Assessment of Molecular Characterization of Mycobacterium avium Subsp. Paratuberculosis Isolates

Abstract

Objective: Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease, which is very common in domestic ruminants. In India, the exact prevalence of MAP genotypes is unknown, and there are no systematic disease control initiatives in place. The goal of this study was to look into the molecular characterisation of MAP isolates. Methods: About 22 MAP isolates were from cattle, sheep, and goat under gone the molecular characterization by three different methods (1) IS1311 polymerase chain reaction (PCR) with restriction enzyme analysis (REA), (2) GyrA and GyrB PCR with sequencing, and (3) DM Collins(DMC)-PCR. The study demonstrated that a) IS1311 PCR with REA (based on point mutations) identified all 22 MAP isolates as “intermediate type” irrespective of a host of origin and also belong to Indian Bison type. Molecular typing based on the gyrA and gyrB genes partial amplification and sequencing revealed that the MAP isolates exhibited more lineages toward the reference Type III, Intermediate strain. Results: The MAP isolate of sheep origin showed more lineages toward the sheep type than the isolates of cattle and goats. This variation may be due to host-pathogen interactions and adaptation to different hosts and environmental conditions in the nature. Sequence analysis of partial nucleotide sequences of  IS1311-PCR products was analyzed for polymorphism in RE sites using the online software NEB cutter. The IS1311 PCR products sequence showed the MAP specific restriction enzyme Hinf1 sites and the BSJ1 site, which is specific for Indian Bison type. Conclusions: The DMC-PCR, which is based on sequence difference at 5` end of IS900 of MAP, differentiated rapidly all the isolates as sheep type. The application of DMC-PCR to differentiate sheep and Intermediate types is limited as the Intermediate type (Type III) and sheep type (Type I) are very closely related to each other and all the MAP isolates were confirmed as Intermediate or Type III by three different methods which are commonly present in India, Spain, and Iceland.