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 TITLE CHG.2-24-97

Authors: Miller, Janice; JENNY, ALLEN - NVSL,USDA,APHIS,AMES,IA; RHYAN, JACK - NVSL,USDA-APHIS,AMES,IA; SAARI, DENNIS - NVSL,USDA,APHIS,AMES,IA; Suarez, David

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/7/1997
Publication Date: N/A
Citation: N/A

Interpretive Summary: Accurate and rapid diagnosis of tuberculosis in animal populations is critical for success of the federal/state tuberculosis eradication program. Suspect animals are identified by pathologists at the time of death, but final diagnosis requires isolation of the causative bacterium. Unfortunately, the culture process takes several weeks to complete and the delay can make it difficult to trace the diseased animal to its herd of origin. In this paper we describe a more rapid method for detection of the tuberculosis bacterium. The new procedure, which uses a technique called polymerase chain reaction (PCR), can be applied to tissue samples collected for examination by pathologists. In PCR, a specific piece of DNA from the tuberculosis-causing bacteria is multiplied many times and the copies are then identified by a method that separates DNA molecules based on size. To determine whether the PCR technique would be a useful diagnostic test, tissues from 99 known cases of tuberculosis in cattle and elk were examined. In 93% of the cases, a definitive diagnosis could be made within 2 to 3 days after receiving the tissue, which represents a marked improvement over the 2 to 3 months previously required for a diagnosis by bacterial culture. This new technique will significantly improve the U. S. tuberculosis eradication program because cases can be traced more quickly to the herd of origin, thus allowing immediate action to prevent further spread of the disease. The PCR test also represents an important contribution to public health because tuberculosis can be transmitted from animals to people. Rapid identification of potential human exposures to the disease will allow medical personnel to initiate preventive treatment as quickly as possible.

Technical Abstract: A presumptive diagnosis of tuberculosis is based on the finding of characteristic histopathologic lesions and acid-fast organisms in a tissue. A definitive diagnosis, however, requires culture and species identification of the causative mycobacterium, a process that usually takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed paraffin-embedded tissue 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 were examined. The primers used for PCR amplified a 123 bp fragment of IS6110, an insertion sequence that is specific for organisms in the M. tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The sequence was detected in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 of these cases the positive results were obtained using a simplified tissue extraction method that did not require paraffin removal, enzyme digestion or DNA purification. For detection of the other 12 cases, DNA purification was required. 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 will provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.