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United States Department of Agriculture

Agricultural Research Service

Title: Pcr Identification of Mycobacterium Avium in Formalin-Fixed Paraffin Embedded Animal Tissues

Authors
item Miller, Janice
item Jenny, Allen - APHIS,NVSL,AMES,IA
item Ellingson, Jay

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 28, 1998
Publication Date: N/A

Interpretive Summary: A rapid diagnostic method for identification of Mycobacterium bovis, which causes tuberculosis in animals, was recently developed in this laboratory. The test, which is applied to tissues submitted to the pathology laboratory, uses polymerase chain reaction (PCR) technology to make multiple copies of bacterial DNA for analysis. Although the test is relatively sensitive, a negative test result can not provide absolute assurance that a tissue does not contain Mycobacterium bovis. To provide additional information in this situation, the PCR method was examined to determine if it could identify Mycobacterium avium, another organism that can cause lesions similar to those of tuberculosis. Several test procedures were evaluated to determine which particular DNA sequences would be the best targets for a PCR assay. Two useful targets were identified, a single-copy gene and a repeated DNA sequence. Although sensitivity of these PCR tests for Mycobacterium avium was lower than that observed for tissues containing Mycobacterium bovis, more than half of the 100 cases examined were diagnosed in 2-3 days, as compared to the 2-6 months typically required for identification by bacterial culture. The availability of rapid diagnostic tests for mycobacterial disease will allow animal health regulatory officials to act more quickly in their efforts to locate infected tuberculous animals and initiate the actions necessary to prevent spread of the disease. These activities are a critical component of the U. S. bovine tuberculosis eradiation program.

Technical Abstract: This study was conducted to determine if a PCR procedure previously developed for identification of Mycobacterium bovis in formalin-fixed tissues could also be used to identify M. avium. Two M. avium subspecies, avium and paratuberculosis, can cause tuberculosis-like lesions, so PCR identification of these organisms would be helpful for rapid differential diagnosis. Tissues were examined from 100 culture-positive cases of M. avium infection, including 86 in which the subspecies was not identified and 14 that were identified as paratuberculosis. Each tissue was tested with several primer sets that detect DNA sequences found in 1 or both M. avium subspecies. The PCR detection rate varied with the primers used and the animal species tested. Among the 86 cases with no subspecies designation, primers for a 16S rRNA gene were clearly the most efficient because they produced amplicons from all samples that reacted to any other primer set. The overall detection rate in these samples was 71percent, being highest in avian tissues, 89percent, followed by swine, 72percent, and lastly ruminants, 57percent. None of the avian or swine tissues reacted with primers for subspecies paratuberculosis, but 7 of 12 ruminant tissues were positive for this subspecies. All 14 culture-positive paratuberculosis cases reacted with primers for IS900, an insertion sequence specific for this subspecies, whereas only 11 of these were positive for 16S rRNA. We conclude that 16S rRNA primers are the most useful for PCR identification of M. avium in non-ruminant species. However, IS900 primers should also be used when ruminant tissues are examined, because these primers provide the greatest sensitivity for detection of subspecies paratuberculosis infections.

Last Modified: 8/19/2014
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