Serodiagnóstico de Paratuberculosis en bovinos del municipio de Sogamoso, Boyacá (Colombia) / Serodiagnosis of Paratuberculosis in cattle of the municipality of Sogamoso, Boyacá (Colombia) / Serodiagnóstico da paratuberculose no gado do municipio de Sogamoso, Boyacá (Colômbia)

RESUMEN La Paratuberculosis Bovina (PTB) o Enfermedad de Johne, es una infección del tracto gastrointestinal causada por Mycobacterium avium, subespecie paratuberculosis (Map), que se caracteriza por generar enteritis granulomatosa crónica y linfadenitis en rumiantes. La preocupación más relevante en relación con la importancia de la PTB es su posible vínculo con la Enfermedad de Crohn (EC) en humanos, sin embargo, esta asociación aún está bajo investigación. Se determinó la seroprevalencia de PTB en el municipio de Sogamoso (Boyacá), donde se recolectaron 604 muestras de sangre, cuyo suero fue procesado mediante la técnica de ELISA indirecta con el kit comercial PARACHEK® 2 KIT (Prionics, Suiza). La seroprevalencia fue de 10,9% (66/604), donde el grupo etario de 2 a 3 años y la raza Jersey fueron los de mayor seroprevalencia. Se encontró asociación estadística significativa (p≤0,05) entre la edad de los individuos evaluados y el suministro de concentrado. La seroprevalencia encontrada indica que se está produciendo una transmisión activa de la enfermedad y que las medidas de control disponibles no están siendo llevadas a cabo o no son lo suficientemente efectivas.

ABSTRACT Bovine Paratuberculosis (BPT), or Johne's Disease, is an infection of the gastrointestinal tract caused by Mycobacterium avium, subspecies paratuberculosis (Map), which is characterized by chronic granulomatous enteritis and lymphadenitis in ruminants. The most relevant concern regarding the importance of BPT is its possible link to Crohn's disease (CD) in humans, however this association is still under investigation. The seroprevalence of BPT was determined in the municipality of Sogamoso (Boyacá), where 604 blood samples were collected, their serum was processed by the indirect ELISA technique with the commercial PARACHEK® 2 KIT (Prionics, Switzerland), following the manufacturer's instructions. The seroprevalence was 10,9% (66/604), with the 2 to 3 years age group and the Jersey breed having the highest seroprevalence. A significant statistical association was found (p≤0,05) with the age of the individuals tested and the supply of concentrate. The seroprevalence found indicates that active transmission of the disease is taking place, and that the available control measures are not being carried out or are not effective enough.

RESUMO A Paratuberculose Bovina (PTB) ou Doença de Johne é uma infecção do trato gastrointestinal causada pela Mycobacterium avium subespécie paratuberculosis (Map), caracterizada por gerar enterite granulomatosa crónica e linfadenite em ruminantes. A preocupação mais relevante em relação à importância do PTB é seu possível vínculo com a Doença de Crohn (DC) em seres humanos, no entanto, essa associação ainda está sob investigação. A soroprevalência do PTB foi determinada no município de Sogamoso (Boyacá), onde foram coletadas 604 amostras de sangue, cujo soro foi processado pela técnica ELISA indireta com o kit comercial PARACHEK® 2 KIT (Prionics, Suíça). A soroprevalência foi de 10,9% (66/604), onde a faixa etária de 2 a 3 anos e a raça Jersey foram as que apresentaram maior soroprevalência. Foi encontrada associação estatisticamente significante (p≤0,05) com a idade dos indivíduos avaliados e o suprimento de concentrado. A soroprevalência encontrada indica que a transmissão ativa da doença está ocorrendo e que as medidas de controle disponíveis não estão sendo realizadas ou não são eficazes o suficiente.

Molecular isolation and identification of Mycobacterium avium subsp. hominissuis in Didelphis virginiana from Hidalgo, Mexico

Objective: To isolate and identify the exact species of the genus Mycobacterium from Didelphis (D.) virginiana, and the direct implications of this bacterium to public health and veterinary medicine. Methods: Thirty-one D. virginiana were captured and necropsied in Hidalgo, Mexico. Tissue samples were collected to culture mycobacteria present and examine individual specimens' histopathology. Mycobacterium identification was obtained through the application of amplification and sequencing of 16S rDNA techniques. Results: Three strains were isolated and identified as Mycobacterium (M.) avium subsp. hominissuis by utilizing M. avium complex- specific primers. Granulomatous lesions were observed in the subpleural zone (granuloma grade Π ) and bronchial (granuloma grade I ) of the lungs of D. virginiana with positive isolation. Conclusions: Three strains of M. avium subsp. hominissuis, from lung tissue samples of D. virginiana were identified. This subspecies of M. avium has important implications in public health and veterinary medicine.

