Genotypic Characterization of Mycobacterium bovis Isolates From Dairy Cattle Diagnosed With Clinical Tuberculosis.

Molecular diagnosis of bovine tuberculosis plays an essential role in the epidemiological knowledge of the disease. Bovine tuberculosis caused by Mycobacterium bovis represents a risk to human health. This study aimed to perform the genotypic characterization of M. bovis isolated from bovines diagnosed as tuberculosis from dairy herds in the state of Pernambuco, Brazil. Granulomas from 30 bovines were sent for microbiological culture, and colonies compatible with Mycobacterium spp. were obtained in at least one culture from 17/30 granulomas. All isolates were confirmed to be M. bovis by spoligotyping and 24loci MIRU-VNTR typing. While spoligotyping characterized the isolates as SB0121, SB0295, SB0852, SB0120, and an unclassified genotype, 24loci MIRU-VNTR rendered two clusters of two isolates each and 13 unique profiles. Loci ETR-A showed higher discriminatory power, and loci (ETR-B, ETR-C, MIRU16, MIRU27, and QUB26) showed moderate allelic diversity. This is the first study on the genetic variability of the infectious agent cause of bovine TB in Pernambuco and demonstrates variability of strains in the state. Thus, it corroborates the importance of this microorganism as agent of bovine tuberculosis and its zoonotic potential, this epidemiological tool being a determinant in the rigor of the sanitary practices of disease control in dairy herds.

Nontuberculous mycobacteria in milk from positive cows in the intradermal comparative cervical tuberculin test: implications for human tuberculosis infections.

Although the tuberculin test represents the main in vivo diagnostic method used in the control and eradication of bovine tuberculosis, few studies have focused on the identification of mycobacteria in the milk from cows positive to the tuberculin test. The aim of this study was to identify Mycobacterium species in milk samples from cows positive to the comparative intradermal test. Milk samples from 142 cows positive to the comparative intradermal test carried out in 4,766 animals were aseptically collected, cultivated on Lowenstein-Jensen and Stonebrink media and incubated for up to 90 days. Colonies compatible with mycobacteria were stained by Ziehl-Neelsen to detect acid-fast bacilli, while to confirm the Mycobacterium genus, conventional PCR was performed. Fourteen mycobacterial strains were isolated from 12 cows (8.4%). The hsp65 gene sequencing identified M. engbaekii (n=5), M. arupense (n=4), M. nonchromogenicum (n=3), and M. heraklionense (n=2) species belong to the Mycobacterium terrae complex. Despite the absence of M. tuberculosis complex species in the milk samples, identification of these mycobacteria highlights the risk of pathogen transmission from bovines to humans throughout milk or dairy products, since many of mycobacterial species described here have been reported in pulmonary and extrapulmonary diseases both in immunocompetent and immunocompromised people.

Diagnosis of mycobacteria in bovine milk: an overview.

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.

Genotyping and rifampicin and isoniazid resistance in Mycobacterium bovis strains isolated from the lymph nodes of slaughtered cattle.

In developing nations, 10-20% of the human cases of tuberculosis are caused by Mycobacterium bovis. However, this percentage may be underestimated because most laboratories in developing countries do not routinely perform mycobacterial cultures, and only a few have the systems in place to identify M. bovis. There are few studies investigating genotypic diversity and drug resistance in M. bovis from animal and/or human infections. The genotypic diversity of M. bovis strains obtained from bovine lymph nodes were investigated by spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR). The phenotypic resistance to isoniazid and rifampicin and MIC values of the isolates were determined using the resazurin microtiter assay plate method (REMA). The evaluation of the possible genetic basis for such resistance was performed with GenoType MTBDRplus. Sixty-seven isolates were obtained, of which 11 (16%) were MDR-TB, 8 (12%) were isoniazid-resistant, and 2 (3%) were rifampicin-resistant. Mutations associated with drug resistance were not found. Genotyping techniques enabled the grouping of the strains into 12 clusters and 21 isolates with unique profiles. The high frequency of M. bovis reinforces the impact of the pathogen as a major causal agent of bovine tuberculosis in the study area. The resistance of the strains to drugs used for first-line treatment of human tuberculosis raises public health concerns. Further studies are required to elucidate the basis of drug resistance and genotypic diversity in M. bovis.

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.

Deforming mandibular osteomyelitis in a cow caused by Trueperella pyogenes / Osteomielite mandibular deformante em vaca causada por Trueperella pyogenes

This study reports an unusual case of deforming mandibular osteomyelitis in a cow caused by Trueperella (Arcanobacterium) pyogenes, on the face of the ventrolateral caudal portion of the right branch of the mandible. Fragment aspired of lesion by fine needle allowed cytological characterization, isolation and identification of T. pyogenes. Radiographic examination showed marked periosteal reaction in the right mandible, numerous lytic areas and cortical bone destruction. Despite of treatment based on in vitro antimicrobial sensitivity test, it was recommended the euthanasia due to progressive worsening of the cow's condition. Multiple abscesses were observed in the mandibular region at necropsy. Pyogranuloma was characterized in histological exam. Sampled material collected from the lesion after necropsy resulted in microbiological reisolation of T. pyogenes .

