Stonebrink Media Research Articles

SANITARY QUALITY OF MILK FROM COWS WITH A NEGATIVE RESALT ROUTINE BACTERIAL RESEARCH FOR MYCOBACTERIUM BOVIS

Pathogenic and potentially pathogenic mycobacteria are able to persist in t he body of mammals and birds in the nominal form and in the non-cultivating state - NKS and L-transformed variant. Such physiologically and morphologically altered forms of existence of mycobacterial infectious pathogens are not identified by routine laboratory methods, and the pathologies induced by them have atypical or latent clinical and epizootological signs. This is a very dangerous phenomenon, because livestock products from cows latently infected with mycobacteria will be biologically dangerous for consumers.
 M. bovis was isolated and identified in one of the samples of milk obtained at a non-sanctioned point of trade during a selective unsystematic examination of the milk of cows of the individual sector. The isolated epizootic culture of M. bovis possessed typical morpho-tinctorial properties, induced HST and was highly virulent for ants and rabbits, apathogenic for chickens. A cultural study gave a negative result. It was possible to isolate the culture only in a bioassay on houseflies with intratesticular contamination of a concentrated milk sample. And already from the positive biomaterial of ants that died from the tbc genoform, a pure culture of M. bovis was obtained on the Stonebrink medium of Polish production. There was no primary growth on other media.
 The milk had high-quality organoleptic properties, was very tasty and fermented well at room temperature, and was of good quality in appearance. But it was infected with an emerging pathogen of lethal anthropozoonosis. This case shows the urgent need for express indication of pathogenic and potentially pathogenic mycobacterial prokaryotes in each milk sample.

Spatial distribution of Mycobacterium bovis spoligotypes in cattle from the state of Minas Gerais, Brazil.

Genotyping methods have led to a better understanding of the epidemiology of Mycobacterium bovis (M. bovis) infection, and its transmission dynamics, as well as the possible phylogenetic relationships between Mycobacterium strains, thus making bovine tuberculosis control programs more efficient. The goal of this study was to characterize the main spoligotypes of M. bovis isolated from cattle in the State of Minas Gerais, Brazil. It was carried out in 28 municipalities of "Triângulo Mineiro" and "Alto Paranaíba" regions of the state. Viscera samples were obtained from 58 bovines positive for tuberculosis according to comparative cervical tests, and from another 100 bovines with lesions suggestive of tuberculosis, which were donated by the National Agricultural Laboratory of Pedro Leopoldo, Minas Gerais. Microbiological isolation was performed in Stonebrink medium, and molecular identification of mycobacteria was performed by PCR. Genotyping was performed using the spoligotyping method at the Agrobiotechnology and Molecular Biology Institute of National Agricultural Technology Institute-National Scientific and Technical Research Council, Buenos Aires, Argentina. Among the 158 viscera samples, we obtained 40 (25%) isolates of M. bovis, and detected 11 spoligotype patterns, with a predominance of SB1142 (37.5%), SB0121 (25.0%), and SB1145 (10.0%). Other standards, SB0295, SB1050, SB0881, SB1144, SB1802, SB0140, SB0120, and SB0849, varied from 2.5 to 7.5%, heterogeneously distributed among the municipalities. The presence of spoligotypes shared with other Brazilian states and different countries indicates their possible exchange through epidemiological relationships, such as the transit of live animals and/or genetic similarity between strains that share a common ancestor.

Could the type and severity of gross lesions in pig lymph nodes play a role in the detection of Mycobacterium avium?

