Resumo
Raising backyard birds is a common practice in Brazil, mainly in the countryside or suburban areas. However, the level of respiratory pathogens in these animals is unknown. We sampled two hundred chickens from 19 backyard flocks near commercial poultry farms and performed ELISA to Infectious Bronchitis Virus, avian Metapneumovirus, Mycoplasma synoviae and Mycoplasma gallisepticum. We evaluated the association between the predictive ability of ELISA and Hemagglutination-inhibition (HI)by comparing results from eight flocks positive to Mycoplasma gallisepticum on ELISA. Besides, we assessed essential biosecurity measures in the properties (multiple species birds, rodent control, hygienic conditions, and water quality for the bird`s consumption). We could access the vaccination program only on four properties; in three of them, the birds were supposedly vaccinated for IBV. Overall the properties had a poor score for the biosecurity measures, and the seroprevalence in backyard poultry flocks for IBV, a MPV, MS, and MG were respectively 87.5% (14/16), 89.5% (17/19), 100 (19/19) and MG 84.21% (16/19). We found low specificity and predictive value between ELISA and HI in MG analysis and a positive correlation between the presence of clinical symptoms and mean MG titers. Backyard chicken are pathogens reservoirs and pose a risk for the commercial poultry farms in the region, and further efforts of the governmental entities and private sector of poultry production should consider these information to avoid future economic losses.(AU)
Assuntos
Animais , Aves/anormalidades , Aves/anatomia & histologia , Contenção de Riscos Biológicos , Hemaglutinação , Metapneumovirus , Vírus da Bronquite InfecciosaResumo
Raising backyard birds is a common practice in Brazil, mainly in the countryside or suburban areas. However, the level of respiratory pathogens in these animals is unknown. We sampled two hundred chickens from 19 backyard flocks near commercial poultry farms and performed ELISA to Infectious Bronchitis Virus, avian Metapneumovirus, Mycoplasma synoviae and Mycoplasma gallisepticum. We evaluated the association between the predictive ability of ELISA and Hemagglutination-inhibition (HI)by comparing results from eight flocks positive to Mycoplasma gallisepticum on ELISA. Besides, we assessed essential biosecurity measures in the properties (multiple species birds, rodent control, hygienic conditions, and water quality for the bird`s consumption). We could access the vaccination program only on four properties; in three of them, the birds were supposedly vaccinated for IBV. Overall the properties had a poor score for the biosecurity measures, and the seroprevalence in backyard poultry flocks for IBV, a MPV, MS, and MG were respectively 87.5% (14/16), 89.5% (17/19), 100 (19/19) and MG 84.21% (16/19). We found low specificity and predictive value between ELISA and HI in MG analysis and a positive correlation between the presence of clinical symptoms and mean MG titers. Backyard chicken are pathogens reservoirs and pose a risk for the commercial poultry farms in the region, and further efforts of the governmental entities and private sector of poultry production should consider these information to avoid future economic losses.
