Molecular epidemiology of Mycobacterium bovis isolates from South America.

One hundred seventy-eight isolates of Mycobacterium bovis were subjected to DNA restriction fragment length polymorphism (RFLP) analysis, using the direct repeat element (DR) and the polymorphic GC-rich repeat sequence (PGRS) as probes. By combining the patterns generated by the two repeat DNA elements, 93 different patterns were observed. One hundred-one isolates were grouped in clusters, which include 25 different clusters. One pattern was the most frequently observed, clustering 18.5% of isolates. It was only found in the Center and northeast regions of Argentina and in one isolate from Paraguay. The isolates from Brazil analyzed here presented exclusive patterns (only found in a particular region). The number of exclusive patterns was high in all argentine regions: northeast 78%, center 81%, and Buenos Aires 81%.

Granulomatous meningomyeloencephalitis in a calf caused by Mycobacterium bovis / Meningomieloencefalite granulomatosa em um bezerro, causada por Mycobacterium bovis

Bovine tuberculosis is a chronic disease caused by Mycobacterium bovis, characterized by respiratory disorders, emaciation and lymphadenomegaly. Neurological signs associated with granulomatous lesions in the central nervous system are rarely seen. In Brazil, the National Program for Control and Eradication of bovine tuberculosis has begun in 2001, and notification of the disease is compulsory. A case of tuberculous meningomyeloencephalitis is reported in a dairy calf less than one year old presenting neurological signs (irritability, aggressiveness, weakness, dizziness and recumbency) which progressed to death in three days. Brain, spinal cord, and lung tissue samples were examined histopathologically. The spinal cord showed granulomatous meningomyelitis with Langhans giant cells in the meninges and perivascular mononuclear cuffing in the white and gray matter. There were a few granulomas in the cerebral cortex with a central area of necrosis surrounded by mononuclear infiltrate. Mononuclear perivascular cuffing, satellitosis, hyperemia, and severe granulomatous meningitis with Langhans giant cells were observed in the neuropile. Samples of brain were inoculated into Petragnani and Stonebrink media. M. bovis was isolated and identified by PCR with JB21-JB22 primers. Considering the 2,888 samples of bovine CNS sent for surveillance of neurological syndrome and negative for Rabies, this was the only case of tuberculous meningomyeloencephalitis (0,035%) diagnosed from 2004 to 2008 by the Laboratory of Anatomopathology of Instituto Biológico. Although tuberculous meningomyeloencephalitis is not so frequent, it's important to diagnose M. bovis as a causative agent of bovine neurological disorders. In accordance with Brazilian regulations, sanitary measures in the infected herd (test-and-slaughter) should be adopted.

A tuberculose bovina é uma doença crônica causada por Mycobacterium bovis, caracterizada por desordens respiratórias, emaciação e linfadenomegalia. Em raros casos, sinais neurológicos associados com lesões granulomatosas no sistema nervoso central são observados. O Brasil iniciou o Programa Nacional para Controle e Erradicação dessa doença em 2001, tornando a notificação compulsória. Um caso de meningomieloencefalite tuberculosa é relatado em uma bezerra leiteira com um ano de idade, que apresentou sinais neurológicos (irritabilidade e agressividade, fraqueza e tontura, decúbito) que progrediram para morte em três dias. A medula espinhal apresentou meningomielite granulomatosa, com células gigantes tipo Langhans nas meninges e manguito perivascular mononuclear na substância branca e cinzenta. Havia alguns granulomas no córtex cerebral, com área central de necrose circundada por infiltrado mononuclear. No neurópilo observou-se manguito perivascular mononuclear, satelitose, hiperemia e severa meningite granulomatosa com células gigantes tipo Langhans. Amostras de cérebro foram inoculadas nos meios de cultivo Petragnani e Stonebrink e M. bovis foi isolado e identificado por PCR com primers JB21-JB22. Considerando as 2.888 amostras de SNC bovino enviadas para a vigilância epidemiológica de síndrome neurológica e negativas para raiva, este foi o único caso (0,035%) de meningomieloencefalite tuberculosa diagnosticado pelo Laboratório de Anatomia Patológica do Instituto Biológico no período de 2004 a 2008. Embora a meningomieloencefalite tuberculosa não seja tão freqüente, é importante incluir a infecção por M. bovis no diagnóstico diferencial de lesões do sistema nervoso central de bovinos. De acordo com a legislação brasileira, medidas sanitárias no rebanho infectado (teste e abate) devem ser adotadas.

Molecular typing of Mycobacterium bovis isolates from south-east Brazil by spoligotyping and RFLP.

