Mycobacterium avium subspecies paratuberculosis and bovine leukemia virus seroprevalence and associated risk factors in dairy herds in Minas Gerais state, Brazil / [Soroprevalência do Mycobacterium avium subespécie paratuberculosis e vírus da leucemia bovina e fatores de risco associados em rebanhos leiteiros de Minas Gerais, Brasil]

Mycobacterium avium subesp. paratuberculosis (MAP) e o vírus da leucemia bovina (BLV) são agentes que causam grandes perdas econômicas nos rebanhos. O objetivo deste estudo foi avaliar a situação epidemiológica da paratuberculose bovina (PTB) e leucose enzoótica bovina (EBL) em rebanhos leiteiros de Lagoa Formosa, Minas Gerais, Brasil. Foram coletadas 236 amostras de sangue de vacas, as quais foram submetidas aos testes ELISA e imunodifusão em gel de ágar para detecção de anticorpos contra MAP e BLV. A soroprevalência de anticorpos contra MAP e BVL foi de 20% para os rebanhos e 6% para os animais e de 85% para os rebanhos e 50,42% para os animais, respectivamente. A presença dessas enfermidades deve servir como um alerta para os produtores e veterinários, para que concentrem maior atenção na implementação de medidas higiênico-sanitárias, incorporando elementos de vigilância com base nos riscos identificados no estudo.(AU)

Occurrence of Mycobacterium avium subsp. paratuberculosis in coalho cheese in the State of Pernambuco, Brazil / Ocorrência de Mycobaterium avium subsp. paratuberculosis em queijo coalho do Estado de Pernambuco, Brasil

Paratuberculosis is a chronic and incurable disease that affects ruminants and other domestic animals. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) that may also be involved in some human diseases such as Crohn's disease, type 1 diabetes, sarcoidosis, multiple sclerosis, and Hashimoto's thyroiditis. The objective of this study was to investigate the occurrence of MAP DNA in samples of artisanal coalho cheese purchased in the State of Pernambuco. Forty samples of coalho cheese submitted to the Real Time Polymerase Chain Reaction (qPCR) technique were analyzed for the detection of the MAP region IS900. 11 (27.5%) were positive with a mean of 195.9 MAP colony forming unit (CFU) per gram of each sample, with a minimum of 30.3 CFU/g and a maximum of 324.2 CFU/g. Thus, this type of cheese that is one of the most consumed in this region of Brazil constitutes a source of human exposure to MAP. Further research in this area should be performed to evaluate the viability of the bacteria in this cheese type.(AU)

Paratuberculose é uma enfermidade crônica e incurável que acomete ruminantes e outras espécies de animais domésticos. É causada pelo Mycobacterium avium subsp. paratuberculosis (MAP) e ainda há a suspeita do seu envolvimento em enfermidades nos humanos como a doença de Crohn, diabetes tipo 1, sarcoidose, esclerose múltipla e tireoidite de Hashimoto. Objetivou-se com esta pesquisa investigar a ocorrência do DNA de MAP em amostras de queijo coalho artesanal adquiridas em estabelecimentos comerciais do Estado de Pernambuco. 40 amostras de queijo coalho artesanal foram submetidas a técnica de Reação em Cadeia da Polimerase em Tempo Real (qPCR) para detecção da região IS900 do MAP. 11 (27,5%) foram positivas com uma média de 195,9 unidades formadoras de colônia (UFC) de MAP por grama de queijo, com detecção mínima de 30,3UFC/g e máxima de 324,2UFC/g. Sendo assim, esse tipo de queijo que é um dos mais consumidos nesta região do Brasil constitui uma fonte de exposição humana ao MAP. Mais pesquisas nessa área devem ser realizadas para avaliar a viabilidade dessa bactéria no queijo coalho.(AU)

First molecular typing of Mycobacterium avium subspecies paratuberculosis identified in animal and human drinking water from dairy goat farms in Brazil

Abstract Mycobacterium avium subspecies paratuberculosis, the etiologic agent of Johne's disease or paratuberculosis, was identified by culture and/or polymerase chain reaction (PCR) in 50% and 30% of water samples for animal and human consumption, respectively, from ten dairy goat farms in Brazil. IS1311 restriction fragment length polymorphism analysis identified the isolates as cattle type C.

