Avian Encephalomyelitis Virus Research Articles

Isolation and characterisation of cytopathic avian enteroviruses from broiler chicks

Cytopathic viruses were isolated from the faeces, pancreas and caecal contents of broiler chicks in two cases of a stunting syndrome. Two representative strains, designated AAF7 and M-8, were identified as enteroviruses on the basis of their size (25 nm in diameter), RNA in the viral core, virus growth in the cytoplasm, resistance to chloroform, trypsin and acid, and partial heat-stabilisation to molar magnesium chloride. They formed irregular plaques in chick kidney cell cultures. Numerous granula eosinophilic inclusions were induced in the cytoplasm of chicken kidney cell cultures infected with the viruses. Both isolates reacted partially with the G4260 strain of avian nephritis virus but not with the VR strain of avian encephalomyelitis virus.

Development and application of an elisa technique for the detection of antibody to avian encephalomyelitis viruses

A rapid sensitive enzyme-linked immunosorbent assay for the detection of antibody to avian encephalomyelitis viruses (AEVs) in chickens using purified antigen is described. The procedure differed from others which have been described for AEV, in that it involved a negative antigen subtraction step which accounted for the variable adhesiveness of chicken sera to plastic surfaces. The procedure was reproducible (between-assay coefficient of variation 8.95 per cent) and a good correlation was observed with results obtained by neutralisation index tests (r = 0.91, P less than 0.1). The assay detects only AEV-specific antibody and allows monitoring of the spread of AEV in flocks.

An Enzyme-Linked Immunosorbent Assay for the Detection of Avian Encephalomyelitis Virus Antigens

An enzyme-linked immunosorbent assay for detection of the antigens of avian encephalomyelitis viruses (AEVs) was developed, using an antigen capture method with affinity-purified anti-AEV immunoglobulins prepared in rabbits. Both the Van Roekel strain and a field strain of AEV could be detected at concentrations of 4 x 10(3) ng/g tissue in the presence or absence of an uninfected chicken embryo extract. The procedure was used to determine the distribution of antigens to the Van Roekel strain after inoculation of 6-day-old chicken embryos. Antigen concentrations were highest in the embryo brain and lower in the heart and other tissues. When the same strain was inoculated intramuscularly into day-old chickens, highest levels of antigen were noted in the pancreas, gizzard, and brain. Patterns of fecal antigen excretion and antibody production were studied after inoculation of the field strain by the intramuscular, intracerebral, and oral routes. Excretion was most prolonged following oral inoculation (4-10 days), although antigen could be detected by 2 days after intracerebral inoculation. Detectable antibody was present by 10 days following infection by each route.

Avian encephalomyelitis virus antibodies in New Zealand chicken flocks

Abstract Extract Madam:— Avian encephalomyelitis (AE) virus is a picornavirus which produces a clinical disease only in young chickens, especially those 2–3 weeks old. Affected birds exhibit ataxia, tremors of the head and neck and there is an average mortality of 10%. (1) Andrews, C. and Pereira, H.G. 1972. Viruses of Vertebrates, 3rd ed, London: Bailliere Tindall. In [Google Scholar] Clear epidemiological evidence linked the clinical form of the disease with a series of egg production drops in 1977 in several North Island multi-age laying farms, which were rearing affected batches of chickens. (2) Howell L.J. (Unpublished data) [Google Scholar] Between 1980 and 1982 Howell tested pooled samples from 40 New Zealand laying chicken flocks (which had not been Ae vaccinated) using the serum neutralization (SN) test. (3) Van der Heide, L. 1975. Isolation and identification of avian pathogens, Edited by: Hitchner, S.B., Domermith, C.H., Purchase, H.G. and Williams, J.E. 197–204. College Station, Texas: AAAP. [Google Scholar] Neutralizing antibody levels indicative of infection with AE virus were detected in 47% of these flocks. (2) Howell L.J. (Unpublished data) [Google Scholar] Recently on agar gel precipitin (AGP) test was described which appeared to offer a more convenient method for detecting antibodies. (4) Girshick, T. and Crary, C.K. Jr. 1982. Preparation of an agar gel precipitating antigen for avian encephalomyelitis and its use in evaluating the antibody status of poultry. Av. Dis., 26: 798–804. [Google Scholar] We present the results obtained with this test, after assaying a selection of samples routinely submitted to the Ruakura Animal Health Laboratory during 1983–84.

