Comparative study of Mycobacterium bovis primary isolation methods

Abstract For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants - 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media - 7H11 Middlebrook with additives and OADC supplement “A” (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement “B”.

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

Assessment of phagosomes infected with Mycobacterium tuberculosis as a vaccine candidate against tuberculosis.

The present study describes a novel and simple vaccination strategy that involve culturing of M. tuberculosis in the macrophage cells. Isolation of phagosome from macrophage (cell line J774) infected with M. tuberculosis (H37) and M. bovis (BCG) at early and late phase of infection was done ensuing the identification and characterization of these phagosome. In vitro study of apoptosis induced by phagosome infected with (H37) and (BCG) was performed. The vaccine candidate with H37 MOI- 1:10 at 3 h, MOI- 1:20 at 1, 1.5, 2.5 and 3 h and BCG MOI- 1:20 at 3.5 h showed percentage apoptosis as 38.64, 39.93, 34.66, 22.56,34.59 and 37.81% respectively. The results designates that macrophages provide cellular niche during infection and illustrate considerable immunogenic property. Novel antigens expressed or secreted by H37 in infected macrophages can provide evidence to be a successful vaccine candidate as it endures enhanced immune response than BCG.

Identificação de Mycobacterium bovis em cepas micobacterianas isoladas espécimes clínicos humanos em um complexo hospitalar na cidade do Rio de Janeiro / Identification of Mycobacterium bovis among mycobacterial isolates from human clinical specimens at a university hospital in Rio de Janeiro, Brazil

Entre 2005 e 2006, 8.121 espécimes clínicos enviados ao Laboratório de Micobactérias do Hospital Universitário Clementino Fraga Filho/Instituto de Doenças do Tórax, no Rio de Janeiro, RJ, foram inoculados em meio Löwenstein-Jensen contendo glicerol e piruvato. Desses espécimes, 79 isolados de micobactérias tiveram crescimento somente em meio com piruvato, sendo selecionados para a identificação presuntiva de Mycobacterium bovis. Esses isolados foram submetidos à identificação por testes bioquímicos, amplificação por PCR com primers específicos (Rv0577 e Rv1510) e teste de suscetibilidade à pirazinamida. Todas as cepas apresentaram padrões fenotípicos e genotípicos de M. tuberculosis, não sendo detectado M. bovis.

In 2005 and 2006, 8,121 clinical specimens submitted to the Mycobacteriology Laboratory of the Clementino Fraga Filho University Hospital/Thoracic Diseases Institute, in the city of Rio de Janeiro, Brazil, were inoculated on Löwenstein-Jensen medium containing glycerol and pyruvate. There were 79 mycobacteria isolates that presented growth only on pyruvate-containing medium, and those isolates were selected for the presumptive identification of Mycobacterium bovis. The selected isolates were screened with biochemical tests, PCR amplification (with the specific primers Rv0577 and Rv1510), and pyrazinamide susceptibility tests. All of the strains isolated showed specific phenotypical and genotypical patterns characteristic of M. tuberculosis, and no M. bovis strains were detected.

Diagnóstico bacteriológico de tuberculosis renal: experiencia del Laboratorio Regional de Tuberculosis de la provincia de Córdoba

Dada la considerable incidencia de tuberculosis renal entre enfermos con tuberculosis pulmonar, nos propusimos estudiar la frecuencia de esta asociación en pacientes atendidos en centros de salud públicos y privados de Córdoba a lo largo del período 1997-2009. Se tomó en consideración la incidencia según el sexo y las especies del complejo Mycobacterium tuberculosis identificadas. El análisis de 948 muestras de orina de 383 pacientes indicó tuberculosis renal en 24 casos (6,3 %), con presencia mayoritaria de Mycobacterium tuberculosis (95,8 %) y presencia de Mycobacterium bovis en 4,2 % de los casos. La asociación tuberculosis renal-tuberculosis pulmonar activa se encontró en 6 casos. En esta investigación quedó demostrada la importancia del cultivo seriado de muestras de orina y la conveniencia de cultivar en medios sólidos y líquidos. Asimismo, el aislamiento de Mycobacterium bovis pone de relieve la importancia de usar el medio Stonebrink junto con el medio de Lowenstein-Jensen. El medio líquido no tuvo un aporte significativo al diagnóstico de tuberculosis renal; sin embargo, el cultivo de muestras seriadas aumentó la sensibilidad de la detección.