The identification of 163 strains of Mycobacterium bovis by polymerase chain reaction (PCR) and microbiological tests was carried out on 252 tuberculous-like lesions (TLLs) collected from slaughtered cattle in south-east Brazil. This study compared the usefulness of three genotyping techniques, IS6110-restriction fragment length polymorphism (RFLP), polymorphic guanine-cytosine-rich sequence (PGRS)-RFLP and direct repeat (DR)-spoligotyping, as applied to M. bovis isolates. Based on IS6110-RFLP genotyping we selected a group of 23 isolates containing more than one IS6110 copy, along with 16 samples containing one IS6110 copy from different geographical areas, evenly distributed among dairy (eight) and beef cattle (eight). These selected isolates were analysed by PGRS-RFLP and DR-spoligotyping genotyping. Dairy cattle (17%) display a higher frequency of multiple IS6110 copies than beef cattle (10%). A comparison between the genotype data obtained fails to show a correlation between the main clusters found by the three techniques. However, the clustering of each genotyping procedure revealed that the majority of strains are closely related. The RFLP-PGRS patterns showed a sizable group (20.5%) containing a 5.5 kb fragment and the predominant spoligotype is similar to that from the BCG vaccine strain. Unexpectedly, four strains (2.4%) showed drug resistance to 0.2 microg/ml isoniazid and 20 microg/ml ethionamide, but none of them was resistant to rifampicin or other antibiotics tested.

Ants' role (Hymenoptera: Formicidae) as potential vectors of mycobacteria dispersion / Comportamento de formigas (Hymenoptera: Formicidae) como vetores potenciais de dispersão de micobactérias

Ants are found worldwide playing an important environmental role. Some species are considered as agricultural pests and potential risk to human life and public health acting as pathogens carriers. Ants as Paratrechina longicornis and Camponotus spp. have been found inside hospitals. The aim of this study was the research of mycobacteria in 138 samples of ants (137 Paratrechina longicornis and only one Camponotus spp.) which got into the laboratories of tuberculosis diagnosis. These ants were suspended in sterile saline solution and inoculated into Petragnani and Stonebrink media, incubated at 37° C until 90 days and the isolates were identified as environmental mycobacteria (1 Mycobacterium fortuitum peregrinum, 1 Mycobacterium smegmatis) and 1 Mycobacterium tuberculosis complex. These results showed that ants should also act as mechanical vectors of mycobacteria dissemination in risk environments, reinforcing their significance in public health.

As formigas têm uma distribuição mundial e representam importante papel no ecossistema. Algumas espécies são consideradas pragas para a agricultura e um risco potencial à vida humana e à saúde pública veiculando mecanicamente agentes patogênicos. Formigas como Paratrechina longicornis e Camponotus spp. têm sido encontradas em ambientes hospitalares. O foco do presente estudo foi a identificação de micobactérias em 138 amostras de formigas (137 Paratrechina longicornis e apenas uma Camponotus sp.), que tiveram acesso a áreas de laboratórios de diagnóstico de tuberculose. Essas formigas foram suspensas em solução salina estéril que foi semeada em meios de Petragnani e Stonebrink, incubadas a 37º C por até 90 dias e as estirpes de micobactérias isoladas foram identificadas pelas técnicas clássicas como micobactérias ambientais (sendo 1 Mycobacterium smegmatis, 2 Mycobacterium fortuitum peregrinum e 1 Mycobacterium tuberculosis). Esses resultados mostram que as formigas podem também se constituir vetores de dispersão de micobactérias em ambientes de risco, reforçando sua importância em saúde pública.

Usefulness of spoligotyping in molecular epidemiology of Mycobacterium bovis-related infections in South America.

Two hundred twenty-four Mycobacterium bovis isolates, mainly from South American countries, were typed by spoligotyping, and 41 different spoligotypes were identified. A total of 202 M. bovis isolates (90%) were grouped into 19 different clusters. The largest cluster contained 96 isolates (42.8%) on the basis of the most frequently observed spoligotype, spoligotype 34. Nineteen M. bovis isolates from humans in Argentina had spoligotypes and polymorphic GC-rich repetitive sequence (PGRS) types that represented the most common types found among isolates from cattle. All five isolates from Uruguay and three of the six isolates from Paraguay had spoligotypes that were also detected for isolates from Argentina. The spoligotypes of isolates from Brazil, Costa Rica, and Mexico and of some of the isolates from Paraguay could not be found in Argentina. A total of 154 M. bovis isolates were selected in order to compare the discriminative power of spoligotyping and restriction fragment length polymorphism (RFLP) analysis with direct repeat (DR) and PGRS probes. By spoligotyping, 31 different types were found, while AluI-digested DR probe-associated RFLP analysis identified 42 types, and RFLP analysis with the PGRS probe also detected 42 types; these were partly independent of the DR types. By combining the results obtained by spoligotyping and by RFLP analysis with the DR and PGRS probes, 88 different types were obtained. Although the differentiation of M. bovis by spoligotyping was less discriminatory than differentiation by RFLP analysis with the DR and PGRS probes, spoligotyping is easier to perform and its results are easier to interpret. Therefore, for the purpose of typing of M. bovis isolates, spoligotyping could be performed first and the isolates could be grouped into clusters and then analyzed by RFLP analysis with the DR and PGRS probes.

