Studies on diagnosis and differentiation of pathogenic mycobacterial species from non-tuberculous mycobacterial species in cattle and buffaloes

A multiplex PCR developed in the department was used for differential detection of Mycobacterium avium subsp. paratuberculosis (MAP), M. bovis (pathogenic mycobacterial species) and M. smegmatis (non-tuberculous mycobacteria). A total of 211 faecal samples from cattle and buffaloes with history of chronic intermittent diarrhoea were collected and processed for acid fast staining, isolation and molecular characterization by ISMap02 targeted nested PCR, IS900 real-time PCR using TaqMan assay and the in-house developed multiplex PCR. Milk (n=111) and blood (n=119) samples from cattle and buffaloes were also processed and subjected to the in-house developed multiplex PCR, IS900 PCR and esx-B PCR. Seventeen tissue samples from animals suspected to have died of TB and Johne's disease (JD) were also collected and subjected to in-house developed multiplex PCR besides IS900 and esx-B PCR. Out of 211 faecal samples, 187 (88.6%) were positive by acid fast staining, among which 127 samples were highly (3+ or 4+) positive. All the highly positive faecal samples (n=127) were then processed for isolation, out of which only 5 faecal samples were positive for MAP colonies, which were confirmed by IS900 PCR and ISMap02 PCR. ISMap02 targeted nested PCR detected 18 faecal samples to be positive out of 187 faecal samples processed, which were also confirmed by IS900 real time PCR, giving a CT value between 22-34. The sensitivity of ISMap02 PCR was found to be 7.6 fg/?l of the standard genomic DNA. The in-house developed multiplex PCR detected 11 positive faecal samples for MAP. Multiplex PCR detected one MAP positive sample in each milk and blood samples, while 2 and 3 samples were positive for M. bovis in milk and blood respectively. The samples positive for MAP by in-house multiplex PCR were also positive by IS900 PCR. The samples positive for M. bovis in in-house multiplex PCR were confirmed positive by esx-B PCR. Tissue samples subjected to in-house multiplex PCR resulted in 2 MAP and 3 M. bovis positive samples, which were confirmed positive by IS900 and esx-B PCR, respectively. None of the blood, milk and tissue samples were positive for M. smegmatis in the in-house developed multiplex PCR. In our study it was seen that the ISMap02 PCR and IS900 PCR were equally comparable in the detection of MAP.


Keywords: Mycobacterium avium subsp. paratuberculosis, M. bovis, M. smegmatis, Faecal, blood, tissue sample, IS900 PCR, ISMap02 PCR, esxB (CFP-10), Real Time PCR, Multiplex PCR, acid fast staining. M.V.Sc.