Assessing the efficacy of a live vaccine against avian encephalomyelitis virus.

Avian encephalomyelitis virus (AEV) causes typical neurological symptoms in young chicks and a transient drop in egg production and hatchability in adult laying birds, resulting in huge economic losses in the poultry industry. An effective way to control and prevent this disease is vaccination of the flocks. Here, we assessed the efficacy of the live vaccine candidate strain GDt29 against avian encephalomyelitis virus. The GDt29 strain has low virulence, was confirmed safe, and showed no signs of pathogenicity. High titers of AEV-specific antibodies were detected in GDt29-vaccinated hens (S/P > 3.0) and their progeny (S/P > 2.0). Moreover, the eggs of GDt29-vaccinated hens with high levels of maternal antibodies were hatched successfully regardless of challenge with a heterologous AEV strain, and the GDt29 attenuated vaccine showed higher protective efficacy against AEV than the commercial vaccine. Furthermore, contact-exposed chicks bred with GDt29-vaccinated birds generated high titers against AE virus (S/P > 2.8). Collectively, our studies are proof of the principle that GDt29 might be an ideal vaccine candidate to prevent AEV infection, and they highlight the utility of using a live vaccine against AEV.

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.

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.

Effects of blood sample mishandling on ELISA results for infectious bronchitis virus, avian encephalomyelitis virus and chicken anaemia virus.

This study determined the effect of sample mishandling on the performance of ELISAs for detection of antibodies against infectious bronchitis virus (IBV), avian encephalomyelitis virus (AEV) and chicken anaemia virus (CAV) in the serum of chickens. The effects of five different sample mishandling treatments were assessed: heat treatment, repetitive freezing and thawing and three levels of severity of haemolysis. These mishandling treatments simulated different conditions that might occur during routine blood collection, transport or storage in a clinical practice setting. Each mishandling treatment was experimentally applied under laboratory conditions and then samples were assayed for antibodies against IBV, AEV and CAV using commercial ELISA kits. Severe haemolysis had the most consistent detrimental effect on ELISA performance, producing results that were significantly different from the reference standard in all three ELISAs, although the direction of the effect varied (less positive for the IBV and CAV assays; more positive for the AEV assay). Moderate levels of haemolysis had a similar, but less consistent, effect to that of severe haemolysis, producing results that were significantly different from the reference standard only for the IBV (less positive) and AEV (more positive) ELISAs. Repetitive freeze-thawing also produced a significant effect on ELISA results for IBV (less positive) and AEV (more positive). The IBV ELISA appeared to be most susceptible to the effects of serum maltreatment. The findings from this study suggest that unpredictable variation in the results of ELISAs can occur due to different sample mishandling treatments.

The VP1 protein of avian encephalomyelitis virus is a major host-protective immunogen that serves as diagnostic potential.

Avian encephalomyelitis virus (AEV) is an important pathogen of poultry and is classified as a member of Picornaviridae. To investigate the protective immunity induced by AEV structural proteins, recombinant VP1, VP0, and VP3 proteins were expressed in a baculovirus system. The result of in vivo protection assays shows that the VP1 protein is a major host-protective immunogen against AEV challenge and demonstrates further that the antibody raised against VP1 protein could neutralize more effectively AEV infection than antibody against VP3 or VP0 protein in a virus neutralization test. These purified recombinant proteins were subsequently evaluated as enzyme-linked immunosorbent assay (ELISA) antigens for detection of AEV infection. A total number of 50 positive sera and 30 negative sera were tested for ELISA validation. Results obtained by testing 193 sera from chickens suspected of being infected AEV further showed that the diagnostic sensitivities of the VP1, VP3, and VP0 protein-based ELISAs were 98.1, 80.6, and 51.9%, and their specificities were 100, 87.9, and 81.8%, respectively. Both sensitivity and specificity of the VP1 protein-based ELISA were comparable with a commercially available test, indicating that the VP1 protein has a highly promising and reliable diagnostic potential, and thus is a suitable antigen for ELISA detection of AEV antibodies in chickens.

