RESUMO
The Brachyspira (formerly Serpulina) species rrl gene encoding 23S ribosomal RNA (rRNA) was used as a target for amplification of a 517bp DNA fragment by polymerase chain reaction (PCR). The primers for PCR amplification had sequences that were conserved among Brachyspira 23S rRNA gene and were designed from nucleotide sequences of Brachyspira hyodysenteriae, Serpulina intermedia, Brachyspira innocens and Brachyspira pilosicoli available from the GenBank database. Digestion of PCR-generated products from reference and field isolates of swine intestinal spirochetes with restriction enzymes Taq I and Alu I revealed five restriction fragment length polymorphism (RFLP) patterns. Each RFLP pattern corresponded to previously established genetic groups including B. hyodysenteriae (I), S. intermedia/B. innocens (II), Brachyspira murdochii (III), B. pilosicoli (IV) and B. alvinipulli (V). The 23S rRNA PCR/RFLP provided a relatively simple genotypic method for identification of porcine pathogenic B. hyodysenteriae and B. pilosicoli.
Assuntos
Enteropatias/veterinária , RNA Ribossômico 23S/genética , RNA Ribossômico/química , Spirochaetaceae/isolamento & purificação , Infecções por Spirochaetales/veterinária , Doenças dos Suínos/diagnóstico , Animais , Primers do DNA/química , DNA Bacteriano/química , DNA Bacteriano/isolamento & purificação , Desoxirribonucleases de Sítio Específico do Tipo II/química , Eletroforese em Gel de Ágar/veterinária , Eletroforese em Gel de Poliacrilamida , Enteropatias/diagnóstico , Enteropatias/microbiologia , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , RNA Ribossômico 23S/química , Alinhamento de Sequência , Spirochaetaceae/classificação , Spirochaetaceae/genética , Infecções por Spirochaetales/diagnóstico , Infecções por Spirochaetales/microbiologia , Suínos , Doenças dos Suínos/microbiologiaRESUMO
Pathogenic intestinal spirochaetes of pigs include Brachyspira (formerly Serpulina) hyodysenteriae, the cause of swine dysentery, and Brachyspira pilosicoli, the cause of porcine colonic spirochetosis (PCS). The purpose of this study was to assess the relative importance of Brachyspira species in diarrhoeal disease of growing pigs on farms in southern Brazil. The intensity and pattern of haemolysis, the production of indole and the hydrolysis of hippurate by reference and field porcine intestinal spirochaetes were compared with 16S-ribosomal RNA (mRNA)- and 23S-rRNA-based polymerase chain reaction assays for the identification of B hyodysenteriae and B pilosicoli. Between July and October 1998, 206 rectal swabs were taken from pigs on 17 farms with a history of diarrhoea developing within 30 days after they had been moved from nursery to growing facilities. Of 49 beta-haemolytic spirochaetes that were cultured, 29 (59.2 per cent) were grown in pure culture for phenotypic and genotypic characterisation, leaving 20 untyped. Of the 29 typed isolates, eight isolates obtained from six farms were identified as B hyodysenteriae, and 15 isolates obtained from seven other farms were identified as B pilosicoli; the remaining six isolates were identified as weakly beta-haemolytic commensal spirochaetes. There was complete agreement between the results of the phenotypic and genotypic analyses.