DETECTION OF MYCOBACTERIUM-BOVIS IN FORMALIN-FIXED, PARAFFIN-EMBEDDEDTISSUES OF CATTLE AND ELK BY PCR AMPLIFICATION OF AN IS6110 SEQUENCE-SPECIFIC FOR MYCOBACTERIUM-TUBERCULOSIS COMPLEX ORGANISMS

A presumptive diagnosis of tuberculosis can be made if a tissue has ch aracteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of t he causative mycobacterium. a process that takes several weeks to comp lete. The purpose of work reported here was to determine if formalin-f ixed, paraffin-embedded tissues could be tested by polymerase chain re action (PCR) to provide a more rapid diagnosis of tuberculosis. Nondec alcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined, The primers used for PCR amplified a 123-bp fr agment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tis sues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, fo llowed by alternating boil/freeze cycles. The remaining positive resul ts were obtained with DNA isolated from the crude tissue extracts by p roteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 ti ssues that had either nonmycobacterial granulomas or granulomatous les ions caused by other mycobacteria (M. paratuberculosis or M. avium). T he findings of this study show that a PCR test usually can provide a r apid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.