The Mycobacterium avium complex (MAC) comprises a widespread group of slowly-growing bacteria from the Mycobacteriaceae. These bacteria are responsible for opportunistic infections in humans and animals, including farm animals. The aim of the study was to determine whether it is possible to predict the presence of M. avium in pig lymph nodes based on the size and type of lesions found during post-mortem examination at a slaughterhouse. Lymph nodes were collected from 10,600 pigs subjected to such post-mortem examination. The nodes were classified with regard to their quality, and the number of tuberculosis-like lesions; following this, 86 mandibular lymph nodes with lesions and 113 without visible macroscopic lesions were selected for further study. Cultures were established on Löwenstein-Jensen and Stonebrink media, and a commercial GenoType Mycobacterium CM test was used to identify and differentiate M. avium species. The prevalence of M. avium was 56.98% in the lymph nodes with lesions and 19.47% in the unchanged ones. Statistical analysis indicated that visual assessment of lesions in the mandibular lymph nodes, in particular the number of tuberculous lesions, is a highly-efficient diagnostic tool. Similar results were obtained for estimated percentage area affected by the lesion, i.e. the ratio of the changed area of the lymph node in cross-section to the total cross-sectional area of the lymph node; however, this method is more laborious and its usefulness in slaughterhouse conditions is limited. By incising the lymph nodes and assessing the number of tuberculosis-like lesions, it is possible to limit the inclusion of meat from pigs infected with M. avium into the human food chain.

Infection of a Free-Living Wild Boar (Sus scrofa) with a Bacterium from the Mycobacterium kansasii Complex.

Simple SummaryMycobacteriosis is a collective term for diseases caused by nontuberculous mycobacteria. Wild animals are a frequent source of mycobacteria infection in farm animals and humans; therefore, it is important to monitor the presence of these pathogens in free-living mammals. We isolated bacterium belonging to Mycobacterium kansasii complex from a submandibular lymph node obtained from a wild boar. This mycobacterium is a common cause of severe human lung diseases and is rarely responsible for animal diseases; therefore, its presence in the wild animal population is of great concern. The animal was apparently healthy, and we did not find any internal organ lesions despite the abundant growth of tissue-isolated bacteria on media. Thanks to our research, the specificity of wild boar mycobacteriosis caused by MKC will be better known.The most numerous group of bacteria in the genus Mycobacterium is the nontuberculous mycobacteria. Currently, over 200 species of bacteria have been classified as belonging to this group, of which approximately 30 are pathogenic to humans and animals. Mycobacterium kansasii complex numbers among these pathogenic species. The submandibular lymph nodes of a wild boar shot by a hunter were examined in order to confirm or exclude infection with bacteria of the genus Mycobacterium. In culture, a bacterial isolate was obtained after 12 days of incubation on Petragnani and Stonebrink media. A multiplex PCR clearly indicated that the isolate was a nontuberculous mycobacterium. The results of species identification attempts via both molecular biology methods and mass spectrometry confirmed that the isolated strain belonged to MKC. The described case of a wild boar infection with MKC is the first documented case in Poland and only the second in Europe, and in confirming the presence of this pathogen among free-living animals, this report implies that MKC is of great concern. Our research elucidates some specifics of wild boar mycobacteriosis and may be used to instill awareness in the public of the dangers of dressing hunt prey or consuming its meat in ignorance of safe procedures, which can contribute to the transmission of the pathogen to humans.

Tuberculosis in bovine slaughtered in the semi-arid region of Rio Grande do Norte, Brazil

Between November 2017 and August 2018, in order to determine the occurrence of tuberculosis in cattle slaughtered in the semi-arid region of Rio Grande do Norte, 11,610 bovine carcasses underwent a routine post-mortem inspection. One animal presented suggestive lesions and samples from lung, spleen and heart were submitted to microbiological culture in Stonebrink medium for 90 days and molecular analysis by nested-PCR. For histopathological examination and Ziehl Neelsen staining, the omentum was used. In the cultured samples, two of them (heart and spleen) showed growth, but were not confirmed as M. bovis by conventional PCR. At nested-PCR, the samples showed amplification for the TbD1 region. The presence of numerous granulomas was detected in the histopathological examination characterized by a necrotic center and areas of mineralization, as well as the presence of acid-fast bacilli (AFB) in the Ziehl-Neelsen stain. Microbiological culture can show false negative results, despite being considered a gold standard technique, although it takes time. Nested-PCR and histopathology show fast and effective results for the diagnosis of the disease. The presence of positive animals represents a public health risk in the studied region. Therefore, one of the essential systems applied to the control of bovine tuberculosis is the epidemiological surveillance of animals in slaughterhouses.

