Evidence of in utero infection by Mycobacterium avium subsp. paratuberculosis using Multiple-Locus Variable-number tandem-repeat Analysis: first report in Argentina / Evidência de infecção intra-uterina por Mycobacterium avium subsp. paratuberculosis usando Multiple-Locus Variable-number tandem-repeat Analysis: primeiro relato na Argentina

A pregnant heifer with an advanced clinical stage of paratuberculosis was reported in a herd in Argentina. Thus, the animal was euthanized and samples of organs of the cow and its fetus was taken and cultured for bacteriology in specific medium. Tissues were analyzed by histopathology (hematoxylin-eosin and Ziehl-Neelsen staining). Histopathological analysis of the cow's samples revealed the presence of lesions consistent with paratuberculosis, and Ziehl-Neelsen staining revealed the presence of acid-fast bacilli, whereas the fetal tissues showed absence of lesions but the presence of acid-fast bacilli by Ziehl-Neelsen staining. After growing in specific medium, colonies in tissues from both cow and fetus were positive for IS900-PCR, confirming the presence of Mycobacterium avium subsp. paratuberculosis (MAP). Finally, the isolates were typed by Multiple-Locus Variable-number tandem-repeat Analysis (MLVA), which confirmed the epidemiological link between them. This study is the first in Argentina to report the detection of MAP that shares an identical MLVA type in a pregnant cow and its fetus. The results of this study are consistent with previous reports and highlight the intra-uterine transmission of MAP as an important source of infection within herds.(AU)

Uma novilha prenha em estado clínico avançado de paratuberculose foi observada em um rebanho bovino na Argentina. O animal foi eutanasiado e foram colhidas amostras dos seus órgãos e dos órgãos feto as quais foram cultivadas para bacteriologia em meio específico. Os tecidos foram examinados por histopatologia (coloração de hematoxilina-eosina e Ziehl-Neelsen). Na histopatologia das amostras colhidas da novilha foram observadas lesões compatíveis com paratuberculose e a coloração de Ziehl-Neelsen revelou a presença de bacilos álcool-ácido resistentes, nos tecidos fetais não foram observadas lesões, porém a coloração de Ziehl-Neelsen revelou a presença de bacilos álcool-ácido resistentes. Após o crescimento em meio específico, as colônias foram positivas para o teste IS900-PCR nos tecidos de ambos, vaca e feto, confirmando a presença de Mycobacterium avium subsp. paratuberculosis. Por fim, os isolados foram tipados por Multiple-Locus Variable-number tandem-repeat Analysis, confirmando a relação epidemiológica entre eles. Este estudo relata a primeira detecção de Mycobacterium avium subsp. paratuberculosis na Argentina em que houve o compartilhamento de um tipo idêntico de MLVA em uma vaca prenhe e no seu feto. Os resultados deste estudo são consistentes com relatos anteriores e destacam a transmissão intra-uterina de Mycobacterium avium subsp. paratuberculosis como importante fonte de infecção nos rebanhos de bovinos.(AU)

Paratuberculosis: new histopathological findings in red deer (Cervus elaphus) and fallow deer (Dama dama) in Chile / Paratuberculosis: novos achados histopatológicos em Veado Vermelho (Cervus elaphus) e Gamo (Dama dama) no Chile

Paratuberculosis is a disease caused by Mycobacterium avium subsp. paratuberculosis (MAP) that affects domestic and wild ruminants. The most common gross lesions are emaciation and corrugation and thickening of the mucosa of the small intestine. Mesenteric lymph nodes might be enlarged. For the present study, 14 red deer and 9 fallow deer from game reserves or venison farms were analyzed. The lesions found correspond to those found by other authors in other geographic locations, except for some differences in histopathological examinations. Among these differences, stands out that intestinal lesions were concentrated mostly in the ileum and granulomas were shown to be more frequent in this section of the intestine than in the corresponding lymph node. Furthermore, in multibacillary lesions the inflammatory infiltrate in the lymph nodes was mainly composed of macrophages. These differences may be due to individual variations of the animals, the stage of disease or a different strain of the pathogen. This study allowed to obtain basic information about the disease and to describe patterns of lesions found in red deer and fallow deer with prediagnosis of clinical paratuberculosis which were not described in the literature before.(AU)

