Molecular characterization of Mycobacterium avium subspecies paratuberculosis Types II and III isolates by a combination of MIRU–VNTR loci

Mycobacterial interspersed repetitive units and variable number tandem repeats typing (MIRU–VNTR) is a useful technique that has been recently applied to characterize members of the Mycobacterium avium complex (MAC). The aim of this study was to examine the genetic variability among a collection of Spanish M. avium subspecies paratuberculosis (M. a. paratuberculosis) isolates with a combination of MIRU–VNTR loci. For this purpose we tested six MIRU–VNTR loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32, VNTR-292 and VNTR-259) in 70 M. a. paratuberculosis isolates of Types II and III that were recovered from 22 Spanish localities during a nine-year period (1998–2007). The combination of five loci (MIRU-2, MIRU-3, VNTR-25, VNTR-32 and VNTR-259) enabled the differentiation of 12 allelic profiles, with a resulting Hunter and Gaston discriminatory index (HGDI) of 0.84. Moreover, we obtained MIRU–VNTR patterns that were unique for each of the M. a. paratuberculosis types analyzed (II and III); other patterns were host-related or restricted to geographic areas. Therefore, this MIRU–VNTR approach could be a useful sub-typing molecular tool in order to get a better sense of the epidemiology of Johne's disease.