Replication of avian encephalomyelitis virus in chick embryo neuroglial cell cultures. Brief report.

Avian encephalomyelitis virus replicated to relatively high titres in chick embryo neuroglial cell cultures, as measured by the indirect fluorescent antibody test, but induced few cytopathic effects. This cell system should provide a good source of virus for serological tests and vaccines.

Improved potency test for live avian encephalomyelitis vaccines

Potency tests were carried out on live avian encephalomyelitis virus (AEV) vaccines. Vaccines were titrated in chick embryo brain cell cultures using an indirect fluorescent antibody test just before vaccination. Groups of chickens were inoculated orally with graded doses of each vaccine. After three weeks serum was taken from the chickens and examined for antibodies to AEV by indirect enzyme-linked immunoassay (ELISA). The chickens were then challenged by intracerebral inoculation of virulent AEV and monitored for signs attributable to clinical AE. The results showed a relationship between virus content, protection and antibody development. It is recommended that serological evaluation using ELISA replace the challenge step in potency tests for live AEV vaccines.

The application of the ELISA to the diagnosis and control of avian encephalomyelitis.

An ELISA for measuring serum antibody against avian encephalomyelitis virus (AEV) was evaluated for its application to the diagnosis and control of avian encephalomyelitis (AE). A scoring system was developed for this ELISA (AE ELISA-Index) so that the overall level of antibody in the flock could be presented in a single, convenient number. During suspected outbreaks of disease thought to have been caused by AEV infection, the AE ELISA-Index increased in sequential serum samples. High levels of antibody against AEV were measured in 13 flocks experiencing egg productivity problems. Variable levels of antibody activity against infectious bronchitis virus (IBV) were also observed in 11 of these flocks. The AE ELISA-Index was correlated with the embryo susceptibility test. Application of the AE ELISA has indicated that natural exposure to the virus does not occur in all flocks, and vaccination failures were detected sufficiently early for revaccination to be administered before the onset of lay.

Detection of avian encephalomyelitis virus

Methods for the detection of two strains of avian encephalomyelitis virus (AEV) in chick embryo brain cell cultures and chickens were compared. It was found that the agar gel precipitin test (AGPT) and the enzyme-linked immunosorbent assay (ELISA) carried out on the serum of inoculated chickens were more sensitive than either the indirect fluorescent antibody test in cell cultures or the detection of clinical signs in chicks. On the basis of results obtained in this experiment the effects were then determined of routes and time of inoculation of chickens on the detection of AEV. It was found that birds infected at two weeks old produced higher antibody titres than one-day-old birds and the AGPT and ELISA detected comparable levels of antibody in them. It was recommended that the tests to detect the presence of AEV as a contaminant of vaccines be replaced by a serological test carried out on chicks inoculated intramuscularly at two weeks old.

Morphological changes of neurons in experimental avian encephalomyelitis.

Cerebrospinal lesions of chicks experimentally infected with avian encephalomyelitis (AE) virus were investigated morphologically. Central chromatolysis in motor neurons was mostly reversible, while affected Purkinje's cells revealed necrosis with glial reaction. Direct fluorescent antibody technique revealed various amounts of granular antigen in Purkinje's cells of all chicks showing marked leg paralysis. AE virus particles, 25nm in diameter, were found in crystalline arays in the cytoplasm of the same cells.

Measurement of antibodies to infectious avian encephalomyelitis virus by elisa

An enzyme-linked immunosorbent assay (ELISA) for the measurement of antibody to avian encephalomyelitis virus (AEV) is described. The wells of microtitre trays were coated with antibody to AEV, to which was bound a partially purified AEV preparation as an immunosorbent. The assay described is specific, in that antibody activity was not detectable in specific-pathogen-free (SPF) chickens except following inoculation with AEV. Inoculation of SPF chickens with a number of other viruses did not induce a detectable response. Furthermore, there was agreement between the ELISA and virus neutralisation test in terms of the rank order of a number of test sera and in the positive/ negative classification of sera. Both ELISA and virus neutralisation were capable of detecting an antibody response within 9 days of primary vaccination indicating that the ELISA was of comparable sensitivity to virus neutralisation. The amount of antibody in a sample was estimated relative to a reference serum preparation, and the results recorded as units of antibody activity. Using this method of data expression it has been possible to demonstrate that these assays are reproducible with little within-assay (co-efficient of variation = 5.6%) or between-assay (co-efficient of variation = 9.8%) variation for the mid-range sample.

