Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Previous investigations have revealed a relative lack of genetic diversity amongst isolates. Combined with the slow growth of the organism in pure culture, strain differentiation among Map isolates has proved to be difficult and has limited the study of the molecular epidemiology of the disease. We here compare a set of 268 isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) that have been previously characterized for IS1311 PCR-restriction endonuclease analysis and SnaBI-SpeI pulsed-field gel electrophoresis patterns with the more recently described short sequence repeat (SSR) analysis of locus 1 (G residue) and locus 8 (GGT residue).

The results show that a total of nineteen different multi-locus SSR (SSR1_SSR8) types were identified amongst the 268 isolates. In terms of host species distribution, there were 13 SSR types identified from cattle, 6 from sheep and 3 from goat isolates. Cluster analysis with both PFGE and SSR based typing methods confirmed that Map isolates are genetically divided into two main groups, the cattle type and sheep type groups Amongst isolates recovered from Spain, SSR type type 7_4 accounted for the 54% of cattle isolates, while types 7_3 and 14_3 together accounted for the 29% of sheep isolates. Interestingly, amongst isolates recovered from goats, approximately the same proportion (43%) of isolates were typed as either cattle type (7_4) or sheep type (14_3). While the overall discriminatory power of both methods as calculated by Simpson´s index of diversity (D) was almost the same (0.693 for PFGE and 0.691 for SSR) for both methods, comparative analysis revealed that the most abundant PFGE 1-1, 2-1 and 23-16 profiles were subdivided into 11, 7 and 4 different types, respectively. Similarly, isolates representing the most abundant SSR type (7_4) could be subdivided into 19 different PFGE profiles. Amongst isolates recovered from sheep, there was a slightly higher discrimination with PFGE (D = 0.865) than with SSR (D = 0.775).

Taken together, the results of our studies confirm the utility of the SSR approach as an easy and rapid method based on PCR and sequence analysis that requires only small amounts of sample to perform. The results also suggest that the addition of a third locus to SSR typing may help in increasing the discriminatory power of this method. Overall, the results of our comparative analyses suggest that, based on current methodologies available, a combined approach that includes IS1311 PCR-REA, SSR and PFGE provide the highest level of discrimination for Map strain typing.

*Both authors contributed equally