Pattern of cytokine and chemokine production by THP-1 derived macrophages in response to live or heat-killed Mycobacterium bovis bacillus Calmette-Guérin Moreau strain

Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1β] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria.

Performance of the SD Bioline TB Ag MPT64 Rapid test for quick confirmation of Mycobacterium bovis isolates from animals

Mycobacterium (M.) bovis, a bacterium in the M. tuberculosis complex, is a causative agent of bovine tuberculosis, a contagious disease of animals. Mycobacterial culture is the gold standard for diagnosing bovine tuberculosis, but this technique is laborious and time-consuming. In the present study, performance of the SD Bioline TB Ag MPT4 Rapid test, an immunochromatographic assay, was evaluated using reference bacterial strains and M. bovis field isolates collected from animals. The SD MPT64 Rapid test produced positive results for 95.5% (63/66) of the M. bovis isolates from cattle and 97.9% (46/47) of the isolates from deer. Additionally, the test had a sensitivity of 96.5% (95% CI, 91.2-99.0), specificity of 100% (95% CI, 96.7-100.0), positive predictive value of 100% (95% CI, 96.7-100.0), and negative predictive value of 92.9% (95% CI, 82.7-98.0) for M. bovis isolates. In conclusion, the SD MPT64 Rapid test is simple to use and may be useful for quickly confirming the presence of M. bovis in animals.

Multiplex-PCR for differentiation of Mycobacterium bovis from Mycobacterium tuberculosis complex

We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.

Molecular typing of Mycobacterium bovis isolates: a review

Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.

Molecular typing of Mycobacterium bovis isolated in the south of Brazil

Bovine tuberculosis is a major infectious disease of the cattle. In this study, 85 M. bovis isolates from 162 lymph nodes, obtained from a herd of cattle on a farm in southern Brazil, were evaluated using spoligotyping and VNTR. The strains were grouped into five clusters and five orphans, showing a heterogenic genetic profile, what could represent diverse geographic origins of the introduced cows and/or the frequent movement of cattle between different properties.

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.

Spoligotyping and variable number tandem repeat analysis of Mycobacterium bovis isolates from cattle in Brazil

We performed spoligotyping and 12-mycobacterial interspersed repetitive unit-variable number tandem repeats (MIRU-VNTRs) typing to characterise Mycobacterium bovis isolates collected from tissue samples of bovines with lesions suggestive for tuberculosis during slaughter inspection procedures in abattoirs in Brazil. High-quality genotypes were obtained with both procedures for 61 isolates that were obtained from 185 bovine tissue samples and all of these isolates were identified as M. bovis by conventional identification procedures. On the basis of the spoligotyping, 53 isolates were grouped into nine clusters and the remaining eight isolates were unique types, resulting in 17 spoligotypes. The majority of the Brazilian M. bovis isolates displayed spoligotype patterns that have been previously observed in strains isolated from cattle in other countries. MIRU-VNTR typing produced 16 distinct genotypes, with 53 isolates forming eight of the groups, and individual isolates with unique VNTR profiles forming the remaining eight groups. The allelic diversity of each VNTR locus was calculated and only two of the 12-MIRU-VNTR loci presented scores with either a moderate (0.4, MIRU16) or high (0.6, MIRU26) discriminatory index (h). Both typing methods produced similar discriminatory indexes (spoligotyping h = 0.85; MIRU-VNTR h = 0.86) and the combination of the two methods increased the h value to 0.94, resulting in 29 distinct patterns. These results confirm that spoligotyping and VNTR analysis are valuable tools for studying the molecular epidemiology of M. bovis infections in Brazil.