Correlation of Serum Antibody Titer for Avian Encephalomyelitis Virus (AEV) in Hens with the Resistance of Progeny Embryos to AEV

Serum antibody titers for avian encephalomyelitis virus of vaccinated breeding hens were correlated with the resistance of the hens' progeny embryos to viral challenge. All of the embryos from hens that had enzyme-linked immunosorbent assay titers greater than 400 were resistant to challenge, and two-thirds of the embryos from hens that had titers greater than 300 were resistant. About one-half of the embryos from hens that had titers from 100 to 300 were immune to challenge.

A Rapid Procedure for the Purification of Avian Encephalomyelitis Viruses

A rapid procedure for the purification of egg-grown or field preparations of avian encephalomyelitis virus (AEV) of neural origin is described. Extracts of infected tissues were clarified and then partly purified with trichlorotrifluorethane (Freon TF), and the virus present was concentrated with polyethylene glycol. The concentrates were then re-extracted with Freon, and a portion was labeled with 125iodine. During subsequent purification steps, virus could be readily detected by monitoring for radioactivity, thus eliminating the need to determine the infectivity in individual fractions or to examine for the presence of virions by electron microscopy. Final purification was achieved by cesium-chloride equilibrium or sucrose-velocity-gradient centrifugation. Virus purified in this manner was shown to be free of tissue debris, to be specific for AEV by immune electron microscopy, and to possess structural proteins characteristic of picornaviruses.

An entero‐like virus associated with the runting syndrome in broiler chickens

A small round unenveloped virus, 31 nm in diameter, and with no obvious surface structure, was identified during the first week of life in the gut contents of broiler chickens which later developed runting. This virus grew in the cytoplasm of the villous epithelial cells of the small intestine, with a predilection for the mid small intestine. Broilers orally infected at 1 day old with a crude inoculum containing the small round virus and reovirus passed abnormal faeces, gained weight and feathered more slowly than controls, which were either uninoculated or inoculated with faeces from SPF chicks. The small round virus was passaged four times through broilers using gut contents collected from experimentally infected birds between 2 and 3 days after inoculation. Clinical signs as described above were obtained at each passage. The small round virus could not be grown serially in cell cultures. However, immunofluorescence showed that viral antigen was synthesised in the cytoplasm of infected chicken embryo liver and chick kidney cell cultures. The small round virus was resistant to pH3. It is suggested that the small round virus is an enterovirus but no evidence for an antigenic relationship with avian encephalomyelitis virus was obtained.

Enzyme-Linked Immunosorbent Assay for Detection of Antibody to Avian Encephalomyelitis Virus in Chickens

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibody to avian encephalomyelitis virus (AEV) has been developed for determining whether existing AEV control programs adequately protect breeder hens. A partially purified AEV antigen was bound to microcuvettes for reaction with specific primary antibody. A second antibody, rabbit anti-chicken immunoglobulin G (IgG) conjugated with horseradish peroxidase, was employed to react with bound primary IgG. The relative amount of bound primary IgG was detected using ortho-phenylenediamine as a substrate for enzymatic production of a chromogen by horseradish peroxidase. Intensity of absorbance of the chromogen at 490 nm was related to the bound primary antibody by the titration method. Negative antisera were surveyed to establish an appropriate positive/negative cutoff level at twice the mean absorbance of negative sera at a 1:100 dilution. The test reagents for the ELISA were optimized by reagent titrations utilizing known positive and negative antisera for discrimination. The optimized ELISA had a coefficient of variation of from 1.2 to 3.3 for within-assay titer and of 2.4 for between-assay mean titer. Even though the ELISA detected only specific IgG, it was as accurate as the virus-neutralization test for evaluating the immune status of hens to AEV. Moreover, the ELISA was more economical in the use of reagents, time, and personnel and was free from dependence on susceptible embryos. Since ELISAs can be standardized and measured with manual or automated instruments, the derived ELISA can be easily and economically used to evaluate the immune status of breeder hens in commercial poultry operations.

