Presence of intestinal Mycobacterium avium subspecies paratuberculosis (MAP) DNA is not associated with altered MMP expression in ulcerative colitis

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium avium </it>subspecies <it>paratuberculosis </it>(MAP) is suspected to be a causative agent in human Crohn's disease (CD). Recent evidence suggests that pathogenic mycobacteria and MAP can induce the expression of Matrix Metalloproteinases (MMP), which are the main proteases in the pathogenesis of mucosal ulcerations in inflammatory bowel disease (IBD). Within this study we assessed the prevalence of intestinal MAP specific DNA in patients with Crohn's disease, ulcerative colitis (UC), and healthy controls. We further analysed regulation patterns of MMPs in mucosal tissues of UC patients with and without intestinal MAP DNA detection.</p> <p>Methods</p> <p>Colonic biopsy samples were obtained from 63 Norwegian and German IBD patients and 21 healthy controls. RNA was quantified by quantitative real-time polymerase chain reaction (PCR) to study MMP gene expression in both pathological and healthy mucosal specimens. The presence of MAP DNA in colonic mucosa was examined using MAP specific PCR.</p> <p>Results</p> <p>MAP DNA was detected in 20% of UC patients and 33% of healthy controls but only in 7% of patients with CD. UC patients treated with corticosteroids exhibited a significantly increased frequency of intestinal MAP DNA compared to those not receiving corticosteroids. Expression of MMP-1, -2, -7, -9, -13, -19, -28 and TNF-α did not differ between UC patients with presence of intestinal MAP DNA compared to those without. MMP-2, MMP-9 and MMP-13 were significantly decreased in UC patients receiving corticosteroids.</p> <p>Conclusions</p> <p>The presence of intestinal MAP specific DNA is not associated with altered MMP expression in UC <it>in vivo</it>. Corticosteroids are associated with increased detection of intestinal MAP DNA and decreased expression of certain MMPs. Frequent detection of MAP DNA in healthy controls might be attributable to the wide environmental distribution of MAP and its presence in the food-chain.</p

Fecal culture and two fecal-PCR methods for the diagnosis of Mycobacterium avium subsp. paratuberculosis in a seropositive herd

Abstract Background: paratuberculosis is a slow-developing infectious disease, characterized by chronic granulomatous enterocolitis. This disease has a variable incubation period from 6 months to over 15 years, and is caused by Mycobacterium avium subsp. paratuberculosis (MAP). Its detection by direct and indirect diagnostic techniques has been of special interest. Objective: to report the diagnosis and detection of MAP using several diagnostic tests in a herd of the Northern region of Antioquia, Colombia. Methods: serum samples from the study herd were analyzed, using a commercial ELISA (enzyme-linked immunosorbent assay) kit. Fecal samples were cultured by duplicate using Herrold´s egg yolk medium (HEYM), and analyzed by an end- point IS900-specific nested PCR protocol, and a commercial F57-real-time PCR kit. Results: eight out of 27 serum samples in the study herd resulted ELISA-positive. None of fecal samples resulted positive to HEYM culture by duplicate and none were found to be positive by F57-real-time PCR. Seven of the 27 fecal samples were found to be positive by end-point IS900-specific nested PCR. Agreement was found between ELISA and end-point IS900-specific nested PCR in one of the animals. Conclusion: the present study gives information about the agreement between direct and indirect MAP-detection techniques, using different matrixes from animals under the same husbandry conditions.Resumo Antecedentes: a paratuberculosis é uma doença infecciosa de evolução lenta, caracterizada por uma enterocolite granulomatosa crônica. Esta doença tem um período de incubação que varia de 6 meses a 15 anos e é causada pelo Mycobacterium avium subsp. paratuberculosis (MAP). Sua detecção por técnicas de diagnóstico diretos e indiretos tem sido de especial interesse. Objetivo: reportar o diagnóstico e a detecção de MAP utilizando várias técnicas de diagnóstico em um rebanho na região norte de Antióquia, Colômbia. Métodos: foram analisadas amostras de soro do rebanho utilizando um kit comercial de ELISA (enzyme- linked immunosorbent assay). As amostras de fezes foram cultivadas em duplicado em Herrold´s egg yolk medium (HEYM) e analisadas utilizando um protocolo de PCR aninhada específico de IS900 e um kit de PCR em tempo real comercial para F57. Resultados: oito das 27 amostras de soro foram positivas para ELISA. Nenhuma das amostras testadas na cultura de fezes HEYM duplicado foram positivas ou na PCR em tempo real para F57. Sete das 27 amostras de fezes foram positivas na PCR aninhada específica para IS900. Foi encontrada concordância entre o resultado de ELISA e PCR aninhada específica para IS900 em um animal. Conclusão: este estudo fornece informações sobre a correlação entre técnicas de detecção direta e indireta do MAP, utilizando diferentes matrizes de animais sob as mesmas condições de condução.Resumen Antecedentes: la paratuberculosis es una enfermedad infecciosa de desarrollo lento, caracterizada por una enterocolitis granulomatosa crónica. Esta enfermedad tiene un periodo de incubación que varía entre los 6 meses hasta por más de 15 años, y es causada por Mycobacterium avium subsp. paratuberculosis (MAP). Su detección por técnicas diagnósticas directas e indirectas ha sido de interés especial. Objetivo: reportar el diagnóstico y detección de MAP utilizando varias técnicas diagnósticas en un hato de la región norte de Antioquia, Colombia. Métodos: se analizaron las muestras de suero del hato de estudio utilizando un kit comercial de ELISA (enzyme-linked immunosorbent assay). Las muestras de materia fecal fueron cultivadas por duplicado en Herrold´s egg yolk medium (HEYM), y analizadas mediante un protocolo de PCR anidado específico de IS900 y un kit comercial de PCR en tiempo real para F57. Resultados: ocho de las 27 muestras de suero resultaron positivas por ELISA. Ninguna de las muestras de materia fecal resultó positiva al cultivo en HEYM por duplicado ni por PCR en tiempo real para F57. Siete de las 27 muestras de materia fecal resultaron positivas a PCR anidado específico de IS900. Se encontró concordancia entre el resultado de ELISA y de PCR anidado específico de IS900 en uno de los animales. Conclusión: el presente estudio brinda información acerca de la concordancia entre técnicas directas e indirectas de detección de MAP, utilizando diferentes matrices a partir de animales bajo las mismas condiciones de manejo

Identification and Characterization of a New Variant of Shiga Toxin 1 in Escherichia coli ONT:H19 of Bovine Origin

A new variant of Shiga toxin 1 (Stx1), designated Stx1d, which deviates considerably more than any other known variant from Stx1 encoded by phage 933J, was identified in an Escherichia coli strain, ONT:H19, isolated from bovine feces. The complete stx(1) gene of this strain was amplified and sequenced. Nucleotide sequence homology with stx(1) from phage 933J was only 91%, resulting in the substitution of 20 amino acids in the A subunit and 7 amino acids in the B subunit of the protein. Cell culture supernatant of this strain, which was negative for stx(2) by PCR testing, was cytotoxic to Vero cells and gave positive results in two commercial enzyme-linked immunosorbent assays for Stx. PCR primers were constructed for the specific detection of the new variant. The findings of this study suggest that Stx1 is not as conserved as thought before and that there might be more variants which cannot be detected by commonly used PCR methods