Bacteriological diagnosis of renal tuberculosis: an experience at the Regional Tuberculosis Laboratory in Córdoba province, Argentina. Given the incidence of renal tuberculosis in patients suffering of pulmonary tuberculosis, we seek to study both the frequency of this association in diagnosed cases of renal tuberculosis and the Mycobacterium tuberculosis complex species that were identified (period 1997-2009), observing its incidence by sex, demonstrating the importance of serial culture of urine samples and evaluating the convenience of using solid and liquid media. The analysis of urine samples from 383 patients indicated renal tuberculosis in 24 cases; in most cases, (95.8 %) Mycobacterium tuberculosis complex species prevailed, whereas the presence of Mycobacterium bovis accounted for 4.2 % of the cases. The association of pulmonary and renal tuberculosis was found in 6 cases. The isolation of Mycobacterium bovis indicates the importance of including Stonebrink medium along with Lowenstein- Jensen medium. The liquid medium made no significant contribution to the diagnosis of renal tuberculosis, but indeed, cultivating serial samples increases sensitivity.

Preparation of a working seed lot of BCG and quality control by PCR genotyping / Preparación de un lote semilla de trabajo de BCG y control de calidad por genotipificación por PCR

The bacillus Calmette-Guérin (BCG) was obtained in 1920 after successive passages leading to the attenuation of a Mycobacterium bovis strain. For the following 40 years, BCG had been replicated, resulting in substrains with genotypic and phenotypic differences. Several genomic studies have compared two BCG strains, M. bovis and Mycobacterium tuberculosis, and observed that deleted regions in the different strains could be related to differences in antigenic properties. In this work, a working seed lot was obtained from a lyophilized secondary seed lot from the BCG Pasteur strain 1173 P2 and genetically characterized. The genome was analyzed by PCR directed to five regions (RD1, RD2, RD14, RD15, DU2), using the seed lot and different available strains as templates. No genetic differences were found in the fragments studied as compared to the Pasteur strain. A total of 20 passages were carried out and no differences were found in the size of the fragments amplified by PCR. In conclusion, this method allows to control a working seed lot genotypically and to assess the stability of the BCG genome.

El bacilo de Calmette-Guérin (BCG) se obtuvo en 1920, después de sucesivos pasajes que llevaron a la atenuación de una cepa de Mycobacterium bovis. A lo largo de los 40 años subsiguientes la cepa BCG fue replicada y surgieron subcepas con diferencias fenotípicas y genotípicas. Se realizaron varios estudios de comparación genómica de diferentes cepas de BCG, M. bovis y Mycobacterium tuberculosis, y se observó que las deleciones de regiones en las diferentes cepas podrían estar relacionadas con diferencias en las propiedades antigénicas. En este trabajo se describe la preparación y caracterización genética de un lote semilla de trabajo obtenido a partir de un lote semilla secundaria liofilizado de la cepa BCG Pasteur 1173 P2. Se analizaron por PCR cinco regiones (RD1, RD2, RD14, RD15, DU2) en el lote semilla de trabajo utilizando como control las diferentes cepas disponibles. No se hallaron diferencias genéticas en los fragmentos estudiados al comparar el lote semilla de trabajo con la cepa BCG Pasteur 1173 P2. Asimismo, se efectuaron hasta 20 pasajes y no se encontraron diferencias en el tamaño de los fragmentos amplificados por PCR. En conclusión, se ha puesto a punto un método que permite controlar el genotipo de un lote semilla de trabajo y evaluar la estabilidad del genoma del BCG.

Prevalência de tuberculose no rebanho bovino de Mossoró, Rio Grande do Norte / A survey of tuberculosis in the cattle from Mossoro, Rio Grande do Norte State

A tuberculose bovina é uma doença infecciosa crônica e debilitante que pode infectar humanos. Uma importante etapa para o controle é a determinação da prevalência nos rebanhos. O objetivo do presente trabalho foi determinar a prevalência da tuberculose bovina em Mossoró, RN. Inicialmente foi realizada uma avaliação de risco para a ocorrência da tuberculose bovina em 21 propriedades da região avaliada. Foram testadas 150 vacas leiteiras por meio do teste da prega caudal. Também foram avaliados outros 120 bovinos pelo teste da tuberculinização cervical comparada. Os resultados revelaram falta de conhecimento dos proprietários sobre o controle da doença, bem como o descuido no momento da aquisição de animais. A prevalência foi de 8,66% e 3,33% nos testes da prega caudal e cervical comparativo, respectivamente. Assim, foi verificada uma prevalência de tuberculose bovina bastante elevada, sendo superior à média nacional.

Bovine tuberculosis is an infectious chronic and debilitating disease that can infect humans. A key step for its controls is to determine the prevalence in the herd. The present study aimed to determine the prevalence of bovine tuberculosis at Mossoró, RN. Initially, it was done an evaluation for infection hazard by tuberculosis in bovines at 21 farms. It were tested 150 dairy cows by caudal-fold tuberculin test. Other 120 bovines were also tested by comparative cervical tuberculin test. The results revealed lack of knowledge by owners about the prevention of the disease, as well as negligence when they buy animals.The prevalence was 8.66% at the cervical test and 3.33% at caudal-fold tuberculin test and comparative cervical tuberculin test, respectively.Thus, it was verified a high prevalence of bovine tuberculosis, which is greater than the national mean prevalence.

