Genotyping of Mycobacterium avium subsp. paratuberculosis isolates from Argentinian cattle using MIRU-VNTRs and SSR (shorts sequence repeats).

IMPERIALE, B.R.; SANTANGELO MARÍA DE LA PAZ; MOYANO DAMIÁN; MARÍA FIORELLA, ALVARADO PINEDO; MARÍA ISABEL ROMANO; MAGALÍ ANDREA ROMERO; TRAVERÍA GABRIEL EDUARDO

Riviera Maya

Congreso; 14th International Colloquium on Paratuberculosis.; 2018

Asociación Internacional de Paratuberculosis

Genotyping is a very important tool of epidemiology that helps to trace back the source of infection in case of outbreaks and surveillance programs OBJECTIVE. Simultaneous genotyping of MAP strains using polymerase chain reaction (PCR)-based detection methods: MIRU-VNTR and SSRSimultaneous genotyping of MAP strains using polymerase chain reaction (PCR)-based detection methods: MIRU-VNTR and SSR.Isolates of MAP from faeces were achieved on Herrold´s medium with mycobactin J. The IS900-PCR was used to identify MAP and the IS1311-PCR-REA to classify them as C/S type. Genotyping was performed using 8 polymorphic MIRU-VNTR loci (292, X3, 3, 7, 10, 25, 32, 47) and polymorphisms using multilocus short sequence repeats (MLSSR) of four loci (L1, L2, L8 and L9). MIRU-VNTR patterns (INMV) were assigned according with the MAC-INMV database. The strain K10 was used as reference control.A total of 40 MAP isolates belonging to C type were obtained. These isolates were differentiated into 6 profiles: INMV 1 (18 isolates); INMV 2 (10 isolates); INMV 11 (3 isolates); and the remain isolates were INMV 8; INMV 5; INMV 16. MLSSR revealed loci L1 and L2 as the most polymorphics. SSR locus L1 showed isolates with 7 and 14 repeats and 10 in K10 whereas locus L2 showed isolates with 8, 9, 10, 11, 12, 13 and 17 repeats. Loci L8 and L9 only showed 4 repeats among isolates; only K10 had 5 repeats in those loci. SSR L2 allowed further differentiation of isolates with the same INMV pattern. The 18 MAP isolates with INMV1 were separated in 6 different patterns using SSR L2, each one with different number of G repeats. The 10 MAP isolates with INMV 2, were separated in 4 different genotypes with SSR L2.We could increase the differentiation among isolates using both genotypic methods. According with our results, MIRU-VNTR assay could be used as a screening tool to differentiate isolates not very close related and then SSR for isolates sharing the same INMV pattern.