Detection of Mycobacterium avium subsp. paratuberculosis in bovine milk from the state of Pernambuco, Brazil

Abstract The aim of this study was to detect the IS900 region of Mycobacterium avium subsp. paratuberculosis (MAP) in bovine milk samples using real-time polymerase chain reaction (qPCR) and conventional PCR, and to study the agreement between these tests. A total of 121 bovine milk samples were collected from herds considered positive for MAP, from the State of Pernambuco, Brazil. MAP DNA was detected in 20 samples (16.5%) using conventional PCR and in 34 samples (28.1%) using qPCR. MAP DNA was detected in all of the 6 animal farms studied. Moderate agreement was found between qPCR and conventional PCR results, where the sensitivity and specificity of conventional PCR in relation to qPCR were 50% and 96.6%, respectively. Thus, the IS900 region of MAP was found in bovine milk samples from the State of Pernambuco. To the best of our knowledge, this is the first report of MAP DNA found in bovine milk in Northeast Brazil. We also demonstrated the qPCR technique is more sensitive than conventional PCR with respect to detection of MAP in milk samples.

Mycobacterium paratuberculosis detection in cow's milk in Argentina by immunomagnetic separation-PCR

Abstract The aim of this study was to standardize a diagnosis procedure to detect Mycobacterium avium subsp. paratuberculosis (Map) DNA in raw cow milk samples under field conditions. A procedure that combines both immunomagnetic separation and IS900 -PCR detection (IMS-IS1 PCR) was employed on milk samples from 265 lactating Holstein cows from Map infected and uninfected herds in Argentina. IMS-IS1 PCR results were analyzed and compared with those obtained from milk and fecal culture and serum ELISA. The extent of agreement between both tests was determined by the Kappa test. IMS-IS1 PCR showed a detection limit of 101 CFU of Map/mL of milk, when 50:50 mix of monoclonal and polyclonal antibodies were used to coat magnetic beads. All of the 118 samples from the Map uninfected herds were negative for the set of the tests. In Map infected herds, 80 out of 147 cows tested positive by milk IMS-IS1 PCR (55%), of which 2 (1.4%) were also positive by milk culture, 15 (10%) by fecal culture, and 20 (14%) by serum ELISA. Kappa statistics (95% CI) showed a slight agreement between the different tests (<0.20), and the proportions of agreement were ≤0.55. The IMS-IS1 PCR method detected Map in milk of the cows that were not positive in other techniques. This is the first report dealing with the application of IMS-IS1 PCR in the detection of Map in raw milk samples under field conditions in Argentina.

Presence of Mycobacterium avium subsp. paratuberculosis (MAP) in Brazilian patients with inflammatory bowel diseases and in controls / Presença de Mycobacterium avium subsp. paratuberculosis (MAP) em pacientes brasileiros com doença inflamatória intestinal e em controles

CONTEXT AND OBJECTIVE: Mycobacterium avium subsp. paratuberculosis (MAP) has attracted the interest of researchers because of similarities between paratuberculosis and Crohn's disease (CD). The aim of this study was to evaluate the frequency of MAP through cultures, histology and polymerase chain reaction (PCR) on intestinal biopsies from Brazilian CD patients. Quantitative real time PCR (qRT-PCR) was performed on positive samples. DESIGN AND SETTING: Analytical cross-sectional study with control group at two federal universities. METHODS: Fresh samples were collected from 25 patients; five with CD, eight with ulcerative colitis (UC) and 12 controls with non-inflammatory bowel disease (nIBD). Formalin-fixed paraffin-embedded (FFPE) samples from 143 patients were also collected: 44 CD, 49 UC and 56 nIBD. RESULTS: None of the fresh samples was positive for MAP. Five FFPE samples (one CD, two UC and two nIBD) and three fresh samples (one in each group) were positive through IS900-PCR. qRT-PCR was performed on these eight samples. Among the FFPE samples, there were 192.12 copies/μl in the CD group, 72.28 copies/μl in UC and 81.43 copies/μl in nIBD. Among the fresh samples, there were 432.99 copies/μl, 167.92 copies/μl and 249.73 copies/μl in the CD, UC and nIBD groups, respectively. The highest bacterial load was in the CD group. CONCLUSION: This study does not provide evidence for a role of MAP in the etiology of CD, although MAP DNA was detected in all three patient groups. This is the first report of MAP presence in human intestinal biopsies in Brazil.

