Polymerase chain reaction identification of Mycobacterium avium in formalin-fixed, paraffin-embedded animal tissues

A PCR procedure previously developed for identification of Mycobacterium bovis in formalin- fixed tissues was used to identify mycobacteria of the M. avium complex. Tissues were examined from 100 culture-positive cases of M. avium complex infection, including 86 in which the subspecies was not identified and 14 that had been identified as M. avium subsp. paratuberculosis. Each sample was tested with 5 primer sets, 16S ribosomal RNA (rRNA), IS900 ,I S901 ,I S1245, and a heat shock protein (hspX), that detect 1 or both M. avium subspecies. The success rate of PCR detection varied with the primers used and the animal species tested. Among the 86 cases with no M. avium subspecies designation, primers for the 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted with any other primer set. The overall detection rate in this group of samples was 71%: highest in avian tissues (89%) followed by swine (72%) and ruminants (57%) None of the avian or swine tissues reacted with primers for IS900 or hspX, which identify M. a. paratuberculosis. In contrast, 7 of the 12 ruminant samples that were 16S rRNA positive reacted with 1 or both of these primers. All of the 14 cases shown by culture to be M. a. paratuberculosis infections were positive with IS900 primers, whereas only 11 were positive for 16S rRNA. These results indicate that 16S rRNA primers are the most useful for PCR identification of M. avium in formalin-fixed tissues of nonruminant species. However, IS900 primers should also be used when ruminant tissues are examined because these primers provide the greatest sensitivity for detection of M. a. paratuberculosis infections. Eradication of bovine tuberculosis in the United States has been a long-term goal that is now nearing attainment. Early in the campaign, the major effort was directed toward identification of infected herds by sys- tematic skin testing. As incidence of the disease de- clined, the focus of attention shifted to a surveillance program that is based on detection of suspect lesions in slaughter animals, with subsequent histopathologic examination and culture of the tissues. If culture re- sults indicate the presence of Mycobacterium bovis, epidemiologic investigations are begun to locate the potential source of infection and to identify other an- imals that might have been exposed. Unfortunately, several weeks may elapse before this effort can be ini- tiated because M. bovis is such a slow-growing organ- ism. To provide a more rapid diagnostic method, a polymerase chain reaction (PCR) procedure was de- veloped that allows specific identification ofM. tuber- culosis complex bacteria (M. tuberculosis, M. bovis, M. africanum, and M. microti) in formalin-fixed, par- affin-embedded tissues. 14 When positive results are ob- tained with this procedure, it is possible to make a