Detection of mycobacteria in coalho cheese sold in Northeastern Brazil

The aim of the present study was to detect and identify Mycobaterium spp. in 50 samples of coalho cheese sold in the Northeast region of Brazil. Of the 50 analyzed samples, 35 were produced by the artisanal process, using raw milk, and 15 originated from industrialized establishments that pasteurize milk. Conventional and real-time nested PCR for the rv2807 gene of the M. tuberculosis complex was performed directly from the 50 analyzed samples. Samples of coalho cheese were grown simultaneously in Stonebrink medium, and conventional PCR was performed from the bacterial isolates with primers that flank differentiation region 4 (DR4), specific to M. bovis, mb400F. Bacterial isolates negative by PCR for RD4 were subjected to PCR for hsp65 of Mycobacterium spp., with subsequent DNA sequencing. The cultures were negative for the M. tuberculosis complex, but two samples (4%) from the artisanal process (with raw milk) exhibited identity with hsp65 of Mycobacterium lehmanii (Sequence ID: KY933786.1, identities: 312/363 [86%]); and Mycobacterium rutilum (sequence ID: LT629971.1, identities: 331/371 [89%]), showing to be indicative environmental contamination. Non-tuberculous mycobacteria are emergent and ubiquitous microorganisms; therefore, they deserve greater attention in the cheese production chain, both in terms of Good Agricultural Practices (GAP) and food Good Manufacturing Practices (GMP).

Bovine tuberculosis in safari park in Brazil

ABSTRACT: Bovine tuberculosis (bTB) is an infectious disease caused by Mycobacterium bovis, affecting domestic animals, wild animals and humans. In captivity, for wild animals, bTB represents a risk to animal keepers and zoo visitors, in addition to the possibility of spreading the infection to domestic animals or through the trade of infected wild animals. Sambar (Cervus unicolor), red deer (Cervus elaphus) and fallow deer (Dama dama) from a safari park in the State of Rio Grande do Sul, Brazil, showed a clinical condition of dyspnea and weight loss. Some animals died and showed lesions suggestive of tuberculosis (LST), which were confirmed by histopathology. After the interdiction of the safari park by the state veterinary authorities, 281 deer were euthanized with the authorization of the “Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis” (IBAMA). Retropharyngeal and submandibular lymph nodes and viscera were collected from 21 animals, which were grown in Stonebrink medium for up to 90 days. After DNA extraction from the bacterial colonies, PCR was performed for targets flanking the region of differentiation 4 (RD4). Of the 21 samples, 14 (66.7%) presented LST with a granulomatous appearance, a whitish coloration, and caseous or calcified consistency, and seven samples (33.3%), showed no lesions. In the culture of 14 samples with LST, 13 (92.8%) presented bacterial growth compatible with M. bovis. In the cultivation of the seven samples without LST, four (57.1%) presented colonies compatible with M. bovis. PCR and DNA sequencing of the PCR amplicons detected as positive all the 17 (100%) bacteriological cultures suggestive of M. bovis, thus confirming the outbreak of bTB in deer. Decisions about positive tested and suspicious animals should be taken based on the evaluation of the risk of transmission to the rest of the zoological animals, animal welfare, conservation considerations and, the zoonotic potential of this pathogen.

Epidemiological characterization of Mycobacterium caprae strains isolated from wildlife in the Bieszczady Mountains, on the border of Southeast Poland