Murine model of BCG lung infection: dynamics of lymphocyte subpopulations in lung interstitium and tracheal lymph nodes.

C57Bl/6 female mice were infected with an intrapulmonary dose of 2.5 x 10(4) BCG (Mycobacterium bovis Bacillus Calmette-Guerin). Lymphocyte populations in lung interstitium and lung-associated tracheal lymph nodes (LN) were examined at 1, 2, 4, 5, 6, 8 and 12 weeks after infection. BCG load in lungs peaked between 4-6 weeks post-infection and declined to very low levels by the 12th week of infection. Lung leukocytes were obtained over the course of infection by enzyme digestion of lung tissue followed by centrifugation over Percoll discontinuous density gradients. By 4 to 6 weeks after infection, numbers of lung leukocytes had more than doubled but the proportions of lymphocytes (about 70%), macrophages (about 18%) and granulocytes (about 12%) remained essentially unaltered. Flow cytometric studies indicated: (i) the total number of CD3+ T cells in lungs increased by 3-fold relative to uninfected controls at 5 to 6 weeks post-infection, but the relative proportions of CD4 and CD8 cells within the T cell compartment remained unaltered; (ii) relative proportion of NK cells in lungs declined by 30% but the total number of NK cells (NK1.1+) per lung increased by about 50%, 5-6 weeks post infection; (iii) tracheal LN underwent marked increase in size and cell recoveries (6-10-fold increase) beginning 4 weeks after infection. While both T and B cells contributed to the increase in cell recoveries from infected tracheal LNs, the T/B ratio declined significantly but CD4/CD8 ratio remained unaltered. In control mice, IFNgamma producing non-T cells outnumbered T cells producing IFNgamma. However, as the adaptive response to infection evolves, marked increase occur in the number of IFNgamma producing T cells, but not NK cells in the lungs. Thus, T cells are the primary cell type responsible for the adaptive IFNgamma response to pulmonary BCG infection. Few T cells in tracheal LN of BCG infected mice produce IFNgamma, suggesting that maturational changes associated with migration to the lungs or residence in the lungs enhance the capability of some T cells to produce this cytokine.

A single strain of Mycobacterium bovis bacillus Calmette-Guérin (BCG) grown in two different media evokes distinct humoral immune responses in mice

Two attenuated bacillus Calmette-Guérin (BCG) preparations derived from the same Moreau strain, Copenhagen but grown in Sauton medium containing starch and bacto-peptone (onco BCG, O-BCG), or asparagine (intradermal BCG, ID-BCG), exhibited indistinguishable DNA sequences and bacterial morphology. The number of viable bacilli recovered from spleen, liver and lungs was approximately the same in mice inoculated with the vaccines and was similarly reduced (over 90 percent) in mice previously immunized with either BCG vaccine. The humoral immune response evoked by the vaccines was, however, distinct. Spleen cell proliferation accompanying the growth of bacilli in tissue was significantly higher in mice inoculated with O-BCG. These cells proliferated in vitro upon challenge with the corresponding BCG extract. Previous cell treatment with mAb anti-CD4 T cells abolished this effect. Anti-BCG antibodies, as assayed either in serum by ELISA or by determining the number of antibody-producing spleen cells by the spot-ELISA method, were significantly higher in mice inoculated with ID-BCG. Anti-BCG antibodies were detected in all immunoglobulin classes, but they were more prevalent in IgG with the following distribution among its isotypes: IgG1>(IgG2a = IgG2b)>IgG3. When some well-characterized Mycobacterium tuberculosis antigens were used as substitutes for BCG extracts in ELISA, although antibodies against the 65-kDa and 96-kDa proteins were detected significantly, antibodies against the 71-kDa, 38-kDa proteins and lipoarabinomannan were only barely detected or even absent. These results indicate that BCG bacilli cultured in Sauton-asparagine medium permitted the multiplication of bacilli, tending to induce a stronger humoral immune response as compared with bacilli grown in Sauton-starch/bacto-peptone-enriched medium

Acción del agua del Volcán Copahue (Neuquén, Argentina) sobre micobacterias / Action of the Copahue volcano waters on mycobacteries

Se efectúa un estudio de la acción del agua del Volcán Copahue (AVC) sobre 14 cepas de micobacterias. Los tres ensayos utilizados para determinar resistencia o sensibilidad al agua dieron resultados coincidentes. Las Micobacterias no tuberculosas presentaron distintos grados de resistencia, en cambio M.tuberculosis, M. bovis y M.marinum mayor sensibilidad. Se complementa con breves estudios de la acción del AVC sobre el efecto pH, destrucción del "factor cuerda" de los bacilos, y pérdida de su actividad enzimática (catalasa y peroxidasa)