Molecular detection of Mycobacterium orygis in lung tissues and lymph nodes using conventional and TaqMan qRT-PCR...

Bovine Tuberculosis (bTB) is a widespread infectious disease of domestic animals and humans. The study aimed to detect mycobacteria at the molecular level in bovine lung tissue and lymph node samples using conventional and TaqMan qRT-PCR assay. Ninety-nine samples of lung tissue (n = 90) and lymph nodes (n = 9) were collected from cattle and buffaloes. All the collected samples were subjected to microscopic examination, decontaminated with 0.75% hexadecylpyridinium chloride (HPC), and inoculated onto Middlebrook7H10 media, followed by PCR screening. In total, 26 isolates were obtained from 99 tissue samples. Nineteen out of twenty-six tissue isolates tested positive for the hsp65 gene, indicative the genus Mycobacterium. However, of the nineteen-genus positive isolates, six samples yielded M. orygis, and 8 yielded M. kansasii on culturing, which were subsequently identified by species-specific PCR. Subsequently six M. orygis isolates were confirmed by the real-time PCR with Cycle Threshold (C_T) values ranging from 18.43 and 34.74. Detection of M. orygis from lung tissue and lymph nodes is reported using conventional and TaqMan qRT-PCR assay. The findings of the current study provide evidence of the presence of non-tuberculous mycobacterial infection in lung tissue, along with tuberculous mycobacteria, which may hamper in diagnosis of bTB reactor-positive animals.