than 6 months, are identified as virus carriers (below 6 months of age, serological reactions can be due to maternal antibodies). They can potentially transmit the infection to other horses.
Identification of the agent: Virus from a horse can be isolated by inoculating suspect blood into a susceptible horse or on to leukocyte cultures prepared from susceptible horses. Recognition of infection in horses that have been inoculated experimentally may be made on the basis of clinical signs, haematological changes and a positive antibody response determined by an immunodiffusion test or enzyme-linked immunosorbent assay (ELISA). A successful virus isolation in horse leukocyte cultures is confirmed by the detection of specific EIA antigen, by immunofluorescence assay, polymerase chain reaction, reverse-transcriptase assay, or by the inoculation of culture fluids into susceptible horses. Virus isolation is rarely attempted because of the time, difficulty and expense involved.
Serological tests: Agar gel immunodiffusion (AGID) tests and ELISAs are simple, reliable tests for the demonstration of EIA virus infection. When ELISAs are positive they should be confirmed using the AGID test. The precipitating antigen can be prepared from infected tissue cultures or by using recombinant DNA technology.
Requirements for vaccines and diagnostic biologicals: There are no biological products currently available.
A. INTRODUCTION
Equine infectious anaemia (EIA) occurs world-wide. The infection is limited to equids. The disease is characterised by recurrent febrile episodes, thrombyctopenia, anaemia, rapid loss of weight and oedema of the lower parts of the body; it tends to become an inapparent infection if death does not result from one of the acute clinical attacks. The incubation period is normally 1-3 weeks, but may be as long as 3 months. In acute cases, lymph nodes, spleen and liver are hyperaemic and enlarged. Histologically these organs are infiltrated with nests of immature lymphocytes and plasma cells. Kupffer cells in the liver often contain haemosiderin or erythrocytes. The enlarged spleen may be felt on rectal examination.
Once a horse is infected with EIA virus, its blood remains infectious for the remainder of its life. This means that the horse is a viraemic carrier and can potentially transmit the infection to other horses (4). Transmission occurs by transfer of blood from an infected horse. In nature, spread of the virus is most likely via interrupted feeding of bloodsucking horseflies on a clinically ill horse and then on susceptible horses, or from the use of contaminated needles. However, in utero infection of the fetus does occur (8). The virus titre is higher in horses with clinical signs and the risk of transmission is higher from these animals than the carrier animals with a lower virus titre.
B. DIAGNOSTIC TECHNIQUES
Agar gel immunodiffusion (AGID) tests (7) and enzyme-linked immunosorbent assays (ELISAs) (15) are accurate, reliable tests for the detection of EIA in horses, except for animals in the early stages of infection and foals of infected dams. In other rare circumstances, misleading results may occur when the level of virus circulating in the blood during an acute episode of the disease is sufficient to bind available antibody, and if initial antibody levels never rise high enough to be detectable (16). Although the ELISA will detect antibodies somewhat earlier and at lower concentrations than the AGID test, positive ELISAs are confirmed using the AGID test. This is because false-positive results have been noted with the ELISA. The AGID test also has the advantage of distinguishing between EIA and non-EIA antigen-antibody reactions by lines of identity.
The EIA virus is a lentivirus, a subfamily of the Retroviridae, which also includes maedi-visna, caprine arthritis/encephalitis, bovine immunodeficiency and the human immunodeficiency viruses. Nucleic acid sequence comparisons have demonstrated a marked relatedness among these viruses.
| 1. | Identification of the agent
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| | Virus isolation is usually not necessary to make a diagnosis.
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| | Isolation of the virus from suspect horses may be made by inoculating their blood on to leukocyte cultures prepared from horses free of infection. Virus production in cultures can be confirmed by detection of specific EIA antigen by ELISA (14), by imunofluorescence assay (18), or by subinoculation into susceptible horses. Virus isolation is rarely attempted because of the difficulty of growing horse leukocyte cultures.
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| | A nested polymerase chain reaction assay to detect EIA proviral DNA from the peripheral blood of horses has been described (12).
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| | When the exact status of infection of a horse cannot be ascertained, the inoculation of a susceptible horse with suspect blood should be employed. In this case a horse that has previously been tested for antibody and shown to be negative is given an immediate blood transfusion from the suspect horse, and its antibody status and clinical condition are monitored for at least 45 days. Usually, 1-25 ml of whole blood given intravenously is sufficient to demonstrate infection, but in rare cases it may be necessary to use a larger volume of blood (250 ml) or washed leukocytes from such a volume (5).
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| 2. | Serological tests
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| | Due to the persistence of EIA virus in infected equids, detection of serum antibody to EIA virus confirms the diagnosis of EIA virus infection.
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Agar gel immunodiffusion test (the prescribed test for international trade)
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Precipitating antibody is rapidly produced as a result of EIA infection, and can be detected by the AGID test. Specific reactions are indicated by precipitin lines between the EIA antigen and the test serum and confirmed by their identity with the reaction between the antigen and the positive standard serum. Horses in the first 2-3 weeks after infection will usually give negative serological reactions. In rare cases the post-infection time prior to the appearance of detectable antibody may extend up to 60 days.
