Elaboración y prueba de potencia de sensitinas correspondientes a Mycobaterium phlei y Micobacterium fortuitum aisladas de suelos pampeanos en la República Argentina

In countries that have managed to eradicate bovine tuberculosis, has been shown to interfere with certain MNT tuberculosis diagnosis, when applied to the intradermal purified protein derivative Mycobacterium bovis PPD. It has also been established that some species can invade and replicate MNT inside Acanthamoeba parasites such as Dictyostelium discoideum, these protozoa may act as reservoirs of MNT mammals parasitized. Exist in mammals in the natural environment of lakes, sewage, soil, environmental dust,etc.,and manmade artificial enviroments such as drinking water, aquariums, plactonica and biofilms. The objectives of the proyect were to develop the sensitines (PPD) from the isolates of NTM isolated from soils of the province of La Pampa and check possible cross-reaction with PPD bovine.Two sensitines series of Mycobacterium fortuitum corresponding to and another Mycobacterium phlei, with protein concentration of 1.5 mg/mL. Guinea pigs were sensitized with Mycobacterum phlei, Mycobacterium fortuitum and Mycobacterium bovis power with sterile vaseline and pumice. After 120 days tests potency were carried out on the flanks of guinea pigs. The relative activity in guinea pigs sensitized with Mycobacterium bovis was of 2.18% with M.phlei and of 0.4% for the PPD Mycobacterium fortuitum ,wich shows that the environmental mycobacteria had no relevant actions in guinea pigs tuberculin intradermal reactionEn países que han logrado erradicar la tuberculosis bovina, se ha comprobado que ciertas MNT interfieren en el diagnóstico de la tuberculosis, cuando se aplica la intradermorreacción con el derivado proteico purificado de Mycobacterium bovis (PPD). Además se ha establecido que algunas especies de MNT pueden invadir y replicar en el interior de parásitos tales como Acanthamoeba y Dictyostelium discoideum pudiendo actuar estos protozoarios como reservorios de MNT en los mamíferos parasitados. Existen MNT en el ambiente natural de lagos, aguas de alcantarillas, suelos, polvo ambiental, étc y en los ambientes artificiales construídos por el hombre tales como : agua potable, acuarios, ya sea en forma planctónica o formando biofilm. Los objetivos del proyecto consistieron en elaborar las sensitinas (PPD) a partir de las cepas de MNT aisladas de los suelos de la provincia de La Pampa y comprobar la posible reacción cruzada con el PPD bovino. Se elaboraron dos series de sensitinas una correspondiente a M. fortuitum y otra a M. phlei, con una concentración de proteína en ambas de 1.5mg/mL. Se sensibilizaron cobayos con polvo de M. phlei, M.fortuitum y M. bovis juntamente con vaselina estéril y piedra pómez. A los 120 días se efectuaron las pruebas de potencia en los flancos de los cobayos. La actividad relativa en cobayos sensibilizados con M.bovis fue de 2.18% con el PPD de M.phlei y de 0.4% para el PPD de M.fortuitum lo cual demuestra que las micobacterias ambientales no presentaron un accionar relevante en la reacción intradérmica tuberculínica en cobayos

Susceptibilidad in vitro a los medicamentos anti-tuberculosos de aislados de cepas del complejo Mycobacterium tuberculosis obtenidos a partir de lobos marinos