CONTEXTO E OBJETIVO: Mycobaterium avium subsp. paratuberculosis (MAP) tem atraído o interesse de pesquisadores devido às semelhanças entre a paratuberculose e a doença de Crohn (CD). Este estudo objetivou avaliar a frequência de MAP por meio de cultura, histologia e reação da polimerase em cadeia (PCR), em biópsias intestinais de pacientes brasileiros com CD. PCR quantitativa em tempo real (qRT-PCR) foi realizada nas amostras positivas. TIPO DE ESTUDO E LOCAL: Estudo transversal analítico com grupo controle realizado em duas universidades federais. MÉTODOS: Amostras frescas foram coletadas de 25 pacientes; cinco com CD, oito com colite ulcerativa (UC) e 12 controles sem doença inflamatória intestinal (nIBD). Também foram coletadas 149 amostras fixadas em parafina (FFPE): 44 CD, 49 UC e 56 nIBD. RESULTADOS: Nenhuma das amostras frescas foi positiva para MAP. Cinco amostras FFPE (uma CD, duas UC e duas nIBD) e três amostras frescas (uma de cada grupo) foram positivas por IS900-PCR. qRT-PCR foi realizada nessas oito amostras. Nas amostras FFPE, havia 192,12 cópias/μl no grupo CD, 72,28 cópias/μl no UC e 81,43 cópias/μl no nIBD. Nas amostras frescas, havia 432,99 cópias/μl, 167,92 cópias/μl e 249,73 cópias/μl nos grupos CD, UC e nIBD, respectivamente. A maior carga bacteriana foi encontrada no grupo CD. CONCLUSÃO: Este estudo não fornece evidências do papel de MAP na etiologia da CD, embora DNA de MAP tenha sido detectado em pacientes dos três grupos. Este é o primeiro relato da presença de MAP em biópsias intestinais humanas no Brasil.

LAMP technology: Rapid identification of Brucella and Mycobacterium avium subsp. paratuberculosis

In this study, we developed new sets of primers to detect Brucella spp. and M. avium subsp. paratuberculosis (MAP) through isothermal amplification. We selected a previously well-characterized target gene, bscp31, specific for Brucella spp. and IS900 for MAP. The limits of detection using the loop-mediated isothermal amplification (LAMP) protocols described herein were similar to those of conventional PCR targeting the same sequences. Hydroxynaphtol blue and SYBR GreenTM allowed direct naked-eye detection with identical sensitivity as agarose gel electrophoresis. We included the LAMP-based protocol in a rapid identification scheme of the respective pathogens, and all tested isolates were correctly identified within 2 to 3 h. In addition, both protocols were suitable for specifically identifying the respective pathogens; in the case of Brucella, it also allowed the identification of all the biovars tested. We conclude that LAMP is a suitable rapid molecular typing tool that could help to shorten the time required to identify insidious bacteria in low-complexity laboratories, mainly in developing countries.

Molecular typing of Argentinian Mycobacterium avium subsp. paratuberculosis isolates by multiple-locus variable number-tandem repeat analysis

Multiple-locus variable number-tandem repeat analysis (MLVA) of Mycobacterium avium subspecies paratuberculosis (MAP) isolates may contribute to the knowledge of strain diversity in Argentina. Although the diversity of MAP has been previously investigated in Argentina using IS900-RFLP, a small number of isolates were employed, and a low discriminative power was reached. The aim of the present study was to test the genetic diversity among MAP isolates using an MLVA approach based on 8 repetitive loci. We studied 97 isolates from cattle, goat and sheep and could describe 7 different patterns: INMV1, INMV2, INMV11, INMV13, INMV16, INMV33 and one incomplete pattern. INMV1 and INMV2 were the most frequent patterns, grouping 76.3% of the isolates. We were also able to demonstrate the coexistence of genotypes in herds and co-infection at the organism level. This study shows that all the patterns described are common to those described in Europe, suggesting an epidemiological link between the continents.

Application of IS1311 locus 2 PCR-REA assay for the specific detection of ‘Bison type’ Mycobacterium avium subspecies paratuberculosis isolates of Indian origin.

