Comparison of multiplex and ordinary PCR for diagnosis of paratuberculosis and tuberculosis in blood samples (buffy coat) of cattle and buffaloes

Background: Paratuberculosis and tuberculosis (TB) caused by Mycobacterium avium paratuberculosis (MAP) and Mycobacterium tuberculosis complex (MTC), respectively are economically important, chronic debilitating diseases affecting the dairy herds and are also potential zoonotic threats. Aims: Differential diagnosis of paratuberculosis and TB in blood samples of cattle and buffaloes. Methods: In this study, an in-house developed multiplex polymerase chain reaction (PCR) targeting MAP, Mycobacterium bovis and Mycobacterium smegmatis was used in blood samples (buffy coat) parallel with IS900 PCR and esxB PCR for diagnosis of paratuberculosis and TB, respectively; in a total of 202 cattle and buffaloes. Results: Out of 202 animals, 12 (5.9%) and 17 (8.4%) animals were positive for MAP by multiplex PCR and IS900 PCR, respectively; from which only 8 (4%) animals were positive by both tests; whereas 4 and 9 animals were exclusively positive by multiplex PCR and IS900 PCR, respectively. None of the animals were found to be positive for M. bovis and M. smegmatis by the multiplex PCR. However, the esxB PCR detected 13 (6.4%) animals positive for TB. In fact, 3 (1.5%) animals were found to be co-infected by both paratuberculosis and TB. Conclusion: The in-house multiplex PCR detected MAP in buffy coat and there was a fair degree of agreement between the multiplex PCR and IS900 PCR in detection of MAP DNA though the latter detected more number of animals to be positive for MAP. Besides, esxB PCR showed a high diagnostic potential and can be used for diagnosis of TB from blood.