Molecular isolation and identification of Mycobacterium avium subsp. hominissuis in Didelphis virginiana from Hidalgo, Mexico

Objective: To isolate and identify the exact species of the genus Mycobacterium from Didelphis (D.) virginiana, and the direct implications of this bacterium to public health and veterinary medicine. Methods: Thirty-one D. virginiana were captured and necropsied in Hidalgo, Mexico. Tissue samples were collected to culture mycobacteria present and examine individual specimens' histopathology. Mycobacterium identification was obtained through the application of amplification and sequencing of 16S rDNA techniques. Results: Three strains were isolated and identified as Mycobacterium (M.) avium subsp. hominissuis by utilizing M. avium complex- specific primers. Granulomatous lesions were observed in the subpleural zone (granuloma grade Π ) and bronchial (granuloma grade I ) of the lungs of D. virginiana with positive isolation. Conclusions: Three strains of M. avium subsp. hominissuis, from lung tissue samples of D. virginiana were identified. This subspecies of M. avium has important implications in public health and veterinary medicine.

Genetic diversity of bovine Mycobacterium avium subsp. paratuberculosis discriminated by IS1311 PCR-REA, MIRU-VNTR, and MLSSR genotyping

The aim of this study was to describe the genetic diversity of Mycobacterium avium subsp. paratuberculosis (MAP) obtained from individual cows in Korea. Twelve MAP-positive fecal DNA samples and 19 MAP isolates were obtained from 10 cattle herds located in 5 provinces in Korea. In addition, 5 MAP isolates obtained from the Czech Republic and Slovakia and 3 isolates from Australia were genotyped for comparison with the domestic isolates. The most prevalent strains in Korea were of the “bison-type” genotype (23 of 31 fecal DNA/isolates) and were distributed nationwide. The remaining MAP isolates (8) and all of the foreign isolates were identified as “cattle-type”. The bison-type strains which were discriminated only as INMV 68 in variable-number tandem repeats of mycobacterial interspersed repetitive units (MIRU-VNTR) typing. Multilocus short sequence repeat (MLSSR) typing differentiated the bison-type strains into 3 different subtypes. The cattle-type strains were divided into 3 subtypes by MIRU-VNTR and 8 subtypes by MLSSR. The allelic diversities in the MIRU-VNTR and MLSSR results were calculated as 0.567 and 0.866, respectively. These results suggest that MIRU-VNTR typing cannot provide a sufficient description of the epidemiological situation of MAP. Therefore, an alternative method, such as MLSSR, is needed for typing of MAP strains to elucidate the molecular epidemiology of MAP infections. Overall, this study is the first epidemiological survey report in Korea using both MIRU-VNTR and MLSSR typing methods, and it has provided basic data necessary to elucidate the characteristics of MAP infections in Korea.

Rapid visual detection of Mycobacterium avium subsp. paratuberculosis by recombinase polymerase amplification combined with a lateral flow dipstick

Paratuberculosis (Johne's disease) is a chronic debilitating disease of domestic and wild ruminants. However, widespread point-of-care testing is infrequent due to the lack of a robust method. The isothermal recombinase polymerase amplification (RPA) technique has applied for rapid diagnosis. Herein, RPA combined with a lateral flow dipstick (LFD) assay was developed to estimate DNA from Mycobacterium avium subsp. paratuberculosis. First, analytical specificity and sensitivity of the RPA-nfo primer and probe sets were assessed. The assay successfully detected M. paratuberculosis DNA in 30 min at 39℃ with a detection limit of up to eight copies per reaction, which was equivalent to that of the real-time quantitative polymerase chain reaction (qPCR) assay. The assay was specific, as it did not amplify genomes from five other Mycobacterium spp. or five pathogenic enteric bacteria. Six hundred-twelve clinical samples (320 fecal and 292 serum) were assessed by RPA-LFD, qPCR, and enzyme-linked immunosorbent assay, respectively. The RPA-LFD assay yielded 100% sensitivity, 97.63% specificity, and 98.44% concordance rate with the qPCR results. This is the first report utilizing an RPA-LFD assay to visualize and rapidly detect M. paratuberculosis. Our results show this assay should be a useful method for the diagnosis of paratuberculosis in resource-constrained settings.