Paratuberculosis é uma doença causada por Mycobacterium avium subsp. paratuberculosis (MAP) que afecta ruminantes selvagens e domésticos. As lesões macroscópicas mais comuns são ondulação e espessamento da mucosa do intestino delgado. Os linfonodos mesentéricos podem aparecer com volume aumentado. Para este estudo, foram analisados 14 veados vermelhos e 9 veados gamo de reservas de caça e fazendas de carne. As lesões encontradas correspondem à encontrada por outros autores em outras localizações geográficas, com exceção de algumas diferenças no exame histopatológico. Entre essas diferenças, sobressai que as lesões intestinais se concentraram principalmente no íleo, os granulomas ocorreram com maior frequência nesta seção do intestino que no seu correspondente linfonodo. Além disso, nas lesões bacterianas, o infiltrado inflamatório linfonodos linfáticos era composta principalmente por macrófagos. Estas diferenças podem ser devidas a variações individuais dos animais, o estádio da doença ou de uma estirpe diferente do agente patogénico. Este estudo permitiu obter informação básica sobre a doença e descrever padrões de lesões encontradas em veados e em gamos com pré-diagnóstico, de paratuberculosis clínica nunca antes descritas na literatura.(AU)

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.

Diagnóstico de paratuberculose por biópsia retal em búfalos / Rectal biopsy for the diagnosis of paratuberculosis in buffaloes

Foram realizadas biópsias retais de 140 búfalos, machos e fêmeas, das raças Murrah e mestiços de Murrah com Mediterrâneo, com idade acima de três anos, em uma propriedade no município de São Mateus, Maranhão, Brasil. Adicionalmente foram realizadas necropsias de 11 búfalos, para realizar um estudo comparativo entre os achados das biópsias retais e de tecidos de íleo e linfonodo mesentérico. A propriedade apresentava histórico de animais com emagrecimento progressivo e diarreia não responsiva a antimicrobianos. Os búfalos apresentavam sinais clínicos caracterizados por diarreia, estado nutricional regular a ruim, desidratação e edema submandibular. Nas biópsias retais seis búfalos apresentaram lesões sugestivas da paratuberculose na Hematoxilina-Eosina (HE), sendo estas caracterizadas por inflamação granulomatosa multifocal moderada na lâmina própria com macrófagos epitelioides. Em quatro animais foram observadas adicionalmente células gigantes do tipo Langhans. Em 15 búfalos foi observado infiltrado linfocitário multifocal leve na lâmina própria. Pela coloração de Ziehl-Neelsen (ZN), 4,3% (6/140) apresentaram bacilos álcool-ácido resistentes (BAAR) e na PCR em tempo real (qPCR), 5,71% (7/140) tiveram amplificação do material genético. Foram necropsiados 11 búfalos, à necropsia foram observados aumento de linfonodos mesentéricos com áreas esbranquiçadas na superfície de corte; intestino delgado e grosso com dobras transversais evidentes, mucosa espessada e irregular, de aspecto reticulado, placas de Peyer evidentes e conteúdo líquido e marrom. Ainda se viam áreas espessadas em torno da válvula ileocecal e vasos linfáticos evidentes. As lesões histológicas localizadas no intestino delgado e linfonodos mesentéricos de quatro búfalos foram compatíveis com lesões já descritas na literatura, e apresentaram BAAR e amplificação de material genético na qPCR. A concordância entre a biópsia retal e a análise dos tecidos de íleo e linfonodo mesentérico, segundo...

Paratuberculosis in a herd of buffaloes was studied in the municipality of São Mateus, Maranhão, Brazil. Rectal biopsies were performed in 140 male and female Murrah, Mediterranean and crossbreed buffaloes older than 3 years. Postmortem examination of 11 buffaloes was performed to compare the rectal biopsies with possible lesions in mesenteric nodes and the intestine. The history of the herd and clinical examination revealed progressive weight loss and non-responsive antimicrobial diarrhea, dehydration and submandibular edema. Rectal biopsies showed in six buffaloes microscopically suggestive lesions for paratuberculosis through hematoxilin-eosin staining (HE), characterized by moderate multifocal granulomatous enteritis with epithelioid cell infiltration. In four buffaloes Langhans giant cells were found. In 15 buffaloes lymphocytic infiltrate was observed in the lamina propria of the large intestine. Ziehl-Neelsen staining (ZN) revealed in 4.3% (6/140) acid-fast bacilli in the rectal mucosa. Real time PCR amplified to 5.71% (7/140) Mycobacterium avium subsp. paratuberculosis (Map) DNA. 11 buffalos were submitted to postmortem examination, gross examination revealed augmented mesenteric nodes with whitish areas in the cut surface. The mucosa of the small intestine was irregular and thickened, with evident traverse folds and Peyer plates. The brownish intestinal content was fluid, the ileocecal valve area thickened and edematous with evident lymphatic vessels. Histological lesions in the mesenteric lymph node and small intestine four buffalo were compatible with those already described in the literature, and presented acid-fast bacilli by ZN staining and amplification of Map genetic material in qPCR. The concordance between the rectal biopsy and the postmortem samples was in agreement with the Kappa test (K=0.792) and was considered substantial or high. The rectal biopsy showed to be promising and can be used by practitioners, together with other...

