Evaluation of mycobacterial interspersed repetitive-unit-variable-number tandem-repeat analysis and spoligotyping for genotyping of Mycobacterium bovis isolates and a comparison with restriction fragment length polymorphism typing

Abstract

Common strain typing methods for differentiation of Mycobacterium bovis isolates include restriction endonuclease analysis (REA), restriction fragment length polymorphism (RFLP) analysis, spoligotyping, and, more recently, mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. MIRU-VNTR typing and spoligotyping were evaluated in this study, and these typing methods were compared with RFLP typing. A total of 386 M. bovis isolates from cattle, badgers, and deer in the Republic of Ireland that had previously been typed by IS6110, polymorphic GC-rich sequence (PGRS), and direct-repeat (DR) RFLP were included in the study. Spoligotyping and analysis of six VNTR loci (QUB 11a, QUB 11b, ETR A, 4052, MIRU 26, and 1895) were performed on the samples. RFLP analysis was the method that gave the greatest differentiation of strains, with a Hunter-Gaston discriminatory index (HGDI) of 0.927; the HGDI recorded for MIRU-VNTR typing was marginally lower at 0.918, and spoligotyping was the least discriminatory method, with an HGDI of 0.7. Spoligotype SB0140 represented approximately 50% of the isolates. Within the group of isolates represented by SB0140, there was a much lower level of concordance between RFLP and MIRU-VNTR typing than for groups represented by other spoligotypes. A combination of spoligotyping and MIRU-VNTR typing offered advantages over MIRU-VNTR typing alone. In a combined spoligotyping and MIRU-VNTR typing protocol, the number of VNTR loci could be reduced to four (QUB 11a, QUB 11b, ETR A, and 4052) while maintaining a high level of strain differentiation.