3rd Annual BMRP Investigator Meeting - Abstract

Mycobacterium Paratuberculosis in Crohn’s:  A Study Using Tissue PCR, In Situ PCR and Gamma-Interferon Responses

Marcel A. Behr^a

Department of Medicine, McGill University (Montreal, Quebec, Canada)

For nearly a century, there has been debate about whether M. avium paratuberculosis, the etiologic agent of Johne’s disease in cattle, may also be responsible for Crohn’s disease in humans.  While some reports on this hypothesis have been limited in terms of study design, it is this investigator’s contention that the major limitation in such studies is the technical capacity of the assays employed.  Critical laboratory issues are the sensitivity of the assay at detecting microscopically undetectable organisms and the microbiologic specificity – do assays for related organisms give completely negative results?  In an effort to address these issues, the investigator’s lab has been developing and validating assays on tissue from sheep with paucibacillary paratuberculosis that, unlike bovine Johne’s, shares considerable histopathological similarity with Crohn’s.  These samples have been used to develop both enhanced DNA extraction methods for tissue PCR and an in situ PCR methodology.  In parallel, the investigator’s laboratory has developed a cell-mediated immune assay to look for a γ-interferon response to paratuberculin and other Mycobacterial antigens in peripheral blood mononuclear cells (PBMC).  To date, 77 subjects have been recruited; with recruitment ongoing, the lab is blinded to disease classification.  Tissue PCR has been delayed for technical reasons due to the development of in situ PCR, but results on the first 14 biopsies are now pending.  In situ PCR has just recently been shown to produce a M. paratuberculosis-specific signal in ovine tissue, so testing on the first 14 human subjects will now begin.  PBMC have been obtained for γ-interferon on the 77 subjects and blinded analysis reveals an unexpectedly high prevalence of exposure to mycobacterial antigens used, including two paratuberculin preparations.  The results of DNA-based testing of gastrointestinal tissue will now provide further data on whether this veterinary pathogen is founded in samples of diseased human tissue.  Once recruitment is complete, we will begin analysis to determine the prevalence of positive results to the three assays and association studies with subject classification.

^aPresenter is Makeda Semret, Co-investigator