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.

Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice / Expressão de citocinas e detecção molecular de Mycobacterium avium subspecies paratuberculosis em órgãos de camundongos infectados experimentalmente

Mycobacterium avium subspecies paratuberculosis (MAP) can infect ruminants and remain subclinical for long periods within herds. The identification of organs that are more susceptible to infection and the evaluation of cytokine expression at the site of infection are important to understand the pathogenesis of MAP. In this study, the probability of detection of MAP-DNA and the expression of cytokines in organs of C57BL/6 mice infected intraperitoneally for 120 days were evaluated. Among the evaluated organs, the spleen (85%), colon (75%) and liver (60%) had the highest frequency of positivity. When compared these frequencies between organs, it has been found that the spleen had 1.54 times as likely to be positive in relation to the ileum, and 2.0 times more likely in relation to the Peyer's patches. In addition, at 60 days post-infection, the spleen and the liver were responsible for upregulation of IFN-γ , and the ileum by TNF-α and IL-4. The results indicate that the spleen is the best organ for evaluating an experimental infection by MAP, especially in the initial stages of the infection. Moreover, it showed that the spleen, liver and ileum have a direct role in the inflammatory response in experimental models.

Mycobacterium avium subespécie paratuberculosis (MAP) pode infectar ruminantes e permanecer subclínica por longos períodos nos rebanhos. A identificação de órgãos mais susceptíveis à infecção e a avaliação da expressão das citocinas no local da infecção são importantes para compreender a patogênese de MAP. Neste estudo foi avaliada a probabilidade de detecção de DNA de MAP e a expressão de citocinas em órgãos de camundongos C57BL/6 infectados por via intraperitoneal durante 120 dias. Dentre os órgãos avaliados, o baço (85%), cólon (75%) e fígado (60%) tiveram as maiores frequências de positividade. Quando comparadas essas frequências entre os órgãos, verificou-se que o baço teve 1,54 vezes mais probabilidade de ser positivo em relação ao íleo, e 2,0 vezes mais probabilidade em relação às placas de Peyer. Além disso, aos 60 dias pós infecção, o baço e o fígado foram responsáveis pela maior expressão de IFN-γ e o íleo pela TNF-α e IL-4. Os resultados indicam que o baço é o melhor órgão para avaliar uma infecção experimental por MAP, principalmente nos períodos iniciais da infecção. Além disso, demonstrou que o baço, fígado e íleo têm importância direta na resposta inflamatória de modelos experimentais.

Paratuberculose em bovinos de corte na região Sul do Rio Grande do Sul / Paratuberculosis in beef cattle in Southern Brazil

Descrevem-se os aspectos epidemiológicos e clínico-patológicos de paratuberculose diagnosticada no sul do Rio Grande do Sul em uma propriedade de bovinos de corte. Dois bovinos criados extensivamente que apresentavam emagrecimento progressivo e diarreia crônica foram necropsiados. Os linfonodos mesentéricos estavam aumentados e edematosos. A mucosa do intestino estava espessada e enrugada com aspecto cerebroide principalmente na porção final do íleo, válvula íleo-cecal e ceco. Fragmentos dos órgãos foram fixados em formalina 10%, incluídos em parafina, cortados e corados pela técnica de hematoxilina e eosina (HE) e Ziehl-Neelsen (ZN). Fezes foram encaminhadas ao Departamento de Medicina Veterinária, Área de Medicina Veterinária Preventiva da Universidade Federal Rural de Pernambuco para o cultivo de Mycobacterium aviumsubsp.paratuberculosis em meio Lowenstein Jensen com micobactina e para realização da PCR. Histologicamente, havia enterite granulomatosa no jejuno, íleo, ceco e reto, afetando multifocalmente, também, o duodeno e o cólon. Havia, ainda, linfangite e adenite granulomatosa. Pela coloração de ZN foram observados numerosos bacilos álcool-ácido resistentes (BAAR) no interior de macrófagos, células gigantes de Langhans e nos linfonodos mesentéricos no jejuno, íleo ceco e reto. Não houve crescimento bacteriano nas amostras de fezes e cinco amostras amplificaram a sequência genética IS900 específica do Mycobacterium aviumsubesp. paratuberculosis. Pelo presente trabalho pode-se concluir que a paratuberculose apesar dos poucos relatos ocorre também em bovinos de corte criados extensivamente no sul do Rio Grande do Sul e pode ter uma prevalência maior do que se supõe na região. Alerta-se para a necessidade do diagnóstico e da tomada de medidas efetivas de controle para esta doença que, por muitos, ainda é considerada uma doença exótica no Brasil.

