The most widely used method to type M. avium ssp. paratuberculosis (Map) strains is RFLP based on IS900. This is, however, only applicable to culturable strains, technically demanding and requires analysis of complex banding patterns. Development of molecular typing methods available for other mycobacteria can provide rapid and more discriminatory procedures for studying the diversity of the Map strains.
Objective
The aim of this study was to identify new genetic markers, consisting of variable number tandem repeat (VNTR) of genetic elements called mycobacterial interspersed repetitive units (MIRUs), to study the clonal distribution and the degree of diversity of Map isolated from different geographic origins and from different hosts.
Methods
A panel of 35 strains including bovine, caprine, ovine, deer, and human isolates from six different countries were used. Thirty-five MIRU-VNTR were selected by using the TRF software and the genomic sequence of Map K10, and by blast analysis using MIRU loci identified in M. tuberculosis. The MIRU-VNTR types were determined by PCR amplification of the different MIRU-VNTR loci, and sizing of the PCR products. The numbers of tandem repeat motifs in the MIRU-VNTR loci were deduced from the sizes of the corresponding PCR products. The IS900-RFLP strain profiles were determined by the standard method and compared to the MIRU-VNTR types.
Results
Among the 35 MIRU-VNTR loci, only 6 were polymorphic. Compared to the IS900 RFLP, MIRU-VNTR typing subdivided the predominant RFLP type, into five subtypes suggesting that this approach provides an additional tool to study the genetic diversity of Map.
Conclusion
although this preliminary study indicates that MIRU-VNTR typing may provide additional discrimination to distinguish different clones of Map, it further highlights the overall genetic homogeneity of this subspecies. Therefore, a combination of microsatellite and minisatellite-based typing might prove to be optimal for PCR-based molecular epidemiological studies of this pathogen.