Assuntos
Animais , Aves/anatomia & histologia , Aves/anormalidades , Contenção de Riscos Biológicos , Hemaglutinação , Metapneumovirus , Vírus da Bronquite InfecciosaResumo
A vacinação é a forma mais utilizada para prevenir a bronquite infecciosa causada pelo vírus da bronquite infecciosa das galinhas (IBV). Contudo, as vacinas convencionais são incapazes de diferenciar aves infectadas de vacinadas. No presente trabalho foi construído, caracterizado, e avaliado como candidato vacinal, um adenovírus recombinante expressando o gene N do IBV. O gene N foi clonado em um adenovírus humano tipo 5 defectivo e transfectado para as células HEK-293A para gerar rAd5_N. Após o vetor ser obtido como esperado e a confirmação da expressão da proteína N em HEK-293ª, foi realizada inoculação pela via oculo-nasal na dose de 10 7 TCID 50 /0,1mL para imunização de galinhas livres de patógenos específicos (SPF). A resposta imunológica do Ad5_N e a proteção contra o desafio ao IBV foram avaliadas e comparadas com uma vacina viva comercial. Não foram detectados anticorpos anti-IBV em aves vacinadas com o Ad5_N. A vacina comercial induziu anticorpos detectáveis a partir do 7º dia pós-vacinal. Em aves vacinadas com o Ad5_N não houve aumento na expressão de IFNγ. Neste estudo, o rAd5_N obtido não conferiu proteção contra desafio com IBV-M41. Os resultados indicam a necessidade de avaliar adenovírus recombinantes expressando outros genes do IBV.(AU)
Assuntos
Animais , Vacinas Sintéticas , Galinhas , Infecções por Coronavirus/prevenção & controle , Vírus da Bronquite Infecciosa , Nucleoproteínas , Proteínas do NucleocapsídeoResumo
A vacinação é a forma mais utilizada para prevenir a bronquite infecciosa causada pelo vírus da bronquite infecciosa das galinhas (IBV). Contudo, as vacinas convencionais são incapazes de diferenciar aves infectadas de vacinadas. No presente trabalho foi construído, caracterizado, e avaliado como candidato vacinal, um adenovírus recombinante expressando o gene N do IBV. O gene N foi clonado em um adenovírus humano tipo 5 defectivo e transfectado para as células HEK-293A para gerar rAd5_N. Após o vetor ser obtido como esperado e a confirmação da expressão da proteína N em HEK-293ª, foi realizada inoculação pela via oculo-nasal na dose de 10 7 TCID 50 /0,1mL para imunização de galinhas livres de patógenos específicos (SPF). A resposta imunológica do Ad5_N e a proteção contra o desafio ao IBV foram avaliadas e comparadas com uma vacina viva comercial. Não foram detectados anticorpos anti-IBV em aves vacinadas com o Ad5_N. A vacina comercial induziu anticorpos detectáveis a partir do 7º dia pós-vacinal. Em aves vacinadas com o Ad5_N não houve aumento na expressão de IFNγ. Neste estudo, o rAd5_N obtido não conferiu proteção contra desafio com IBV-M41. Os resultados indicam a necessidade de avaliar adenovírus recombinantes expressando outros genes do IBV.(AU)
Assuntos
Animais , Vacinas Sintéticas , Galinhas , Infecções por Coronavirus/prevenção & controle , Vírus da Bronquite Infecciosa , Nucleoproteínas , Proteínas do NucleocapsídeoResumo
A Brazilian field isolate (IBV/Brazil/PR05) of avian infectious bronchitis virus (IBV), associated with development of nephritis in chickens, was previously genotyped as IBV variant after S1 gene sequencing. The aim of this study was to evaluate the levels of IL-6 in kidneys and trachea of birds vaccinated and challenged with IBV/Brazil/PR05 strain, correlating these results with scores of microscopic lesions, specific IBV antigen detection and viral load. The up-regulation of IL-6 and the increased levels of viral load on renal and tracheal samples were significantly correlated with scores of microscopic lesions. Reduced levels of viral load were detected in kidneys of birds previously vaccinated and challenged, compared to non-vaccinated challenged group, although markedly microscopic lesions were observed for both groups. The expression of IL-6, present both in the kidney and in the tracheas, was dependent on the load of the virus present in the tissue, and the development of lesions was related with IL-6 present in the tissues. These data suggest that variant IBV/Brazil/PR05 can induce the expression of proinflammatory cytokines in a manner correlated with viral load and increased IL-6 is involved in the tissue with the influx of inflammatory cells and subsequent nephritis. This may contribute with a model to the development of immunosuppressive agents of IL-6 to prevent acute inflammatory processes against infection with IBV and perhaps other coronaviruses, as well as contribute to the understanding of the immunopathogenesis of IBV nephropatogenic strains.(AU)