Potential biomarkers as an indicator of vertical transmission of Johne's disease in a Korean native cattle farm

Paratuberculosis (PTB) is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is one of the most widespread and economically important diseases in cattle. After birth, calves are raised with natural breast feeding without separation from their mothers in most Korean native cattle (Hanwoo breed) farms. Vertical transmission of PTB has been reported, but the exact PTB infection route has not been revealed in Hanwoo farms. Calves of MAP seropositive dams were tested for MAP presence and MAP antibodies in feces and tissues. MAP was detected in calf tissues by using polymerase chain reaction. Expressions of genes reported to be prognostic biomarkers of MAP infection changed in both calves and cows (p < 0.05). Expression of two genes (HGF and SERPINE1) were significantly decreased in MAP-infected cattle and their offspring (p < 0.01). The results suggest that biomarker gene expression profiles can be useful in detecting early stage MAP infection. Based on the results, complete eradication of MAP may be possible if accurate diagnostic methods to detect infected calves are added to the current PTB eradication strategy, which, because infected individuals are likely to develop into fecal MAP shedders at any time, includes isolation of new born calves and feeding sterilized colostrum.

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.

Estudio epidemiológico de paratuberculosis bovina en hatos lecheros del sur de Nariño, Colombia / Epidemiological Study of Bovine Paratuberculosis in Dairy Herds in Southern Narino, Colombia / Estudo epidemiológico de paratuberculose bovina em gado leiteiro do sul de Nariño, na Colômbia

Introducción: la paratuberculosis (PTBC), también llamada enfermedad de Johne, es un trastorno gastrointestinal crónico de rumiantes domésticos y silvestres causado por Micobacterium avium subespecie paratuberculosis (MAP). Está distribuida mundialmente y genera un alto impacto en la ganadería, debido a que disminuye la producción, se pierde potencial genético por reemplazos tempranos de animales infectados y se incrementa la mortalidad. Objetivos: evaluar la presencia de anticuerpos anti-MAP en fincas lecheras del sur de Nariño y describir su distribución de acuerdo con características poblacionales. Métodos: se realizó un estudio transversal descriptivo en 958 vacas mayores de 2 años en 16 fincas lecheras. Para determinar la presencia de anticuerpos, se usó la prueba diagnóstica de Elisa indirecto (kit comercial Svanova®). La seropositividad fue asociada con las variables de raza, edad, ubicación, condición corporal, número de lactancias y estadio clínico mediante la prueba estadística de chi cuadrado. Resultados: se encontraron 15 fincas (94 %) con al menos un animal positivo y 77 vacas (8 %) con anticuerpos anti-MAP. Se estableció una asociación significativa (p < 0,05) con la condición corporal del animal. Conclusiones: en los principales municipios lecheros se encontraron vacas con anticuerpos anti-MAP y no se encontró relación entre edad, raza, ubicación y estatus clínico con la seroprevalencia-MAP, pero sí con la condición corporal.

Introduction: Paratuberculosis (PTB), or Johne's disease, is a chronic gastrointestinal disorder in domestic and wild ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP). It is distributed worldwide and generates a high impact on livestock, due to a decrease in production, loss of genetic potential by early replacement of infected animals, and increase in mortality. Objectives: To evaluate the presence of anti-MAP antibodies in dairy farms in southern Narino and to describe their distribution according to population characteristics. Methods: A descriptive cross-sectional study was performed on 958 cows over 2 years of age in 16 dairy farms. To determine the presence of antibodies, the indirect Elisa diagnostic test was used (Svanova® commercial kit). Seropositivity was associated with variables of race, age, location, body condition, lactation number, and clinical state by using the chi-square test statistic. Results: There were found 15 farms (94%) with at least one positive animal and 77 cows (8%) with anti-MAP antibodies. A significant association (p < 0.05) with the animal's body condition was established. Conclusions: Cows with anti-MAP antibodies were found in major dairy municipalities; it was found that MAP-seroprevalence is not connected to age, race, location and clinical status, but it is linked to body condition.