The epidemiological, clinical and pathological aspects of paratuberculosis diagnosed in southern Rio Grande do Sul in a beef cattle property are described. Two cattle raised extensively and that presented progressive weight loss and chronic diarrhea were necropsied. The mesenteric lymph nodes were enlarged and edematous. The intestinal mucosa was thickened and wrinkled with cerebroid aspect, especially in the final portion of the ileum, ileocecal valve and cecum. Fragments of the organs were fixed in 10% formalin, embedded in paraffin, sectioned and stained with hematoxylin and eosin (HE) and Ziehl-Neelsen (ZN). Feces samples were referred to Departamento de Medicina Veterinária, Área de Medicina Veterinária Preventiva da Universidade Federal Rural de Pernambuco, for the cultivation of Mycobacterium avium subsp. paratuberculosis with Lõwenstein Jensen for mycobacterial and PCR analysis. Histologically, granulomatous enteritis was observed in the jejunum, ileum, cecum and rectum, and in multifocal areas the duodenum and colon. Lymphangitis and granulomatous adenitis was also observed. In ZN staining numerous resistant acid-fast bacilli (AFB) within macrophages, giant cells of Langhans and mesenteric lymph nodes in the jejunum, ileum cecum and rectum were observed. There was no bacterial growth in stool samples. Five samples amplified the gene sequence IS900 specific for Mycobacterium aviumsubsp. paratuberculosis. It can be concluded that paratuberculosis, despite the few reports, occurs in beef cattle raised extensively in southern Rio Grande do Sul, Paratuberculosis may have a higher prevalence in the region than it is assumed. There is need for diagnosis of this disease and to assume effective measures for its control, as for many it is still considered an exotic malady in Brazil.

Evaluation of ‘cattle’ and ‘Indian Bison’ type antigens of Mycobacterium avium subspecies paratuberculosis for diagnosis of Bovine Johne’s Disease using ‘indigenous ELISA’ and AGPT .

Two antigens (‘cattle’ type and ‘Indian Bison’ type) of Mycobacterium avium subspecies paratuberculosis were evaluated for diagnosis of Johne’s disease (JD) in a gaushala (cattle herd). Of the 160 cows of Sahiwal and Hariana breeds screened, 81 (50.6%) tested positive in ELISA and 66 (41.8%) in AGPT test. Using the two antigens, 33.5% tested positive in both the tests while 41.1% tested negative. Exclusively, only 8.2% tested positive in ELISA while 17.1% tested positive in AGPT. Two antigens together detected 58.9% prevalence of MAP in the gaushala. Individually, indigenous ELISA using antigen from native source of MAP proved superior to AGPT in the diagnosis of JD in cows.

A novel low-cost method for Mycobacterium avium subsp. paratuberculosis DNA extraction from an automated broth culture system for real-time PCR analysis

PCR is a highly accurate technique for confirming the presence of Mycobacterium avium subsp. paratuberculosis (Map) in broth culture. In this study, a simple, efficient, and low-cost method of harvesting DNA from Map cultured in liquid medium was developed. The proposed protocol (Universidad Austral de Chile [UACH]) was evaluated by comparing its performance to that of two traditional techniques (a QIAamp DNA Stool Mini Kit and cethyltrimethylammonium bromide [CTAB] method). The results were statistically assessed by agreement analysis for which differences in the number of cycles to positive (CP) were compared by Student's t-test for paired samples and regression analysis. Twelve out of 104 fecal pools cultured were positive. The final PCR results for 11 samples analyzed with the QIAamp and UACH methods or ones examined with the QIAamp and CTAB methods were in agreement. Complete (100%) agreement was observed between data from the CTAB and UACH methods. CP values for the UACH and CTAB techniques were not significantly different, while the UACH method yielded significantly lower CP values compared to the QIAamp kit. The proposed extraction method combines reliability and efficiency with simplicity and lower cost.

