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.

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.

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.

Avian encephalomyelitis virus in chicken pancreatic cell cultures.

Monolayer cell cultures consisting of epithelioid cells were made from pancreatic tissue of 10-to-13-day-old chicks. The maximum virus titer of the cell-culture fluid was obtained 8 days after inoculation with an embryo-adapted avian encephalomyelitis virus (AEV). Virus titers also increased in cell cultures inoculated with a chick-pancreas-passed AEV or a field isolant. Cell cultures inoculated with 3 strains of AEV maintained virus titers of 10(2.9)-10(3.7) 50% embryo-infective doses/ml for 15-20 days. In other cell cultures from pancreatic tissues of chicks preinfected orally with the chick-pancreas-passed AEV or the field isolant, the virus titers decreased for several days after cultivation and thereafter increased and persisted until at least the 25th or 30th day. Neither a cytopathic effect nor any inclusion body was observed in the cell cultures infected with AEV. No AEV-antigen-positive cell was detected by direct fluorescent-antibody technique.

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.

Immunity levels against avian encephalomyelitis in vaccinated poultry breeder flocks in Maine.

Four commercial poultry breeder flocks that were vaccinated under field conditions against avian encephalomyelitis (AE) with commercial live or inactivated vaccine were monitored periodically by virus-neutralization testing of blood serum samples and by challenge of their progeny eggs and chicks. The history of the flocks and results of the tests indicate that field exposure might occur during the laying period, thereby boosting immunity titers without causing clinical AE in the progeny chicks. The data also indicate field exposure of certain flocks before AE vaccination.

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.

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.

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.

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.

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.

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.

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.

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.

Pancreas-passaged avian encephalomyelitis virus and its immunogenicity.

A serial 34-chicken pancreas passage of avian encephalomyelitis virus by oral administration was successful. Oral inoculation test with 4 passaged viruses showed rapid infection of the duodenal wall and unchangeable infection of pancreas diminishing the viral invasiveness to other organs. The passaged virus caused neither detectable viremia nor clinical avian encephalomyelitis signs and produced neutralizing antibody of high titers.

Immunofluorescent study on egg-adapted avian encephalomyelitis virus infection in chickens.

Fluorescent antibody study showed persistent infection of egg-adapted avian encephalomyelitis virus in the central nervous system and pancreatic tissues of infected embryos and chickens hatched from them. The limited organ tropism of the egg-adapted virus in hatched chickens was in striking contrast to the systemic infection that occurs with a field strain. In chidkens orally infected with egg-adapted virus strains, transient infection of a few organs was found despite occurrence of viremia.

Horizontal transmission of a pancreas-passaged avian encephalomyelitis virus in chicks.

Pancreas-passaged avian encephalomyelitis (AE) virus was transmitted horizontally in a group of 40 (1-day-old) chicks within 3 weeks after they were intermingled with two orally infected 1-day-old chicks. Viral antigen was detected in the pancreas of these contact-exposed chicks. After 5 weeks, contact-exposed chicks developed high titers against AE virus, but the chicks did not develop clinical signs of AE. The passaged virus could not be recovered from feces of six immunized chicks.