Detection of Mycobacterium avium subspecies paratuberculosis in Crohn's diseased tissues by in situ hybridization

OBJECTIVES: Reports about the association between Crohn's disease (CD) and cell wall-deficient (CWD) forms of Mycobacterium avium subspecies paratuber culosis (M. paratuberculosis) are controversial. This may be due to the het erogeneous nature of CD where only about 50% of the patients show granuloma tous inflammation. Detection of CWD forms of M. paratuberculosis in tissues from patients with CD would support its association with the disease. To h elp identify these forms in inflamed tissues, a previously developed and op timized nonradioactive in situ hybridization method was applied on well-def ined tissue materials obtained from patients with CD, ulcerative colitis (U C), and controls. METHODS: Specimens from 37 patients with CD (15 with epitheloid cell granul omas and 22 without granulomas), 21 UC, and 22 noninflammatory bowel diseas e (IBD) patients were analyzed by the in situ hybridization method based on the digoxigenin-labeled M. paratuberculosis IS900 fragment, previously sho wn to be species specific. Samples were counterstained with hematoxylin and eosin to show the location of the positive signal. Positive controls made of beef cubes injected with CWD and acid-fast M. paratuberculosis and negat ive controls were included in each experiment to monitor for nonspecific hy bridization or staining. RESULTS: Six of 15 (40%) patients with CD and granulomas showed positive si gnals in myofibroblasts and macrophages. interestingly, no positive signals were observed within granulomas. Only 4.5% of 22 CD samples from patients with nongranulomatous disease, 9.5% of 21 UC, and remarkably, none of the 2 2 non-IBD patients were M. paratuberculosis positive. CONCLUSION: The demonstration of DNA from CWD forms of M. paratuberculosis in this limited number of CD tissues further supports and confirms previous reports of its association with the granulomatous type of the disease. (C) 2001 by Am. Cell. of Gastroenterology.