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.

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.

Obtención y evaluación de un derivado proteico purificado de una cepa argentina de Mycobacterium avium subsp. paratuberculosis / Production and evaluation of a purified protein derivative from an Argentine strain of Mycobacterium avium subsp. paratuberculosis (MAP)

Los derivados proteicos purificados (PPD) son mezclas antigénicas no definidas obtenidas de distintas micobacterias. Los PPD bovino (PPDb) y PPD aviar (PPDa) son los antígenos que se emplean para evaluar la respuesta inmunitaria celular en infecciones como tuberculosis y paratuberculosis en el bovino. El PPDa comercial se produce a partir de Mycobacterium avium subsp. avium, y no a partir de la subespecie paratuberculosis. En este trabajo se seleccionó una cepa local de Mycobacterium avium subsp. paratuberculosis cuyo patrón molecular por RFLP es el más frecuente entre los aislamientos de nuestro país que han sido estudiados, y a partir de esta, se obtuvo un derivado proteico purificado: PPDj-IB. Se emplearon tanto el PPDa comercial como el PPDj-IB como antígenos en la prueba de liberación de gamma-interferón en animales de un tambo con paratuberculosis y en animales control. Aun cuando ambos PPD fueron capaces de estimular diferencialmente la liberación de la citoquina en el tambo infectado (respecto de los tambos control), no hubo diferencias significativas en los niveles de estimulación producidos y solo dos animales fueron positivos mediante el empleo de PPDj-IB. A partir del análisis por Western blot se demostró que el contenido de lipoarabinomano y del antígeno Apa/ModD era distinto en los PDD evaluados. Estas diferencias podrían explicar, en parte, las diferencias en los niveles de estimulación en términos individuales. Si bien el empleo de PPDj-IB no mejoró significativamente los resultados de la prueba de liberación de ?IFN, es importante destacar que se logró producir en el laboratorio un PPD apto para su empleo en ensayos in vitro.

Purified Protein Derivatives (PPDs) are non-defined antigens prepared from mycobacteria cultures. They are usually employed to evaluate the specific cellular immune response both in animals and humans. Bovine and avian PPDs are usually employed as antigens in mycobacterial infections such as tuberculosis and paratuberculosis. Nevertheless, PPD from Mycobacterium avium subsp. paratuberculosis, (PPDj) is neither commonly used nor frequently available. However, PPD from Mycobacterium avium subsp. avium is in fact used. We aimed to obtain and evaluate the performance of a PPDj from a local isolate of MAP using the ãInterferon-release assay. The stimulation of ãInterferon-release was significantly different between infected and control cattle when this antigen, named PPDj-IB, was used. Stimulation in the infected animals was similar with both antigens (PPDa and PPDj-IB). However, some animals were positively stimulated with PPDj-IB and not with PPDa. We demonstrated by Western blot that two antigenic molecules, lipoarabinoman and APA/ModD antigen were differentially represented in both PPDs. This could explain the difference in stimulation induction of ?IFN observed at individual level. Although PPDj-IB could not improve PPDa performance, we could easily produce an effective purified protein derivative for in vitro assays.

Diagnosis of paratuberculosis in cattle: microbiological culture, serology and PCR

The aim of this study was to confirm clinical diagnosis of paratuberculosis in two cows showing suggestive clinical signs of the disease. Based on clinical signs, in culture and in IS900 PCR results from the individual milk samples it was possible to diagnose paratuberculosis in the cows studied.

Paratuberculose em caprinos e ovinos no Estado da Paraíba / Paratuberculosis in goats and sheep in the state of Paraiba

