Genotypic and phenotypic characterisation of Mycobacterium avium subsp. paratuberculosis (MAP) cultures from different states of Germany

In the present work, MAP field cultures (n=147) from three German federal states (Hesse, Thuringia and Saxony) were subjected to genotypic and phenotypic characterisation. Different combinations of genetic markers were tested to formulate a typing approach that would provide sufficiently high and accurate MAP discrimination at a reasonable cost. First, a cost effective typing approach combining different variable number tandem repeat (VNTR) panels (INMV + MV) provided a significantly lower discriminatory index (DI) value (0.895) compared with an established typing approach (INMV + SSR; DI 0.931). Furthermore, two novel MAP VNTR loci were discovered. One locus (VNTR 1798) enabled the formulation of two cost effective and fairly discriminating (DI 0.916 and 0.927) MAP typing approaches by avoiding the higher costs associated with sequencing short sequence repeat (SSR) loci. Lately, however, phylogenomic studies have provided evidence against using MIRU-VNTR for MAP typing. Therefore, a novel, relatively cost effective hierarchical typing approach (SNP + INMV) was formulated that guaranteed both a phylogenetically robust and highly discriminating (DI 0.938) genotyping. Even more discrimination was achieved when further markers including the novel locus (VNTR 1798) were extra combined. Based on the hierarchical approach, all the investigated MAP cultures were characterised as type C and were found to display a relatively high heterogeneity. Eight phylogenetic groups were identified among the cultures, from which four are described for the first time for MAP in Germany. One group (clade 6) was almost exclusively identified in Hesse (40%; 21/52) which could reflect a common infection source. Multiple strains were detected in 13-25% of the herds that had provided multiple faecal and/or environmental samples. Interestingly, two strains with a minor genetic variation were identified in one cow and are assumed to be a result of intra-animal mutation in the sense of “micro-evolution”. By investigating the VNTR 7 sequence data, novel TRs (16 and 56 bp) and two group-discriminating SNPs were identified and sequencing is recommended to correctly assign the underlying alleles at this locus. Moreover, promising results were obtained upon testing melting curve analysis (MCA) as a possible MIRU-VNTR allele determining technique based on the differential Tm values. Viable cultures were also phenotypically characterised using Fourier transform infrared spectroscopy (FT-IR) as a rapid, simple and cost-effective bacterial typing tool based on the whole biomolecular composition. Discrimination between the two main phylogenetic groups of MAP type C (subgroups A and B) was accomplished. Within subgroup A, clade 6 as the most frequently identified group in Hesse was successfully discriminated from all other tested clades. To conclude, the practicability, accuracy, cost effectiveness and high discrimination provided by the typing methodologies and approaches developed in this work will improve future epidemiological MAP investigations at regional or national levels. Furthermore, the high genetic diversity of MAP in the investigated regions should be considered in local control programs.