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”.

Diagnosis of mycobacteria in bovine milk: an overview

ABSTRACT Tuberculosis remains as the world’s biggest threat. In 2014, human tuberculosis ranked as a major infectious disease by the first time, overcoming HIV death rates. Bovine tuberculosis is a chronic disease of global distribution that affects animals and can be transmitted to humans by the consumption of raw milk, representing a serious public health concern. Despite the efforts of different countries to control and eradicate bovine tuberculosis, the high negative economic impact on meat and milk production chains remains, given the decreased production efficiency (approximately 25%), the high number of condemned carcasses, and increased animal culling rates. This scenario has motivated the establishment of official programs based on regulations and diagnostic procedures. Although Mycobacterium tuberculosis and Mycobacterium bovis are the major pathogenic species to humans and bovines, respectively, nontuberculous mycobacteria within the Mycobacterium genus have become increasingly important in recent decades due to human infections, including the ones that occur in immunocompetent people. Diagnosis of mycobacteria can be performed by microbiological culture from tissue samples (lymph nodes, lungs) and secretions (sputum, milk). In general, these pathogens demand special nutrient requirements for isolation/growth, and the use of selective and rich culture media. Indeed, within these genera, mycobacteria are classified as either fast- or slow-growth microorganisms. Regarding the latter ones, incubation times can vary from 45 to 90 days. Although microbiological culture is still considered the gold standard method for diagnosis, molecular approaches have been increasingly used. We describe here an overview of the diagnosis of Mycobacterium species in bovine milk.

Direct detection of Mycobacterium tuberculosis complex in bovine and bubaline tissues through nested-PCR

Post-mortem bacterial culture and specific biochemical tests are currently performed to characterize the etiologic agent of bovine tuberculosis. Cultures take up to 90 days to develop. A diagnosis by molecular tests such as PCR can provide fast and reliable results while significantly decreasing the time of confirmation. In the present study, a nested-PCR system, targeting rv2807, with conventional PCR followed by real-time PCR, was developed to detect Mycobacterium tuberculosis complex (MTC) organisms directly from bovine and bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other Actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. Regarding the analytical sensitivity, DNA of the M. bovis AN5 strain was detected up to 1.5 pg by nested-PCR, whereas DNA of M. tuberculosis H37Rv strain was detected up to 6.1 pg. The nested-PCR system showed 100% analytical specificity for MTC when tested with DNA of reference strains of non-tuberculous mycobacteria and closely-related Actinomycetales. A clinical sensitivity level of 76.7% was detected with tissues samples positive for MTC by means of the culture and conventional PCR. A clinical specificity of 100% was detected with DNA from tissue samples of cattle with negative results in the comparative intradermal tuberculin test. These cattle exhibited no visible lesions and were negative in the culture for MTC. The use of the nested-PCR assay to detect M. tuberculosis complex in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

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.

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.

Epidemiología molecular de las tuberculosis bovina y humana en una zona endémica de Querétaro, México / Molecular epidemiology of cattle and human tuberculosis in Mexico

OBJETIVO: Determinar el papel de la tuberculosis bovina en la tuberculosis humana. MATERIAL Y MÉTODOS: Se analizaron 255 muestras de pacientes sintomáticos, sembradas en medios de Stonebrink y Lõwenstein-Jensen y analizadas por PCRMPB70 anidada y luego por spoligotyping. RESULTADOS: De las 255 muestras, 74 fueron positivas a la PCR y 20 al aislamiento: de las primeras, 58 (78 por ciento) mostraron espoligotipo de M. tuberculosis y 5 (6.7 por ciento) de M. bovis; de las segundas, 8 (47 por ciento) revelaron espoligotipo de M. tuberculosis y 8 (47 por ciento) de M. bovis. De las 94 muestras positivas al aislamiento o PCR, 66 (70 por ciento) correspondieron a M. tuberculosis y 13 (13.8 por ciento) a M. bovis. Los patrones moleculares de cuatro muestras de M. bovis de seres humanos fueron idénticos a los de las cepas de M. bovis de ganado. CONCLUSIONES: Se demuestra que M. bovis juega un papel importante en la epidemiología de la tuberculosis humana y representa un riesgo para la salud pública.