Background & objectives: Of the three major genotypes of Mycobacterium avium subspecies paratuberculosis (MAP), ‘Bison type’ is most prevalent genotype in the domestic livestock species of the country, and has also been recovered from patients suffering from Crohn’s disease. Recently, a new assay based on IS1311 locus 2 PCR- restriction endonuclease analysis (REA) was designed to distinguish between ‘Indian Bison type’ and non-Indian genotypes. The present study investigated discriminatory potential of this new assay while screening of a panel of MAP isolates of diverse genotypes and from different geographical regions. Methods: A total of 53 mycobacterial isolates (41 MAP and 12 mycobacterium other than MAP), three MAP genomic DNA and 36 MAP positive faecal DNA samples from different livestock species (cattle, buffaloes, goat, sheep and bison) and geographical regions (India, Canada, USA, Spain and Portugal) were included in the study. The extracted DNA samples (n=92) were analyzed for the presence of MAP specific sequences (IS900, ISMav 2 and HspX) using PCR. DNA samples were further subjected to genotype differentiation using IS1311 PCR-REA and IS1311 L2 PCR-REA methods. Results: All the DNA samples (except DNA from non-MAP mycobacterial isolates) were positive for all the three MAP specific sequences based PCRs. IS1311 PCR-REA showed that MAP DNA samples of Indian origin belonged to ‘Bison type’. Whereas, of the total 19 non-Indian MAP DNA samples, 2, 15 and 2 were genotyped as ‘Bison type’, ‘Cattle type’ and ‘Sheep type’, respectively. IS1311 L2 PCR-REA method showed different restriction profiles of ‘Bison type’ genotype as compared to non-Indian DNA samples. Interpretation & conclusions: IS1311 L2 PCR-REA method successfully discriminated ‘Indian Bison type’ from other non-Indian genotypes and showed potential to be future epidemiological tool and for genotyping of MAP isolates.

Diagnóstico imuno-histopatológico da paratuberculose subclínica em bovinos no estado do Rio de Janeiro / Imuno-histopatologic diagnosis of subclinic bovine paratuberculosis in the state of Rio de Janeiro

O diagnóstico precoce e específico da paratuberculose ainda é um desafio. Isto pode estar associado à baixa sensibilidade dos testes laboratoriais e ou à variação da resposta imunológica frente à infecção por Mycobacterium avium subsp. paratuberculosis. Mundialmente, é uma enfermidade que causa importantes prejuízos econômicos, em especial, à bovinocultura leiteira, devido ao caráter crônico da infecção. No Brasil, a paratuberculose já foi descrita em diversas espécies de ruminantes domésticos e em vários estados, o que demonstra que a enfermidade está presente em território nacional e há a necessidade de elaboração de técnicas de diagnóstico para a confirmação da infecção. O objetivo deste trabalho foi caracterizar os achados anátomo-histopatológicos e imuno-histoquímicos em intestino e linfonodos mesentéricos de bovinos assintomáticos, provenientes de rebanhos positivos para paratuberculose localizados no estado do Rio de Janeiro, Brasil. O estudo macroscópico revelou alterações inespecíficas tais como áreas avermelhadas na mucosa do intestino, aumento do volume das placas de Peyer e dos linfonodos mesentéricos, além disso, observou-se que vasos linfáticos mesentéricos estavam dilatados e esbranquiçados. Do total de 52 vacas leiteiras avaliadas, a histopatologia revelou infiltração granulomatosa, por vezes com formação de células gigantes multinucleadas, em mucosa e ou submucosa de jejuno, íleo e em linfonodos mesentéricos, principalmente na região cortical, em 32 animais. Estes bovinos foram submetidos à coloração de Ziehl-Neelsen cujo teste não demonstrou reação positiva, no entanto, quando analisados pelo teste imunohistoquímico para Mycobacterium spp. observou-se imunorreação em 6 animais. Desta forma, a histopatologia e imunohistoquímica pode ser uma importante ferramenta para diagnóstico da paratuberculose subclínica.