Uma estirpe variante do vírus da bronquite infecciosa (VBI) associada com o desenvolvimento de nefrite em galinhas, foi isolado e identificado como variante por análise do gene S1. A estirpe IBV/Brazil/PR05 foi testada quanto à sua capacidade de induzir a expressão de interleucina-6 (IL-6) nos tecidos renais e traqueais. Galinhas vacinadas com a estirpe Massachusetts H120 e não vacinadas foram desafiadas com a estirpe IBV/Brazil/PR05. Cinco dias após a infecção, traquéias e rins foram coletados para análise por RT-qPCR, imunohistoquímica e histopatologia. Foi determinada a expressão relativa de IL-6 e da carga viral. A expressão de IL-6 e carga viral foram correlacionadas com o desenvolvimento de nefrite e lesão traqueal. A expressão de IL-6 foi maior quando houve aumento da carga viral na traqueia e nos rins. A carga viral presente nos rins foi inferior quando as aves foram vacinadas, entretanto foi observada nefrite acentuada. Houve alta correlação entre o desenvolvimento de nefrite e o nível de expressão de IL-6, bem como a expressão de IL-6 e a carga viral. A expressão de IL-6, presente tanto nos rins e nas traqueias, foi relacionada a carga viral presente nestes tecidos, e o desenvolvimento das lesões foi relacionado com a expressão de IL-6. Estes dados sugerem que a variante IBV/Brazil/PR05 pode induzir a expressão de citocinas pró-inflamatórias de forma correlacionada com a carga viral, e o aumento de IL-6 está envolvido com o influxo de células inflamatórias no tecido, o que evolui para o desenvolvimento de nefrite. Isto pode contribuir como um modelo para o desenvolvimento de agentes imunossupressores da IL-6 para evitar processos inflamatórios agudos contra infecção com o VBI e talvez outros coronavírus, bem como contribuir para o entendimento da imunopatogênese das estirpes nefropatogênicas deste vírus.(AU)
Assuntos
Animais , Galinhas/virologia , Vírus da Bronquite Infecciosa/isolamento & purificação , Nefrite/veterinária , Interleucina-6/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Rim/patologia , Traqueia/patologiaResumo
In the United States, more than 90% of chicken meat is produced in the southeastern states, and most egg production resides in the eastern half of the country and Texas. Several molecular epidemiological studies have indicated that most infectious bronchitis (IB) virus (IBV) isolates obtained from outbreaks of respiratory disease in these regions correspond to Ark-type IBV in spite of extensive vaccination programs which include IBV ArkDPI-derived vaccines. Accumulating evidence suggests that Ark-type strains may have a distinct capacity to circumvent preventive measures. Two strategies by which Ark-type IBV strains may maintain a high prevalence in commercial chickens are: (1) Unusually high genetic and phenotypic variability, and (2) synergism with concurrent viral immunodeficiency. Support for the first strategy includes epidemiological findings showing continued isolations of Ark-like viruses from respiratory disease affecting flocks vaccinated with serotype-specific homologous (ArkDPI-derived) vaccines, experimental data demonstrating selection of new predominant phenotypes occurring rapidly after a single passage in the host, and recent findings indicating changes of the predominant IBV population occurring within the host during the invasion process. The second strategy is supported by epidemiological data indicating increased isolations of Ark-type IBV showing minor geno-/phenotypic variation occurring in chickens simultaneously affected by immunosuppressive viruses. In addition, experimental results have shown that viral immunodeficiency leads to more severe and prolonged IB signs and lesions, delayed and reduced specific antibody responses, and increased and persistent IBV shedding. Finally, accumulating evidence confirms high genetic and phenotypic heterogeneity in commercial ArkDPI-derived vaccines. The rapid selection of new predominant phenotypes occurring in these vaccines may be facilitating the emergence of Ark-like strains. Thus, improvement of Ark-type vaccines and prevention of viral immunodeficiency's seem to be essential for an effective control of the disease.