Mediante o teste de ELISA foi determinada a presença de anticorpos para Mycobacterium avium subsp. paratuberculosis (map) em amostras de soro de 734 caprinos e 392 ovinos, sem sinais clìnicos aparentes, provenientes de 14 Municipios do semiárido Paraibano. Em caprinos, a frequência média de anticorpos de 44,86± 22,91% e em ovinos foi de 52,96±31,49. Das 46 propriedades estudadas, 44 (95,65%) apresentaram pelo menos um animal soropositivo. Nos 14 municípios avaliados houve presença de animais sorologicamente positivos, variando de 20% a 70%. Em caprinos sem raça definida e mestiços a frequência (48,56%) foi significativamente inferior (P<0,0270) que a de caprinos de raças puras (57,24%). Em ovinos com escore de 1 a 3 a frequência (59,39%) foi significativamente maior (P=0,0034) que a frequência em ovinos com escore superior a 3 (42,42%). Não houve diferença significativa nas frequências em caprinos e ovinos de diferentes idades, em caprinos com diferente escore corporal e em ovinos de diferentes raças. No exame microbiológico, em cultivos em meio HEYM com micobactina J, foram observadas colônias semelhantes às do Map em 9 (6,58%) das 180 amostras de fezes, sendo uma de caprinos e 8 de ovinos. Na coloração de Ziehl Nieelsen as bactérias apresentavam características morfo-tintorias de Map. Os resultados obtidos indicam alta frequência de caprinos e ovinos infectados com Map no semiárido da Paraíba, tornando-se necessário desenvolver pesquisas sobre a epidemiologia e forma de controle da doença nas condições de criação da região.

Antibodies to paratuberculosis were determined by ELISA in serum samples of 734 goats and 392 sheep, without clinical signs, from 14 municipalities of the semiarid region of Paraíba state, Brazil. In goats, average frequency of antibodies was 44.86±22.91% and in sheep was 52.96±31.49%. All municipalities had affected animals with a frequency varying from 20% to 70%. In goats of unknown breed and crossbreds the prevalence (48.56%) was significantly lower (P=0.0270) than in pure breed goats (57.24%). In sheep with body scores of 1 to 3, the frequency (59.39%) was significantly higher (P=0.0034) than in sheep with body score from 3 to 5 (42.42%). No significant differences were observed in goats and sheep of different ages, in goats with different body score, and in sheep of different breeds. In the microbiologic examination, after 16 weeks of incubation in HEYM medium with mycobactin J, colonies similar to those of do Mycobacterium avium subsp. paratuber culosis were observed in cultures of 9 (6.58%) out of 180 fecal samples, being one from goats and 8 from sheep. In the Ziehl Nieelsen the bacteria had morphologic and staining characteristics similar to M. avium subsp. paratuberculosis. These results suggest that paratuberculosis is endemic with high prevalence in the semiarid region of Paraíba. It is necessary to develop research on the epidemiology and control measures of the disease in the conditions of this region. Also the risk for human beings should be determined, mainly in relation with milking goats.

Aspectos clínico-patológicos e controle da paratuberculose em rebanho bovino leiteiro / Clinic-pathological aspects and control of paratuberculosis in a dairy cattle herd

A paratuberculose ou doença de Johne é uma enterite granulomatosa causada por Mycobacterium avium subsp. paratuberculosis. Descrevem-se os aspectos epidemiológicos, clínico-patológicos e laboratoriais da paratuberculose em rebanho bovino leiteiro no município de Rio Claro, região Sul do Estado do Rio de Janeiro. No período de 2006 a 2009, oito vacas adultas da raça Girolanda apresentaram diarreia crônico-intermitente e perda progressiva de peso. À necropsia, observaram-se linfonodos mesentéricos aumentados de volume e úmidos ao corte, vasos linfáticos subserosos das alças intestinais proeminentes, serosa do intestino com aspecto anelado e cerebroide e a mucosa espessada, pregueada e com aspecto microgranular. À microscopia havia, desde o duodeno até o reto, inflamação granulomatosa difusa, marcada dilatação dos vasos linfáticos no ápice das vilosidades, linfangiectasia e linfangite granulomatosa na submucosa, muscular e serosa. A inflamação granulomatosa também foi vista nos linfonodos mesentéricos. A coloração de Ziehl-Neelsen revelou variável quantidade de bacilos álcool-ácido resistentes no interior de macrófagos, de células gigantes de Langhans e livres na mucosa e submucosa dos intestinos delgado e grosso e em linfonodos mesentéricos. Em alguns animais, a lâmina própria da mucosa, principalmente do jejuno e íleo exibia acentuada hipertrofia. Mycobacterium avium subsp. paratuberculosis foi isolado em cultivo bacteriano de Herrold com micobactina, a partir de amostras de fezes, de raspado de mucosa intestinal e de leite e identificado pela técnica de PCR IS900. Através da avaliação sorológica semestral, foram analisadas 298 vacas do mesmo rebanho a partir de três anos de idade, observou-se cerca de 40 por cento de animais reagentes ao teste ELISA indireto no período estudado. O diagnóstico da paratuberculose foi baseado nos dados clínico-patológicos, na sorologia, no isolamento e identificação do agente através de cultivo bacteriano e PCR IS900. Após implementação de medidas de controle, tais como eliminação de animais doentes, abate seletivo dos animais soropositivos, separação dos bezerros ao nascer e utilização de banco de colostro, observou-se, nos três anos de estudo, diminuição da ocorrência de casos clínicos no rebanho, de seis casos por ano para cerca de um caso por ano.

