Molecular typing of Mycobacterium bovis strains isolated in Italy from 2000 to 2006 and evaluation of variable-number tandem repeats for geographically optimized genotyping

Abstract

Spoligotyping and exact tandem repeat (ETR) analysis of Mycobacterium bovis and M. caprae isolated strains has been routinely carried out in Italy since 2000 to obtain a database of genetic profiles and support traditional epidemiological investigations. In this study, we characterized 1,503 M. bovis and 57 M. caprae isolates obtained from 2000 to 2006 in 747 cattle herds mainly located in northern Italy. We identified 81 spoligotypes and 113 ETR profiles, while the combination of spoligotyping/ETR analysis differentiated 228 genotypes, with genotypic diversity indices of 0.70 (spoligotyping), 0.94 (ETR-A to -E typing), and 0.97 (spoligotyping/ETR-A to -E typing), respectively. Despite the high degree of resolution obtained, the spoligotyping/ETR methods were not discriminative enough in the case of genotypes characterized by the combination of SB0120, the predominant spoligotype in Italy, with the most common ETR profiles. To obtain a more informative subset of typing loci, 24 mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) markers were evaluated by analyzing a panel of 100 epidemiologically unrelated SB0120 isolates. The panel was differentiated into 89 profiles with an overall genotypic diversity of 0.987 that could be also achieved by using a minimal group of 13 loci: ETR-A, -B, and -E; MIRU 26 and 40; and VNTR 2163a, 2163b, 3155, 1612, 4052, 1895, 3232, and 3336. The allelic diversity index and the stability of single loci was evaluated to provide the most discriminative genotyping method for locally prevalent strains.

FIG. 1.

Dendrogram and schematic representation of 81 different spoligotypes obtained from the analysis of 747 isolates. Most spoligotypes are clustered in one major BCG-like group of similarity. A divergent group includes M. caprae patterns and similar M. bovis spoligotypes (indicated by asterisks). The dendrogram shows genetic distances and was generated by using the categorical character option and UPGMA clustering method (Bionumerics software). When available, the spligotype code is given.

FIG. 2.

Geographical distribution of isolates from the Piedmont, Lombardy, Emilia-Romagna, and Veneto regions of Italy. The most predominant spoligotypes are SB120 (55.4%), SB0134 (5.7%), and SB0841 (4.1%). Thirty strains showed spoligotypes consistent with M. caprae identification.

FIG. 3.

Dendrogram and schematic representation of 100 SB0120 M. bovis strains typed for 24 MIRU-VNTR loci. The panel was differentiated into 89 different profiles with 9 clusters only. The dendrogram shows genetic distances and was generated as described in Materials and Methods. *, epidemiologically linked isolates.