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Reagents for AGID are available commercially from several companies. Alternately, AGID antigen and reference serum may be prepared as described below.
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Preparation of antigen
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Specific EIA antigen may be prepared from the spleen of acutely infected horses (6), from infected equine tissue culture (10), from a persistently infected canine thymus cell line (3), or from proteins expressed in bacteria or baculovirus using the recombinant DNA technique (2, 9). Preparation from infected cultures or from recombinant DNA techniques gives a more uniform result than the use of spleen cells and allows for better standardisation of reagents.
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To obtain a satisfactory antigen from spleen, a horse must be infected with a highly virulent strain of EIA virus. The resulting incubation period should be 5-7 days, and the spleen should be collected 9 days after inoculation, when the virus titre is at its peak and before any detectable amount of precipitating antibody is produced. Undiluted spleen pulp is used in the immunodiffusion test as antigen (6). Extraction of antigen from the spleen with a saline solution and concentration with ammonium sulphate does not give as satisfactory an antigen as selection of a spleen with a very high titre of EIA antigen.
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Alternatively, equine fetal kidney or dermal cells or canine thymus cells are infected with a strain of EIA virus adapted to grow in tissue culture (American Type Culture Collection). Virus is collected from cultures by precipitation with 8% polyethylene glycol or by pelleting by ultracentrifugation. The diagnostic antigen, p26, is released from the virus by treatment with detergent or ether (10). EIA virus core proteins, expressed in bacteria or baculovirus, are commercially available and find practical use as high quality antigens for serological diagnosis.
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The p26 is an internal structural protein of the virus that is coded for by the gag gene. The p26 is more stable among EIAV strains than the virion glycoproteins gp45 and gp90 (11). There is evidence of minor strain variation in the p26 amino acid sequence; however there is no evidence to indicate that this variation influences any of the diagnostic tests (19).
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Preparation of standard antiserum
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A known positive antiserum may be collected from a horse previously infected with EIA virus. This serum should yield a single dense precipitation line that is specific for EIA, as demonstrated by a reaction of identity with a known standard serum. It is essential to balance the antigen and antibody concentrations in order to ensure the optimal sensitivity of the test. Reagent concentrations should be adjusted to form a narrow precipitation line approximately equidistant between the two wells containing antigen and serum.
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Test procedure (1, 65, 13)
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Immunodiffusion reactions are carried out in a layer of agar in Petri dishes. For Petri dishes that are 100 mm in diameter, 15-17 ml of 1% Noble agar is used. Six wells are punched out of the agar surrounding a centre well of the same diameter. The wells are 5.3 mm in diameter and 2.4 mm apart. Each well must contain the same volume of reagent.
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The antigen is placed in the central well and the standard antiserum is placed in alternate exterior wells. Serum samples for testing are placed in the remaining three wells.
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The dishes are maintained at room temperature in a humid environment.
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After 24-48 hours the precipitation reactions are examined over a narrow beam of intense, oblique light and against a black background. The reference lines should be clearly visible at 24 hours, and at that time any test sera that are strongly positive may also have formed lines of identity with those between the standard reagents. A weakly positive reaction may take 48 hours to form and is indicated by a slight bending of the standard serum precipitation line between the antigen well and the test serum well. Sera with high precipitating antibody titres may form broader precipitin bands that tend to be diffuse. Such reactions can be confirmed as specific for EIA by dilution at 1/2 or 1/4 prior to retesting; these then give a more distinct line of identity. Sera devoid of EIA antibody will not form precipitation lines and will have no effect on the reaction lines of the standard reagents.
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Interpretation of the results: Horses that are in the early stages of an infection may not give a positive serological reaction in an AGID test. Such animals should be bled again after 3-4 weeks.
In order to make a diagnosis in a young foal, it may be necessary to determine the antibody status of the dam. If the mare passes EIA antibody to the foal though colostrum, then a period of 6 months or longer after birth must be allowed for the maternal antibody to wane. Sequential testing of the foal at monthly intervals may be useful to observe the decline in maternal antibody. To conclude that the foal is not infected, a negative result must be obtained (following an initial positive result) at least 2 months after separating the foal from contact with the EIA positive mare or any other positive horse.
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| | b) | Enzyme-linked immunosorbent assay
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| | | There are three ELISAs that are approved by the United States Department of Agriculture for the diagnosis of equine infectious anaemia and are available internationally; a competitive ELISA and two non-competitive ELISAs. The competitive ELISA and one non-competitive ELISA detect antibody produced against the p26 core protein antigen. The other non-competitive ELISA incorporates both p26 core protein and gp45 (viral transmembrane protein) antigens. Typical ELISA protocols are used in all tests. If commercial ELISA materials are not available, a non-competitive ELISA using p26 antigen purified from cell culture material may be employed (14).