Mycobacterium bovis: polymerase chain reaction identification in bovine lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism.

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70% of the samples. PCR confirmed the identification of 23 samples (100%) that grew in culture, 9 samples (60%) that failed to grow in culture, plus 6 (37.5%) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes.

Estudos de prevalência da brucelose bovina no âmbito do Programa Nacional de Controle e Erradicação de Brucelose e Tuberculose: Introdução / Prevalence studies on bovine brucelosis according to Brazilian National Program for the Control and Eradication of Bovine Brucellosis and Tuberculosis: Introduction

As estratégias de combate à brucelose bovina são bastante conhecidas e, até o momento, os resultados são divergentes. No Brasil, o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) implementou, em 2001, o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose. Conhecer a situação epidemiológica da brucelose no início de um programa de controle permite: 1 - escolher as melhores estratégias de controle em função da frequência e padrão de distribuição da doença na população e 2 - acompanhar o programa com a finalidade de promover correções e evitar o desperdício de tempo e de recursos. Em razão disso, é necessário realizar estudos para dar suporte à escolha das melhores estratégias para os vários estados e regiões brasileiras e criar um mecanismo racional de verificação da efetividade das ações implementadas. Para tanto, o MAPA estabeleceu um Termo de Cooperação Técnica com a Faculdade de Medicina Veterinária da Universidade de São Paulo e envolveu também a Faculdade de Agronomia e Veterinária da Universidade de Brasília. Até o momento, foram concluídos os estudos de 15 unidades federativas, cujos resultados são apresentados nos artigos subsequentes. Além disso, há um 16º artigo que explora o impacto da vacinação de bezerras com a B19 na redução da prevalência da brucelose.

Although strategies for controlling and eradicating of bovine brucellosis are well known, the achievements vary widely. In 2001, the Brazilian Ministry of Agriculture, Livestock, and Food Supply (MAPA) started a new National Program for the Control and Eradication of Brucellosis and Tuberculosis (NPCEBT). In the beginning of a brucellosis control program, it is of utmost importance to gain insights into the epidemiological status of the disease in order to: (1) determine the sanitary measures according to the frequency and distribution patterns of the disease in the population; (2) monitor the development of the program and decide when to make adjustments, avoiding the waist of time and resources. As the epidemiological status of bovine brucellosis is not adequately known in Brazil, there was a case for undertaking cross-sectional studies targeted at identifying the best strategies for each state and region, as well as providing a basis for future appraisal of the NPCEBT. For this purpose, MAPA established a collaborative project with FMVZ-Universidade de São Paulo, which also included FAV-Universidade de Brasília. So far, 15 states completed the survey, whose results are described in the following papers. In addition, it is presented a paper that deals with modeling the impact of vaccination of heifers with strain 19 upon the reduction of brucellosis prevalence.

Estudos de prevalência da brucelose bovina no âmbito do Programa Nacional de Controle e Erradicação de Brucelose e Tuberculose: Introdução / Prevalence studies on bovine brucelosis according to Brazilian National Program for the Control and Eradication of Bovine Brucellosis and Tuberculosis: Introduction

As estratégias de combate à brucelose bovina são bastante conhecidas e, até o momento, os resultados são divergentes. No Brasil, o Ministério da Agricultura, Pecuária e Abastecimento (MAPA) implementou, em 2001, o Programa Nacional de Controle e Erradicação da Brucelose e Tuberculose. Conhecer a situação epidemiológica da brucelose no início de um programa de controle permite: 1 - escolher as melhores estratégias de controle em função da frequência e padrão de distribuição da doença na população e 2 - acompanhar o programa com a finalidade de promover correções e evitar o desperdício de tempo e de recursos. Em razão disso, é necessário realizar estudos para dar suporte à escolha das melhores estratégias para os vários estados e regiões brasileiras e criar um mecanismo racional de verificação da efetividade das ações implementadas. Para tanto, o MAPA estabeleceu um Termo de Cooperação Técnica com a Faculdade de Medicina Veterinária da Universidade de São Paulo e envolveu também a Faculdade de Agronomia e Veterinária da Universidade de Brasília. Até o momento, foram concluídos os estudos de 15 unidades federativas, cujos resultados são apresentados nos artigos subsequentes. Além disso, há um 16º artigo que explora o impacto da vacinação de bezerras com a B19 na redução da prevalência da brucelose.

