RESUMO
Background: Brucella sp. are the causative agents of brucellosis, an infectious disease that affects various species ofanimals and can be transmitted to humans through direct contact with infected animals, indirectly by the ingestion of rawmilk products, and during the handling of strains or infected material in the laboratory. Being a zoonosis, the detectionof Brucella species in animals is essential for the prevention of the disease in humans and to perform a good program ofcontrol in infected herds. This study aimed at identifying Brucella field strains isolated from 1976 to 2013 in Brazil, usingthe modified Bruce-Ladder method, to evaluate the performance of this technique.Materials, Methods & Results: Eighty-three strains of Brucella sp. were included in the study, i.e. 21 reference strains(nine B. abortus, one B. canis, four B. melitensis, two B. ovis and five B. suis) and 62 field strains (six B. canis, oneB. suis and 55 B. abortus). For the identification of the genus and/or species of Brucella, biochemical and physiologicaltests, including MacConkey-agar growth, glucose fermentation, haemolysis, catalase, oxidase and urease tests, nitratereduction, citrate utilization, H2S production and CO2 requirement, were performed. Genomic DNA was extracted frompure cultures through heat-lysis of bacterial cultures and the genus was confirmed by a genus-specific PCR (bcsp31 targetgene), before performing the modified Bruce-Ladder PCR for the confirmation of the Brucella species. No problems ofspecificity were observed with the Bruce-Ladder PCR. However, the 1,682 bp fragment was not systematically amplified,even after several modifications such as the concentration of mix components, annealing temperatures and time. Therefore,an individual PCR using primers specific to this fragment was needed for complete identification of some strains. Also,only one kind of Polymerase gave the best results...(AU)
Assuntos
Brucella/isolamento & purificação , Métodos , Brucelose/diagnóstico , Reação em Cadeia da Polimerase/métodos , BrasilRESUMO
Background: Brucella sp. are the causative agents of brucellosis, an infectious disease that affects various species ofanimals and can be transmitted to humans through direct contact with infected animals, indirectly by the ingestion of rawmilk products, and during the handling of strains or infected material in the laboratory. Being a zoonosis, the detectionof Brucella species in animals is essential for the prevention of the disease in humans and to perform a good program ofcontrol in infected herds. This study aimed at identifying Brucella field strains isolated from 1976 to 2013 in Brazil, usingthe modified Bruce-Ladder method, to evaluate the performance of this technique.Materials, Methods & Results: Eighty-three strains of Brucella sp. were included in the study, i.e. 21 reference strains(nine B. abortus, one B. canis, four B. melitensis, two B. ovis and five B. suis) and 62 field strains (six B. canis, oneB. suis and 55 B. abortus). For the identification of the genus and/or species of Brucella, biochemical and physiologicaltests, including MacConkey-agar growth, glucose fermentation, haemolysis, catalase, oxidase and urease tests, nitratereduction, citrate utilization, H2S production and CO2 requirement, were performed. Genomic DNA was extracted frompure cultures through heat-lysis of bacterial cultures and the genus was confirmed by a genus-specific PCR (bcsp31 targetgene), before performing the modified Bruce-Ladder PCR for the confirmation of the Brucella species. No problems ofspecificity were observed with the Bruce-Ladder PCR. However, the 1,682 bp fragment was not systematically amplified,even after several modifications such as the concentration of mix components, annealing temperatures and time. Therefore,an individual PCR using primers specific to this fragment was needed for complete identification of some strains. Also,only one kind of Polymerase gave the best results...