Rapid Differential Detection of Selected Mycobacterial Species from Faecal Samples of Cattle and Buffaloes by Using In-house Designed Multiplex PCR, IS900 PCR and TaqMan Real-time PCR

Abstract

Aim: Mycobacterial infections due to Mycobacterium avium paratuberculosis (MAP) (Paratuberculosis), and M. bovis (Tuberculosis) in dairy animals are potential public health risk that needs to be diagnosí at the earliest for effective prevention. In addition, M. smegmatis (nontuberculous mycobacteria) is a saprophytic mycobactera that may interfere with the diagnosis of mycobacterial infection besides causing opportunistic infections. This study aims at rapid differential detection of these organisms from faecal samples of cattle and buffaloes using in-house designed multiplex PCR, besides other techniques for aiding in the accuracy of diagnosis. Methodology: Faecal samples (n=268) were collected per-rectum from suspected animals and screened by Ziehl-Neelsen staining and graded as low, medium and high shedders; Then faecal culture in Middlebrook 7H11 media for isolation of MAP from faecal samples of medium to high shedders (n=150). All the samples were subjected to in-house designed multiplex PCR targeting MAP, M. bovis and M. smegmatis; besides using IS900 conventional PCR and TaqMan real-time PCR for detection of MAP. Results: A total of 237 (88.43%) samples were found to be positive for Acid-fast bacilli, out of which 49 (20.68%), 106 (44.73%) and 82 (34.60%) samples were from low, medium and high shedders respectively. Only 4 (2.67%) samples were culture positive for MAP. Whereas, multiplex PCR detected 16 (5.97%) faecal samples as positive for MAP, and none were positive for M. bovis and M. smegmatis. However, IS900 conventional PCR and IS900 TaqMan real-time PCR detected 19 (7.09%) and 29 (10.82%) faecal samples as positive for MAP respectively. Conclusion: Thus, the in-house multiplex PCR can be used for screening mycobacterial infections and also for the rapid detection of MAP like the IS900 PCR. After all, a large proportion of the Acid-fast bacilli in the faecal samples may be from other mycobacteria or non-mycobacterial Acid-fast bacilli that need to be studied further.