RESUMO
Thirty-five strains of Bordetella bronchiseptica, recovered primarily from pigs, rabbits, dogs, cats and humans, were characterized by phenotypic and genotypic markers. Biochemical typing only showed variation in the ability to reduce nitrate to nitrite. OMP profiles from virulent strains showed variations in the region of 85-95kDa, which lead us to describe five OMP-types alpha, beta, gamma, delta and epsilon. Genotypic markers included the presence of IS1001, and polymorphisms in the flagellin gene (flaA) and pertussis toxin (PT) promoter region. The IS1001 was detected in 16 isolates (2 from humans and 10 from pigs) but was absent in rabbit isolates. The restriction profiles of the flaA gene allowed us to differentiate the strains into types A-C. The PT types were characterized by an RFLP assay and could be typed through patterns III-V. There was no apparent association between the flaA or PT types and the origin of the isolates. Eleven groups of isolates were identified on the basis of specific combinations of the analyzed markers. The combination of phenotypic and genotypic tests used could be useful in characterizing isolates and differentiating between certain clonal types of B. bronchiseptica.
Assuntos
Técnicas de Tipagem Bacteriana/veterinária , Bordetella bronchiseptica/classificação , Bordetella bronchiseptica/genética , Variação Genética , Polimorfismo de Fragmento de Restrição , Animais , Proteínas da Membrana Bacteriana Externa , Western Blotting/veterinária , Bordetella bronchiseptica/patogenicidade , Gatos , Cães , Eletroforese em Gel de Poliacrilamida/veterinária , Flagelina/genética , Genótipo , Humanos , Peso Molecular , Nitratos/metabolismo , Toxina Pertussis/genética , Fenótipo , Filogenia , Reação em Cadeia da Polimerase/métodos , Reação em Cadeia da Polimerase/veterinária , Coelhos , Especificidade da Espécie , SuínosRESUMO
In the present study biochemical tests and outer membrane protein profile (OMP) capacity for typing Bordetella bronchiseptica field isolates were evaluated. The biochemical tests were performed by API 20NE system. OMP enriched fractions were obtained from cultures under virulent and modulated conditions and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have identified five patterns by differences in the bands in the 85-95 kDa region (alpha, beta, gamma, delta and epsilon) from virulent cultures; and three different patterns by the flagellin expressed isotype from modulated cultures (A: 40 kDa, B: 45 kDa, and C: 40 and 45 kD simultaneously). Isotypes alpha, beta and gamma were linked to isotpye A, isotype delta to B and C, and isotypes epsilon to B. There is no evident correlation between characterized isotypes and the origin of the isolate. Nitrate reduction was the unique variable biochemical characteristic, only observed in rabbit isolates. It was possible to differentiate seven groups with the traits included in the study. The capacity of discrimination of the traits analyzed using the Hunter and Gaston index was 0.829.
Assuntos
Técnicas de Tipagem Bacteriana , Bordetella bronchiseptica/classificação , Animais , Proteínas da Membrana Bacteriana Externa/análise , Bordetella bronchiseptica/genética , Bordetella bronchiseptica/patogenicidade , Eletroforese em Gel de Poliacrilamida , Flagelina/análise , Fenótipo , Coelhos , VirulênciaRESUMO
In the present study biochemical tests and outer membrane protein profile (OMP) capacity for typing Bordetella bronchiseptica field isolates were evaluated. The biochemical tests were performed by API 20NE system. OMP enriched fractions were obtained from cultures under virulent and modulated conditions and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have identified five patterns by differences in the bands in the 85-95 kDa region (alpha, beta, gamma, delta and epsilon) from virulent cultures; and three different patterns by the flagellin expressed isotype from modulated cultures (A: 40 kDa, B: 45 kDa, and C: 40 and 45 kD simultaneously). Isotypes alpha, beta and gamma were linked to isotpye A, isotype delta to B and C, and isotypes epsilon to B. There is no evident correlation between characterized isotypes and the origin of the isolate. Nitrate reduction was the unique variable biochemical characteristic, only observed in rabbit isolates. It was possible to differentiate seven groups with the traits included in the study. The capacity of discrimination of the traits analyzed using the Hunter and Gaston index was 0.829.
RESUMO
In the present study biochemical tests and outer membrane protein profile (OMP) capacity for typing Bordetella bronchiseptica field isolates were evaluated. The biochemical tests were performed by API 20NE system. OMP enriched fractions were obtained from cultures under virulent and modulated conditions and were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We have identified five patterns by differences in the bands in the 85-95 kDa region (alpha, beta, gamma, delta and epsilon) from virulent cultures; and three different patterns by the flagellin expressed isotype from modulated cultures (A: 40 kDa, B: 45 kDa, and C: 40 and 45 kD simultaneously). Isotypes alpha, beta and gamma were linked to isotpye A, isotype delta to B and C, and isotypes epsilon to B. There is no evident correlation between characterized isotypes and the origin of the isolate. Nitrate reduction was the unique variable biochemical characteristic, only observed in rabbit isolates. It was possible to differentiate seven groups with the traits included in the study. The capacity of discrimination of the traits analyzed using the Hunter and Gaston index was 0.829.