Paratuberculosis (Johne's disease) is a granulomatous enteritis of ruminants caused by Mycobacterium avium subsp. paratuberculosis. Epidemiology, clinic-pathological and laboratorial aspects of paratuberculosis in a dairy cattle herd are described. The disease was diagnosed from 2006 to 2009 in eight cows that presented chronic-intermittent diarrhea and chronic weight loss, in the Rio Claro municipality, Rio de Janeiro. At necropsy, the subserosal lymphatic vessels were proeminent and dilated, mesenteric nodes were enlarged and intestinal mucosa was corrugated, thickened and of microgranular aspect. From duodenum to the rectum, histopathology revealed severe and diffuse granulomatous inflammation of the lamina propria and submucosa, broadened and distorted villi, dilatation of the lymphatic vessels in their apex, lymphangioectasia and granulomatous lymphangitis in the submucosa. Ziehl-Neelsen stain showed variable amounts of acid-fast bacilli in macrophages, in Langhans giant cells and freely in the mucosa and submucosa of the small intestine, colon and lymphnodes. In some cows, the lamina propria presented severe hypertrophy, mainly in the jejunum and ileum. Mycobacterium avium subsp. paratuberculosis was isolated through bacterial cultivation of samples taken from feces, intestinal mucosa and milk, and identified through IS900 PCR. From 298 cows older than three years, the percentage of reactive animals was 40 percent by indirect ELISA test. The diagnosis of paratuberculosis was based on clinic-epidemiological data, serology, bacterial isolation in Herrold egg yolk medium with micobactin and on IS900 PCR. After the adoption of control measures, as slaughter of cows with clinical signs, selective slaughter of seropositive cows, removal of the calf from the dam at birth, and use of the colostrum bank, we observed a reduction from six clinical cases to only one case per year, in the last three years of the study.

Paratuberculose em caprinos e ovinos no Brasil / Paratuberculosis in goats and sheep in Brazil

Este trabalho relata, pela primeira vez no Brasil, no Estado da Paraíba, paratuberculose em dois rebanhos com criação conjunta de caprinos e ovinos. Na Fazenda 1, de um rebanho de 33 caprinos e 13 ovinos, uma cabra adulta apresentou emagrecimento progressivo por aproximadamente um ano e fezes pastosas um mês antes da morte. Todos os animais do rebanho foram tuberculinizados com a prova comparativa. Um ovino (2,2 por cento) teve resultado positivo à tuberculina aviar e em dois o teste foi inconclusivo. Na Fazenda 2, com 200 ovinos e 80 caprinos, foi afetada uma ovelha adulta que apresentou emagrecimento progressivo por aproximadamente um ano e fezes pastosas por aproximadamente 20 dias. Todos os ovinos com mais de 4 meses de idade e 23 caprinos foram tuberculinizados com tuberculina aviar; em 47 (25,4 por cento) o resultado foi positivo, em 115 (61,5 por cento) inconclusivo e em 25 (13,4 por cento) negativo. Entre as cabras não houve nenhuma positiva à tuberculina aviar, mas em 11 (47,8 por cento) o teste foi inconclusivo e em 12 (52,2 por cento) foi negativo. Na necropsia dos dois animais com sinais clínicos os linfonodos mesentéricos estavam aumentados de tamanho e edemaciados. O ovino afetado da Fazenda 2, apresentou espessamento e enrugamento da mucosa do intestino, principalmente no íleo e válvula íleo-cecal. Microscopicamente o caprino e o ovino com sinais clínicos apresentaram lesões semelhantes, caracterizadas por granulomas com predominância de macrófagos espumosos, na lâmina própria e submucosa do intestino e linfonodos mesentéricos. O ovino positivo à tuberculina e um caprino negativo na Fazenda 1 foram eutanasiados e apresentaram discreto espessamento da mucosa do íleo. Na histologia foi observado infiltrado preferentemente linfocítico. Em todos os casos dentro de macrófagos e linfócitos havia bacilos ácool-ácidos resistentes, positivos na imunohistoquímica para Mycobacterium spp. Sugere-se a necessidade de estudar a prevalência ...