Introdução: a paratuberculose (PTBC), também conhecida como doença de Johne, é um transtorno gastrointestinal crônico de ruminantes domésticos e silvestres causado por Mi-cobacterium avium subespécieparatuberculose (MAP). Está distribuída mundialmente e gera um alto impacto na pecuária, devido a que diminui a produção, se perde potencial genético por substituições precoces de animais infectados e aumenta a mortalidade. Objetivos: avaliar a presença de anticorpos anti-MAP em fazendas de gado do sul de Nariño e descrever sua distribuição de acordo com características populacionais. Métodos: realizou-se um estudo transversal descritivo em 958 vacas maiores de 2 anos em 16 fazendas produtoras de gado. Para determinar a presença de anticorpos, se usou a prova diagnóstica de Elisa indireto (kit comercial Svanova®). A soro positividade foi associada com as variáveis de raça, idade, localização, condição corporal, número de lactancia e estádio clínico através da prova estatística de chi quadrado. Resultados: se encontraram 15 fazendas (94 %) com pelo menos um animal positivo e 77 vacas (8 %) com anticorpos anti-MAP. Estabeleceu-se uma associação significativa (p < 0,05) com a condição corporal do animal. Conclusões: nos principais municípios produtores de leite se encontraram vacas com anticorpos anti-MAP e não se encontrou relação entre idade, raça, localização e status clínico com a soro prevalência-MAP, mas sim com a condição corporal.

Development of vaccines to Mycobacterium avium subsp. paratuberculosis infection

Johne's disease or paratuberculosis is a chronic debilitating disease in ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). The disease causes significant economic losses in livestock industries worldwide. There are no effective control measures to eradicate the disease because there are no appropriate diagnostic methods to detect subclinically infected animals. Therefore, it is very difficult to control the disease using only test and cull strategies. Vaccination against paratuberculosis has been considered as an alternative strategy to control the disease when combined with management interventions. Understanding host-pathogen interactions is extremely important to development of vaccines. It has long been known that Th1-mediated cellular immune responses are play a crucial role in protection against MAP infection. However, recent studies suggested that innate immune responses are more closely related to protective effects than adaptive immunity. Based on this understanding, several attempts have been made to develop vaccines against paratuberculosis. A variety of ideas for designing novel vaccines have emerged, and the tests of the efficacy of these vaccines are conducted constantly. However, no effective vaccines are commercially available. In this study, studies of the development of vaccines for MAP were reviewed and summarized.

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 goat based ‘Indigenous vaccine’ against Bovine Johne’s Disease in endemically infected native cattle herds.

‘Indigenous vaccine’ prepared from ‘Indian Bison Type’ a native bio-type of Mycobacterium avium subspecies paratuberculosis strain ‘S5’ of goat origin (goat based) was evaluated in indigenous cattle herds located in gaushalas (cow shelters), endemic for Bovine Johne’s disease. Cows (893) were randomly divided into vaccinated (702 = 626 adults + 76 calves) and control (191 = 173 adults + 18 calves) groups. Response to vaccination was evaluated on the basis of health (mortality, morbidity), productivity (growth rate, reproductive performance, total milk yield), immunological parameters (LTT, ELISA titer), survivability of animals naturally infected with MAP, bacterimia (by specific blood PCR), sero-conversion (by indigenous ELISA) and status of shedding of MAP in feces (by microscopy) in the two groups before and after vaccination. Reduction in MAP shedding [to the extent of 100% in Herd A; and from 82.1% (0 DPV) to 10.7% (270 DPV) in Herd C] was the major finding in vaccinated cows. Whereas, the control group cows have shown no improvement. As the first indicator of vaccine efficacy, MAP bacilli disappeared from the blood circulation as early as 15 days post vaccination, however, peak titers were achieved around 90 DPV. Peak titers initially declined slightly but were maintained later throughout the study period. Control animals did not show any pattern in antibody titers. Mortality was low in vaccinated as compared to the control groups. Vaccination of endemically infected native cattle herds with inactivated whole-cell bacterin of novel ‘Indian Bison Type’ bio-type of goat origin strain ‘S5’ effectively restored health and productivity and reduced clinical BJD. Application of goat based ‘indigenous vaccine’ for therapeutic management of BJD in native cattle herds (gaushalas) is the first of its kind.

Effective DNA extraction method to improve detection of Mycobacterium avium subsp. paratuberculosis in bovine feces / 대한수의학회지

Paratuberculosis caused by Mycobacterium avium subsp. paratuberculosis (MAP) has extended latent periods of infection. Due to this property, difficulties in the detection of fecal shedder have been raised. A newly designed method for DNA extraction from fecal specimens, mGITC/SC was evaluated in terms of diagnostic efficiency. The detection limit of IS900 real-time PCR was about 50 MAP (1.5 cfu) in 250 mg of feces (6 cfu per g). Also, this DNA extraction method was faster and cheaper than that using commercial kit or other methods. Consequently, the mGITC/SC is an economical DNA extraction method that could be a useful tool for detecting MAP from fecal specimens.

