Mycobacteria research in water buffalo milk

Resumo

Milk is an important nutritional source to man, but it canalso carry pathogenic mycobacteria, especially Mycobacteriumbovis. In this study, we tried to evaluate the classical technique(culture) and molecular technique PCR (Polymerase ChainReaction) and PRA (PCR - Restriction Enzyme Analysis) in theisolation and identification of mycobacteria from water buffalo´smilk samples. First of all, it was standardized an efficient PCRmethod for M. bovis identification from milk. In this sense, aknown number of M. bovisAN5 were inoculated in the milk thatwas submitted to a serial dilution. Then, two different PCRprotocols were done to evaluate the threshold of M. bovisdetection. Two pair of primers were tested, one to detectMycobacterium spp (INS1 e INS2) and other to detect M. bovis(JB21 e JB22). Dilutions were also submitted for culture usingStonebrink medium and incubated at 37°C/90 days, to determinethe number of bacilli in the milk. After this, 23 samples of milkcollected from water buffalos (Bubalus bubalis), 7 PPD + and 16PPD-, were analyzed by PCR technique and culture (Stonebrinkmedium) to detect the presence of M. bovis. Isolation of othermycobacteria were done by culture (Lowenstein-Jensen medium)and identified by mycolic acids and PRA. The results of PCRshowed that the protocol using primer par INS1/ INS2 waspositive until 10-3 dilution (800 CFU/mL). The protocol withprimers JB21/JB22 was more sensitive and specific, detectingM. bovis until 10-4 dilution (80 CFU/mL). M. bovis could not beidentified by PCR and isolated in culture from milk samples ofwater buffalo. Other species of mycobacteria were identified as:M. flavenscens (1 sample), M. simiae (3 samples) and M.intracellulare (1 sample). The last two species are consideredpathogenic mycobacteria to human.