Resumo
In the United States, more than 90% of chicken meat is produced in the southeastern states, and most egg production resides in the eastern half of the country and Texas. Several molecular epidemiological studies have indicated that most infectious bronchitis (IB) virus (IBV) isolates obtained from outbreaks of respiratory disease in these regions correspond to Ark-type IBV in spite of extensive vaccination programs which include IBV ArkDPI-derived vaccines. Accumulating evidence suggests that Ark-type strains may have a distinct capacity to circumvent preventive measures. Two strategies by which Ark-type IBV strains may maintain a high prevalence in commercial chickens are: (1) Unusually high genetic and phenotypic variability, and (2) synergism with concurrent viral immunodeficiency. Support for the first strategy includes epidemiological findings showing continued isolations of Ark-like viruses from respiratory disease affecting flocks vaccinated with serotype-specific homologous (ArkDPI-derived) vaccines, experimental data demonstrating selection of new predominant phenotypes occurring rapidly after a single passage in the host, and recent findings indicating changes of the predominant IBV population occurring within the host during the invasion process. The second strategy is supported by epidemiological data indicating increased isolations of Ark-type IBV showing minor geno-/phenotypic variation occurring in chickens simultaneously affected by immunosuppressive viruses. In addition, experimental results have shown that viral immunodeficiency leads to more severe and prolonged IB signs and lesions, delayed and reduced specific antibody responses, and increased and persistent IBV shedding. Finally, accumulating evidence confirms high genetic and phenotypic heterogeneity in commercial ArkDPI-derived vaccines. The rapid selection of new predominant phenotypes occurring in these vaccines may be facilitating the emergence of Ark-like strains. Thus, improvement of Ark-type vaccines and prevention of viral immunodeficiency's seem to be essential for an effective control of the disease.
Resumo
Avian infectious bronchitis virus (IBV) isolates have been widely characterized by reverse transcription followed by polymerase chain reaction and DNA sequencing. In present study, these techniques were applied to three viral genomic regions comprising the complete and/or a partial S1 segment, S2 and nucleocapsid genes. DNA sequences from viral isolates obtained from 1972 to 1989 and from 2006 to 2008 were compared. High similarity (>90%) was observed among some of the genomic segments, including S1 hypervariable region, which could suggest a common origin or ancestry. DNA sequences from S2 and N protein genes obtained from different infected tissues of the same flock were analyzed, and a clear segregation between respiratory and intestinal tract was observed. Therefore, these data suggest co-circulation of more than one viral strain in the same flock. 57.1% of DNA sequences from the S1 complete segment samples, 53.3% from the S2 fragment and 62.5% from the partial N gene were found to be different from analyzed sequences from reference strains leading to the conclusion that parte of viral isolates included in this study may be considered region specific. Considering the simultaneous analysis of the three genes, a large IBV genetic profile was observed in both old and recent isolates groups. However, most prominent diversity between viral isolates was obtained in the period from 1972 and 1989, showing the presence of a large number of variants in the state of Minas Gerais before the official approval of vaccination (1980).
Resumo
Avian infectious bronchitis virus (IBV) isolates have been widely characterized by reverse transcription followed by polymerase chain reaction and DNA sequencing. In present study, these techniques were applied to three viral genomic regions comprising the complete and/or a partial S1 segment, S2 and nucleocapsid genes. DNA sequences from viral isolates obtained from 1972 to 1989 and from 2006 to 2008 were compared. High similarity (>90%) was observed among some of the genomic segments, including S1 hypervariable region, which could suggest a common origin or ancestry. DNA sequences from S2 and N protein genes obtained from different infected tissues of the same flock were analyzed, and a clear segregation between respiratory and intestinal tract was observed. Therefore, these data suggest co-circulation of more than one viral strain in the same flock. 57.1% of DNA sequences from the S1 complete segment samples, 53.3% from the S2 fragment and 62.5% from the partial N gene were found to be different from analyzed sequences from reference strains leading to the conclusion that parte of viral isolates included in this study may be considered region specific. Considering the simultaneous analysis of the three genes, a large IBV genetic profile was observed in both old and recent isolates groups. However, most prominent diversity between viral isolates was obtained in the period from 1972 and 1989, showing the presence of a large number of variants in the state of Minas Gerais before the official approval of vaccination (1980).
