Effectiveness of combination of Mini-and Microsatellite loci to sub-type Mycobacterium avium subsp. paratuberculosis Italian type C isolates

Abstract

BackgroundMycobacterium avium subsp. paratuberculosis (Map) is the etiological agent of paratuberculosis. The aim of our study was to combine Mini-and Microsatellite loci analysis in order to explore the effectiveness of this sub-typing method in a group of Map isolates. For this purpose, 84 Italian Type C Map isolates, each from a different cattle herd, were submitted to MIRU-Variable-Number Tandem-Repeats (VNTRs) typing and Short Sequence repeats (SSRs) sequencing. Moreover, the method was used to analyse the variability inside 10 herds (from three to 50 isolates per herd).ResultsThe molecular sub-typing, carried out using three SSR and 10 MIRU-VNTR loci, differentiated the 84 isolates into 33 clusters, reaching a Simpson's Discriminatory Index (SID) value of 0.952 (0.933 to 0.972, 95% confidence intervals). Among all considered loci, six (SSR2, MIRU2, SSR1, SSR8, VNTR3527 and VNTR1067) showed relevant allelic variability. Thirty-eight% of the isolates were clustered into four genotypes, differing from each other for the SSR2 locus. The other isolates, characterised by differences in two or more loci, were spread among the rest of the clusters. The intra-herd analysis revealed more than one genotype in most herds with a similar distribution of clusters.ConclusionsOur results revealed the advantage of using both Mini-and Microsatellite approaches for successfully discriminating among Map Type C isolates from the same geographic area, host species and herd. These data suggest that the combination of loci here proposed could be a useful molecular tool for regional epidemiological studies.

Survey on the presence of Mycobacterium avium subsp. paratuberculosis in ground beef from an industrial meat plant

“…The genotyping analysis revealed an unknown locus profile not previously found in Italy and an already described locus profile, which is largely diffused in Northern Italy (Ricchi et al, 2011).…”

“…For the allelic profile, the loci analysed were, respectively, MIRU 1, MIRU 2, MIRU 3, VNTR 25, VNTR 32, VNTR3, VNTR 7, VNTR 47, MLVA 1067 and MLVA 3527 for Mini satellites loci and SSR 1, SRR 2 and SSR 8 for Micro satellites. One sample (sample 51) showed a profile already described (3 7 5 3 8 2 2 3 2 2 7 >11 4) (Ricchi et al, 2011), denominated as MSV27 in the original publication. This allelic profile was found to be widespread in the Northern Italian herds.…”

“…The LOD of cultural assay was calculated taking into account the result of F57-qPCR independently of the call (positive or negative) obtained after the incubation in the VersaTREK1 instruments. The two positive cultures confirmed by F57-qPCR, were typed according to Marsh et al (1999) and sub-typed according to a 13 Micro and Minisatellites scheme already reported (Ricchi et al, 2011).…”

Genetic Structure of Mycobacterium avium subsp. paratuberculosis Population in Cattle Herds in Quebec as Revealed by Using a Combination of Multilocus Genomic Analyses

“…This might be due to random dispersal of the bacteria following large-scale processes such as cattle movements between herds. It has been reported that herds with an elevated turnover of cows show multiple genotypes (25,61), underscoring the importance of live animal trade in the transmission of M. avium subsp. paratuberculosis between herds.…”

“…paratuberculosis is a highly monomorphic species, and using a combination of multilocus typing methods is necessary to increase their overall discriminatory power, as previously shown for the M. tuberculosis complex (21,22) and M. avium subsp. paratuberculosis strains from different host species and different geographic locations (17,(23)(24)(25)(26). A typing scheme based on 8 MIRU-VNTR loci (called INMV typing) has been previously proposed for the M. avium complex (26).…”