Molecular and immunohistochemical detection of Mycobacterium bovis in formalin-fixed tissues from animals with spontaneous bovine tuberculosis

Bovine tuberculosis (TB) is an infectious and emerging but neglected disease of cattle, caused by Mycobacterium bovis. Diagnostic techniques including histopathology, acid fast staining, immunohistochemistry, polymerase chain reaction (PCR) and real-time PCR were compared for extent of accurate diagnosis of bovine TB. Study was conducted on 16 archival tissue samples collected from suspected cases of bovine tuberculosis. Histopathological studies revealed both well-organized and poorly-organized granulomas in the lungs and lymph nodes. Acid fast bacilli were detected by Ziehl Neelsen's staining in 13 (81.25%) of the 16 suspected cases. Immunohistochemistry using specific polyclonal antibody against M. bovis detected mycobacterial antigen extracellularly in caseous areas as well as intracellularly in macrophages and giant cells. DNA was extracted from formalin-fixed, paraffin-embedded tissues and subjected to IS6110 PCR using T4/T5 and INS1/INS2 primers specific for Mycobacterium tuberculosis complex (MTC) with an expected band size of 123 bp and 245 bp, respectively. The IS6110 PCR assay was positive in 12 out of 16 cases (75%). IS6110 real-time PCR using IS6110_T primers confirmed 6 (37.5%) samples positive for M. bovis. The present investigation indicated that real-time PCR using IS6110_T primers had increased specificity over conventional IS6110 PCR assay and can be used for detecting specific M. bovis infection in animals suspected for bovine tuberculosis.