BackgroundThe majority of animal tuberculosis (TB) cases reported in wildlife in Poland over the past 20 years have concerned the European bison inhabiting the Bieszczady Mountains in Southeast Poland: an area running along the border of Southeast Poland. As no TB cases have been reported in domestic animals in this region since 2005, any occurrence of TB in the free-living animals inhabiting this area might pose a real threat to local livestock and result in the loss of disease-free status. The aim of the study was to describe the occurrence of tuberculosis in the wildlife of the Bieszczady Mountains and determine the microbiological and molecular characteristics of any cultured strains. Lymph node samples were collected for analysis from 274 free-living animals, including European bison, red foxes, badgers, red deer, wild boar and roe deer between 2011 and 2017. Löwenstein–Jensen and Stonebrink media were used for culture. Molecular identification of strains was performed based on hsp65 sequence analysis, the GenoType®MTBC (Hain Lifescience, Germany) test, spoligotyping and MIRU-VNTR analysis.ResultsMycobacterium caprae was isolated from the lymph nodes of 21 out of 55 wild boar (38.2%; CI 95%: 26.5%, 51.4%) and one roe deer. Since 2014, no new TB cases have been reported in the Bieszczady European bison population.ConclusionsThe identification of TB in wild boar in the Bieszczady is an alarming phenomenon, which requires further investigation. The Bieszczady mountains are a precious, unique area, home to many protected species. However, it is also the only area in Poland where TB cases have been reported in free-living animals. The occurrence of TB in wild boar inhabiting this area might pose a real threat to local livestock and many of the protected species (for example European bison that can share feeding places with wild boar). Given this situation, ongoing monitoring of the prevalence of TB should be conducted, and protective measures should be considered.

A rare case of spinal tuberculosis due to Mycobacterium bovis. Is zoonotic tuberculosis underdiagnosed?

We report a rare presentation of Pott's disease caused by M. bovis, suggesting transmission from infected cattle, and only the second case described so far in scientific reports. Noteworthy of this case was that the strain was only isolated on Stonebrink medium, a sodium pyruvate-containing culture medium for the isolation of mycobacteria. This medium is frequently ignored in diagnostic laboratories and in the laboratory manuals of most international health organizations. In general laboratories use a culture medium that contains glycerol, a carbon substrate considered inhibitory for the growth of M. bovis. The use of glycerol-containing medium therefore likely contributes towards underestimating zoonotic tuberculosis. Our case suggests that, in order to improved surveillance efforts for zoonotic TB and increase the notification rate for M. bovis to human TB, the use of pyruvate-containing media should be promoted, particularly in developing countries with a high prevalence of bovine TB, but also through the World Health Organization' (WHO) End TB Strategy and the Roadmap for Zoonotic TB.

Epidemiological Study of Mycobacterium bovis Infection in Buffalo and Cattle in Amazonas, Brazil

Bovine Tuberculosis (BTB) is an endemic disease in about one hundred countries, affecting the economy causing a decrease in productivity, condemnation of meat, and damaging the credibility on international trade. Additionally, Mycobacterium bovis the major causative agent for BTB can also infect humans causing a variety of clinical presentations. The aim of this study was to determine BTB prevalence and the main risk factors for the Mycobacterium bovis prevalence in cattle and buffalos in Amazonas State, Brazil. Tissue samples from 151 animals (45 buffalo and 106 cattle from five herds with buffalo only, 22 herds with cattle only, and 12 herds with buffalo and cattle) were obtained from slaughterhouses under State Veterinary Inspection. M. bovis were isolated on Stonebrink medium. The positive cultures were confirmed by polymerase chain reaction (PCR) testing. The apparent herd and animal prevalence rates were 56.4 and 5.40%, respectively. Regarding animal species, the apparent prevalence rates were 3% in cattle and 11.8% in buffalo. Generalized Linear Mixed Models (GLMM) with random effect were used to assess the association with risk factors on the prevalence. Species (buffalo), herds size (>100 animals) and the presence of both species (buffalo and cattle) in the herd were the major risk factors for the infection by Mycobacterium bovis in the region. The findings reveal an urgent need for evidence-based effective intervention to reduce BTB prevalence in cattle and buffalo and prevent its spread to the human population. Studies are needed to understand why buffalo are more likely to be infected by M. bovis than cattle in Amazon. Recommendations for zoning, use of data from the inspection services to generate information regarding BTB focus, adoption of epidemiological tools, and discouragement of practices that promote the mixing of cattle and buffalo, were made.