Correlation between seventy-three patients with Crohn's disease and Mycobacterium avium subspecies paratuberculosis infection / 中华消化杂志

Objective To elucidate the correlation between Chinese patients with Crohn's disease (CD) and Mycobacterium avium subspecies paratuberculosis (MAP) infection.Methods The peripheral blood of 73 patients with CD (ileocolon type) and 40 healthy individuals was obtained.The monocyte layer was isolated for MAP culture.The growth of bacteria colony in the culture tube was observed every two weeks and totally for 16 weeks.By the end of culture,specific DNA of MAP (IS900) in the culture tube was detected by nested polymerase chain reaction.ANOVA was performed for measurement data analysis and chi square test was used for count data.Results After cultured for 16 weeks,no bacteria colony of MAP had been observed in 113 mycobacteral growth indicator tube (MGIT).No IS900 had been detected in the peripheral blood of all patients with CD and all healthy controls.Conclusion MAP is not correlated with the pathogenesis of CD (ileocolon type) in Chinese patients.

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.

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.

Frequência de anticorpos para paratuberculose em bovinos no semiarido paraíbano / Frequency of antibodies against paratuberculosis in cattle in the state of Paraiba

A paratuberculose é uma doença importante em bovinos na Paraíba, tendo sido diagnosticados, pelo Hospital Veterinário da Universidade Federal de Campina Grande, cinco focos da doença nos últimos quatro anos. Neste trabalho objetivou-se realizar um estudo sorológico em rebanhos com e sem histórico da doença para estimar a frequência da infecção por Mycobacterium avium subsp. paratuberculosis (Map) em diferentes regiões do semiárido Paraibano. Utilizando o teste de ELISA pesquisou-se a frequência de animais soropositivos contra Map em duas fazendas onde tinha sido diagnosticada a doença, encontrando-se 72,22% (13/18) e 68,75% (11/16), respectivamente de bovinos sorologicamente positivos. Amostras de soro de 486 bovinos de 36 fazendas sem histórico da doença de diferentes regiões da Paraíba (sertão, cariri e agreste), também foram examinados por ELISA. A frequência de animais soropositivos foi de 10,08±1,07% (49/486). Foram encontrados animais positivos em 21 (58.33%) das 36 fazendas estudadas. Os resultados sugerem que o agente da paratuberculose está disseminado em bovinos na Paraíba e que são necessárias medidas de controle para diminuir a frequência de casos clínicos e subclínicos da doença.

Paratuberculosis is an important disease of cattle in the state of Paraíba, northeastern Brazil. In the Veterinary Hospital of the Federal University of Campina Grande, five outbreaks of paratuberculosis were diagnosed in the last four years. The objective of this paper is to report the frequency of antibodies against paratuberculosis in different regions of the state of Paraíba, in farms with previous diagnosis of the disease and in farms without diagnosis. The prevalence of antibodies against paratuberculosis, examined by ELISA, in two farms with cases of the disease, was of 72.22% (13/18) and 68.75% (11/16), respectively. Serum samples from 486 healthy cattle from 36 farms without paratuberculosis diagnosis, from three different regions of Paraíba (sertão, cariri, and agreste), were also examined by ELISA. The frequency of antibodies was 10.08±1,07% (49/486). Antibodies against paratuberculosis were found in 21 (58.33%) out of 36 farms examined. These results suggest that paratuberculosis is an important disease of cattle in the state of Paraíba and that control measures to decrease the prevalence of the disease are necessary.

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.

Effect of lipoarabinomannan from Mycobacterium avium subsp avium in Freund’s incomplete adjuvant on the immune response of cattle

The aim of the present study was to determine whether lipoarabinomannan (LAM), in combination with Freund’s incomplete adjuvant (FIA), was able to improve cell-mediated and antibody-mediated immune responses against ovalbumin (OVA) in cattle. Twenty-three calves were assigned to four treatment groups, which were subcutaneously immunized with either OVA plus FIA, OVA plus FIA and LAM from Mycobacterium avium subsp avium, FIA plus LAM, or FIA alone. Lymphoproliferation, IFN-γ production and cell subpopulations on peripheral blood mononuclear cells before and 15 days after treatment were evaluated. Delayed hypersensitivity was evaluated on day 57. Specific humoral immune response was measured by ELISA. Inoculation with LAM induced higher levels of lymphoproliferation and IFN-γ production in response to ConA and OVA (P < 0.05). Specific antibody titers were similar in both OVA-immunized groups. Interestingly, our results showed that the use of LAM in vaccine preparations improved specific cell immune response evaluated by lymphoproliferation and IFN-γ production by at least 50 and 25 percent, respectively, in cattle without interfering with tuberculosis and paratuberculosis diagnosis.