Resumo
Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of infectious bronchitis outbreaks. These antibodies are homogeneous, highly specific and fully characterizable, allowing the improvement of immunological techniques detection and antigenic characterization of avian infectious bronchitis virus strains (IBV). A phage display library was used, which was prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments specific for heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning, a set of 15 scFv antibodies were expressed in phages and cross-reacted in ELISA with these three viral strains. Western-blotting analysis showed that two of the clones were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41. In conclusion, the recombinant fragments of monoclonal antibodies expressed in phage have a great potential for future use in immunodiagnostic techniques and to study the evolution of infectious bronchitis virus.
Anticorpos monoclonais constituem a base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por phage display contra a estirpe vacinal (H120) do VBI foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01 e IBVPR05, isoladas de surtos a campo no Brasil e a estirpe SE-17, isolada nos Estados Unidos. Após três ciclos de panning, foi identificado, pelo ELISA, um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que dois desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzidos em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus.
Resumo
Monoclonal antibodies are the basis of various techniques used for antigen detection or characterization, and their use is specially recommended for the identification of viral strains involved in the etiology of infectious bronchitis outbreaks. These antibodies are homogeneous, highly specific and fully characterizable, allowing the improvement of immunological techniques detection and antigenic characterization of avian infectious bronchitis virus strains (IBV). A phage display library was used, which was prepared previously against the IBV vaccine strain (H120) for the selection of new scFv antibody fragments specific for heterologous IBV strains isolated from outbreaks in Brazil (IBVPR01, IBVPR05) and USA (SE-17). After three cycles of panning, a set of 15 scFv antibodies were expressed in phages and cross-reacted in ELISA with these three viral strains. Western-blotting analysis showed that two of the clones were expressing scFv specific for the nucleoprotein of these IBV strains, as well as to the recombinant form of this protein derived from M41. In conclusion, the recombinant fragments of monoclonal antibodies expressed in phage have a great potential for future use in immunodiagnostic techniques and to study the evolution of infectious bronchitis virus.
Anticorpos monoclonais constituem a base de vários testes usados na detecção e na identificação de antígenos. Nesse contexto, tais imuno-reagentes têm sido extensivamente empregados na identificação de estirpes virais envolvidas na etiologia de surtos de bronquite infecciosa a campo, permitindo o aperfeiçoamento das técnicas de detecção e caracterização antigênica do vírus da bronquite infecciosa das galinhas (VBI). No presente estudo, uma biblioteca de fragmentos de anticorpos de galinha originalmente preparada por phage display contra a estirpe vacinal (H120) do VBI foi usada para a seleção de fragmentos de anticorpos recombinantes com reatividade cruzada para as estirpes heterólogas IBVPR01 e IBVPR05, isoladas de surtos a campo no Brasil e a estirpe SE-17, isolada nos Estados Unidos. Após três ciclos de panning, foi identificado, pelo ELISA, um conjunto de 15 anticorpos scFv expressos em fagos e com reatividade cruzada para essas mesmas estirpes do VBI. A análise por Western-blotting revelou que dois desses clones apresentavam fagos expressando fragmentos de anticorpos monoclonais com reatividade cruzada para a nucleoproteína N das três estirpes do VBI e também para a forma recombinante dessa nucleoproteína derivada da estirpe M41. Concluindo, os fragmentos de anticorpos monoclonais recombinantes scFv-N produzidos em fagos interagem com um epítopo mais conservado da proteína N do VBI e apresentam um grande potencial para utilização na detecção e no diagnóstico direto desse vírus.
Resumo
O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.