The early and specific diagnosis of paratuberculosis remains a challenge due to the low sensitivity of the currently available laboratory tests and also because of variations in the immune response towards infection with Mycobacterium avium subsp. paratuberculosis. Globally this disease causes significant economic losses, primarily in dairy cattle, owing to the chronic nature of the infection. Paratuberculosis has been described in a number of Brazilian states and from a diversity of domestic ruminant species clearly demonstrating that the disease is present in the country and highlighting the requirement for the development of diagnostic techniques for confirmation of infection and for epidemiological analyses. The aim of this study was to characterize the anatomo-histopathological and immunohistochemical findings in the bowel and mesenteric lymph nodes of assymptomatic cattle, derived from paratuberculosis positive herds located in state of Rio de Janeiro, Brazil. Macroscopic examination during necropsy revealed nonspecific changes including reddening of the gut mucosa, increased volumes for the Peyer's patches and mesenteric lymph nodes and in some case dilation and whitening of the mesenteric lymphatic vessel. Histopathology revealed granulomatous infiltration, occasionally with the formation of giant cells in the jejunal and ileal mucosa or sub-mucosa, and/or in the cortical region of the mesenteric lymph nodes, in 32 of the 52 cattle examined. Tissue sections from these animals were subjected to Ziehl-Neelsen staining, but the presence of acid-fast bacilli was not observed. Subsequent analysis, employing genus specific immunohistochemisty for Mycobacterium, revealed areas of immunoreactivity in sections prepared from a total of six animals. The results of this investigation highlighted the value of histopathology and particularly immunohistochemistry as tools for the diagnosis of subclinical paratuberculosis.

First identification of Mycobacterium avium paratuberculosis sheep strain in Argentina

We here identified for the first time the presence of Mycobacterium avium paratuberculosis (MAP) sheep (S) strain in Argentina. IS900 polymerase chain reaction (PCR) was positive. The S strain was compared with MAP cattle (C) strains by using IS1311 PCR-restriction endonuclease analysis (PCR-REA), multiplex PCR and restriction fragment length polymorphism (RFLP) analysis.

Avaliação sorológica e de fatores de risco para a infecção por Mycobacterium avium subsp. paratuberculosis em rebanhos leiteiros da Microrregião de Garanhuns, Pernambuco / Serological evaluation and risk factors for Mycobacterium avium subsp. paratuberculosis infection in dairy herds of Microregion Garanhuns, Pernambuco

Objetivou-se com esse trabalho realizar um inquérito epidemiológico da infecção por Mycobacterium avium subsp. paratuberculosis (MAP) em bovinos leiteiros da microrregião de Garanhuns, Pernambuco, Brasil. Para este estudo foram coletadas amostras sanguíneas de 408 animais, provenientes de 19 rebanhos localizados em 15 municípios. O exame sorológico foi realizado por Ensaio Imunoenzimático (ELISA) indireto para detecção de anticorpos frente ao MAP. Em todas as propriedades, foi aplicado um questionário investigativo para análise dos fatores de risco, e as coordenadas geográficas coletadas por um aparelho de Global Position System (GPS) para realização da distribuição espacial. A prevalência da infecção por MAP foi de 2,7% (11/408; I.C. 1,4-4,9). O número de focos foi 47,4% (9/19). Na análise de regressão logística foi identificado como fator de risco a taxa anual de nascimentos superior a 51 bezerros/ano (OR 3,8; I.C. 1,1-13,1). Desta forma, conclui-se que a infecção por MAP encontra-se presente nos rebanhos bovinos leiteiros da microrregião estudada e que medidas de controle baseadas nos fatores de risco identificados devem ser implementadas com o objetivo de reduzir o número de focos da infecção.

The present study aimed to conduct an epidemiological investigation of Mycobacterium avium subsp. paratuberculosis (MAP) infection in dairy cattle of the Garanhuns microregion, in Pernambuco, Brazil. Blood samples were collected from 408 animals from 19 herds located in 15 cities. Serological tests were performed by indirect immunoenzymatic assay (ELISA) for antibodies against MAP. In all farms, a questionnaire to investigate risk factors was used, and Global Position System (GPS) receivers were used to collect geographic coordinates to show the spatial distribution of the animals. The prevalence of MAP infected cattle was 2.7% (11/408; I.C. 1.4-4.9). The rate of infection was 47.4% (9/19). An annual birth rate over 51 calves/year (OR 3.8; I.C. 1.1-13.1) was identified as a risk factor in logistic regression analysis. Thus, it is concluded that MAP infection is present in dairy cattle of the microregion studied here, and control measures based on the identified risk factors should be implemented in order to reduce the sources of infection.

