Vaccination of chicken embryos with escape mutants of La Sota Newcastle disease virus induces a protective immune response.

To reduce the embryonic pathogenicity of Newcastle disease virus (NDV), escape mutants of the La Sota strain were produced with selected monoclonal antibodies. Immunoselection resulted in the elimination of an epitope by single amino acid substitution (F and HN molecule) or in a conformational change (HN molecule). The embryonic pathogenicity of these escape mutants was reduced and their dose was optimised for in ovo vaccination. Because antibody responses and protection of in ovo vaccinated chicks were similar to controls vaccinated at hatch with the La Sota strain, immunoselection appears a valuable technique to produce attenuated NDV strains, which are candidate in ovo vaccines.

Phylogenetic analysis of fowl adenoviruses.

The sequences of the L1 loop of the hexon protein from representative fowl adenovirus (FAdV) strains of the different European and American collections were determined and compared. This study highlighted the lack of consensus in the numbering of the individual serotypes between the American and the European classifications. An identification system is proposed based on restriction fragment length polymorphism of the hexonA/hexonB polymerase chain reaction product. In addition, new insights into the relationships among FAdV strains are presented and discussed on the basis of phylogenetic analysis of the L1 loops sequences. Six clusters of strains that are supported by high bootstrap values were identified. Three of them are clearly independent, forming groups A, B and C, whereas the three others are clustered in a single 'supergroup', denominated D. Interestingly, the Japanese strain TR22 that is presently classified as European type 5 (species B) could not be assigned to any of the aforementioned clusters and might therefore constitute the sole representative of a seventh cluster.

Assessment of the cell-mediated immune response in chickens by detection of chicken interferon-gamma in response to mitogen and recall Newcastle disease viral antigen stimulation.

The potential of a capture enzyme-linked immunosorbent assay (ELISA) specific for chicken interferon-gamma (ChIFN-gamma) has been evaluated as a tool to assess cell-mediated immunity (CMI) in the chicken. In a first step, ChIFN-gamma production and cell proliferation of mitogen-activated chicken splenocytes have been compared. In general, for each of the stimulation conditions where significant proliferation was observed, production of ChIFN-gamma could be measured by ELISA. In our hands, the combination of ionomycin and phorbol-12-myristate 13-acetate or the use of recombinant chicken interleukin-2 gave the most satisfactory results. Then, the CMI response induced by live or killed Newcastle disease virus (NDV) vaccines has been evaluated sequentially by ex vivo antigen-specific ChIFN-gamma production and cell proliferation of splenocytes from immune chickens. The ex vivo data showed that both types of NDV vaccines are capable of stimulating CMI responses to NDV in chickens as measured by the ChIFN-gamma ELISA. However, most of the chickens vaccinated with the live vaccine produced ChIFN-gamma after antigen recall stimulation, from 2 to 4 weeks after vaccination, when only some chickens vaccinated with the inactivated vaccine showed a specific response 4 weeks after vaccination. No significant proliferative responses to either NDV vaccine were detectable during the 4 weeks of the study. From our results, it appears that antigen-specific ChIFN-gamma production can be used as a good indicator of actively acquired immunity to NDV and that the sensitivity range of the capture ELISA test is well adequate to measure ex vivo release of ChIFN-gamma.