The nucleocapsid protein (N) gene (1,230 bp) of the M41 strain of infectious bronchitis virus (IBV) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and cloned in two systems; pET28a Escherichia coli and pFLD Pichia pastoris. The recombinant expression constructs (pET28a-N or pFLD-N) were identified by PCR and sequencing analysis. The transformant clones of BL21 strain of E. coli or GS115 of P. pastoris were submitted to appropriate inducting protocols. Expression of histidine-tagged fusion N proteins with a molecular mass of 54 kDa was determined by SDS-PAGE and Western blotting analysis, confirming that both recombinant N proteins were comparable in size and antigenicity to native IBV N protein. The E. coli system overexpressed the recombinant N protein, while the P. pastoris system produced a low yield of this recombinant protein. The bacteria expressed N protein was purified by chromatography on nickel-sepharose resin. These results indicated that the pET28a E. coli expression system is more effective to generate N recombinant protein for using as an antigen to detect anti-IBV antibodies in immuno-assays for this viral infection.
Assuntos
Pichia/genética , Vírus da Bronquite Infecciosa/ultraestrutura , Proteínas do Nucleocapsídeo/ultraestrutura , Escherichia coli/genética , Ensaio de Imunoadsorção Enzimática , Clonagem MolecularResumo
ABSTRACT The nucleocapsid protein (N) gene (1,230 bp) of the M41 strain of infectious bronchitis virus (IBV) was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), and cloned in two systems; pET28a Escherichia coli and pFLD Pichia pastoris. The recombinant expression constructs (pET28a-N or pFLD-N) were identified by PCR and sequencing analysis. The transformant clones of BL21 strain of E. coli or GS115 of P. pastoris were submitted to appropriate inducting protocols. Expression of histidine-tagged fusion N proteins with a molecular mass of 54 kDa was determined by SDS-PAGE and Western blotting analysis, confirming that both recombinant N proteins were comparable in size and antigenicity to native IBV N protein. The E. coli system overexpressed the recombinant N protein, while the P. pastoris system produced a low yield of this recombinant protein. The bacteria expressed N protein was purified by chromatography on nickel-sepharose resin. These results indicated that the pET28a E. coli expression system is more effective to generate N recombinant protein for using as an antigen to detect anti-IBV antibodies in immuno-assays for this viral infection.
RESUMO O gene da proteína de nucleocapsídeo (1.230 pb) da estirpe M41 do vírus da bronquite infecciosa (VBI) foi amplificado pelas reações de transcrição reversa e em cadeia da polimerase (RT-PCR) e clonado, em seguida, em dois sistemas; pET28a - Escherichia coli e pFLD -Pichia pastoris. Os produtos recombinantes construídos para expressão (pET28a-N ou pFLD-N) foram identificados por análises de PCR e de sequenciamento de nucleotídeos. Os clones transformantes da linhagem BL21 de E. coli e da linhagem GS115 de P. pastoris foram submetidos aos protocolos apropriados de indução. A expressão da proteína N de fusão com etiqueta de poli-histidina e com massa molecular de 54 kDa foi determinada pelas técnicas de SDS-PAGE e de Western blotting, confirmando-se que ambas proteínas N recombinantes apresentaram tamanhos e antigenicidade compatíveis com a proteína N nativa do próprio VBI. O sistema E. coli expressou uma quantidade relevante da proteína N recombinante, enquanto que o sistema P. pastoris produziu uma baixa recuperação dessa proteína recombinante. A proteína N recombinante gerada pelo sistema bacteriano foi purificada em resina de níquel-sepharose. O conjunto de resultados indica que o sistema de expressão constituído por pET28a E. coli é mais efetivo para produzir a proteína N recombinante do VBI destinada ao uso como antígeno para detectar anticorpos anti-virais específicos em ensaios de imunodiagnóstico para essa infecção viral.