Paratuberculose em ruminantes no Brasil / Paratuberculosis in ruminants in Brasil: a review

A paratuberculose ou doença de Johne é uma enterite granulomatosa causada por Mycobacterium avium subsp. paratuberculosis (Map) e comumente afeta ruminantes domésticos, no entanto, pode infectar várias espécies de mamíferos. Está presente nos cinco continentes e é considerada endêmica em algumas regiões pela Organização Internacional de Epizootias (OIE). Pertence à lista de enfermidades notificáveis, que compreende as doenças transmissíveis de importância sócio-econômica e/ou em saúde-pública, cujo controle é necessário para o comércio internacional de animais e alimentos de origem animal. A importância da doença de Johne não se restringe somente aos prejuízos econômicos causados à indústria animal, mas também na possível participação do Map na íleocolite granulomatosa que afeta seres humanos, conhecida como doença de Crohn. No Brasil, a paratuberculose já foi descrita em diversas espécies de ruminantes e em vários estados. Embora os relatos naturais da enfermidade sejam pontuais, acredita-se na possibilidade da transmissão interespecífica e na disseminação do agente através da compra e venda de animais infectados. O objetivo deste artigo foi reunir as informações disponíveis referentes aos aspectos epidemiológicos, clínico-patológicos e laboratoriais da paratuberculose em bovinos, bubalinos, caprinos e ovinos no Brasil, e salientar a necessidade de implementação de medidas de controle sanitário da enfermidade no país, o que possibilitaria a melhoria da qualidade e valorização dos produtos de origem animal no mercado internacional.

Paratuberculosis also known as Johne's disease, is a granulomatous enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP), an acid-fast bacillus that preferentially resides within host intestinal macrophages. The condition is most commonly seen in domestic ruminants, however MAP can also infect other mammalian species. Paratuberculosis shows a global distribution and is considered endemic in some regions. The World Organization for Animal Health (OIE, Office International des Epizooties), have classified paratuberculosis as a notificable disease; considered to be of socio-economic and/or public-health importance, the control of which is necessary for the international trade of animal and animal products. The importance of paratuberculosis is related primarily to economic losses in the animal industry and also because of a potential role for this bacterium in the pathogenesis of Crohn´s disease, a debilitating condition affecting the digestive tract of humans. In Brazil, paratuberculosis has been reported in a variety of ruminant species and shows a broad geographic distribution. The reported incidence of natural cases in Brazil has been limited, but it is believed that interespecific transmission of MAP and dissemination of the agent is driven by the commercialization of infected animals. The main objective of this paper was to collate the published epidemiological, clinic-pathological and diagnostic information in relation to paratuberculosis in cattle, buffaloes, goats and sheep in Brazil. Moreover, it served as a platform to emphasize the requirement to implement sanitary policies for control of MAP in the county, which may serve to improve the quality and value of animal products on international markets.

First isolation of Mycobacterium avium subsp paratuberculosis from commercial pasteurized milk in Argentina

Mycobacterium avium subsp paratuberculosis was isolated from two out of seventy samples (2.86 %) of pasteurized and ultra-pasteurized milk. The isolates were positives to IS900 PCR and showed a C17 RFLP pattern, the most prevalent in Argentina. The present study is the first report of Mycobacterium avium subsp paratuberculosis culture from pasteurized milk in Argentina.

Isolamento de micobactérias provenientes de amostras clínicas da região de Rio Claro: análise da frequência / Mycobacterial isolates from patients clinical samples from Rio Claro region: frequency analysis

