Diversidad de especies de micobacterias no tuberculosas aisladas en ambientes acuáticos de la ciudad de General Pico, La Pampa, Argentina / Species diversity of non-tuberculous mycobacteria isolated from aquatic environments of General Pico city, Province of La Pampa (Argentina)

Las micobacterias no tuberculosas (MNT) no solo se estudian por su importancia como patógenos oportunistas, sino también por sus aplicaciones en biotecnología y biorremediación. Nuestro objetivo fue determinar la presencia de micobacterias en los distintos hábitats acuáticos de la ciudad de General Pico (provincia de La Pampa), así como su diversidad. Los porcentajes de muestras positivas a micobacterias fueron los siguientes: 37,5% en el sistema de distribución de agua de red, 32,6% en el acuífero que abastece dicho sistema, 36,8% en el agua proveniente de las precipitaciones, 53,1% en los humedales del área de influencia, 80% en los natatorios cubiertos y 33,3% en las fuentes decorativas ubicadas en plazas públicas. De los 90 aislamientos de MNT obtenidos el 8,9% no logró ser identificado a nivel de especie con los métodos utilizados, que incluyeron pruebas fenotípicas y métodos moleculares. Las especies más frecuentemente aisladas fueron Mycobacterium fortuitum y Mycobacterium gordonae. Algunas especies identificadas han sido reportadas en casos de micobacteriosis en nuestro país, entre ellas M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum y M. nonchromogenicum. No se aislaron MNT en muestras de agua de red con concentraciones de cloro activo residual mayores de 0,8mg/l, mientras que en los natatorios la presencia de hasta 1,5mg/l de cloro activo residual no fue una limitante para la proliferación de estos microorganismos. Se puede considerar que la incidencia de micobacterias en los ambientes acuáticos de General Pico es cercana al 35%, y que la presencia de estos microorganismos y su diversidad se ve afectada por el contacto con el hombre y sus actividades, como así también por la existencia de vida animal.

Non-tuberculous mycobacteria (NTM) are studied not only for their importance as emerging opportunistic pathogens but also for their applications in biotechnology and bioremediation. Our aim was to determine the occurrence and diversity of mycobacteria in different aquatic habitats of General Pico city, Province of La Pampa. The percentage of samples with positive cultures for mycobacteria were the following: 37.5% recovered from the water supply distribution system; 32.6% from the aquifer that supplies water to the distribution system; 36.8% from rain water; 53.1% from the two wetlands in the area of influence; 80% from indoor swimming pools; and 33.3% from water fountains in downtown public squares. Of the 90 NTM isolates, 8.9% could not be identified at the species level with any of the used methods, phenotypic tests and molecular methods. Mycobacterium fortuitum and Mycobacterium gordonae were the most frequently isolated species. Some of the identified species such as, M. fortuitum, M. gordonae, M. intracellulare, M. vaccae, M. lentiflavum and M. nonchromogenicum, have been reported in cases of mycobacteriosis in Argentina. Mycobacteria with values higher than 0.8mg/ml of residual active chlorine were not recovered from the drinking water supply network, whereas in the swimming pools the presence of up to 1.5 mg/l was not a constraint. Based on our results, the presence of mycobacteria in aquatic environments is close to 35% and their occurrence and diversity is affected both by contact with man and his activities as well as by the existence of animal life.

Mycobacterium bovis en fauna silvestre de la cuenca lechera de Santa Fe, Argentina / Mycobacterium bovis in wildlife of the dairy regions of Santa Fe (Argentina)

El control y la erradicación de la tuberculosis bovina basados en la detección de los animales infectados y su inmediata faena permitió lograr progresos satisfactorios en varios países y regiones, pero no todos pudieron lograrlo debido principalmente a la presencia de fauna silvestre infectada con Mycobacterium bovis. La Argentina aplica desde 1999 estas mismas premisas y ha logrado avances en los rodeos lecheros, aunque no se ha evaluado el factor ambiental como la fauna silvestre. El objetivo de este trabajo fue determinar si la fauna silvestre de la cuenca lechera de Santa Fe está infectada con M. bovis. Se realizó la captura/sacrificio de fauna silvestre presente en 5 rodeos lecheros con altos niveles de reaccionantes positivos a la prueba de tuberculina. Sobre 95 mamíferos silvestres examinados, se aisló M. bovis de 7 individuos de comadreja overa (Didelphis albiventris), de uno de zorro gris (Lycolapex gimnocercus) y de uno de rata (Rattus norvegicus). Los sitios anatómicos que produjeron estos aislamientos variaron de acuerdo con las especies; en ninguno de los ejemplares evaluados se observaron lesiones macroscópicas de tuberculosis. Los espoligotipos de M. bovis aislados con mayor frecuencia de los animales silvestres correspondieron a los tipos 34 (4 aislamientos) y 12 (3 aislamientos); el primero es el más corrientemente aislado del ganado en Argentina. Se discute en este estudio el papel de la comadreja overa (D. albiventris) como hospedador circunstancial de M. bovis