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| | | A positive test result by ELISA should be retested using the AGID test to confirm the diagnosis because some false-positive results have been noted with the ELISA. The results can also be confirmed by the immunoblot technique. A standard antiserum for immunodiffusion, which contains the minimum amount of antibody that should be detected by laboratories, is available from the OIE Reference Laboratories. Uniform methods for EIA control have been published (17).
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C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS
No biological products are available currently.
REFERENCES
| 1. | Association française de normalisation (AFNOR) (2000). Animal Health Analysis Methods. Detection of Antibodies against Equine Infectious Anaemia by the Agar Gel Immunodiffusion Test. NF U 47-002. AFNOR, 11 avenue Francis de Pressensé, 93571 Saint-Denis La Plaine Cedex, France.
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| 2. | Archambault D., Wang Z., Lacal J.C., Gazit A., Yaniv A., Dahlberg J.E. & Tronick S.R. (1989). Development of an enzyme-linked immunosorbent assay for equine infectious anaemia virus detection using recombinant Pr55gag. J. Clin. Microbiol., 27, 1167-1173.
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| 3. | Bouillant A.M.P., Nelson K., Ruckerbauer C.M., Samagh B.S. & Hare W.C.D. (1986). The persistent infection of a canine thymus cell line by equine infectious anaemia virus and preliminary data on the production of viral antigens. J. Virol. Methods, 13, 309-321.
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| 4. | Cheevers W.M. & McGuire T.C. (1985). Equine infectious anaemia virus; immunopathogenesis and persistence. Rev. Infect. Dis., 7, 83-88.
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| 5. | Coggins L. & Kemen M.J. (1976). Inapparent carriers of equine infectious anaemia (EIA) virus. In: Proceedings of the IVth International Conference on Equine Infectious Diseases. University of Kentucky, Lexington, Kentucky, USA, 14-22.
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| 6. | Coggins L., Norcross N.L. & Kemen M.J. (1973). The technique and application of the immunodiffusion test for equine infectious anaemia. Equine Infect. Dis., III, 177-186.
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| 7. | Coggins L., Norcross N.L. & Nusbaum S.R. (1972). Diagnosis of equine infectious anaemia by immunodiffusion test. Am. J. Vet. Res., 33, 11-18.
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| 8. | Kemen M.J. & Coggins L. (1972). Equine infectious anaemia: transmission from infected mares to foals. J. Am. Vet. Med. Assoc., 161, 496-499.
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| 9. | Kong X. K., Pang H., Sugiura T., Sentsui H., Onodera T., Matsumoto Y. & Akashi H. (1997). Application of equine infectious anaemia virus core proteins produced in a Baculovirus expression system, to serological diagnosis. Microbiol. Immunol., 41, 975-980.
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| 10. | Malmquist W.A., Barnett D. & Becvar C.S. (1973). Production of equine infectious anaemia antigen in a persistently infected cell line. Arch. Gesamte Virusforsch., 42, 361-370.
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| 11. | Montelaro R.C., Parekh B., Orrego A. & Issel C.J. (1984). Antigenic variation during persistent infection by equine infectious anaemia, a retrovirus. J. Biol. Chem., 16, 10539-10544.
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| 12. | Nagarajan M.M. & Simard C. (2001). Detection of horses infected naturally with equine infectious anemia virus by nested polymerase chain reaction. J. Virol. Methods, 94, 97-109.
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| 13. | Pearson J.E. & Coggins L. (1979). Protocol for the immunodiffusion (Coggins) test for equine infectious anaemia. Proc. Am. Assoc. Vet. Lab. Diagnosticians, 22, 449-462.
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| 14. | Shane B.S., Issel C.J. & Montelaro R.C. (1984). Enzyme-linked immunosorbent assay for detection of equine infectious anemia virus p26 antigen and antibody. J. Clin. Microbiol., 19, 351-355.
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| 15. | Suzuki T., Ueda S. & Samejina T. (1982). Enzyme-linked immunosorbent assay for diagnosis of equine infectious anaemia. Vet. Microbiol., 7, 307-316.
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| 16. | Toma B. (1980). Réponse sérologique négative persistante chez une jument infectée. Rec. Med. Vet., 156, 55-63.
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| 17. | United States Department of Agriculture Animal and Plant Health Inspection Service (2002). Equine Infectious Anemia Uniform Methods and Rules. http://www.aphis.usda.gov/oa/pubs/eiaumr.pdf
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| 18. | Weiland F., Matheka H.D. & Bohm H.O. (1982). Equine infectious anaemia: detection of antibodies using an immunofluorescence test. Res. Vet. Sci., 33, 347-350.
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| 19. | Zhang W., Auyong D.B., Oaks J.L. & McGuire T.C. (1999). Natural variation of equine infectious anemia virus Gag-protein cytotoxic T lymphocyte epitopes. Virology, 261, 242-252.
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NB: There are OIE Reference Laboratories for Equine infectious anaemia (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).
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