Bovine Tuberculosis in the Republic of Macedonia: Postmortem, Microbiological and Molecular Study in Slaughtered Reactor Cattle

Abstract Bovine tuberculosis is a chronic infectious disease in cattle caused mainly by Mycobacterium bovis and to a lesser extent by Mycobacterium caprae. The other members of the Mycobacterium tuberculosis complex (MTBC) can also cause the disease in domestic and wild animals and all of them have a zoonotic potential. The main purpose of the study was to determine the presence and distribution of the tuberculous lesions in reactor cattle, and to isolate and identify the causative agents of bovine tuberculosis in the Republic of Macedonia. Lymph nodes and affected organs from 188 reactor cattle slaughtered due to a positive intradermal comparative cervical tuberculin test were analyzed by detection of tuberculous lesions, followed by isolation and molecular identification of the isolated mycobacteria. The isolation was performed on selective media - Lowenstein Jensen with glycerol, Lowenstein Jensen without glycerol and Stonebrink medium supplemented with pyruvate. The molecular identification of the MTBC members was performed by analysis of the Regions of difference (RD1, RD9 and RD4) and detection of single nucleotide polymorphisms in the lepA gene for Mycobacterium caprae. Typical tuberculous lesions were detected in 62 animals (33.0%) and the lesions were most prevalent in the mediastinal lymph nodes (47.5%). The isolated mycobacteria in the MTBC were identified as Mycobacterium bovis and Mycobacterium caprae and were found in both animals with visible lesions (82.2%) and animals without visible lesions (27.7%). The slaughterhouse postmortem examinations and laboratory investigations should be included on regular bases in order to improve the National eradication program.

Mycobacterium bovis in a European bison (Bison bonasus) raises concerns about tuberculosis in Brazilian captive wildlife populations: a case report

BackgroundTuberculosis caused by Mycobacterium bovis is an important worldwide zoonosis and has been reported to cause clinical disease in several animal species, including captive wildlife. This report describes a case of M. bovis infection in a European bison from a Brazilian zoo and compiles a number of literature reports that raise concern regarding tuberculosis among captive wildlife in Brazil.Case presentationA 13 year-old captive-born male bison (Bison bonasus) from a Brazilian zoo began presenting weight loss, diarrhea and respiratory symptoms, which inevitably led to his death. At the animal’s necropsy, inspection of the thoracic and abdominal cavities revealed multiple enlarged lymph nodes, ranging from 4 to 10 cm, and pulmonary nodules containing caseous masses with firm white materials consistent with mineralization. Histopathology findings showed a significant amount of acid-alcohol resistant bacilli compatible with Mycobacterium spp. Specimens from lymph nodes and lungs were cultured on Petragnani and Stonebrink media, and specific PCR assays of the bacterial isolate identified it as M. bovis.ConclusionThe European bison reported herein died from a severe form of disseminated tuberculosis caused by M. bovis. A review of the available literature indicates possible widespread occurrence of clinical disease caused by M. bovis or M. tuberculosis affecting multiple animal species in Brazilian wildlife-related institutions. These likely underestimated numbers raise concern regarding the control of the disease in captive animal populations from Brazil.

Cryopreservation of Mycobacterium bovis isolates

Research, development of new biotechnological methods, diagnostic tests, confirmation of results, and reinvestigations are possible because of the availability of well-preserved living organisms maintained without any changes. Cryopreservation is a simpler, more reliable and long-term stable method for culture maintenance. Storage temperature and composition of the suspending vehicle are factors that affect the viability of mycobacterial strains. Three vehicles and three storage temperatures were evaluated to define a suitable cryoprotective medium for the preservation of Mycobacterium bovis strains. Colonies of sixteen M. bovis isolates were used to prepare the suspensions, which were then added to three vehicles: sterile 0.85% saline solution (SS), Middlebrook 7H9 broth (7H9), and Middlebrook 7H9 broth with sodium pyruvate (7H9p) replacing glycerol. Aliquots of these suspensions were frozen by three different methods, directly in the -20°C freezer, directly in the -80°C freezer, and at -196°C by immersion in liquid nitrogen (LN). The frozen aliquots were thawed at room temperature after 45, 90 and 120 days. Mycobacterial viability was assessed by counting the living cells on plates of Stonebrink medium before and after the freezing procedure. Storage at -20°C exhibited a lower recovery of M. bovis compared to storage at -80°C (Dunn’s test, p=0.0018) and LN (Dunn’s test, p=0.0352). There was no statistically significant difference between storage at -80°C and in LN (Dunn’s test, p=0.1403), yet -80°C showed better results than LN. All three suspending vehicles showed no statistically significant difference in terms of viability (Friedman’s test, p=0.7765). Given the low loss proportion of 5% during storage at -20°C and the high cost equipment required for storage at -80°C and LN, we recommend storage at -20°C or -80°C, when this is available, for preservation of M. bovis field strains.