Control eradication campaigns of bovine tuberculosis based on the «test and slaughter¼ approach were successful in many countries and regions; however, in some areas the infection persists and one of the main reasons is Mycobacterium bovis infection in wild life species. Argentina has applied the same approach since 1999, achieving progress in dairy cattle herds. Nonetheless, the wildlife role has never been investigated. The objective of this study was to determine if wildlife from the Santa Fe dairy area is infected with M. bovis. Wildlife species having a positive tuberculin skin test were captured in five dairy farms. Ninety five wildlife mammals were captured; M. bovis was recovered from 7 possums (Didelphys albiventris), from one fox (Lycolapex gimnocercus) and from one rat (Rattus norvegicus). None of the animals exhibited macroscopic lesions. The most frequently isolated M. bovis spoligotypes were types 34 (4 isolates) and 12 (3 isolates). Spoligotype 34 is the most frequently isolated type in Argentine cattle. The role of D. albiventris as spillover host of M. bovis is discussed in this study

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

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.

Identificação e genotipagem de mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul / Identification and genotyping of mycobacterium bovis from positive cattle in skin test for tuberculosis in the state of Mato Grosso do Sul, Brazil

Neste estudo, realizou-se genotipagem de isolados de Mycobacterium bovis, provenientes de amostras de tecidos de bovinos positivos no teste cervical comparativo (TCC) para tuberculose em Mato Grosso do Sul, por meio da técnica de spoligotyping. Tecidos de 13 bovinos positivos, oriundos de diferentes municípios do estado, foram cultivados em meio de Stonebrink. As colônias resultantes foram submetidas à coloração de Ziehl-Neelsen e todos os isolados apresentaram características tintoriais de BAAR. Os 13 isolados de BAAR foram identificados por PCR multiplex (mPCR). O gene hsp65 foi alvo para identificação de Mycobacterium spp, a sequência de inserção IS6110 foi alvo para identificação de complexo Mycobacterium tuberculosis (CMT) e a região rvd1rv2031c foi explorada para detecção de M. bovis. Os isolados micobacterianos foram genotipados pela técnica de spoligotyping. Dos 13 bovinos, sete tinham pelo menos uma lesão sugestiva de tuberculose em linfonodos retrofaríngeos, parotídeos e pulmonares ou no pulmão, e em seis não foram encontradas lesões visíveis sugestivas da doença. Na mPCR, 11/13 (84,6%) isolados foram positivos para Mycobacterium spp, 8/13 (61,5%) positivos para CMT e 7/13 (53,8%) positivos para M. bovis. Com base no spoligotyping, oito isolados de BAAR foram agrupados dentro de três diferentes agrupamentos de genótipos e uma amostra remanescente apresentou perfil único, sendo quatro isolados com padrão de espoligotipo SB0121, dois SB1145, dois SB0881 e um SB0140. A técnica de spoligotyping demonstrou que há diversidade genética entre os espoligotipos presentes no estado de Mato Grosso do Sul, embora predomine o perfil SB0121.

Spoligotyping was performed in the present study to genotype Mycobacterium bovis isolates obtained from tissues of cattle that were positive in the comparative intradermal tuberculin test (CITT) in the state of Mato Grosso do Sul (Brazil). Tissue samples from 13 positive cattle from different municipalities of the state were cultured using a Stonebrink medium. The resulting colonies were subjected to Ziehl-Neelsen staining and all isolates exhibited the staining characteristics of AFB. The 13 isolates of AFB were identified by means of a multiplex PCR (mPCR) assay. The hsp65 gene was targeted for the identification of Mycobacterium spp., whereas the IS6110 insertion sequence was targeted for the identification of the Mycobacterium tuberculosis complex (MTC) and the rvd1rv2031c region was explored for the detection of Mycobacterium bovis. The spoligotyping assay was performed to genotype mycobacterial isolates. Of the 13 cattle, seven had at least one lesion suggestive of tuberculosis in the retropharyngeal, parotid and lung lymph nodes or lung. The remaining six exhibited no lesions suggestive of the disease. In the mPCR, 11 of the 13 isolates (84.6%) were positive for Mycobacterium spp., 8/13 (61.5%) were positive for the MTC and 7/13 (53.8%) were positive for M. bovis. Based on the spoligotyping, eight isolates were grouped into three different groups of genotypes and one isolate exhibited an orphan type. Four isolates exhibited spoligotype pattern SB0121, while two isolates were associated with the pattern SB1145, another two were associated with pattern SB0881 and one was associated with pattern SB0140. Spoligotyping confirmed the genetic diversity present among isolates found in the state of Mato Grosso do Sul. In addition, SB0121 was confirmed as the predominant profile.

