SPECIFIC DETECTION OF MYCOBACTERIUM-PARATUBERCULOSIS DNA ASSOCIATED WITH GRANULOMATOUS TISSUE IN CROHNS-DISEASE

The role of mycobacteria, specifically Mycobacterium paratuberculosis, in Crohn's disease has aroused considerable controversy for many year s. Using the ultra sensitive polymerase chain reaction some studies ha ve reported detection of M paratuberculosis DNA in as many as 65% of C rohn's disease patients but also in patients without disease. Other st udies have been negative for both groups. We therefore designed a doub le blind control trial to investigate the presence of mycobacterial DN A in age, sex, and tissue matched paraffin wax embedded tissues from 3 1 Crohn's disease tissues, 20 diseased gut control tissues, and 10 ulc erative colitis tissues. The specimens were coded and analysed blind w ith three separate polymerase chain reactions (PCR) based on DNA seque nces specific for M paratuberculosis (IS900), M avium (RFLP type A/1) (IS901), and the Mycobacterium genus (65 kDa gene, TB600). The number of granulomata and presence of acid fast bacilli in each Crohn's disea se tissue was also investigated. The sensitivity of the system was det ermined using similarly prepared gut tissue from an animal infected wi th M paratuberculosis. Four of 31 Crohn's disease tissues and none of the 30 control and ulcerative colitis derived tissues amplified M para tuberculosis DNA. Crohn's disease tissues containing granulomata were significantly more likely to amplify M paratuberculosis specific DNA o n PCR than the non-Crohn's disease tissues (p=0.02). All the positive Crohn's disease tissues contained granulomata, and none contained acid fast bacilli. Equivalent numbers of Crohn's and non-Crohn's disease t issues amplified the region of the 65 kD gene on PCR for nonspecific m ycobacterial DNA (11/31 and 9/30 respectively). No sections produced a n amplified product with the IS901 PCR. These results suggest that few Crohn's disease gut biopsy sections contain M paratuberculosis DNA in association with granulomata. The absence of such DNA in any control and ulcerative colitic tissue strengthens the case for it having a spe cific association, which may be pathogenic, with Crohn's disease in th is minority of patients.