RESUMO
Ornithobacterium rhinotracheale is a pathogen involved in respiratory infection and systemic disease in poultry. Previously, eight potential vaccine candidates were identified that induced cross-protective immunity when administered to chickens as a multi-component vaccine. In this study, we analyzed the immunogenicity of these eight recombinant proteins by subunit vaccination, and characterized the different proteins and corresponding genes more thoroughly by sequencing, in vitro expression analysis, and cellular localization experiments. We found, that all genes encoding the eight antigens were highly conserved among different O. rhinotracheale serotypes, but the different antigens were not expressed by all serotypes. Cellular fractionation experiments indicated that the majority of the antigens are predominantly located in the outer membrane fraction. Vaccination of chickens with single-antigen vaccines demonstrated that the Or77 antigen was protective against serotypes that expressed Or77 in vitro, suggesting that the protein has strong potential as a vaccine antigen. Furthermore, immunization with four-component subunit vaccines indicated the existence of immunogenic synergism between the candidate vaccine antigens.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas/imunologia , Infecções por Flavobacteriaceae/veterinária , Ornithobacterium/imunologia , Doenças das Aves Domésticas/prevenção & controle , Sacos Aéreos/patologia , Animais , Anticorpos Antibacterianos/sangue , Antígenos de Bactérias/administração & dosagem , Antígenos de Bactérias/análise , Antígenos de Bactérias/genética , Proteínas da Membrana Bacteriana Externa/administração & dosagem , Proteínas da Membrana Bacteriana Externa/análise , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Western Blotting , Membrana Celular/química , Galinhas , Sequência Conservada , Reações Cruzadas , DNA Bacteriano/química , DNA Bacteriano/genética , Infecções por Flavobacteriaceae/patologia , Infecções por Flavobacteriaceae/prevenção & controle , Expressão Gênica , Variação Genética , Dados de Sequência Molecular , Ornithobacterium/química , Ornithobacterium/genética , Doenças das Aves Domésticas/patologia , Proteínas Recombinantes/imunologia , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Vacinas de Subunidades Antigênicas/administração & dosagem , Vacinas de Subunidades Antigênicas/genética , Vacinas de Subunidades Antigênicas/imunologia , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologiaRESUMO
Ornithobacterium rhinotracheale is a bacterial pathogen known for causing respiratory disease in poultry. In this study, we demonstrate for the first time that cross-protective immunity against different O. rhinotracheale serotypes can be induced by live vaccination. Sera from these live-vaccinated and cross-protected birds were used to identify new vaccine targets by screening an O. rhinotracheale expression library. Out of 20,000 screened plaques, a total of 30 cross-reactive clones were selected for further analysis. Western blot analysis and DNA sequencing identified eight different open reading frames. The genes encoding the eight cross-reactive antigens were amplified, cloned in an expression vector, and expressed in Escherichia coli. Purified recombinant proteins with a molecular mass ranging from 35.9 kDa to 62.9 kDa were mixed and tested as a subunit vaccine for (cross-)protection against challenge with homologous and heterologous O. rhinotracheale serotypes in chickens. Subunit vaccination resulted in the production of antibodies reactive to the recombinant proteins on Western blot, and this eight-valent vaccine conferred both homologous and heterologous protection against O. rhinotracheale challenge in chickens.
Assuntos
Antígenos de Bactérias/imunologia , Vacinas Bacterianas , Infecções por Flavobacteriaceae/veterinária , Ornithobacterium/imunologia , Doenças das Aves Domésticas/prevenção & controle , Animais , Anticorpos Antibacterianos/imunologia , Antígenos de Bactérias/genética , Vacinas Bacterianas/imunologia , Sequência de Bases , Clonagem Molecular , Reações Cruzadas , Infecções por Flavobacteriaceae/imunologia , Infecções por Flavobacteriaceae/prevenção & controle , Biblioteca Genômica , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Doenças das Aves Domésticas/imunologia , Subunidades Proteicas/genética , Subunidades Proteicas/imunologia , VacinaçãoRESUMO
Unravelling of the protective immunity acquired during a natural infection may contribute to vaccine development. To assess the role of antibody-mediated immunity in protection against Ornithobacterium rhinotracheale infection in chickens, a novel experimental method was applied that combined immune depletion and passive transfer of immunity within the same host. Administration of cyclophosphamide (CY) to broiler chickens successfully suppressed B lymphocyte development, and therefore humoral immunity, as confirmed by histological and serological analysis. Challenge of CY-treated birds with O. rhinotracheale revealed a significantly higher pathology score in comparison to immune-competent birds that received the same bacterial challenge. Measurement of serum immunoglobulin levels of immune-competent birds revealed a positive correlation between IgA and/or IgG production and protection against infection. Passive transfer of O. rhinotracheale-specific antiserum to the immune-suppressed birds prior to pathogen challenge significantly decreased morbidity. This protective effect was not observed after administration of control sera containing similar concentrations of immunoglobulins. Together, these results provide firm evidence that chicken humoral immunity to O. rhinotracheale is a key component in protection against infection. Our data confirm that the applied immune depletion and reconstitution approach is an attractive tool to analyse the nature of the protective immune response.
