Detection of avian encephalomyelitis virus in chickens in Japan using RT-PCR.

A reverse transcription-polymerase chain reaction (RT-PCR) method was developed for broadly detecting the avian encephalomyelitis virus (AEV). The new primers were based on conserved sequences of the 5'-untranslated region of AEV, because the virus was not detected using previous reported RT-PCR. By applying this method to the chicken samples with suspected AEV infection in Japan, we successfully obtained PCR products of the predicted size from all samples, and we confirmed the presence of AEV via sequence analysis.

Serological evidence of avian encephalomyelitis virus and Pasteurella multocida infections in free-range indigenous chickens in Southern Mozambique.

A total of 398 serum samples from free-range indigenous chickens originating from four villages in Southern Mozambique were tested for the presence of avian encephalomyelitis virus (AEV) and Pasteurella multocida (PM) antibodies through commercial enzyme-linked immunosorbent assay (ELISA) kits. AEV and PM antibodies were detected in all villages surveyed. The proportion of positive samples was very high: 59.5% (95% confidence interval (CI) 51.7-67.7%) for AEV and 71.5% (95% CI 67.7-77.3%) for PM. Our findings revealed that these pathogens are widespread among free-range indigenous chickens in the studied villages and may represent a threat in the transmission of AEV and PM to wild, broiler or layer chickens in the region. Further research is warranted on epidemiology of circulating strains and impact of infection on the poultry industry.

A survey for selected avian viral pathogens in backyard chicken farms in Finland.

Backyard poultry are regaining popularity in Europe and increased interest in the health and management of non-commercial farms has resulted. Furthermore, commercial poultry farm owners have become concerned about the risk represented by contagious avian diseases that nearby backyard poultry could transmit. Fifty-one voluntary backyard chicken farms were visited between October 2012 and January 2013. Blood samples and individual cloacal swabs were collected from 457 chickens. In 44 farms (86%), one or more of the tested chickens had antibodies against avian encephalomyelitis and chicken infectious anaemia viruses, 24 farms (47%) had chickens seropositive for infectious bronchitis virus, 10 farms (20%) had chickens seropositive for infectious bursal disease virus, six farms (12%) had chickens seropositive for infectious laryngotracheitis virus and two farms (5.4%) had chickens seropositive for avian influenza virus. No farms had chickens seropositive for Newcastle disease virus. Of the 51 farms, five (10%) had chickens positive for coronavirus reverse transcription polymerase chain reaction. A phylogenetic analysis showed that all backyard chicken coronaviruses collected were QX type infectious bronchitis viruses. All chickens tested for avian influenza and Newcastle disease viruses using real time reverse transcription polymerase chain reaction were negative. To our knowledge, there is no evidence to date to suggest that these diseases would have been transmitted between commercial and non-commercial flocks.

Health evaluation of African penguins (Spheniscus demersus) in southern Africa.

The African penguin (Spheniscus demersus) is an endangered seabird that breeds along the coast of Namibia and South Africa, and disease surveillance was identified as a priority for its conservation. Aiming for the establishment of baseline data on the presence of potential pathogens in this species, a comprehensive health assessment (blood smear examination, haematology, biochemistry and serology) was conducted on samples obtained from 578 African penguins at 11 breeding colonies and a rehabilitation centre. There were 68 penguins that were seropositive for at least one of seven pathogens tested: avian encephalomyelitis virus, avian infectious bronchitis virus, avian reovirus, infectious bursal disease virus, Newcastle disease virus, Mycoplasma gallisepticum and Mycoplasma synoviae. All samples were seronegative for avian influenza virus subtypes H5 and H7 and infectious laryngotracheitis virus. The apparent prevalence of Babesia sp. and Borrelia sp. in blood smears was consistent with previous studies. Babesia-infected individuals had a regenerative response of the erythrocytic lineage, an active inflammatory response and hepatic function impairment. These findings indicate that African penguins may be exposed to conservation-significant pathogens in the wild and encourage further studies aiming for the direct detection and/or isolation of these microorganisms.

Serological evidence of avian encephalomyelitis virus infection associated with vertical transmission in chicks.

Avian encephalomyelitis virus (AEV) can be transmitted both horizontally and vertically. In the present study, we report a typical case of AEV infection in broiler breeder chickens and their progeny identified by clinical survey of the disease, antibody detection, and reverse transcription (RT)-polymerase chain reaction (PCR) assay.

Development of a SYBR Green real-time RT-PCR assay for the detection of avian encephalomyelitis virus.

Avian encephalomyelitis virus (AEV) causes epidemic diseases in poultry worldwide. A SYBR Green real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay was developed for the rapid detection and quantitation of AEV in this study. A pair of specific primers was designed in the highly conserved VP1 gene of this virus. When comparing this assay with conventional RT-PCR, the rRT-PCR assay was 100 times more sensitive and could detect levels as low as 10 standard DNA copies of the AEV SX strain. The specificity of this technique was evaluated in five other avian pathogens. The AEV RNA was detected as early as three days post-infection in chicken embryos. All 18 clinical chicken brains collected from an AEV outbreak in Northwestern China were detected to be positive (100%) using the rRT-PCR assay. However, only 5 of the 18 samples were positive (28%) using the conventional RT-PCR. The results were confirmed by virus isolation in chicken embryos. This high sensitivity, specificity, and simplicity of the SYBR Green rRT-PCR approach can be a more effective method than the conventional one for AEV diagnosis and surveillance.

