Diagnosis of Mycobacterial infections (Tuberculosis and Paratuberculosis) in tissue samples using molecular (inhouse...

An in-house multiplex PCR designed for differential detection of M. bovis, M. avium paratuberculosis (MAP) and M. smegmatis in tissue samples, besides PCR and TaqMan real-time PCR targeting esxA (ESAT-6) and esxB (CFP-10) gene of M. tuberculosis complex (MTC) and IS900 gene targeting MAP were carried out in the present study. Tissue samples comprising of lung, mediastinal lymph node, intestine and mesenteric lymph node from cattle and buffaloes (n=16) suspected to have died of tuberculosis (TB) or paratuberculosis (JD) were collected at post-mortem. Out of 16 animals, only 4 (25%) cases were positive for M. bovis by in-house designed multiplex PCR, where as 9 (56.25%) cases were positive for MTC by both conventional and real-time PCR targeting esxA (ESAT-6) and esxB (CFP-10) genes. Out of 9 TB positive animals, 2 (22.22%) had co-infection with MAP as confirmed by IS900 PCR, real-time PCR and also by in-house multiplex PCR. No samples were found positive for M. smegmatis by the in-house multiplex PCR. Analytical sensitivity of in-house multiplex primers targeting M. bovis, MAP and M. smegmatis were 170 fg/µl, 300 fg/µl and 51 fg/µl of genomic DNA respectively. Analytical sensitivity of primers for both conventional and real time PCR tareting esxA were 8 pg/µl and 800 fg/µl, esxB were 800 fg/µl and 80 fg/µl and IS900 were 30 fg/µl and 3 fg/µl of M. tuberculosis and MAP genomic DNA respectively. The tissue samples were also subjected to histopathology and Immunohistochemical (IHC) staining using ESAT-6 and CFP-10 antibodies for TB and anti-JD anti-serum (raised in rabbit) for JD. All the animals positive for TB (9 of 9) and JD (2 of 2) in PCR and real-time PCR were also positive in histopathology and IHC. Thus, combination of molecular, histopathology and IHC techniques increased the accuracy of TB and JD diagnosis, all of which had same sensitivity and specificity except the in-house multiplex PCR which was comparatively less sensitive in M. bovis detection. However, the molecular methods were found to be more convenient and rapid, capable of making the diagnosis within 6 hours. The study indicated that Real-time PCR was ten times more sensitive than the conventional PCR.