New mini-satellite tandem repeat markers for strain typing of Mycobacterium avium subsp. paratuberculosis

The multiplicity of infection sources along with the genetically homogenous nature of Mycobacterium avium subsp. paratuberculosis (MAP) necessitates the application of high-discriminative fingerprinting techniques to establish epidemiological links and control of paratuberculosis in ruminants. The present study aimed to develop a simple Multiple-Locus Variable-Number Tandem-Repeat Analysis (MLVA) assay suitable for low-budget laboratory settings.

A bioinformatics screening was conducted on three annotated MAP genomes (K10, MAP4, and MAP-JIII-386) leading to the detection of 7 potential polymorphic variable-number tandem repeats (VNTRs). A MAP collection of the three aforementioned strains, 30 field isolates and 2 laboratory strains from Iran plus 59 global Mycobacterium avium complex strains were subsequently genotyped at laboratory or in situ. Findings were validated against the French INRAE MLVA typing system (Thibault's scheme) dedicated to typing Mycobacterium avium subsp. paratuberculosis, hominissuis, avium, and silvaticum.

Fourty-one MLVA types were detected by our suggested MLVA scheme, compared to 45 types found by Thibault's scheme. The calculated discriminatory power of the MLVA for the former and the latter was 0.93 and 0.95, respectively.

The suggested MLVA scheme discriminated between isolates with identical types determined by Thibault's scheme. Noting the fact that strains grouped in INMV types 2, 3, 4, 5, 33, 39, 68, 82, 131, 219, 220, New1 and New 25 were further resolved into sub-groups, we suggest considering VNTR 1067 (DI = 0.71), MAP 4356 (DI = 0.68), MATR 7 (DI = 0.53), MAP 3824 (DI = 0.56) and MATR 6 (DI = 0.49), in any future upgrading of the current version of the Thibault's scheme.