Genotyping Mycobacterium bovis from cattle in the Central Pampas of Argentina: temporal and regional trends

Mycobacterium bovis is the causative agent of bovine tuberculosis (TB), a disease that affects approximately 5% of Argentinean cattle. Among the molecular methods for genotyping, the most convenient are spoligotyping and variable number of tandem repeats (VNTR). A total of 378 samples from bovines with visible lesions consistent with TB were collected at slaughterhouses in three provinces, yielding 265 M. bovis spoligotyped isolates, which were distributed into 35 spoligotypes. In addition, 197 isolates were also typed by the VNTR method and 54 combined VNTR types were detected. There were 24 clusters and 27 orphan types. When both typing methods were combined, 98 spoligotypes and VNTR types were observed with 27 clusters and 71 orphan types. By performing a meta-analysis with previous spoligotyping results, we identified regional and temporal trends in the population structure of M. bovis. For SB0140, the most predominant spoligotype in Argentina, the prevalence percentage remained high during different periods, varying from 25.5-57.8% (1994-2011). By contrast, the second and third most prevalent spoligotypes exhibited important fluctuations. This study shows that there has been an expansion in ancestral lineages as demonstrated by spoligotyping. However, exact tandem repeat typing suggests dynamic changes in the clonal population of this microorganism.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Disease caused by non-tuberculous mycobacteria: diagnostic procedures and treatment evaluation in the North of Buenos Aires Province

Non-tuberculous mycobacteria (NTM) have emerged as pathogens frequently associated to HIV co-infection. The aims of this study were to describe the clinical importance of NTM in patients from the North of Buenos Aires Province and the drug-susceptibility patterns in relation with the therapy used. A total of 23,624 clinical specimens were investigated during the period 2004-2010. Ziehl-Neelsen stain and cultures were used for diagnosis. Molecular and biochemical tests were performed to identify the mycobacteria. TB and mycobacterioses cases were 2 118 and 108 respectively. Sixteen NTM species were found: Mycobacterium avium and Mycobacterium intracellulare as the main causative agents. Infections produced by more than one species at the same time were confirmed (4 cases). Macrolides and fluoroquinolones were the most active in vitro drugs. Treatment evaluation showed that 68.0 % of the cases completed the therapy, 20 % died; and 12 % were relapses. The cases in which the treatment outcome was evaluated received an individual tailor-made therapeutic scheme including those drugs showing in vitro activity and presumed in vivo usefulness. More than a quarter of the patients had HIV co-infection and the majority of the deaths were associated with this co-infection.

Enfermedad causada por micobacterias no tuberculosas: diagnóstico y evaluación del tratamiento en el norte del Gran Buenos Aires. Las micobacterias no tuberculosas (MNT) emergieron como patógenos frecuentemente asociados a la co-infección con el HIV. EL objetivo del estudio fue describir la importancia clínica de las MNT en pacientes de la región norte de la provincia de Buenos Aires y los patrones de drogo-sensibilidad en relación con la terapia empleada. Se investigó un total de 23.624 especímenes clínicos durante, el período 2004-2010. La tinción de Ziehl-Neelsen y los cultivos se utilizaron para diagnóstico. Las micobacterias fueron identificadas mediante pruebas bioquímicas y moleculares. Los casos de tuberculosis y micobacteriosis fueron 2 118 y 108, respectivamente. Se encontraron 16 especies de MNT, siendo las principales, Mycobacterium avium y Mycobacterium intracellulare. En 4 casos se confirmaron infecciones producidas por más de una especie al mismo tiempo. Los macrólidos y las fluoroquinolonas tuvieron mayor actividad in vitro. La evaluación del tratamiento confirmó que el 68 % de los casos completó la terapia; 20 % murió y el 12 % recayó. Los casos en los que se evaluó el tratamiento recibieron un esquema terapéutico individual incluyendo aquellas drogas que mostraron actividad in vitro. Más de un cuarto de los pacientes tuvieron co-infeccion con el HIV y la mayoría de las muertes estuvieron asociadas con esta co-infección.

