PCR for the reliable detection of M. paratuberculosis in gut samples from sheep with paucimicrobial enteritis requires ribolysation of the tissue lysate

Abstract

G4540 PCR FOR THE RELIABLE DETECTION OF M.PARATUBERCULOSIS IN GUT SAMPLES FROM SHEEP WITH PAUCIMICROBIAL ENTERITIS REQUIRES RIBOLYSATION OF THE TISSUE LYSATE. A. Verstocken, D. Winterbourne, J.Ford, C.Clarke, J.Hermon-Taylor. Department of Surgery, St. George's Hospital Medical School, London. U.K. M.paratuberculosis (Map) is a pathogen which causes chronic enteritis in many different animals including primates. The animal disease ranges from pluribacillary to paucimicrobial like leprosy in humans. In paucimicrobial paratuberculosis in sheep there is florid granulomatous inflammation of the intestine but Map may not be seen microscoPically or isolated by laboratory culture, and IS900 PCR for Map is frequently negative. Preliminary tests on culturable strains of bacillary-form Map from mature liquid cultures showed that boiling in detergent or chaotropic agents for 30 min, or treatment with SDS proteinase K, methods which reliably release DNA from other bacteria, were suboptimal for Map. Access to Map DNA was improved by exposure of these cultured organisms to vibration at 6.5 m/sec for 45 sec in a mixed slurry of silica and ceramic particles using Hybaid Ribolyser blue (HRB) tubes. Tests on 20-40mg samples of fresh frozen inflamed intestine from 2 sheep with paucimicrobial Map enteritis showed that tissue dissolution was best achieved by incubation in HRB tubes for 24 hours at 37°C in 800~tl lysis buffer TEN (2raM EDTA, 400mM NaCI, 10mM Tris HC1 pH 8.0) SDS 0.6% containing 20~ag proteinase K (Qiagen). After centrifugation of the lysate at 13,000 rpm for 10 rain, the supematant was removed, the pellet was ribolysed and DNA was isolated from both fractions by phenol chloroform extraction. IS900 PCR was consistently positive on both fractions. Serial dilutions, however, showed that the ribolysed pellet contained 1-2 logs more Map DNA than the supernatant suggesting that a majority of the organisms had remained intact. These findings show that digestion with SDS proteinase K alone is insufficient for assured access to Map DNA in the form that this organism exists within the intestine of sheep suffering from paucimicrobial chronic granulomatous enteritis. Reliable detection of these robust pathogens by PCR requires physical disruption by ribolysation.