Detection of non-tuberculous mycobacterial species using PCR-RFLP analysis in trans-tracheal washes in cattle and buffaloes with respiratory distress

Bovine tuberculosis (bTB) is a chronic disease of cattle with high economic importance in livestock farming caused by Mycobacterium bovis and bears a zoonotic potential. There are some non-tuberculous mycobacteria (NTM) which cause disease similar to bTB and interfere with diagnosis of bTB. Non-tuberculous mycobacteria are saprophytic in nature but some of them may cause pulmonary infections, mastitis, lesions in respiratory tract and lymph nodes of cattle, due to which they are being recognized worldwide and interfere with the diagnosis of bTB.

The aim of the study was to detect NTM species from cattle and buffaloes with respiratory distress using biochemical test and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis (PRA).

A total of 50 trans-tracheal washes were collected from cattle (n=41) and buffaloes (n=9) with respiratory distress. The samples were inoculated on Middlebrook 7H10 media after proper decontamination with 4% NaOH. The isolate obtained was identified by biochemical testing. Extracted DNA from samples and isolate was subjected to PRA which involved hsp65 gene amplification (439 bp) and RFLP analysis of amplified product.

Out of 50 trans-tracheal washes only one isolate of Mycobacterium kansasii (n=1) (2%) was obtained which was confirmed by biochemical testing and PRA. Mycobacterium kansasii (n=4) (8%), Mycobacterium intracellulare (n=1) (2%), and Mycobacterium vaccae (n=1) (2%) were identified by PRA.

The study emphasizes the importance of NTM in animals. Polymerase chain reaction-restriction fragment length polymorphism analysis is a more reliable and rapid method for identification of NTM than conventional methods.