Strain differentiation of 13 indigenous Mycobacterium bovis isolates from infected cattle by restriction fragment...

Mycobacterium bovis is mainly detected in cattle throughout the world. This bacterium is considered the main causative agent of tuberculosis in man and animals. M. bovis is also reported to be endemic in badgers and in farmed and feral deer. The disease caused by M. bovis is a slow progressive disease with clinical signs not apparent until late in the disease process. key factors for effective control of tuberculosis includes rapid detection, adequate therapy, and contact tracing to halt further transmission. However, in order to locate the source and route of transmission of M. bovis infection, a thorough epidemiological analysis is routinely carried out, that could lead to the control of disease in the herds and avoid economic losses. Recent developments in DNA technology and molecular biology have led to methods for the rapid detection of mycobacterium DNA. Among a number of described molecular methods, IS6110 fingerprinting is the recommended standard primary genotyping method and the most widely used worldwide. In this study, we used restriction fragment length polymorphism analysis with probes derived from the insertion element IS6110, the polymorphic GC-rich sequence (PGRS), and the direct repeat (DR) sequence which proved to be a useful method for differentiating M. bovis strains. A total of 13M. bovis samples from infected cattle in the West Azerbaijan Province of Iran were included in the study. The samples were submitted to the Tuberculosis Reference Laboratory at the Razi Vaccine and Serum Research Institute, Karaj. All isolates were cultivated on Lowenstein Jensen media with pyruvate (pyruvic acid), and then identified according to The World Organization for Animal Health (OIE) recommendations. The extracted genomic DNA samples of the isolates in the study were subjected to IS6110 primers, digested with restriction enzyme PvuII, and hybridized by oligonucleotide probes PGRS and DR. Polymorphic banding patterns obtained after hybridization discriminated the M. bovis strains and a database of strain types was established. Based on our results, the 13 isolates showed five different DNA patterns with a PGRS probe and similarly five patterns were obtained with the DR probe. PP-1 pattern was found almost among all the isolates while a distinct DNA pattern PP-3 was seen specifically from the West Azerbaijan Province.