Evaluation of the efficacy of a live fowlpox-vectored infectious laryngotracheitis/avian encephalomyelitis vaccine against ILT viral challenge.

Infectious laryngotracheitis (ILT) is caused by an alphaherpesvirus, and latency can be produced by previous exposure to vaccine virus. The main sites of latency for the ILT virus have been shown to be the trigeminal ganglion and the trachea. Reactivation of latent virus is one factor related to the production of clinical signs. The development of a genetically engineered ILT vaccine has been suggested for many years as a tool to eliminate viral latency. Several approaches have been suggested. Included among them is the development of a thymidine kinase-deficient mutant or the insertion of ILT viral glycoproteins into a viral vector such as a poxvirus. A commercially available, live, fowlpox-vectored infectious laryngotracheitis + avian encephalomyelitis (FP-LT+AE) vaccine was used in field trials in leghorn pullet flocks and evaluated by tracheal challenge in a laboratory setting with the use of the National Veterinary Services Laboratory (Ames, IA) ILT challenge virus. Interference of the pigeon pox vaccine, which is often administered concurrently with fowlpox vaccine, was also evaluated when given in conjunction with the FP-LT+AE vaccine. Overall, the results indicate that the FP-LT+AE vaccine provides adequate protection against ILT viral challenge. Proper administration is essential. In one flock, inadequate protection was most likely a result of either poor vaccine administration or previous exposure to pox virus. In addition, the simultaneous administration of pigeon pox vaccine did not appear to interfere with protection against ILT viral challenge.

Prevalence of infectious diseases in ring-necked pheasant flocks in Poland.

The health status of ring-necked pheasants in view of the prevalence of infectious diseases was estimated in Polish pheasantries in the years 1997-2000. Anatomicopathological, microbiological and serological examinations were carried out on birds derived from 26 pheasantries, including birds randomly selected from 18 flocks and sick or dead birds sent from 8 pheasantries. Antibodies specific to the following viruses were detected in serum blood samples: HE, AE, AP, REO, AI, Adeno group 1, MD, ND, as well as Mycoplasma gallisepticum specific antibodies. However, in none of the examined flocks was the presence of antibodies against reticuloendoteliosis virus found. Marble spleen disease and salmonellosis proved to be the most frequent cause of death during the growing period.

Serological monitoring on layer farms with specific pathogen-free chickens.

To monitor the existence of avian pathogens in laying chicken flocks, specific pathogen-free (SPF) chickens were introduced into two layer farms and reared with laying hens for 12 months. SPF chickens were bled several times after their introduction and examined for their sero-conversion to avian pathogens. As a result, antibodies to eight or ten kinds of pathogens were detected in SPF chickens on each farm. Antibodies to infectious bronchitis virus (IBV), avian nephritis virus, Mycoplasma gallisepticum and M. synoviae were detected early within the first month. Antibody titer to IBV suggested that the laying chickens were infected with IBV repeatedly during the experiment on both farms. However, antibodies to infectious bursal disease virus and 6 pathogens were not detected.

[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.

Approaches to food-based vaccines for domestic chickens.

Domestic chickens were fed viral vaccines that were applied to the surface of food pellets. Responses were judged by the production of specific antibodies, and compared with the responses obtained when the same vaccines were given by conventional routes. Chickens responded similarly to commercial avian infectious encephalomyelitis vaccine given on food or by eyedrop when antibodies were measured by ELISA, and the vaccine virus spread by contact. Increasing the dose of oral vaccine tenfold gave a more rapid serological response but the levels of antibody were not increased. There was no serological response to commercial infectious laryngotracheitis virus vaccine given on food. An experimental avian adenovirus vaccine produced a serological response when given on food, but higher levels of antibody were produced in response to vaccination by eyedrop. The vaccine virus spread by contact. It was concluded that current avian infectious encephalomyelitis vaccines, and prospective recombinant vaccines based on avian adenovirus vectors, could be delivered on food.