This paper reports paratuberculosis in sheep and goats for the first time in Brazil. On Farm 1 in a flock of 33 goats and 13 sheep, one adult female goat presented progressive weight loss during about one year, and soft feces during one month before death. All the animals from the flock were submitted to the comparative tuberculin test. One sheep was positive to the avian tuberculin (2.2 percent) and in two the results were inconclusive. On Farm 2, in a flock of 200 sheep and 80 goats, one adult ewe presented progressive weight loss during about one year, and soft feces during 20 days before death. All sheep older than four months of age and 23 goats were submitted to tuberculin test with avian tuberculin; 47 (25.4 percent) sheep were positive, 115 (61.5 percent) had inconclusive results, and 25 (13.4 percent) were negative. No positive tuberculin test were observed in the goats, but in 11 (47.8 percent) the test was inconclusive and in 12 (52.2 percent) was negative. At necropsy both affected animals had enlarged mesenteric lymph nodes. In the affected sheep, the gut wall was thickened and corrugated, mainly in the ileum and ileocecal valve. Histologically, granulomas with mainly foamy macrophages were observed in the lamina propria and submucosa of the gut, in mesenteric lymph nodes, and in the goat also in the liver. One tuberculin positive sheep and one tuberculin negative goat from Farm 1 were euthanized and necropsied. A mild thickened of the intestinal wall was observed grossly in both animals. Inflammatory infiltrate with mainly lymphocytes was observed on the submucosa and lamina propria of the gut. In the four cases acid-alcohol positive rods, positive for Mycobacterium spp. on immunohistochemical examination were observed in macrophages and lymphocytes. New studies are necessary to know the prevalence and economical impact of paratuberculosis in goats and sheep in Brazil, to determine control measures, and to evaluate the risk ...

Infección por Mycobacterium avium subsp. paratuberculosis en un rebaño criollo limonero / Mycobacterium avium subsp. paratuberculosis infection in a criollo limonero herd

El objetivo de este estudio consistió en definir, analizar y evaluar la situación de un rebaño Criollo Limonero elite en relación a la paratuberculosis bovina. La investigación se llevó a cabo utilizando las variables epidemiológicas y el análisis de muestras (leche y suero) mediante inmunoensayo enzimático (ELISA) comercial. Posteriormente, se tomaron como base estos resultados y utilizando otras herramientas diagnósticas como la exploración clínica, tinción directa, cultivos, identificación mediante ensayo de reacción en cadena de la polimerasa (PCR) y estudio histomorfológico, todas ellas dirigidas a confirmar el estado de la infección. Los resultados permiten aseverar la existencia de infección por Mycobacterium avium subsp. paratuberculosis (MAsP) en tasas de positividad variable. La técnica de ELISA mostró alta capacidad en la identificación de animales subclinicamente afectados, lo que demuestra su potencial como prueba primaria de diagnóstico en el establecimiento de futuros programas de control. La técnica de amplificación por PCR reveló la presencia de genoma de subespecies de Mycobacterium avium, lo cual apoya fuertemente la presunción de infección en estos animales por MAsP, pese a la imposibilidad de lograr claras evidencias clínicas, clinicopatológicas, microbiológicas e histopatológicas que permitieran corroborar este dictamen. La estrecha correlación entre los resultados de ELISA y PCR, ratifica no sólo la confirmación diagnóstica, sino también validan los datos obtenidos a través de la prueba serológica.

The purpose of this research was to define, analyze and evaluate the situation of an elite Criollo Limonero herd related to bovine paratuberculosis. The investigation was carried out using the epidemiological variables and samples analysis (milk and serum) through a commercial enzyme immunoassay (ELISA). The results of the above test were taken as a foundation to use other diagnostic tools, such as clinical examination, direct staining, bacterial culture, identification through polymerase chain reaction (PCR) and histopathologic studies, to find out the status of the disease. The results allowed asserting the infection by the presence of Mycobacterium avium subsp. paratuberculosis (MAsP) in positive variable rates. The ELISA technique showed high capacity in identification of the sub clinical affected animal, which proved its potential as a diagnostic primary test in the establishment of future control programs. The PCR amplification revealed Mycobacterium avium subspecie genome presence, which lead to predict these animals infection by MAsP, in spice of the impossibility to accomplish clear clinical, clinic pathologic, microbiologic and histopathologic evidences that allowed corroborating the diagnosis. The narrow relation between ELISA and PCR results ratified not only the diagnostic confirmation, but also they validated the data collected through the serological test.

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.