Preparation of an Agar-Gel-Precipitating Antigen for Avian Encephalomyelitis and Its Use in Evaluating the Antibody Status of Poultry

An agar-gel-precipitin (AGP) antigen for avian encephalomyelitis virus (AEV) was prepared from infected chicken embryo brains. The antigen could precipitate specific antibodies to AEV. No nonspecific reactions were observed. Results of AGP tests were compared with those of virus-neutralization (VN) tests on both unvaccinated and AEV-vaccinated chickens. The AGP test reliably detected antibodies to AEV as early as four days postinoculation. Antibodies persisted in most vaccinated birds for over one year.

An in vitro Assay for Quantifying the Virus of Avian Encephalomyelitis

Chicken embryo brain (CEB) cell cultures support the replication of embryo-adapted strains, vaccine strains, and field isolates of avian encephalomyelitis virus. A centrifugal force of 1,500 X g was applied during virus adsorption. Viral antigen was detected in the infected cells by using the indirect fluorescent-antibody technique (IFAT). Combining the infectivity of the virus in CEB cell culture with the ability to detect viral antigen by the IFAT resulted in the development of a virus-titration method. This in vitro assay proved to be more sensitive than the standard embryo-inoculation assay. It was concluded that the in vitro assay provides a satisfactory alternative to the embryo-inoculation assay.

Survey of some poultry viruses in Papua New Guinea.

During the years 1974 to 1979 a virological study on domestic poultry throughout Papua New Guinea was conducted involving clinical examination of diseased birds with subsequent attempted virus isolations and serological surveys of free village fowls and commercial poultry. Viruses isolated included those of Newcastle disease, infectious bronchitis, pox, avian encephalomyelitis and adenovirus. Clinical and pathological diagnoses of pox, avian encephalomyelitis, reticuloendotheliosis and Marek's disease were made. The serological survey included tests for Newcastle disease, influenza A, adenovirus, Marek's disease, pox, avian encephalomyelitis and infectious bursal disease virus. Antibody was demonstrated to all of these viruses except for bursal disease.

Studies on the serological detection of antibodies to avian encephalomyelitis virus

An avian encephalomyelitis virus (AEV) antigen, prepared from the gastrointestinal tract of infected chick embryos and partially purified and concentrated by chloroform and polyethylene glycol treatments, exhibited the highest reactivity in the agar-gel precipitin test (AGPT). Antigen used in the passive haemagglutination test (PHAT) that was purified and concentrated yielded higher antibody titres than when untreated crude antigens were used. The use of the AGPT, PHAT and embryo susceptibility test (EST) on chicken breeding flocks with and without a history of previous vaccination against AE revealed that the PHAT was more sensitive in detecting AEV antibodies than the AGPT. The sensitivity of the PHAT was nearly equal to the EST. No correlation was found between the results of the AGPT and the immune status of a flock judged by the EST.

Localization of Viral Protein in Avian-Encephalomyelitis-Virus-Infected Hens

Eighteen 2-year-old hens were infected orally with wild avian encephalomyelitis virus and examined using the immunofluorescent-antibody technique for 30 days. Viral antigen was detected in 10 organ tissues (liver, spleen, kidney, pancreas, and ovary) in 7 birds during the first half of the observation period and sporadically in alimentary tracts of 12 birds throughout this period, especially the lower parts of the intestines, but it was not detected in other organ tissues. Thirteen test-positive hens showed more localization of the antigen than baby chicks of other studies did.

Physico-chemical and morphological characteristics of avian encephalomyelitis virus

Avian encephalomyelitis virus (AEV) was purified from infected chick embryos by a gradient centrifugation in cesium chloride. The virus had a buoyant density of 1.31 to 1.32 g/ml and a sedimentation coefficient of 148 S. The purified AEV was resistant to treatments with chloroform, acid pH or trypsin. The presence of Mg ++ stabilized the virus against heat inactivation (56°C, 1 h). Electron microscopic study showed the virus to be 24 to 32 nm in diameter. The surface structure of the purified virus was not easily discernable. Nevertheless, with uranyl acetate-stained particles, Markham's rotation technique revealed that AEV has five-fold symmetry with 32 or 42 capsomers. Exact classification of AEV awaits characterization of the viral nucleic acid.