Mycobacteria strains belonging to the Mycobacterium tuberculosis complex were isolated from seals found in the South Atlantic. The animals were received in Mundo Marino installations and treated for Mycobacterium tuberculosis complex by conventional therapy of intensive care and enriched food supply; however, in all cases treatment failed. Necropsies of all animals revealed extensive lesions compatible with tuberculosis involving lungs, liver, spleen and lymphatic nodes. Classical biochemical methods as well as molecular techniques using the IS6110 probes were performed for mycobacterial identification. Furthermore, the LCx M. tuberculosis assay (Abbott Laboratories) identified all strains as Mycobacterium tuberculosis complex members. The in vitro susceptibility pattern was examined in mycobacterial strains isolated from seven seals and in 3 reference strains--BCG, H37Rv (M. tuberculosis) and AN5 (Mycobacterium bovis)--to 4 medications--isoniazid, rifampin, streptomycin and ethambutol. Minimal inhibitory drug concentrations were determined by the Mycobacterial Growth Indicator Tube (BD Argentina) method and a microdilution and colorimetric assay using 3-(4-5 dimethyltiazol-2)-2,5 diphenyltetrazolium bromide. All the isolates and the reference strains BCG and AN5 were inhibited by MIC values similar to those of H37Rv with good agreement obtained by both techniques. These findings suggest that a therapeutic regimen aimed to seals diagnosed with tuberculosis play an important role in the prevention of tuberculosis transmission from infected animals to humans that are in routine contact with them.Se han hallado cepas de micobacterias aisladas de lobos marinos del Atlántico sur y pertenecen al complejo de Mycobacterium tuberculosis. Los animales se recibieron en las instalaciones del Oceanario Mundo Marino y fueron tratados apropiadamente para su recuperación con la terapia convencional, cuidados intensivos y suplemento alimentario pero no se observó mejoría en su estado general. Se practicaron necropsias en todos los animales y se observaron lesiones extensas compatibles con tuberculosis en pulmones, hígado, bazo y ganglios linfáticos. Para la identificación de las micobacterias, se realizaron pruebas bioquímicas y técnicas de biología molecular con la sonda IS6110. Además, se identificaron todas las cepas como pertenecientes al complejo M. tuberculosis mediante el equipo LCx M. tuberculosis Assay (Abbott Laboratories). El objetivo de este estudio fue determinar in vitro la sensibilidad de las cepas patrón BCG, H37Rv (M. tuberculosis) y AN5 (Mycobacterium bovis) y la de las siete aisladas de lobos marinos a isoniacida, rifampicina, estreptomicina y etambutol. La concentración inhibitoria mínima (CIM) de las drogas antituberculosas se llevó a cabo con el equipo Mycobacterial Growth Indicator Tube (MGIT, BD, Argentina) y la microdilución con el ensayo colorimétrico con bromuro de 3-(4-5 dimetiltiazol-2)-2,5 difeniltetrazolio. Todos los aislamientos y las cepas de referencia BCG y AN5 se inhibieron con valores CIM de los de H37Rv con buena concordancia entre los resultados obtenidos con ambas técnicas. Los hallazgos permiten sugerir que podrían ser una importante ayuda terapéutica en los lobos marinos con diagnóstico de tuberculosis y evaluar el posible papel sanitario en la prevención y transmisión de la tuberculosis de los animales a los humanos y el trabajo en conjunto

Use of touch-down polymerase chain reaction to enhance the sensitivity of Mycobacterium bovis detection

The confirmatory diagnosis of Mycobacterium bovis (M. bovis) in animal samples is carried out by culture in Stonebrink media. However, culture is very slow because of the extremely long duplication time of the bacillus and difficult because of the scarcity of bacilli in diagnostic samples. This study describes the development of a single-tube touch-down polymerase chain reaction (PCR) protocol for the detection of M. bovis using primers that target the IS6110 element. Spiked water and milk as well as routine diagnostic samples (milk and nasal swabs) from M. bovis–positive cattle were tested. This protocol allows the rapid and sensitive detection of M. bovis in bovine samples by enhancing the sensitivity of standard PCR amplification.Instituto de BiotecnologíaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Meikle, Virginia. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bernardelli, Amelia. Servicio Nacional de Sanidad y Calidad Agroalimentaria. Dirección de Laboratorios y Control Técnico; ArgentinaFil: Abdala, Alejandro Ariel. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Tarabla, Hector Dante. Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Rafaela; ArgentinaFil: Romano, Maria Isabel. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; Argentin

Draft genome sequence of Mycobacterium bovis 04-303, a highly virulent strain from Argentina

Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.Instituto de BiotecnologíaFil: Nishibe, Christiane. Universidade Federal de Mato Grosso do Sul. Faculdade de Computação; BrasilFil: Canevari Castelão, Ana Beatriz. Universidade Federal de Mato Grosso do Sul. Programa de Pós-Graduação em Ciência Animal; BrasilFil: Dalla Costa, Ricardo. Life Technologies do Brasil; BrasilFil: Pinto, Beatriz Jeronimo. Life Technologies do Brasil; BrasilFil: Varuzza, Leonardo. Life Technologies do Brasil; BrasilFil: Cataldi, Angel Adrian. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Bernardelli, Amelia. Ceva Salud Animal; ArgentinaFil: Bigi, Fabiana. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Blanco, Federico Carlos. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Zumarraga, Martin Jose. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Biotecnología; ArgentinaFil: Almeida, Nalvo Franco. Universidade Federal de Mato Grosso do Sul. Faculdade de Computação; BrasilFil: Araujo, Flabio Ribeiro de. Empresa Brasileira de Pesquisa Agropecuária (Embrapa). Gado de Corte; Brasi

Tuberculosis in seals caused by a novel member of the Mycobacterium tuberculosis complex: Mycobacterium pinnipedii sp. nov.

