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

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In this study, 147 MAP field cultures from Hesse, Thuringia, and Saxony were genotypically and phenotypically characterized. Various genetic marker combinations were evaluated to create a cost-effective typing approach that maintains high accuracy. A combination of VNTR panels (INMV + MV) yielded a lower discriminatory index (DI) of 0.895 compared to the established INMV + SSR approach (DI 0.931). Two novel VNTR loci were identified, with one (VNTR 1798) allowing the development of two cost-effective typing methods (DIs of 0.916 and 0.927), avoiding the expense of SSR sequencing. However, phylogenomic studies have questioned the efficacy of MIRU-VNTR for MAP typing. A new hierarchical approach (SNP + INMV) was proposed, achieving a robust and highly discriminating DI of 0.938, which improved further with additional markers, including VNTR 1798. All cultures were classified as type C, revealing significant heterogeneity with eight phylogenetic groups identified, four of which are newly described in Germany. Notably, clade 6 was predominantly found in Hesse, suggesting a common infection source. Multiple strains were observed in 13-25% of herds, indicating intra-animal mutation. Novel TRs and group-discriminating SNPs were discovered, and melting curve analysis was tested as a potential allele determination method. Phenotypic characterization using FT-IR effectively distinguished between MAP type C subgroups. The methodologies d