Relata-se caso incomum de osteomielite mandibular deformante em vaca, causada por Trueperella (Arcanobacterium) pyogenes, na face ventro-lateral da porção caudal do ramo direito da mandíbula. A punção aspirativa de fragmento da lesão permitiu a caracterização citológica, o isolamento microbiano e identificação de T. pyogenes. Exame radiográfico mostrou acentuada reação periodontal na mandíbula direita, com predomínio de áreas líticas e destruição da cortical óssea. Apesar da instituição do tratamento baseado no teste de sensibilidade microbiana in vitro, foi recomendada a eutanásia, em virtude da piora progressiva do estado geral do animal. No exame post-mortem foram observados múltiplos abscessos na lesão que, histologicamente, foi caracterizada como piogranuloma. A colheita de material da região mandibular afetada, após a necropsia, resultou no reisolamento microbiológico de T. pyogenes .

Occurrence of mycobacteria in bovine milk samples from both individual and collective bulk tanks at farms and informal markets in the southeast region of Sao Paulo, Brazil.

BACKGROUND: Mycobacterium spp. is one of the most important species of zoonotic pathogens that can be transmitted from cattle to humans. The presence of these opportunistic, pathogenic bacteria in bovine milk has emerged as a public-health concern, especially among individuals who consume raw milk and related dairy products. To address this concern, the Brazilian control and eradication program focusing on bovine tuberculosis, was established in 2001. However, bovine tuberculosis continues to afflict approximately 1,3 percent of the cattle in Brazil. In the present study, 300 samples of milk from bovine herds, obtained from both individual and collective bulk tanks and informal points of sale, were cultured on Löwenstein-Jensen and Stonebrink media. Polymerase chain reaction (PCR)-based tests and restriction-enzyme pattern analysis were then performed on the colonies exhibiting phenotypes suggestive of Mycobacterium spp., which were characterized as acid-fast bacilli. RESULTS: Of the 300 bovine milk samples that were processed, 24 were positively identified as Mycobacterium spp.Molecular identification detected 15 unique mycobacterial species: Mycobacterium bovis, M. gordonae, M. fortuitum, M. intracellulare, M. flavescens, M. duvalii, M. haemophilum, M. immunogenum, M. lentiflavum, M. mucogenicum, M. novocastrense, M. parafortuitum, M. smegmatis, M. terrae and M. vaccae. The isolation of bacteria from the various locations occurred in the following proportions: 9 percent of the individual bulk-tank samples, 7 percent of the collective bulk-tank samples and 8 percent of the informal-trade samples. No statistically significant difference was observed between the presence of Mycobacterium spp. in the three types of samples collected, the milk production profiles, the presence of veterinary assistance and the reported concerns about bovine tuberculosis prevention in the herds. CONCLUSION: The microbiological cultures associated with PCR-based identification tests are possible tools for the investigation of the presence of Mycobacterium spp. in milk samples. Using these methods, we found that the Brazilian population may be regularly exposed to mycobacteria by consuming raw bovine milk and related dairy products. These evidences reinforces the need to optimize quality programs of dairy products, to intensify the sanitary inspection of these products and the necessity of further studies on the presence of Mycobacterium spp. in milk and milk-based products.

Disseminated Mycobacterium tuberculosis infection in a dog.

An uncommon disseminated Mycobacterium tuberculosis infection is described in a 12-year-old female dog presenting with fever, dyspnea, cough, weight loss, lymphadenopathy, melena, epistaxis, and emesis. The dog had a history of close contact with its owner, who died of pulmonary tuberculosis. Radiographic examination revealed diffuse radio-opaque images in both lung lobes, diffuse visible masses in abdominal organs, and hilar and mesenteric lymphadenopathy. Bronchial washing samples and feces were negative for acid-fast organisms. Polymerase chain reaction (PCR)-based species identification of bronchial washing samples, feces, and urine revealed M. tuberculosis using PCR-restriction enzyme pattern analysis-PRA. Because of public health concerns, which were worsened by the physical condition of the dog, euthanasia of the animal was recommended. Rough and tough colonies suggestive of M. tuberculosis were observed after microbiological culture of lung, liver, spleen, heart, and lymph node fragments in Löwenstein-Jensen and Stonebrink media. The PRA analysis enabled diagnosis of M. tuberculosis strains isolated from organs.