Cytokine gene expression and molecular detection of Mycobacterium avium subspecies paratuberculosisin organs of experimentally infected mice

摘要

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-?3 , and the ileum by TNF-?± 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. Index terms:?? Mycobacterium avium subsp. paratuberculosis ; mice; Johne's disease; IS900 ; experimental infection Resumo Mycobacterium avium subesp??cie paratuberculosis (MAP) pode infectar ruminantes e permanecer subcl?-nica por longos per?-odos nos rebanhos. A identifica?§?￡o de ?3rg?￡os mais suscept?-veis ?? infec?§?￡o e a avalia?§?￡o da express?￡o das citocinas no local da infec?§?￡o s?￡o importantes para compreender a patog?anese de MAP. Neste estudo foi avaliada a probabilidade de detec?§?￡o de DNA de MAP e a express?￡o de citocinas em ?3rg?￡os de camundongos C57BL/6 infectados por via intraperitoneal durante 120 dias. Dentre os ?3rg?￡os avaliados, o ba?§o (85%), c?3lon (75%) e f?-gado (60%) tiveram as maiores frequ?ancias de positividade. Quando comparadas essas frequ?ancias entre os ?3rg?￡os, verificou-se que o ba?§o teve 1,54 vezes mais probabilidade de ser positivo em rela?§?￡o ao ?-leo, e 2,0 vezes mais probabilidade em rela?§?￡o ??s placas de Peyer. Al??m disso, aos 60 dias p?3s infec?§?￡o, o ba?§o e o f?-gado foram respons??veis pela maior express?￡o de IFN-?3 e o ?-leo pela TNF-?± e IL-4. Os resultados indicam que o ba?§o ?? o melhor ?3rg?￡o para avaliar uma infec?§?￡o experimental por MAP, principalmente nos per?-odos iniciais da infec?§?￡o. Al??m disso, demonstrou que o ba?§o, f?-gado e ?-leo t?am import?￠ncia direta na resposta inflamat?3ria de modelos experimentais. Termos de indexa?§?￡o:?? Mycobacterium avium subsp. paratuberculosis ; doen?§a de Johne, IS900 ; murinos; infec?§?￡o experimental Introduction Mycobacterium avium subspeciesparatuberculosis (MAP) is the etiologic agent of paratuberculosis also known as Johne's disease. It is a chronic granulomatous enteritis with clinically progressive diarrhea, emaciation and death that mainly affects domestic and wild ruminants (Chiodini et al. 1984, Clarke 1997). In addition, MAP is important for public health due to its possible relationship with Crohn's disease (Mendoza et al. 2009). Although ruminants are the main natural hosts of MAP, and thus better models for testing, they need to be maintained in evaluation for long periods before clinical manifestation, limiting these animals models only to the few institutions with facilities capable of meeting these needs (Harris & Barletta 2001). For these reasons, different strains of mice have been used in studies related to infection by MAP, having low cost, easy handling and short experimentation time (Mutwiri et al. 1992, Chiodini & Buergelt 1993, Roupie et al. 2008). In experimental infections in cattle, MAP had a predilection for involving parts of the ileum including the ileocecal valve, jejunum and corresponding lymph nodes, maintaining the infection in these tissues (Sweeney et al. 2006, Stabel et al. 2009). Still, the recovery of viable MAP in extra-intestinal tissues such as the spleen and tonsils was possible by experimental infection with high doses of oral inoculums (Sweeney et al. 2006). However, the evaluation of experimental hematogenous dissemination of MAP in various tissues of sheep, reinforced the assertion of the disease's predilection for infection in filtering organs, the spleen, lungs and liver instead of gut-associated tissues, as occurs with systemic involvement (Bower et al. 2013). These patterns of MAP tissue disseminations must also be evaluated in mice, in order to establish a better comparison among these experimental models with ruminants. Furthermore, it is very important to associate this information with the inflammatory potential of the agent, mainly evaluating the ability of this agent to stimulate cytokine expression in these tissues. Pro-inflammatory cytokines such as IFN-?3, IL-2 and TNF-?± are critical for the recruitment of immune c