Resumo
Aves de vida livre podem ser carreadoras e disseminadoras de agentes patogênicos e representarem um risco para galinhas de produção. O objetivo deste trabalho foi verificar em aves de vida livre a presença de anticorpo contra: Salmonella Pullorum (SP), vírus da doença de Newcastle (VDN) e vírus da bronquite infecciosa (VBI), bem como a presença de Salmonella spp. De 48 aves de vida livre capturadas nas imediações de uma granja avícola, no norte do Estado de São Paulo, em 2005 e 2006, foram colhidos soros para realização de testes de soroaglutinação rápida em placa (SAR) para SP, inibição de hemaglutinação (HI) para o VDN e soroneutralização (SN) para VBI. Foram colhidos fragmentos de fígado, baço e ovários/testículos, todos cultivados juntos como um pool e conteúdo intestinal separado, para cultura de Salmonella spp. Todas as amostras foram negativas para a doença de Newcastle e bronquite infecciosa. Quanto à Salmonella Pullorum, pela técnica de SAR, a amostra de Theristicus caudatus foi positiva. No exame bacteriológico, foi isolado o agente em três aves: Theristicus caudatus (Salmonella Muenchen), Zenaida auriculata (Salmonella Enteritidis) e em Cariama cristata tanto Salmonella Muenchen como Salmonella Saintpaul.(AU)
Assuntos
Animais , Salmonella/isolamento & purificação , Vírus da Doença de Newcastle/isolamento & purificação , Vírus da Bronquite Infecciosa/isolamento & purificação , Vetores de Doenças , Testes de Aglutinação , Animais SelvagensResumo
O vírus da bronquite infecciosa das galinhas (VBI) é o agente etiológico da Bronquite Infecciosa (BI), uma enfermidade altamente contagiosa que causa grandes perdas econômicas na avicultura. O VBI é um vírus envelopado, que possui genoma constituído de RNA fita simples, que codifica 4 proteínas estruturais, dentre elas a Nucleoproteína (N), que é produzida em grande quantidade na infecção viral e é reconhecidamente imunogênica. O controle da BI se faz com a imunização das aves através da aplicação de vacinas vivas atenuadas, seguidas de vacinação utilizando antígeno inativado, sendo o sorotipo Massachusetts o único liberado para uso no Brasil. Um dos objetivos do presente trabalho foi realizar um estudo exploratório, afim de conhecer a opinião de diferentes segmentos da avicultura sobre a situação atual da ocorrência de BI nos planteis brasileiros e os custos que ela representa. Diante disso, surge então a necessidade do desenvolvimento de vacinas alternativas e seguras para controle da BI, entre elas a utilização de vacinas vetoriais. Dessa forma, com o objetivo de desenvolver uma vacina efetiva no controle da BI, amostras variantes de VBI foram clonadas em adenovírus humano recombinante e utilizadas para transfectar células HEK293, originando adenovírus recombinantes carreadores do gene N do VBI. Estes vírus foram purificados e utilizados como vacinas recombinantes para imunização de aves SPF. Com base nos dados obtidos, observou-se que apesar das diferentes estratégias de vacinação, a BI ainda é considerada uma doença de alta prevalência que continua causando significativas perdas econômicas na produção avícola de corte e postura no Brasil. Os resultados obtidos demonstraram que a vacina recombinante não induziu uma resposta sorológica detectável pelo teste de Elisa comercial utilizado, bem como não reduziu os escores de lesões nos tecidos das aves vacinadas e desafiadas. Assim, a vacina recombinante carreada por adenovírus defectivo expressando o gene N do VBI foi construída e caracterizada, porém se mostrou ineficaz e não induziu suficiente proteção às aves experimentalmente imunizadas frente ao desafio com VBI.
