Development of a hierarchical typing approach for Mycobacterium avium subsp. paratuberculosis (MAP) and characterization of MAP field cultures from Central Germany

Abstract

Aims: Development of a novel hierarchical Mycobacterium avium subsp. paratuberculosis (MAP) typing approach and characterization of MAP field cultures in Central Germany. Methods and Results: By combining single nucleotide polymorphisms (SNPs) and mycobacterial interspersed repetitive unit-variable number tandem repeat, we developed a highly discriminating and phylogenetically accurate hierarchical MAP typing approach. Moreover, a novel stepwise workflow was employed to reduce the number of SNP reactions required making the typing approach more affordable. MAP field cultures (n = 142) from dairy herds in Central Germany were classified as cattle type and showed a high level of heterogeneity. Intra-herd multiple genotypes were evident in (13-25%) of the investigated herds. Conclusions: The hierarchical MAP typing approach proved to be useful in fine discrimination between MAP cultures within limited geographical regions. This could potentially be used in unravelling MAP transmission chains in the respective regions. The observed heterogeneity in some herds is assumed to be due to either multiple introductions through inter-herd trade or intra-herd evolution over time. Significance and Impact of the Study: Future MAP epidemiological studies will benefit from the advantages of the novel hierarchical typing approach. The SNP number reduction approach employed here could be extrapolated for other analogous pathogens.

Strain diversity in Mycobacterium avium subsp. paratuberculosis-positive bovine fecal samples collected in Switzerland

“…Especially INMV 1 and INMV 2, to a lower extend also INMV 3, INMV 5, INMV 6, and INMV 9, are commonly found in Europe ( 12 , 24 ), while S-type MAP (INMV 72) was only detected before in New Zealand and Spain ( 25 ). INMV 116 was reported to exist in Ireland and Germany ( 26 , 27 ). The two new genotypes INMV 252 (C-type MAP; detected once) and INMV 253 (S-type MAP; detected in two cantons), were not observed in other countries so far.…”

Molecular Diversity of Mycobacterium avium subsp. paratuberculosis in Four Dairy Goat Herds from Thuringia (Germany)

“…various phylogenetic subgroups, together with cattle or sheep isolates [36]. Howev most previous studies investigating the intra-herd or inter-herd diversity of MAP or cussing the possible transmission routes of the disease have focused on cattle, sheep deer, but not on goats [39,[46][47][48][49][50][51][52][53]. Currently, no genotyping data of goat isolates are av able from Germany.…”

“…These SNPs are mutually exclusive, i.e., if an isolate possess one particular SNP, it lacks all the other ones [38]. As recommended in [39] (see supplementary Table S3), a MIRU-VNTR analysis was performed first. Based on the detected INMV types, specific PCRs were prioritized for the SNP-based analysis to reduce the number of SNP-PCRs for each sample.…”

“…Depending on the applied method, previously described or novel distinct genotypes have been identified, or isolates have been assigned to defined or novel phylogenetic groups and subgroups by comparing WGS-based single nucleotide polymorphisms (SNPs) [37,38]. Recently, Fawzy et al [39] recommended a hierarchical genotyping approach including a combination of mycobacterial interspersed repetitive unit variable number tandem repeat (MIRU-VNTR) analysis [40] and an SNP-based assay [38], resulting in fine discrimination of MAP from animals within limited geographical regions.…”

“…In previous studies, goat isolates originating from different herds in Spain or from individual goats in some European countries showed a high diversity [43][44][45] and belonged to various phylogenetic subgroups, together with cattle or sheep isolates [36]. However, most previous studies investigating the intra-herd or inter-herd diversity of MAP or discussing the possible transmission routes of the disease have focused on cattle, sheep, or deer, but not on goats [39,[46][47][48][49][50][51][52][53]. Currently, no genotyping data of goat isolates are available from Germany.…”