[Development and testing of an indirect ELISA for the detection of antibodies against avian encephalomyelitis]. / Entwicklung und Erprobung eines indirekten ELISA zum Nachweis von Antikörpern gegen die Aviäre Encephalomyelitis.

Because of the intensive efforts required and the dissatisfactory sensitivity achieved with the traditional serological diagnostic techniques for Avian Encephalomyelitis, an ELISA was developed and tested to show the presence of antibodies in chicken serum. Using a multi-phase antigen preparation and optimizing the reagents and serum dilutions, a method was developed which shows considerable advantages in sensitivity, specificity, practicability and costs compared with the Embryo Serum Neutralization Test.

Antibody responses to avian encephalomyelitis virus vaccines when administered by different routes.

Antibody responses to a commercial avian encephalomyelitis virus (AEV) vaccine administered by different routes were measured by an enzyme-linked immunosorbent assay (ELISA). Responses to single doses of vaccine administered by the ocular route to 10% of a flock were comparable with those obtained when all birds received a single dose in the drinking water. However, ocular vaccination of 5% of the flock resulted in significantly lower responses than those obtained when 10% were vaccinated. Maternal antibody was shown by the ELISA to persist in chickens from vaccinated flocks for up to 21 days after hatching. Day-old chickens with serum absorbances of < 0.3 at 492 nm, as determined by the ELISA, were shown to be susceptible to intracerebral challenge with the neurotropic Van Roekel strain of AEV.

Development and application of an improved infectivity assay for the standardization of avian encephalomyelitis vaccines.

An improved infectivity assay for avian encephalomyelitis viruses (AEVs) is described in which susceptible 7-day-old chicken embryos were inoculated with dilutions of a particular AEV strain and, after incubation for 12 days, the presence of viral antigen in brains was detected by an enzyme-linked immunosorbent assay (ELISA). The assay (the brain antigen ELISA), which may be used for the titration of both egg-adapted and non-egg-adapted strains, can be completed within 12 days. For the conventional essays of non-egg-adapted vaccine strains, embryos are infected in ovo and hatched chickens examined for neurological signs after 3-4 weeks. No animal handling facilities are required with the brain antigen ELISA and results of comparable sensitivity to the hatch-out assay were obtained.

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.

[Prospect of avian encephalomyelitis antibodies in breeding farms in Chile]. / Prospección de anticuerpos contra encefalomielitis aviar en planteles de reproductoras en Chile.

Ten different layer and broiler breeding farms, located in the central part of the country were sampled to detect antibodies against the Avian Encephalomyelitis Virus (AEV). Fifty embrionated eggs, per house, were examined using the Embryo Susceptibility Test. Samples of eggs from 21 houses of broiler breeder hens and 9 of breeder layers were collected from all the poultry farms studied. The antigen used in the Embryo Susceptibility Test was prepared from the van Roekel strain for AEV. The results indicated a high degree of specific immunity against the AEV in 86.7% of the sections checked. This immunity could be due to the presence of the causal of AEV in natural form, since the birds in these farms had not been vaccinated against this ornithopathy. This is the first study carried out in Chile which detected the presence of antibodies against AEV in layer and broiler breeding farms, leading to a massive vaccination program against the disease in the country.

Clinical encephalitis following avian encephalomyelitis vaccination in broiler breeder pullets.

Twelve-to-fourteen-week-old broiler breeder pullets and cockerels from two different companies were found to have encephalitis 2 weeks after vaccination with avian encephalomyelitis (AE) vaccine. Histologic examination and virus isolation indicated that affected chickens had AE. All chickens had been vaccinated with either one of two serials of AE vaccine approximately 2 weeks before the onset of clinical signs.

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.