Assessment of Molecular Characterization of Mycobacterium avium Subsp. Paratuberculosis Isolates


Johne’s illness is caused by Mycobacterium avium subsp. paratuberculosis (MAP), which is highly frequent in domestic ruminants. There are no systematic disease control programmes in place in India, and the exact prevalence of MAP genotypes is unclear. The purpose of this research was to investigate the molecular characteristics of MAP isolates.

Methods: Molecular characterisation of approximately 22 MAP isolates from cattle, sheep, and goats was performed using three distinct 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 found that a) IS1311 PCR with REA (based on point mutations) classified all 22 MAP isolates as “intermediate type” and also belonged to the Indian Bison type, regardless of the host. The MAP isolates had more lineages toward the reference Type III, Intermediate strain, according to molecular typing based on partial amplification and sequencing of the gyrA and gyrB genes.

Results: Sheep-derived MAP isolates had more sheep-like lineages than cattle- and goat-derived isolates. This diversity could be due to host-pathogen interactions and natural adaptation to various hosts and environmental situations. The online software NEB cutter was used to analyse partial nucleotide sequences of IS1311-PCR products for polymorphism in RE sites. The MAP specific restriction enzyme Hinf1 sites and the BSJ1 site, which is particular for Indian Bison type, were found in the IS1311 PCR products sequence.

Conclusions: The DMC-PCR, which is based on a sequence difference at the 5′ end of the MAP IS900, quickly identified all of the isolates as sheep type. Because the Intermediate type (Type III) and sheep type (Type I) are very closely related, the use of DMC-PCR to differentiate sheep and intermediate types is limited. All of the MAP isolates were confirmed as Intermediate or Type III using three different methods that are commonly used in India, Spain, and Iceland.

Author(S) Details

M. Sobha Rani
Department of Sero-epidemiology, Southern Regional Disease Diagnostic Laboratory, Institute of Animal Health and Veterinary Biological, Hebbal, Bengaluru, Karnataka, India.

T. G. Prabhakar
Department of Veterinary Microbiology, Madras Veterinary College, Chennai, Tamil Nadu, India

B. Samuel Masilmoni Ronald
Department of Veterinary Microbiology, Madras Veterinary College, Chennai, Tamil Nadu, India.

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