A comparison of Mycobacterium tuberculosis complex isolates from seals (pinnipeds) in Australia, Argentina, Uruguay, Great Britain and New Zealand was undertaken to determine their relationships to each other and their taxonomic position within the complex. Isolates from 30 cases of tuberculosis in six species of pinniped and seven related isolates were compared to representative and standard strains of the M. tuberculosis complex. The seal isolates could be distinguished from other members of the M. tuberculosis complex, including the recently defined ‘Mycobacterium canettii’ and ‘Mycobacterium caprae’, on the basis of host preference and phenotypic and genetic tests. Pinnipeds appear to be the natural host for this ‘seal bacillus’, although the organism is also pathogenic in guinea pigs, rabbits, humans, Brazilian tapir (Tapirus terrestris) and, possibly, cattle. Infection caused by the seal bacillus is predominantly associated with granulomatous lesions in the peripheral lymph nodes, lungs, pleura, spleen and peritoneum. Cases of disseminated disease have been found. As with other members of the M. tuberculosis complex, aerosols are the most likely route of transmission. The name Mycobacterium pinnipedii sp. nov. is proposed for this novel member of the M. tuberculosis ..

Assessment of the Sensitivity and Specificity of Enzyme-Linked Immunosorbent Assay (ELISA) for the Detection of Mycobacterial Antibodies in Bovine Tuberculosis

Fil: Ritacco, Viviana. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Universidad de Lujan; Argentina.Fil: De Kantor, Isabel N. Organización Panamericana de la Salud. Centro Panamericano de Zoonosis; Argentina.Fil: Barrera, Lucía. ANLIS Dr.C.G.Malbrán; Argentina.Fil: Nader, Alfredo. Servicio Nacional de Sanidad Animal (SENASA); Argentina.Fil: Bernardelli, Amelia. Servicio Nacional de Sanidad Animal (SENASA); Argentina.Fil: Torrea, Gabriela. Organización Panamericana de la Salud. Centro Panamericano de Zoonosis; Argentina.Fil: Errico, Francisco. Centro de Investigaciones Veterinarias Miguel C. Rubino; Uruguay.Fil: Fliess, Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET). Universidad de Lujan; Argentina.Summary An ELISA which detects bovine circulating IgG mycobacterial antibodies using M. bovis PPD as antigen was assessed. PPDs prepared from unheated cultures of two M. bovis strains were compared with autoclaved bovine PPD; the latter was found to be a more reliable antigen.Ninety per cent of bacteriologically confirmed tuberculous cattle were detected by this method (18/20), whereas 89.8% of negative reactions (44/49) were observed in healthy cattle from a tuberculosis-free area. Antibody levels of tuberculin negative cattle from an endemic area did not differ significantly from antibody levels of cattle from a tuberculosis-free area.An almost complete qualitative correlation was observed between instrumental and visual readings.In the present trial, the sensitivity and specificity of the ELISA were similar to those of the tuberculin skin test at its best. In addition, operative advantages and low cost of the ELISA make of it a valuable tool for the diagnosis of bovine tuberculosis.ZusammenfassungBestimmung der Sensitivität und Spezifität eines enzymgebundenen Immunadsorptionstests (ELISA) zum Nachweis von mykobakteriellen Antikörpern bei boviner TuberkuloseEin ELISA zum Nachweis von zirkulierenden, bovinen IgG-Antikörpern mit M. bovis als Antigen wurde überprüft. Autoklaviertes bovines PPD erwies sich, verglichen mit PPDs aus nicht erhitzten Kulturen zweier M. bovis-Stämme, als geeignetes Antigen.Mit Hilfe dieser Methode konnten bei Rindern mit bakteriologisch abgesicherter Tuberkulose in 90% der Fälle (18/20) Antikörper nachgewiesen werden. Negative Befunde wurden dagegen zu 89,9% bei gesunden Tieren aus einem tuberkulosefreiem Gebiet ermittelt. Zwischen den Antikörpergehalten tuberkulinnegativer Rinder aus einem endemischen Gebiet und Tieren aus dem tuberkulosefreien Gebiet konnte keine signifikante Abweichung festgestellt werden. Eine nahezu vollständige Korrelation bestand zwischen instrumenteller und visueller Ablesung.In der vorliegenden Untersuchung waren die Ergebnisse des ELISA bezüglich Sensitivität und Spezifität ähnlich denen des Tuberkulin-Hauttestes. Darüber hinaus machen die technischen Vorteile und geringen Kosten den ELISA zu einem nützlichen Hilfsmittel für die Diagnostik der bovinen Tuberkulose