New variable-number tandem-repeat markers for typing Mycobacterium avium subsp. paratuberculosis and M. avium strains: comparison with IS900 and IS1245 restriction fragment length polymorphism typing

Abstract

Mycobacterium avium subsp. paratuberculosis, the etiological agent of paratuberculosis, affects a wide range of domestic ruminants and has been suggested to be involved in Crohn's disease in humans. Most available methods for identifying and differentiating strains of this difficult species are technically demanding and have limited discriminatory power. Here, we report the identification of novel PCR-based typing markers consisting of variable-number tandem repeats (VNTRs) of genetic elements called mycobacterial interspersed repetitive units (MIRUs). Eight markers were applied to 183 M. avium subsp. paratuberculosis isolates from bovine, caprine, ovine, cervine, leporine, and human origins from 10 different countries and to 82 human isolates of the closely related species M. avium from France. Among the M. avium subsp. paratuberculosis isolates, 21 patterns were found by MIRU-VNTR typing, with a discriminatory index of 0.751. The predominant R01 IS900 restriction fragment length polymorphism type, comprising 131 isolates, was divided into 15 MIRU-VNTR types. Among the 82 M. avium isolates, the eight MIRU-VNTR loci distinguished 30 types, none of which was shared by M. avium subsp. paratuberculosis isolates, resulting in a discriminatory index of 0.889. Our results suggest that MIRU-VNTR typing is a fast typing method that, in combination with other methods, might prove to be optimal for PCR-based molecular epidemiological studies of M. avium/M. avium subsp. paratuberculosis pathogens. In addition, presumably identical M. avium subsp. paratuberculosis 316F vaccine strains originating from the Weybridge laboratory and from different commercial batches from Mérial actually differed by one or both typing methods. These results indicate a substantial degree of genetic drift among different vaccine preparations, which has important implications for prophylactic approaches.

FIG. 1.

Selected IS900 RFLP profiles represented in our collection of M. avium subsp. paratuberculosis strains. The percentages indicate the proportion of each IS900 RFLP profile in our collection. R types are designated according to the nomenclature of the National Institute of Public Health and the Environment, Bilthoven, The Netherlands.

FIG. 2.

IS900 RFLP profiles of M. avium subsp. paratuberculosis 316F strains. Profiles from five different cultures of Mérial-316F (Néoparasec) corresponding to four batches, Néop 69340, Néop 68451, Néop 4/81, and Néop 8/81, as well as from one culture from a 316F strain from Weybridge, are represented. Néop 68451a and Néop 68451b correspond to cultures from two different Mérial-316F vials identified by the same batch number, 68451. R and C types are designated according to the nomenclature of the National Institute of Public Health and the Environment, Bilthoven, The Netherlands, and Collins et al. (5) and Pavlik et al. (19), respectively. The arrows indicate polymorphic bands among the different profiles.

FIG. 3.

MIRU-VNTR profiles of M. avium subsp. paratuberculosis 316F strains from Weybridge laboratory and Mérial. The PCR products were analyzed by electrophoresis using agarose gels, as described in Materials and Methods. The positions of size standard bands and designations of MIRU-VNTR loci are indicated on the left and at the top, respectively. a, analysis of Weybridge 316F strain; b, analysis of Mérial 316F strain. A large asterisk indicates the locus (X3) that varies between the two strains. *a, provided by P. Willemsen (Central Institute for Animal Disease Control, Department of Bacteriology and TSEs, 8203 AA Lelystad, The Netherlands); **b, provided by K. Stevenson (Moredun Research Institute, Pentlands Science Park, Bush Loan, Penicuik EH26 0PZ, Scotland, United Kingdom). The batches marked ^c and ^d were cultured from two different Néoparasec vials identified by the same batch number on the same date.