Development of a non-radioactive digoxigenin cDNA probe for the detection of avian encephalomyelitis virus.

A non-radioactive digoxigenin cDNA probe for detection of avian encephalomyelitis virus (AEV) was developed. The cDNA probe hybridized specifically with a fragment of VP1 gene of AEV genome was found to be sensitive with as little as 10 pg target DNA fragment in a sensitivity test. Infected chicken embryos were strongly labelled by testing the probe against a range of AEV strains, and no non-specific reaction was observed in non-infected chicken embryos as well as five other avian pathogenic virus-infected samples used as negative controls. Furthermore, the cDNA probe was capable of detecting AEV from chicken embryo brain at 3 days post-inoculation as compared with an immunofluorescence assay, which required up to 5 days of incubation in the embryos. In clinical application, five out of 16 clinical brain samples that were negative by the immunofluorescence assay were positive for AEV by the cDNA probe. Both the sensitivity and specificity of the developed cDNA probe indicated that it is a highly promising and reliable diagnostic tool for the detection of AEV infections.

[Ataxia and paralysis in meat chickens, as a result of an infection with avian encephalomyelitis virus in the breeding flock]. / Ataxie en paralyse bij vleeskuikens, als gevolg van een infectie met trilziektevirus op het vermeerderingsbedrijf.

An outbreak of avian encephalomyelitis (AE) in 7 broiler flocks, with signs of ataxia and paralysis is reported. The diagnosis was made by immunofluorescence, histopathology and virus isolation. The breeding flock had a temporary drop in egg production and reduced hatchability caused by late embryonic mortality. The breeding flock had not been vaccinated against AE. The problems were probably caused by vertical transmission of AE 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.

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.

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.

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.

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.

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.

Purification of avian encephalomyelitis virus by ultracentrifugation in a nonlinear cesium chloride gradient.

A rapid and convenient method is described for purifying avian encephalomyelitis virus. Infected embryo homogenate was treated with polyethylene glycol followed by extraction with fluorocarbon. Extracted sample was pelleted at 73,400 x g, resuspended, and subjected to isodensity centrifugation in preformed nonlinear gradient. A distinctive virus band was observed after centrifugation for 120 min at 192,000 x g. The fraction containing the virus band demonstrated the major portion of infectivity and the presence of the virus particles by electron microscopy. The method was reproducible within a limited number of trials.

The pathogenesis of infectious avian encephalomyelitis. 3. The relationship between viraemia, invasion of the brain by the virus, and the development of specific serum neutralising antibody.

An association was demonstrated between the development of clinical infectious avian encephalomyelitis (IAE), the persistence and titre of infectious avian encephalomyelitis virus (IAEV) in the brain of the chicken, the duration of detectable viraemia and the age of the chicken at the time of infection with the virus. The older the chicken at the time of infection the milder the disease, the lower the virus titre in the brain and the shorter the period of viraemia. IAEV serum neutralising antibody was produced earlier after infection in older chickens, and its detection was associated with decreasing virus titres in the brain and the cessation of detectable viraemia. Treatment of chickens with testosterone in ova, to inhibit the development of antibody synthesis, prevented the onset of age-associated resistance and testosterone treated birds were as susceptible to clinical IAE as baby chickens. The results suggested that the ability to produce IAEV serum neutralising antibody was an important component of age-associated resistance to IAE.

Use of polyethylene glycol and fluorocarbon for the purification of avian encephalomyelitis virus.

A simple method is described for isolating avian encephalomyelitis virus (AEV) from a bulk amount of proteins and lipids in a homogenate of infected whole chicken embryos. More than 90% of the virus in the homogenate was precipitated by 4% polyethylene glycol (PEG), and the precipitate was free of approximately 90% of the total nonviral protein. When the homogenate was extracted with fluorocarbon, the aqueous fraction retained the full virus infectivity and about 70% of the protein but was free from most lipid materials. A two-step procedure (precipitation of the homogenate by 4% PEG, followed by extraction of the precipitate by fluorocarbon) gave a 50-fold purification of AEV over the original homogenate. The procedure is simple, rapid, and effective in removing nonviral proteins and lipids from host tissues. It is useful as an initial treatment of embryonic materials for the purification of AEV.

The immunisation of chickens against infectious avian encephalomyelitis.

An immune response was induced in chickens vaccinated with the NSW-1 virus by the intramuscular, eye drop, wing web or oral methods. The oral procedure was the most sensitive in inducing an immune response with the lowest dose of virus. The wing web method was the least sensitive. Clinical IAE developed in 1 chicken following intramuscular vaccination. The virus was recovered from the faeces of chickens vaccinated orally with 700 CID50 of the virus from the third to the tenth day after vaccination.

Emergence pattern of egg-adapted avian encephalomyelitis virus by alternating passage in chickens and embryos.

The chance of inducing complete egg-adaptation was slight in alternating passage in chickens and embryos with chicken-brain-passaged viruses of wild avian encephalomyelitis viruses: egg-adaptation was accomplished by use of an incipient egg-adapting mutant, but failed with other passaged virus.