P.B. Spradbrow

Mucosal immunity in chickens vaccinated with the V4 strain of Newcastle disease virus

Chickens vaccinated orally with the V4 strain of Newcastle disease virus (NDV) and possessing low levels or undetectable levels of serum haemagglutination-inhibition (HI) antibodies against NDV may resist challenge with virulent virus. Evidence for vaccine-induced mucosal immunity was sought. HI antibodies were detected in serum, lachrymal fluid and tracheal washing after vaccination with V4 virus by intranasal, eyedrop or intracrop routes. IgA was detected by immunodiffusion in lachrymal fluid of both vaccinated and control birds. Lymphoid accumulations were detected in tracheas of chickens after vaccination and there were significant increases in the numbers of plasma cells in sections of Harderian glands from chickens after vaccination. In a second experiment, ELISA was used to demonstrate the production of NDV-specific IgA which was detected in serum, lachrymal fluid, tracheal washing and intestinal washing after intracrop or eyedrop vaccination with V4 virus. It was concluded that oral vaccination of chickens with V4 virus induces a mucosal immune response.

Field isolates of fowlpox virus contaminated with reticuloendotheliosis virus

The polymerase chain reaction (PCR) method was used to examine samples from field cases of fowlpox for the presence of reticuloendotheliosis virus (REV). The S-strain fowlpox vaccine, known to be contaminated with REV, served as a positive control. Fowlpox virus was grown from field samples and vaccines by inoculation of embryonated hen eggs by the chorioallantoic membrane (CAM) route. DNA was extracted from the CAM lesions and examined for REV proviral sequences using primers specific for the long terminal repeats of REV. Amplicons of the expected length were detected in all the 45 field samples from poultry and in the S strain vaccine. Two other vaccines and two isolates from wild birds contained no detectable REV sequences. The PCR products from the vaccine and one field isolate were sequenced and were identical. These products showed 81 to 87.5% homology with the published sequences for the long terminal repeats of REV. It was not determined whether the REV proviral DNA was integrated with cellular DNA, fowlpox DNA or both. Inoculation of day-old chickens with the S-strain vaccine resulted not only in the production of fowlpox lesions but also feathering defects and proventriculitis. This suggests that the REV present in the vaccine is replication competent. Problems being encountered with protection from fowlpox following vaccination in Australia might be attributed to simultaneous challenge with fowlpox virus and REV.

Recent isolates of Newcastle disease virus in Australia

Forty-five recently isolated strains of Newcastle disease virus and the V4 vaccine strain of Newcastle disease virus were used to infect experimental chickens. Neither V4 nor any of the new strains produced detectable clinical disease. All the viruses produced an antibody response and spread by contact. Some of the newly isolated viruses produced a more rapid serological response than V4 virus did. Dual or multiple infections with one of the new strains of Newcastle disease virus, infectious bronchitis virus and Escherichia coli did not enhance the pathogenicity of any of the agents.

Serological response of chickens to oral vaccination with newcastle disease virus

Conventional Newcastle disease vaccines are not suitable for application to village chickens in tropical countries of Asia. Trials with food-based vaccines are being initiated and the following experiments were performed to evaluate oral vaccination with Newcastle disease virus. Experimental chickens were vaccinated orally with the avirulent V4 strain of Newcastle disease virus and haemagglutination-inhibition antibody responses were measured. V4 virus was introduced into the crop by tube and total faecal output was collected daily and assayed for Newcastle disease virus. Virus was recovered on Days 5 and 6 after vaccination from most chickens that had received 10(7.4) and 10(6.4) 50% egg-infectious doses (EID50) of virus. There was no recovery of virus from birds receiving a lower dose of vaccine. Groups of chickens kept in cages with wire floors were given various doses of vaccine into the crop. Higher antibody titres were achieved with higher doses of virus. This dose responsiveness was not observed when various doses of vaccine were presented on food pellets and the groups of chickens were kept on concrete floors. Similar antibody responses were then seen with nominal doses of 10(5.2) and 10(8.2) EID50 per bird, possibly as a result of excretion and re-ingestion of the vaccine virus. Spread of the vaccine virus was demonstrated when control chickens and chickens receiving 10(7.7) EID50 of V4 virus on food pellets were housed together on a concrete floor. Similar antibody titres were achieved in both vaccinated and in-contact chickens.

Cell-mediated immunity in chickens vaccinated with the V4 strain of Newcastle disease virus

A leukocyte migration inhibition assay was used to demonstrate antigen-specific cell-mediated immunity (CMI) in chickens vaccinated with the V4 strain of Newcastle disease virus. Chickens were vaccinated when 5 weeks old, and again 3 and 7 weeks later. CMI was detected 9 days after initial vaccination by eyedrop, but only after the second dose of vaccine delivered into the crop. In some chickens there was a temporary suppression of CMI to Newcastle disease virus, especially after the initial application of vaccine to the crop. Haemagglutination inhibition antibodies developed to similar levels in chickens after vaccination by either route. There was no obvious correlation between antibody and CMI responsiveness.

Molecular studies on avian strains of Pasteurella multocida in Australia.

A collection of 45 strains of Pasteurella multocida was assembled. The strains had been isolated from cases of fowl cholera in eastern Australia over 8 years, and included mainly type A strains. All the strains were examined for plasmids and resistance to 10 antimicrobial agents and most of the strains were examined for restriction fragment length polymorphism. Nine strains were assayed for pathogenicity for mice. Twenty strains yielded no plasmid. Seven contained a single plasmid of 1.3 kbp and 18 contained 2 plasmids, of 2.4 and 7.5 kbp. All the strains were resistant to streptomycin, trimethoprim and lincomycin while one strain was resistant to tetracycline. There was no correlation between plasmid content and resistance to antimicrobial agents. Three strains that lacked plasmids were highly virulent for mice, 6 strains containing plasmids were not. Restriction fragment length polymorphism generated by HpaII allowed the 39 strains that were tested to be divided into 10 groups.

The contribution of lectins to the interaction between oral Newcastle disease vaccine and grains

Successful oral vaccination of chickens with Newcastle disease (ND) depends on the survival of vaccine virus on the grains that are used as carriers. Some interactions between grains and the V4 strain of ND virus (NDV) were studied. Crude saline washings were prepared from several grains - rice (unhusked, brown, white and boiled white), sorghum, millet, wheat, maize and barley - and tested for lectin activity, as indicated by agglutination of chicken erythrocytes. Only washings from unhusked rice, sorghum and millet failed to haemagglutinate. None of the crude washings antagonised the haemagglutinating activity of NDV, and the washing from white rice produced an 8-fold enhancement. The presence of lectins in the washings from rice, wheat and barley was confirmed by purifying a substance with N-acetyl-D-glucosamine specificity. Only the crude extract from white rice had any profound effect on infectivity, reducing the infectivity titre by 99.99%. It is not known if the viricidal substance is identical with the lectin. Of 9 commercial lectins tested, only ConA bound the V4 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.

Australian studies on Newcastle disease virus. The French heritage.

Eric French contributed greatly to the early Australian studies on Newcastle disease virus, producing the foundations on which subsequent Australian studies were based. In 1964 he conducted the first major serological survey for Newcastle disease in the Australian poultry flock, and showed that the pathotypes of the virus recognised at that time were not present. After the isolation of strain V4 in 1966, he initiated some of the first studies on the nature of this stain. In particular, he demonstrated the avirulence of this virus, its ability to infect chickens when delivered orally with food and its potential utility as a vaccine. Subsequent studies by other workers included the development of strain V4 as a conventional vaccine and as a vaccine suitable for use in village chickens.