Mycobacterial isolation from Felis concolor in captivity

This study was made in a wildlife preserve from Argentina where a previous tuberculosis report in Felis concolor has been done. The aim was to identify mycobacterial species isolated from the orpharynx of five American lions using bacteriological and molecular biology techniques on cases with nonspecific clinical signals. Samples were collected after sedation. They were treated in order to isolate Mycobacterium. Bacteriological differentiation was made using biochemical tests. Polymerase chain reaction has been performed to detect hsp65, IS6110 and IS1081. Acid fast bacilli were present infour specimens and from them were isolated slowly growing mycobacteria. The strains were differentiated as M. gordonae in two cases and M. simiae, M. scrofulaceum and M. avium/intracellulare in one case each other. The strains were identified as M. gordonae in three cases and M. avium III or M. simiae in two by PRA. The role of feral cats in the epidemiology of nontuberculous mycobacterial diseases remains to be further investigated

Este trabalho foi realizado em uma reserva natural da Argentina com antecedentes de tuberculose em uma suçuarana adulta. O objetivo foi identificar por meio de técnicas bacteriológicas e de biologia molecular as espécies isoladas da orofaringe de cinco suçuaranas que apresentavam sinais clínicos inespecíficos. As amostras foram colhidas das suçuaranas após sedação. Posteriormente foram processadas para obtenção do isolamento e identificação por meio de provas bioquímicas do gênero Mycobacterium pela técnica de PCR. Investigou-se a presença das seqüências de inserção IS6110 e IS1081 e hsp65. Obtiveram-se resultados positivos à coloração de Ziehl-Neelsen de quatro amostras, isolando cinco cepas de crescimento lento. As cepas foram classificadas como M. gordonae em dois casos e M. simiae, Mscrofulaceum e M. avium/intracellulare em um. Por PRA, identificou-se o padrão de M. gordonae em três cepas e M. avium III ou M.simiae em dois

Diferencias proteómicas entre una cepa argentina de Mycobacterium bovis con la cepa de referencia AN5 / Proteomic differences between an argentine mycobacterium bovis strain and the reference AN5 strain

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

Tipificación de Mycobacterium avium subsp. paratuberculosis a partir de aislamientos de materia fecal y órganos de ciervos / Typing of mycobacterium avium subsp. paratuberculosis from isolations of feces and organs of deer

El desarrollo de la tecnología del P.C.R. y RFLP constituyen metodologías adecuadas para la identificación y tipificación del Mycobacterium avium subsp. paratuberculosis (Mtpbc), microorganismo que produce paratuberculosis animal y que está relacionado a la Enfermedad de Crohn en humanos. Los objetivos del presente trabajo fueron: 1) Aislar cepas de Mptbc a partir de materia fecal y órganos e identificar M. paratuberculosis. 2) Establecer mediante R.F.L.P. el patrón genético de las cepas aisladas. 3) Analizar las proteínas celulares y extracelulares expresadas en Mptbc. Se cultivó materia fecal y órganos de ciervos en los medios Herrold yema-huevo, con y sin el agregado de mycobactina, adicionando piruvato y antibióticos, para el aislamiento de Mptbc. Las cepas aisladas fueron identificadas por P.C.R., y analizadas por R.F.L.P., e Inmunoblotting (IB). Se aislaron 12 cepas de Mptbc, 6 de materia fecal y 6 a partir de los órganos. Las cepas aisladas se desarrollaron únicamente en el medio Herrold con mycobactina evidenciando su dependencia característica. El análisis por P.C.R. realizado a partir de cepas desarrolladas en el medio de cultivo resultó positivo. Los aislamientos revelaron idéntico patrón genético de R.F.L.P. del tipo "A", con la sonda de 217 pares de bases (endonucleasa BstE II y Pst I). Con el inmunoblotting se detectaron antígenos proteicos de 65 KDa (shock térmico), 42 KDa, 35 KDa y 28 KDa, correspondientes a Mptbc de un animal con sintomatología clínica y lesiones histológicas características en órganos. Se identificó un único patrón genético "A", en la población estudiada; se amplificó la secuencia IS900 específica de Mptbc. y se identificaron las proteínas excretadas y contenidas en el soma bacteriano, pero para aumentar la sensibilidad de las pruebas inmunológicas se considera necesario caracterizar mejor los antígenos específicos de Mptbc