Detection of Mycobacterium Bovis in Formalin-Fixed, Paraffin-Embedded Tissues of Cattle and Elk by PCR Amplification of an IS6110 Sequence Specific for Mycobacterium Tuberculosis Complex Organisms

Abstract

Abstract.A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment 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 tissues 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, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.The United States has been actively pursuing the goal of bovine tuberculosis eradication for almost a century, and that objective has nearly been achieved. Most of the initial progress was made by using the caudal fold skin test to identify infected herds, which were then eliminated, but current regulatory efforts are focused on surveillance methods to identify infected animals. The cases are then traced to the herd of origin for further testing or other appropriate resolution of the problem.Tuberculosis surveillance depends on detection of gross lesions by regulatory officials in meat inspection facilities or by pathologists in state diagnostic laboratories. In some cases, suspect tissues are obtained from animals other than domestic cattle, usually farmed elk (Cervus elaphus) and deer, but occasionally from other captive ruminant species or from free-ranging wildlife. Formalin-fixed tissue samples are routinely submitted for histopathologic examination, and in most

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