Identification of Mycobacterium tuberculosis complex by culture and duplex Polymerase Chain reaction in bovines

Abstract

Bovine tuberculosis (bTB) is an economically important zoonotic disease (can spread to human through inhalation or ingestion) caused by Mycobacterium bovis which belongs to Mycobacterium tuberculosis complex (MTC). Control and eradication of infection is difficult even in organized dairy farms. So combinations of tests like culturing and nucleic acid-based diagnostics are used for the isolation and identification of mycobacterial infections in cattle. Even though, there are many advances in diagnosis of bovine TB infection in cattle but till now, isolation identification of the etiological agent from clinical samples stands as a definitive and gold standard test. Nucleic acid based methods like Polymerase Chain Reaction (PCR) which have advantages of speed, sensitivity and specificity can be used for diagnosis of tuberculosis along with isolation. In the present study, isolation of Mycobacterium tuberculosis complex organisms was attempted from nasal swabs and milk of cattle using Lowenstein-Jensen (LJ) media without glycerol. Cattle which were positive for tuberculosis either by skin test or gamma interferon test were selected. Two of the twelve nasal swabs and none of the seven milk samples showed typical mycobacterial colonies on LJ media after 8 weeks of incubation. Ziehl-Neelsen staining of colonies showed slender, rod shaped acid fast organisms suggestive of Mycobacterium. Deoxy ribo nucleic acid (DNA) was extracted by boiling method and amplified by duplex PCR for 245 and 500 bp amplicons specific for MTC (IS6110) and M. bovis (RvD1Rv2031c), respectively. Electrophoresis revealed 245 bp product but not 500 bp which confirmed the identity and relatedness of the isolated mycobacterium to Mycobacterium tuberculosis complex. © 2014 Academic Journals Inc.