The infectious bronchitis virus (IBV) is the etiologic agent of Infectious bronchitis (IB), a highly contagious disease that causes great economic losses in the poultry industry. The IBV is an enveloped virus that has RNA single strand genome, encoding four structural proteins, among them Nucleoprotein (N), which is produced abundantly in viral infection and is known immunogenic. The IB control is done by immunization of birds by applying live attenuated vaccine, followed by vaccination using inactivated antigen, wherein the Massachusetts serotype is the only released for use in Brazil. One of the goals of the present work was to conduct an exploratory study in order to know the opinion of different segments of the poultry industry on the current situation of the occurrence of BI in Brazilian squads and the costs that it represents. Therefore, the development of alternative and safe vaccines to BI control is necessary, including the use of vectors. In order to develop an effective vaccine to IB control, samples from IBV field variants were cloned into recombinant human adenovirus and used to transfect HEK293 cells, resulting in recombinant adenovirus carriers of the N gene of the IBV. These recombinant viruses were purified and used as vaccines to immunization of SPF chickens. Based on the obtained data, it was observed that despite the different vaccination strategies, IB is still considered highly prevalent disease that causes significant economic losses in Brazilian poultry industry. The results here obtained showed that the recombinant vaccine does not causes detectable positive serological responses by commercial Elisa test in vaccinated chickens and does not reduce the tissues damage in vaccinated and challenged chickens. Thus, the recombinant vaccine carried by defective adenovirus expressing N gene of IBV was constructed and characterized, but seemed to be ineffective and did not induce sufficient protection to experimentally immunized chickens against IBV challenge.
Resumo
A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.
Resumo
A semi-nested reverse transcription-polymerase chain reaction (Semi-N-RT-PCR) was developed and used to detect the S glycoprotein gene of infectious bronchitis virus (IBV) strains and to discriminate H120 vaccine strain from other strains. Viral RNA was extracted from the allantoic fluid of chicken embryos and from tissues of chickens experimentally infected with different strains of IBV. Amplification and identification of the viral RNA was performed using two sets of primers complementary to a region of the S glycoprotein gene in the Semi-N-RT-PCR assay. The pair of primers used in the first PCR consisted of universal oligonucleotides flanking a more variable region of S1-S2 gene. The second primer pair was used in the Semi-N-RT-PCR and was comprised of one of the primers from the first universal pair together with either another universal internal oligolucleotide or a oligonucleotide sequence specific for the H120 strain of IBV. The universal primers detected all reference IBV strains and field isolates tested herein. The Semi-N-RT-PCR had high sensitivity and specificity, and was able to differentiate the H120 vaccine strain from other reference IBV strains; including M41 strain. All tissue samples collected from chickens experimentally infected with H120 or M41 strains were positive in the semi-nested RT-PCR using universal primers, while only the H120-infected tissue samples were amplified by the set of primers containing the H120-oligonucleotide. In conclusion, the ability of Semi-N-RT-PCR to detect distinct IBV strains and preliminarily discriminate the vaccine strain (H120) closes a diagnostic gap and offers the opportunity to use comprehensive PCR procedures for the IBV diagnosis.
Resumo
RESUMO A bronquite infecciosa das galinhas (BIG) é uma doença respiratória altamente contagiosa causada por um Coronavírus, o vírus da bronquite infecciosa das galinhas (VBIG). Embora o VBIG seja um patógeno primário do trato respiratório, ele é também uma causa comum de redução da produção e qualidade dos ovos em galinhas. Certos tipos de VBIG causam lesões renais, com significativa mortalidade. Há também mortalidade por conseqüências respiratórias. A doença possui grande importância econômica devida às perdas na produção, sendo estas maiores que as perdas por mortalidade. A ocorrência de múltiplos sorotipos e as características mutantes de seu agente etiológico tem complicado e aumentado os custos de produção e dificultado sua prevenção através da imunização. Recentemente, uma variante do VBIG tem sido descrita associado com a miopatia dos músculos peitorais em muitas partes do mundo, inclusive no Brasil.
ABSTRACT Avian infectious bronchitis (IB) is a highly contagious respiratory disease of chickens caused by a Coronavirus, infectious bronchitis virus (IBV). Although IBV is primarily a respiratory tract pathogen, it is also a common cause of reduced egg production and egg quality in laying hens, and certain strains of IBV cause kidney lesions with significant mortality. Mortality by tracheal blockage also occurs. The disease has high economic importance due the loss of production, which is more important than the loss by mortality. The occurrence of multiple serotypes and mutants of the IBV have complicated and increased the production costs and have impaired its prevention through immunization. Recently, a variant IBV has been reported associated with pectoral muscle myopathy in many parts of world including Brazil.