Investigation of mycobacteria in Crohn's disease tissue by Southern blotting and DNA hybridisation with cloned mycobacterial genomic DNA probes from a Crohn's disease isolated mycobacteria

Abstract

A mycobacterial aetiology for Crohn's disease (CD) has been suggested. Slow growing mycobacteria indistinguishable from M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease) have been isolated from CD tissues. We have used cloned genomic DNA probes derived from a CD isolated mycobacteria strain Ben, to investigate the presence of mycobacterial DNA sequences in CD tissues. DNA was extracted from total tissue from 17 CD and four control gut specimens. DNA was digested with restriction endonucleases, electrophoresed and transferred to nylon membranes by Southern blotting and hybridised to radiolabeled DNA probes. No mycobacterial DNA was detected in any tissue sample studied. Reconstitution experiments with known numbers of in vitro cultured mycobacteria showed sensitive detection of mycobacterial DNA. DNA extracted from mouse liver, infected with M lepraemurium revealed a strong hybridisation signal and showed the applicability of the experimental approach to the detection of mycobacterial DNA in naturally infected tissues. The results do not provide evidence for the involvement of mycobacteria in the pathogenesis of CD but do not exclude the possibility of low levels of infection in subsets of intestinal cells with spheroplast or cell wall deficient forms of mycobacteria.