Comparative study of Mycobacterium bovis primary isolation methods

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”.

Identificação e genotipagem de Mycobacterium bovis em bovinos positivos no teste intradérmico para tuberculose em Mato Grosso do Sul

Neste estudo, realizou-se genotipagem de isolados de Mycobacterium bovis, provenientes de amostras de tecidos de bovinos positivos no teste cervical comparativo (TCC) para tuberculose em Mato Grosso do Sul, por meio da técnica de spoligotyping. Tecidos de 13 bovinos positivos, oriundos de diferentes municípios do estado, foram cultivados em meio de Stonebrink. As colônias resultantes foram submetidas à coloração de Ziehl-Neelsen e todos os isolados apresentaram características tintoriais de BAAR. Os 13 isolados de BAAR foram identificados por PCR multiplex (mPCR). O gene hsp65 foi alvo para identificação de Mycobacterium spp, a sequência de inserção IS6110 foi alvo para identificação de complexo Mycobacterium tuberculosis (CMT) e a região rvd1rv2031c foi explorada para detecção de M. bovis. Os isolados micobacterianos foram genotipados pela técnica de spoligotyping. Dos 13 bovinos, sete tinham pelo menos uma lesão sugestiva de tuberculose em linfonodos retrofaríngeos, parotídeos e pulmonares ou no pulmão, e em seis não foram encontradas lesões visíveis sugestivas da doença. Na mPCR, 11/13 (84,6%) isolados foram positivos para Mycobacterium spp; 8/13 (61,5%) positivos para CMT e 7/13 (53,8%) positivos para M. bovis. Com base no spoligotyping, oito isolados de BAAR foram agrupados dentro de três diferentes agrupamentos de genótipos e uma amostra remanescente apresentou perfil único, sendo quatro isolados com padrão de espoligotipo SB0121, dois SB1145, dois SB0881 e um SB0140. A técnica de spoligotyping demonstrou que há diversidade genética entre os espoligotipos presentes no estado de Mato Grosso do Sul, embora predomine o perfil SB0121

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

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.

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.

BackgroundMycobacterium 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.ResultsOf 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.ConclusionThe 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.

Detection ofMycobacterium bovis–Infected Dairy Herds Using PCR in Bulk Tank Milk Samples

Bovine tuberculosis (bTB) is a chronic and zoonotic disease due to Mycobacterium bovis. The tuberculosis eradication campaign carried out in Argentina has considerably improved the health situation of the herds. Here we evaluated a strategy to detect M. bovis-infected herds by Touch-Down IS6110 polymerase chain reaction (PCR) in bulk tank raw milk from dairy farms. We evaluated 177 samples from herds with the official tuberculosis free certificate (TFC) and 80 from herds without the certificate, non-tuberculosis-free certificate (NTFC), from 10 departments of Santa Fe province, Argentina. To avoid the effect of Taq polymerase inhibitors, a dilution of DNA template was performed. Positive PCR results were obtained in 102 (40%) of the samples, whereas negative ones were obtained in 155 (60%) of the samples. Importantly, 44% of NTFC and 38% of TFC samples were positive. All samples were subjected to culture in Löwenstein Jensen and Stonebrink media with no positive isolation. The negative predictive value (NPV) of PCR in the TFC group was 95%, while the positive predictive value (PPV) of PCR in the NTFC group was 51%. Based on these results, this work proposes a method that should be applied regularly to detect M. bovis--infected dairy herds, complementary to the official test of tuberculin, or purifed protein derivative (PPD), to control dairy herds, especially those free of tuberculosis.