RESUMO
The Semliki Forest virus (SFV) expression system was evaluated as a basis for avian vaccine development. Initial studies indicated that 1-day-old specific pathogen-free (SPF) chicks were susceptible to infection with an infectious strain of SFV, producing SFV-specific antibodies but no clinical disease. One-day-old SPF chicks immunised intramuscularly with recombinant replication-defective SFV (rSFV) particles expressing the Escherichia coli (E. coli) lacZ reporter gene developed high titres of beta-gal- specific antibodies at 4 weeks p.i. after two inoculations. In contrast, significantly lower antibody levels were elicited in chicks immunised with a recombinant SFV-based DNA construct or a conventional CMV promoter-based DNA plasmid. rSFV particles encoding the protective VP2 protein or the VP2/VP4/VP3 polyprotein of infectious bursal disease virus (IBDV) were produced and the expressed antigens were characterised in cell culture. Proteins of the correct size were generated and found to react against a range of IBDV-specific monoclonal antibodies. Immunisation of 1-day-old SPF chicks with rSFV particles encoding the IBDV proteins resulted in specific antibodies being elicited in all birds, neutralising antibodies being induced in some but not all birds.
Assuntos
Infecções por Birnaviridae/veterinária , Vírus da Doença Infecciosa da Bursa/genética , Vírus da Doença Infecciosa da Bursa/imunologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/prevenção & controle , Vírus da Floresta de Semliki/genética , Vírus da Floresta de Semliki/imunologia , Vacinas Sintéticas/farmacologia , Vacinas Virais/farmacologia , Animais , Anticorpos Antivirais/sangue , Antígenos Virais/genética , Sequência de Bases , Infecções por Birnaviridae/imunologia , Infecções por Birnaviridae/prevenção & controle , Linhagem Celular , Galinhas , Cricetinae , Primers do DNA/genética , Escherichia coli/genética , Genes Reporter , Vetores Genéticos , Óperon Lac , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Recombinação Genética , Vacinas Sintéticas/genética , Proteínas Virais/genética , Proteínas Virais/imunologia , Vacinas Virais/genéticaRESUMO
An enzyme-linked immunosorbent assay (ELISA) was developed for the serological diagnosis of big liver and spleen (BLS) disease. The test utilizes a soluble, BLS-specific antigen that can be recovered from the livers of infected hens and that is known to react in the agar gel immunodiffusion (AGID) test. For use in ELISA, the BLS-specific antigen is fractionated by gel filtration chromatography and immobilized on microtiter plates using glutaraldehyde. The ELISA was evaluated using sera from infected and uninfected flocks originating in the United Kingdom and the United States. An ELISA format that incorporated control antigen recovered from the livers of uninfected birds for each serum tested was found to be more sensitive than the AGID test. A less sensitive but more cost-effective format that did not incorporate this control is considered suitable for large-scale flock screening programs.
Assuntos
Galinhas , Ensaio de Imunoadsorção Enzimática/métodos , Hepatomegalia/veterinária , Doenças das Aves Domésticas/diagnóstico , Esplenomegalia/veterinária , Animais , Anticorpos/sangue , Antígenos/isolamento & purificação , Estudos de Avaliação como Assunto , Feminino , Hepatomegalia/diagnóstico , Hepatomegalia/imunologia , Imunodifusão , Programas de Rastreamento , Doenças das Aves Domésticas/imunologia , Testes Sorológicos/métodos , Esplenomegalia/diagnóstico , Esplenomegalia/imunologiaRESUMO
Mice were immunized with partially purified preparations of the Cux-1 isolate of chicken anemia agent (CAA), and their splenocytes were fused with NSO myeloma cells. Three patterns of staining of CAA-infected cells were recognized when the resulting hybridomas were screened by indirect immunofluorescence (IIF). Hybridomas representative of each staining pattern were cloned, and the monoclonal antibodies (MAbs) were characterized. Type 1 staining was indistinguishable from that produced by polyclonal chicken antisera to CAA. Type 2 staining was confined to large nuclear inclusions. Type 3 staining was predominantly nuclear and granular, and differed from type 1 in being more intense and occurring in a higher proportion of nuclei. Three MAbs producing type 1 staining were predominantly Cux-1-specific by IIF; they also reacted to lower titers with the Gifu-1 isolate but not at all with three other CAA isolates. These MAbs had very slight neutralizing activity against Cux-1. Another MAb giving type 1 staining reacted with all CAA isolates tested to high titers in IIF and neutralization tests. MAbs with type 2 and type 3 staining reacted by IIF with all CAA isolates tested but possessed no neutralizing activity. The availability of MABs to CAA should facilitate development of diagnostic tests for the virus.