OBJECTIVE: The purpose of the study was to determine the role of bovine TB in cases of human TB. MATERIAL AND METHODS: Two-hundred and fifty-five samples from symptomatic patients were included in the study. All samples were cultured in Stonebrink and Lowënstein-Jensen media and analyzed using a nested PCRMPB70. The molecular analysis was performed by spoligotyping. RESULTS: From 255 samples, 74 were PCR-positive and 20 were culture-positive. From 94 samples positive to PCR or to isolation, 66 (70 percent) showed a spoligotype compatible with M. tuberculosis, and 13 (13.8 percent) with M. bovis. Four fingerprints of M. bovis from humans were identical to the fingerprints of M. bovis from cattle in the same region. CONCLUSIONS: Our study shows that M. bovis plays an important role in the epidemiology of TB in humans and that TB in cattle represents a risk to public health.

Isolation of Mycobacterium bovis & M. tuberculosis from cattle of some farms in north India--possible relevance in human health.

BACKGROUND & OBJECTIVE: Infection due to Mycobacterium bovis typically occurs in cattle and animals transmit infection to each other. The choice of appropriate clinical specimen is very important for isolation of M. bovis and M. tuberculosis from cattle. The present study reports the isolation of M. tuberculosis and M. bovis from different types of specimens from cattle suspected to be suffering from tuberculosis in certain organized cattle farms in north India. METHODS: A total of 768 specimens (heparinized or EDTA containing blood (162), fine needle aspirates from prescapular lymph gland (PSLG,160), milk (154), pharyngeal swab (PhS, 98), rectal pinch (RP, 97) and faecal sample (97) from 161 cattle of organized cattle farms in north India suspected to be suffering from tuberculosis were analyzed. After decontamination by modified Petroff's method isolation of M.tuberculosis complex was done on Lowenstein-Jensen medium (with and without pyruvate). The culture isolates were identified as M. tuberculosis and M. bovis on the basis of biochemical tests. RESULTS: A total of 54 M. tuberculosis complex isolates were obtained, of them 40 were identified as M.bovis and 14 as M. tuberculosis. M.bovis were isolated from 12 of 38 animals in group A (Tuberculin +ve with signs of tuberculosis), 7 of 37 animals in group B (Tuberculin +ve and apparently healthy), 9 of 21 group C animals in (Tuberculin -ve with clinical signs of tuberculosis), 4 of 26 animals in group D (Tuberculin -ve and apparently healthy), 4 of 27 group E animals (having non-mycobacterial infection) and 4 of 12 animals in group F (having clinical signs such as debilitated condition, cough, decreasing milk production, etc). Maximum number of M. bovis (19/40, 47.5%) and M. tuberculosis (5/14, 35.7%) isolates were grown from prescapular lymph gland biopsy (PSLG) followed by blood from which 9/40 (22.5%) M. bovis and 4/14 (28.5%) M. tuberculosis were isolated. M. bovis [6/40(15%)] and M. tuberculosis [4/14(28.5%)] were also isolated from milk. Only 3/40 (7.5%) isolates of M.bovis could be isolated from 97 rectal pinch followed by 98 pharyngeal swab 2/40 (5%) and 97 fecal samples 1/40 (2.5%) while 1/14 (7.1%) M.tuberculosis isolates were obtained from pharyngeal swab. INTERPRETATION & CONCLUSION: Among the samples analyzed, PSLG was found to be most suitable specimen for isolation of M. tuberculosis complex from cattle and is thus of diagnostic importance. M. bovis in milk indicates the need to investigate the transmission to human in such settings. Isolation of M. bovis and/or M. tuberculosis from apparently healthy cattle indicates sub-clinical infection in the herd. Further, isolation of a significant number of M. tuberculosis from cattle suggests possible human-to-cattle transmission which need to be confirmed by prospective studies including tools like DNA fingerprinting.

Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo)

Mycobacterium was verified in animals from a Brazilian dairy herd, a total of 42 samples from 30 cows were submitted to culture and the isolated strains were analyzed by two polymerase chain reaction (PCR), the first specific for species belonging to the Mycobacterium complex (MTBC) and the other for differentiating M. tuberculosis from M. bovis. Twenty seven samples (64.3 percent) from 18 animals (60 percent) were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5 percent), 9 prescapular lymphnodes (33.3 percent), 2 lungs (7.4 percent), and 1 liver (3.7 percent). All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.

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.

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.