Identification and differentiation of Mycobacterium tuberculosis complex organisms in bovine clinical samples by Polymerase chain reaction

Abstract

Mycobacterium bovis and Mycobacterium tuberculosis are the public health important bacteria that cause diseases in both animals and humans, especially in cattle and dairy farm workers. In this study, 36 tissue samples were collected from 24 dairy cows previously diagnosed as tuberculin positive in Nakhon Pathom province, Thailand. Infection with Mycobacterium organisms was confirmed by histopathologic examination and AFB staining. Species identification was made by using a panel of PCR based on 3 specific genes (16SrRNA, Rv1970, JB fragment) and direct sequencing using oxyR gene. Thirty of 36 (83.33%) clinical tissue samples were identified as Mycobacterium spp., 12 from 30 (40%) were positive for M. tuberculosis while 18 from 30 (60%) were positive for M. bovis strains. DNA fingerprints using variable number tandem repeat (VNTR) was used to differentiate among M. bovis and M. tuberculosis isolates. Eighteen of M. Bovis isolates can be grouped into 6 VNTR patterns. However, VNTR analysis indicated that 12 of M. tuberculosis isolates infected in cattle were identified in only one VNTR pattern. Our result showed that the combination of PCR and gene sequencing are very useful for identification of M. bovis and M. tuberculosis from clinical tissue samples. Moreover, VNTR is a simple useful tool for strain differentiation and for the study of genetic diversity, especially for M. bovis.