Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains

Abstract

We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six 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 M. paratuberculosis isolates representing different multiplex PCR for 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 with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.

FIG. 1.

Sequence analysis of two representative SSR loci. (A) Locus 8 with (GGT)₅ repeats. Strain MAP-K10 contains five copies of GGT, while MAP-08 and MAP-11 contain four and three copies of GGT, respectively. (B) Locus 10 with (GCC)₅ repeats. Strain MAP-K10 contains five copies of GCC, while MAP-11 and MAP-07 have a A-to-C substitution in a single copy of GCC.

FIG. 2.

Allelic variation at 11 SSR loci among 33 M. paratuberculosis isolates. The aligned nucleotide sequences of each of the alleles at the 11 SSR loci discovered and characterized during this investigation, along with adjacent conserved sequences, are shown. The SSRs and polymorphic sites are boxed. The locations of single-nucleotide polymorphisms are indicated with asterisks below the aligned sequences.

FIG. 3.

Dendrogram depicting genetic relationships among 33 M. paratuberculosis isolates on the basis of the 11 SSR loci determined by MLSSR analysis. The dendrogram was generated by the unweighted pair-group method with arithmetic averages with the PAUP program. The results of the bootstrap analysis are represented as percentages and are indicated adjacent to the major nodes when the branch order was supported by >50% of the 1,000 replicate trees. Genetic distance is indicated at the top of the dendrogram. Isolate identifications, sources, geographic locations, and MILP and AFLP types are shown to the right of the dendrogram.