Os agentes etiológicos da tuberculose pertencem ao Complexo Mycobacterium tuberculosis (MT), porém outras espécies do gênero e de importância médica, denominadas micobactérias não causadoras de tuberculose (MNT), são frequentemente isoladas. A identificação correta e rápida das MNT é obrigatória para o diagnóstico, auxiliando o clínico na conduta terapêutica. A frequência da MT e MNT foi avaliada no período de 2003 a 2009, analisando-se 6.460 amostras clínicas, sendo 5.696 (88,2 por cento) culturas negativas para micobactéria, 715 (11,1 por cento) culturas positivas e 49 (0,7 por cento) não avaliadas. Os isolados mais frequentes foram MT 78,6 por cento (562). Dentre as MNT, a espécie mais isolada foi M. gordona e em 3,09 por cento(22) das amostras, seguida por M. fortuitum em 2,39 por cento (17), Complexo M. avium (MAC) em 2,25 por cento (16),M. kansasii em 1,97 por cento (14) e M. abscessus em 1,83 por cento (13). Quando relacionado ao número de pacientes,o complexo MAC foi o mais isolado (19,4 por cento). Foi observado um declínio na frequência de isolamento de MT no período estudado, enquanto a frequência de MNT teve um aumento de 22,8 por cento. A alta frequênciade MT nas amostras é uma constante preocupação para a saúde pública, mas o aumento de doentes com MNT salienta a importância da notificação e do monitoramento epidemiológico desses pacientes.

Strain diversity within Mycobacterium avium subspecies paratuberculosis — A review.

Mycobacterium avium subspecies paratuberculosis (MAP), is the etiological agent of Johne’s disease (or paratuberculosis) in animals and has also been linked with Crohn’s disease of human beings. Extreme fastidious nature of the organism (MAP) has hampered studies on diversity within the organism. Studies based on phenotypic properties like growth rate, pigmentation, lipid profile etc., are unable to provide complete information on diversity of MAP organism in nature. However, with the advent of molecular assays (IS900 RFLP, PFGE, IS1311 PCR-REA, SSR typing, VNTR typing etc.) in last 2 decades, progress has been made to differentiate MAP strains. MAP isolates have been classified into various types and subtypes using these molecular tools. Optimization of these typing assays has led to generation of new information about MAP strains, subtypes, their comparative genomics, relative evolution, comparative virulence etc. Knowledge of strain diversity is important for better understanding of molecular and sero-epidemiology, infection and patho-biology, vaccine development and planning control strategies. The present review provides available information on MAP strains, host adaptations, their virulence, comparative genomics, relative genetic evolution and differentiation.

Comparação de duas técnicas de isolamento do Mycobacterium avium subsp. paratuberculosis em amostras de fezes de ovinos com suspeita clínica de paratuberculose / Comparison of two techniques of isolation of Mycobacterium avium subsp. paratuberculosis in faecal samples of ovine with clinical suspicion of paratuberculosis

A paratuberculose é uma enterite crônica granulomatosa causada por Mycobacterium avium subsp. paratuberculosis que afeta principalmente os ruminantes. A cultura de bactérias a partir de amostras de fezes e tecidos constitui um dos métodos mais eficazes de diagnóstico, sendo ainda o único método disponível para obtenção de isolamentos e estirpes de micobactérias. Contudo, este método apresenta baixa sensibilidade e requer meses de incubação antes do crescimento de colônias. Neste estudo, utilizou-se a cultura fecal como método de diagnóstico em ovinos de diferentes raças portuguesas, com sinais compatíveis com a doença. Fez-se ainda a comparação entre os meios de cultura Lõwenstein Jensen® com micobactina® J e o de Middlebrook® 7H11 com OADC®, utilizados no isolamento da bactéria. As percentagens de isolamento em cada um os meios foram de 2,0 por cento (6/300) para Lõwenstein Jensen® com micobactina J e 1,0 por cento (3/300) para Middlebrook® 7H11/OADC. As três amostras positivas no meio de Middlebrook® 7H11/OADC também foram positivas no meio de Lõwenstein Jensen® com micobactina J e nenhuma foi somente positiva no meio de Middlebrook® 7H11/OADC. Os resultados deste estudo sugerem que o meio de Lõwenstein-Jensen® com micobactina® J é mais efetivo para a obtenção de estirpes ovinas em Portugal.