Mycobacterium bovis in Argentina: isolates from cats typified by spoligotyping / Mycobacterium bovis en Argentina: aislamientos de gatos tipificados por spoligotyping

In the present work, 19 Mycobacterium bovis isolates from different cats were typified by spoligotyping. We detected nine spoligotypes. There was only one cluster, which grouped 11 of the isolates (57.9%), showing the main spoligotype from cattle from Argentina. The rest of the spoligotypes presented only one isolate each. Five of them were not found in cattle, and were unique and exclusive of cats. The isolates studied show that tuberculosis of bovine origin in cats constitutes a potential public health problem in Buenos Aires region. The identification of genotypes from non-natural hosts could contribute to understand the spread of bovine tuberculosis. This is the first report showing genetic profiles of M. bovis isolates in felines from Argentina.

En el presente trabajo se tipificaron por spoligotyping 19 aislamientos de M. bovis de diferentes gatos. Se detectaron 9 espoligotipos y un único agrupamiento o cluster integrado por 11 aislamientos (57,9%) y relacionado con el principal espoligotipo de bovinos de Argentina. El resto de los espoligotipos detectados presentaron solamente un aislamiento cada uno; 5 de ellos no se encontraron en bovinos y fueron únicos y exclusivos de gatos. La presencia de estos aislamientos indica que la tuberculosis bovina en los gatos constituye un potencial problema de salud pública en la ciudad de Buenos Aires. La identificación de genotipos de aislamientos de M. bovis de hospedadores no convencionales podría contribuir a la mejor comprensión de la diseminación de la tuberculosis bovina. Este es el primer informe en el que se muestran los perfiles genotípicos de aislamientos de M. bovis obtenidos de felinos de Argentina.

Characterization of Mycobacterium africanum subtype I among cows in a dairy farm in Bangladesh using spoligotyping.

Acid-fast bacilli (AFB) were detected in the autopsy lung tissue homogenate samples of four cows (variety Frisian cross) in a dairy farm in Bangladesh. Histopathological examination of the lung tissue demonstrated prominent granulomas, caseating necrosis and calcification indicative of tuberculosis (TB) infection. Mycobacteria could not be cultured from the tissue homogenate samples by Lowenstein-Jensen based conventional culture method though AFB were evident by Ziehl-Neelsen (ZN) staining of the smears of tissue homogenate and in paraffin embedded tissue slices. Spoligotyping performed on DNA extracts of paraffin embedded lung tissue samples confirmed the AFB as a member of the M. tuberculosis complex (MTBC) with a pattern assigned to M. africanum subtype I. This characterization by spoligotyping was confirmed by subjecting M. africanum subtype I isolates from other parts of the world to an alternative identification method based on DNA polymorphism in the gyrB gene (Hain Life Science, GmbH, Nehren, Germany). Since M. africanum is believed to be a human pathogen, general infection in cattle may be a public health threat. The presence of these bacteria in the animal reservoir most likely originated from a caretaker.

Evaluación de un ensayo de RPC múltiple para diferenciar micobacterias del complejo Mycobacterium tuberculosis en un laboratorio de referencia / Evaluation of a multiplex PCR assay to differentiate mycobacteria of the Mycobacterium tuberculosis complex in a reference laboratory

Mycobacteria that cause tuberculosis in animals and humans belong to the Mycobacterium tuberculosis complex. Techniques for conventional diagnosis are time-consuming and do not differentiate between different strains belonging to the M. tuberculosis complex. The aim of this study was to evaluate a multiplex PCR assay applicable to mycobacteria in culture with the capacity to differentiate different strains belonging to the M. tuberculosis complex in a reference laboratory. Primers based on genomics regions of difference (RD) consisting in DNA segments that are present in M. tuberculosis, but differentially deleted in several members of M. tuberculosis complex were used in a PCR assay. The test was applied to 86 clinical isolates of mycobacteria. The pattern of amplification allowed differentiating between M. tuberculosis, M. bovis and M. bovis BCG in a single PCR reaction. This PCR multiplex assay may be used in a Reference Laboratory of Tuberculosis Diagnosis as a complementary test to differentiate mycobacteria strains belonging to the M. tuberculosis complex. This test significantly reduces the time period between culture and strain identification, and thus for could favor the adoption of better strain specific antimycobacterial regimens as well as identification of zoonotic transmission of M. bovis to humans.

