Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/2/2004
Publication Date: 4/24/2004
Citation: Amonsin, A., Li, L., Zhang, Q., Bannantine, J.P., Motiwala, A., Sreevatsan, S., Kapur, V. 2004. A multi-locus short sequence repeat sequencing approach for mycobacterium avium subspecies paratuberculosis stain differentiation. Journal of Clinical Microbiology. 42:169-1702. Interpretive Summary: The goal of this study was to find a better way to differentiate between strains of Mycobacterium paratuberculosis, the bacterial agent that causes Johne's disease. By analyzing the complete genome sequence of M. paratuberculosis, we found several short sequence repeats scattered throughout the genome. Further analysis of these short repeats showed that they vary among strains of M. paratuberculosis. The sensitivity of this method of differentiating M. paratuberculosis strains is even better than other published methods. This new technique will allow for better epidemiological tracking of strains, which will lead to clues for better management practices for Johne's disease.
Technical Abstract: We here describe a multi-locus short sequence repeat (MLSSR) sequencing approach for genotyping strains of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis). Preliminary analysis identified 185 mono-, di-, and tri-nucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci in 6 M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs) with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 isolates of M. paratuberculosis representing different multiplex PCR of IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types consistent with geographic and epidemiologic correlates and an Index of Discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and the cattle isolates of M. paratuberculosis, and easily and reproducibly differentiated strains representing the previously described predominant MPIL (A18) and AFLP genotypes (Z1 and Z2) of M. paratuberculosis. Taken together, the results of our studies suggest that MLSSR sequencing will enable facile and reproducible high resolution subtyping of M. paratuberculosis isolates for molecular epidemiological and population genetic analyses.