Paratuberculosis is a chronic enteric disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis. Culture of bacteria from faeces and tissues samples constitutes one of the most effective methods of confirming the diagnosis of para-tuberculosis and the only method available to obtain strains of mycobacteria. However, this method is less sensitive and requires months of incubation before colony growth occurs. In this study, culture method was used on sheep faeces to diagnose paratuber-culosis in animals with compatible signs of the disease. A comparison of two culture media used to isolation was also investigated. Culture was positive in 2.0 percent of faecal samples. Isolation was obtained using Lõwenstein Jensen® with mycobactin® J, and the Middlebrook® 7H11 with OADC®. The Lõwenstein Jensen® with mycobactin® J was that provided highest amount of isolations. The percentages of isolation in each culture media were 2.0 percent (6/300) to Lõwenstein Jensen® with micobactina J, and 1.0 percent (3/300) to Middlebrook® 7H11/OADC. The three positive samples in Middlebrook® 7H11/OADC were also positive in Lõwenstein Jensen® with micobactina J. In the Middlebrook® 7H11/OADC alone there was no sample growth. The results of this study suggest that culture media of Lõwenstein-Jensen® with micobactina® J is more effective for the isolation of sheep strains in Portugal.

Coloração de Ziehl-Neelsen como método rápido de diagnóstico de paratuberculose ovina / Ziehl-Neelsen staining as a fast method in the diagnosis of ovine paratuberculosis

Estudou-se a presença de bacilos álcool-ácido resistentes compatíveis com Mycobacterium avium subsp. paratuberculosis em esfregaços de fezes e tecidos de ovinos. Vinte e seis esfregaços de fezes e 104 de tecidos, pertencentes a 26 animais diagnosticados como paratuberculosos, foram analisados pelo método de Ziehl-Neelsen. Dezesseis (61,5 por cento) esfregaços fecais apresentaram bacilos álcool-ácido resistentes compatíveis no exame microscópico. Vinte animais (76,9 por cento) foram diagnosticados pelo método nos esfregaços de tecidos. Vinte e um animais apresentaram esfregaços positivos nas fezes e nos tecidos, simultaneamente. A sensibilidade de Ziehl-Neelsen para os esfregaços fecais, esfregaços de tecidos e para a combinação de ambos foi de 61,5 por cento, 76,9 por cento e 80,8 por cento, respectivamente.

The presence of acid-fast bacilli compatible with Mycobacterium avium subsp. paratuberculosis in fecal and tissues smears was investigated using the Ziehl-Neelsen staining. A total of 26 fecal smears and 104 tissues smears collected from 26 sheep with confirmed paratuberculosis were analyzed. Sixteen (61.5 percent) fecal smears showed compatible with acid-fast bacilli on microscopic examination after staining. Twenty animals (76.9 percent) were diagnosed based on the positivity of tissues smears. The Ziehl-Neelsen sensitivities to faecal smears, tissues smears, and a combination of both were 61.5 percent, 76.9 percent, and 80.8 percent, respectively.

Respueta humoral a micobacterias en pacientes con enfermedad de Crohn / Humoral response to mycobacterium in patients with Crohn disease

El reciente aislamiento de Mycobacterium paratuberculosis de lesiones de pacientes con enfermedad de Crohn (EC) reavivó el interés en el posible origen micobacteriano de esta enfermedad. Sin embargo, las evidencias inmunológicas acumuladas en favor de esta hipótesis son poco sólidas. La respuestaespecífica en la EC podría estar enmascarada por el amplio cruzamiento antigénico que existe entre microbacterias. En este estudio se aplico un procedimiento propuesto para develar la presencia de anticuerpos anti-M. paratuberculosis en la enfermedad de Johne bovina: la absorción de los anticuerpos con reactividad cruzada mediante la preincubación de los sueros con M. phlei. Se cuantificó por enzimoinmunoensayo IgG anti-M. paratuberculosis en 90 muestras de suero de 17 pacientes con EC, 23 con colitis ulcerosa (CU) y 14 con otras patologías intestinales (OP). Muestras de 86 individuos sanos, con tuberculosis, microbacteriosis o micosis sirvieron como control. Se comparó los resultados con los obtenidos con antígenos de M. avium y M. tuberculosis. Se observó una discreta reactividad humoral a micobacterias en pacientes con EC, Cu y OP. Las alteraciones fueron de muy escasa magnitud y frecuencia en relación con las observadas en pacientes con tuberculosis, micobacteriosis y micosis. La absorción de los sueros no develó una presunta respuesta específica en la EC. Tampoco se detectaron elevaciones en los niveles de anticuerpos de pacientes analizados periodicamente. Estos datos no agregan evidencia sólida a la hipótesis del origen micobacteriano de la EC