Las micobacterias que causan tuberculosis en animales y humanos pertenecen al complejo Mycobacterium tuberculosis. Las técnicas de diagnóstico convencional, además de ser lentas y laboriosas, no permiten diferenciar entre miembros de este complejo. El objetivo de este estudio fue evaluar ensayos de RPC múltiple para contribuir a la identificación diferencial de micobacterias del complejo M. tuberculosis a partir de cultivos, en un laboratorio de referencia. Se utilizaron oligonucleótidos partidores basados en regiones de diferencia (RD) que consisten en segmentos de ADN que están presentes en M. tuberculosis, pero que han sido eliminados diferencialmente del genoma de otros miembros del complejo M. tuberculosis. El ensayo se aplicó sobre 86 aislados clínicos de micobacterias. El patrón de amplificación permitió diferenciar entre cepas de M. tuberculosis, M. bovis y M. bovis variedad BCG en una única RPC. Este ensayo de RPC múltiple puede ser utilizado en el Laboratorio de Referencia de Diagnóstico de Tuberculosis como prueba complementaria para diferenciar micobacterias del complejo M. tuberculosis, contribuyendo a un acortamiento en el período de reporte de resultados y un tratamiento adecuado del paciente, y podría ser aplicado también en estudios epidemiológicos de transmisión zoonótica de M. bovis a humanos.

Mycobacterium bovis identification by a molecular method from post-mortem inspected cattle obtained in abattoirs of Mato Grosso do Sul, Brazil

The presence of Mycobacterium bovis in bovine carcasses with lesions suggestive of tuberculosis was evaluated. Seventy-two carcass samples were selected during slaughter inspection procedures in abattoirs in the state of Mato Grosso do Sul, Brazil. Seventeen (23.6 percent) of samples showed colonies suggestive of mycobacteria that were confirmed to be acid-fast bacilli by Ziehl-Neelsen staining. Polymerase chain reaction (PCR) using primers specific for M. bovis identified M. bovis in 13 (76.5 percent) isolates. The PCR-restriction enzyme pattern analysis using gene encoding for the 65-kDa protein and two restriction enzymes identified the remaining four isolates that were represented by two M. tuberculosis complex and two nontuberculous mycobacteria. The results are indicative of infection of slaughter cattle by M. bovis and other mycobacteria in the state of Mato Grosso do Sul.

Molecular fingerprinting of clinical isolates of Mycobacterium bovis and Mycobacterium tuberculosis from India by restriction fragment length polymorphism (RFLP)

Forty mycobacterial strains comprising clinical Indian isolates of Mycobacterium tuberculosis (28 field isolates +1H37 Rv) and Mycobacterium bovis (10 field isolates +1 AN5) were subjected to restriction fragment length polymorphism analysis (RFLP) using IS6110 and IS1081 probes. Most of these strains originated from dairy cattle herd and human patients from Indian Veterinary research Institute (IVRI) campus isolated from the period of 1986 to 2000. Our study showed presence of 8 copies of IS6110 in most of the M.tuberculosis (96.6%) strains irrespective of their origin with the exception of one M.tuberculosis strain with presence of an extra copy (3.4%). All M.bovis strains showed a single copy of IS6110 on the characteristic 1.9kb restriction fragment. RFLP analysis with IS1081 invariably showed the presence of 5 copies in all isolates of M.bovis and M.tuberculosis at the same chromosomal location. Similarity of IS6110 RFLP fingerprints of M.tuberculosis strains from animals and human suggested the possibility of dissemination of single M.tuberculosis strain among animals as well as human. It was not possible to discriminate within the isolates of either M.tuberculosis or M.bovis, when IS1081 was used as target sequence. The IS6110 RFLP is a valuable tool for disclosing transmission chain of M. tuberculosis and M. bovis among humans as well as animals