ABSTRACT
RESUMEN Objetivo. Determinar la diversidad molecular de Mycobacterium avium subsp. paratuberculosis (MAP) en muestras ambientales de hatos lecheros colombianos. Materiales y métodos. Las muestras ambientales de 25 hatos lecheros positivos a MAP por IS900-qPCR se cultivaron por duplicado en medio de yema de huevo de Herrold con micobactina J para obtener aislamientos. Las colonias sospechosas fueron confirmadas para MAP por IS900-qPCR. El ADN positivo se subtipó utilizando técnicas de unidades micobacterialess repetitivas intercaladas - número variable de repeticiones en tándem (MIRU-VNTR) y técnicas de repeticiones de multilocus de secuencia corta (MLSSR) para analizar las diferencias genéticas entre los aislamientos. Resultados. El subtipado reveló dos genotipos diferentes por MIRU-VNTR (INMV 2 e INMV 36). La técnica de MLSSR se realizó para aumentar el poder discriminatorio de lo obtenido por MIRU-VNTR, pero no se observaron diferencias entre los aislamientos recuperados. Conclusiones. El presente estudio representa un enfoque importante para el conocimiento del estatus epidemiológico de MAP en la población de estudio.
ABSTRACT Objective. To determine Mycobacterium avium subsp. paratuberculosis (MAP) molecular diversity in environmental samples from Colombian dairy herds. Materials and methods. Environmental samples from 25 IS900-qPCR MAP-positive dairy herds were cultured by duplicate in Herrold's egg yolk medium with mycobactin J to obtain isolates. Suspicious colonies were confirmed by MAP-IS900-qPCR. Positive DNA was sub-typed using mycobacterial interspersed repetitive units-variable number of tandem repeat (MIRU-VNTR) and multilocus short sequence repeats (MLSSR) techniques to analyze the genetic differences between the isolates. Results. Sub-typing revealed two different genotypes by MIRU-VNTR (INMV 2 and INMV 36). MLSSR technique was carried out to increase the discriminatory power from what was obtained by MIRU-VNTR, but no differences were observed among the recovered isolates. Conclusions. The present study represents an important approach to the knowledge on MAP epidemiological status in the study population.
ABSTRACT
Introduction: The term non-tuberculous mycobacteria (NTM) includes different ambient species capable of sickening humans and/or animals, even by means of a potential zoonotic transmission. Objectives: To determine: The clinical importance of several species within the genus Mycobacterium and the genetic diversity of the M. avium complex (MAC), the in vitro bacterial sensitivity and the success of the specific treatment. Materials and Methods: Collection of clinical and epidemiologic data and information about isolates of the 2009-2016 period; molecular identification of the isolates; determination of the in vitro bacterial sensitivity and genetic diversity of the MAC; treatment evaluation. Results: 225 mycobacteriosis cases were diagnosed, with a stable prevalence of ≈6% per year and 22 recovered species: 4 rapidly growing species isolated from 66 patients and 18 slowly growing species. The MAC was isolated in 95 cases, M. avium hominissuis - 40 cases, M. intracellulare - 51 cases, M. chimaera - 3 cases and M. colombiense - 1 case. We observed a greater probability of getting sick from M. intracellulare in patients previously treated for tuberculosis (TB). HIV-positive patients had a greater risk of falling ill from M. avium hominissuis. Aminoglycosides, fluoroquinolones and macrolides were the most active drugs against most NTM. Approximately half of the cases healed. Conclusions: M. intracellulare, M. aviumhominissuis with great genetic variability and M. abscessus were the most commonly found pathogens. The cases of TB+NTM mixed disease were an important finding. For treating these patients, it was necessary to add second line drugs to the therapeutic regimen for TB; and most of them healed
Subject(s)
Bacteria , Genetic Variation , Nontuberculous MycobacteriaABSTRACT
Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.
Subject(s)
Animals , Cattle , Genetic Variation , Minisatellite Repeats , Molecular Typing/methods , Mycobacterium avium subsp. paratuberculosis/classification , Mycobacterium avium subsp. paratuberculosis/genetics , Paratuberculosis/microbiology , Argentina/epidemiology , Cattle Diseases/microbiology , Genotype , Goats , Goat Diseases/microbiology , Molecular Epidemiology , Mycobacterium avium subsp. paratuberculosis/isolation & purification , Sheep , Sheep Diseases/microbiologyABSTRACT
La tuberculosis bovina es en Argentina una enfermedad que provoca graves pérdidas económicas y que afecta a un 5 por ciento del ganado. En un trabajo previo de tipificación de cepas por RFLP mediante el uso de las sondas PGRS y DR se identificó una cepa altamente predominante que se llamó AA. A diferencia de la cepa salvaje AA, la cepa de referencia AN5, de origen europeo, que se utiliza para elaborar la tuberculina, es una cepa adaptada al crecimiento en laboratorio, que puede haber sufrido mutaciones en genes de antígenos o de virulencia. Para ello se analizó la producción de proteínas secretadas y del extracto celular, de la cepa salvaje AA comparada con la cepa de referencia AN5, con el propósito de identificar diferencias que puedan dar cuenta de la virulencia y para identificar nuevos antígenos. Se utilizaron técnicas como electroforesis en geles de policrilamida, geles de 2 dimensiones y Western blot utilizando antisueros específicos contra antígenos ya caracterizados y sueros de bovinos infectados con tuberculosis confirmada por aislamiento de M. bovis, empleando proteínas celulares y secretadas (a los 25 y 100 días de cultivo) de ambas cepas. Se pudieron identificar, una proteína secretada de aproximadamente 29 kDa y otra de 28 kDa del estracto celular que parecen ser exclusivas o producidas en mayor cantidad por la cepa AA. También, se identificaron otras pero cuyas bandas eran más débiles. En conclusión, algunas de las proteínas identificadas pueden servir para mejorar el diagnóstico de la tuberculosis bovina