Although strategies for controlling and eradicating of bovine brucellosis are well known, the achievements vary widely. In 2001, the Brazilian Ministry of Agriculture, Livestock, and Food Supply (MAPA) started a new National Program for the Control and Eradication of Brucellosis and Tuberculosis (NPCEBT). In the beginning of a brucellosis control program, it is of utmost importance to gain insights into the epidemiological status of the disease in order to: (1) determine the sanitary measures according to the frequency and distribution patterns of the disease in the population; (2) monitor the development of the program and decide when to make adjustments, avoiding the waist of time and resources. As the epidemiological status of bovine brucellosis is not adequately known in Brazil, there was a case for undertaking cross-sectional studies targeted at identifying the best strategies for each state and region, as well as providing a basis for future appraisal of the NPCEBT. For this purpose, MAPA established a collaborative project with FMVZ-Universidade de São Paulo, which also included FAV-Universidade de Brasília. So far, 15 states completed the survey, whose results are described in the following papers. In addition, it is presented a paper that deals with modeling the impact of vaccination of heifers with strain 19 upon the reduction of brucellosis prevalence.

Dermatite micobacteriana atípica em gato: relato de caso / Atypical mycobacterial dermatitis in cat: a case report

Relata-se, pela primeira vez no Brasil, um caso de dermatite decorrente de infecção por micobactéria atípica do complexo Mycobacterium fortuitum-peregrinum, em espécie felina, sem raça definida, fêmea, com cinco anos de idade. Há oito meses, evoluía com lesões maculares equimóticas, nodulares, erosadas, ulceradas, acompanhadas de fístulas exsudativas, com intenso prurido e algia. Evidenciou-se a presença de micobactéria do complexo Mycobacterium fortuitum-peregrinum (grupo IV de Runyon ) identificada após evidenciação histopatológica, cultivo bacteriano e por testes bioquímicos. Após dois meses de terapia sistêmica com enrofloxacina (5mg/kg/Bid/VO) e tópica (triclosan e rifamicina) houve involução das lesões, com efeitos colaterais discretos.

A case of dermatitis caused by atypical mycobateria of Mycobacterium fortuitum peregrinum complex was observed for the first time in Brazil. A five-year-old female mixed breed cat had a dermatitis process which had started eight months before. Lesions were characterized by macules, nodules, erosions, ulcers and exsudative fistulas, with intensive pruritus and pain. It had failed to respond to clinical treatment and surgical excision. The diagnosis was based on history, physical and dermatologic examination and complementary tests (cytologic, bacterioscopic, bacterial culture, histopathological, hemathological, ultrasonographic, radiographic and electrocardiographic). The presence of mycobacteria of Mycobacterium fortuitum-peregrinum complex (group IV of Runyon) was observed and identified after histopathological evidence, bacterial culture and biochemical tests. After two months of systemic therapy with enrofloxacin (5mg/kg, orally/twice a day) and topic treatment with triclosan and rifamicin, rapid improvement of the clinical appearance of the lesions were observed with low incidence of side-effects.

Mycobacterium bovis: polymerase chain reaction identification in bovine Lymphonode biopsies and genotyping in isolates from Southeast Brazil by spolygotyping and restriction fragment length polymorphism

Diagnosis of the Mycobacterium tuberculosis complex by direct PCR of mediastinal lymphnode DNA and microbiological tests were compared in cattle suspicious of bearing tuberculous-like lesions detected during slaughter. The PCR procedure applied on DNA samples (n=54) obtained by adding alpha -casein into the thiocyanate extraction mix was positive in 70 percent of the samples. PCR confirmed the identification of 23 samples (100 percent) that grew in culture, 9 samples (60 percent) that failed to grow in culture, plus 6 (37.5 percent) samples that resulted in growth of bacterial contaminants. Genotyping by IS6110-RFLP and DR-spoligotyping analysis of seven samples revealed the presence of several polimorphisms. Seven of the isolates contained multiple copies of IS6110, thus defining the existence of five singular genotypes