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Journal of Clinical Microbiology, July 2003, p. 2915-2923, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2915-2923.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection and Verification of Mycobacterium avium subsp. paratuberculosis in Fresh Ileocolonic Mucosal Biopsy Specimens from Individuals with and without Crohn's Disease

Tim J. Bull,1 Elizabeth J. McMinn,1 Karim Sidi-Boumedine,1 Angela Skull,1 Damien Durkin,1 Penny Neild,2 Glenn Rhodes,3 Roger Pickup,3 and John Hermon-Taylor1*

Department of Surgery,1 Department of Gastroenterology, St. George's Hospital Medical School, London SW17 0RE,2 Centre for Ecology and Hydrology, Windermere, Ambleside LA22 0LP, United Kingdom3

Received 29 January 2003/ Returned for modification 25 March 2003/ Accepted 4 April 2003

Mycobacterium avium subsp. paratuberculosis is a robust and phenotypically versatile pathogen which causes chronic inflammation of the intestine in many species, including primates. M. avium subsp. paratuberculosis infection is widespread in domestic livestock and is present in retail pasteurized cows' milk in the United Kingdom and, potentially, elsewhere. Water supplies are also at risk. The involvement of M. avium subsp. paratuberculosis in Crohn's disease (CD) in humans has been uncertain because of the substantial difficulties in detecting this pathogen. In its Ziehl-Neelsen staining-negative form, M. avium subsp. paratuberculosis is highly resistant to chemical and enzymatic lysis. The present study describes the development of optimized sample processing and DNA extraction procedures with fresh human intestinal mucosal biopsy specimens which ensure access to M. avium subsp. paratuberculosis DNA and maximize detection of these low-abundance pathogens. Also described are two nested PCR methodologies targeted at IS900, designated IS900[L/AV] and IS900[TJ1-4], which are uniquely specific for IS900. Detection of M. avium subsp. paratuberculosis in mucosal biopsy specimens was also evaluated by using mycobacterial growth indicator tube (MGIT) cultures (Becton Dickinson). IS900[L/AV] PCR detected M. avium subsp. paratuberculosis in 34 of 37 (92%) patients with CD and in 9 of 34 (26%) controls without CD (noninflammatory bowel disease [nIBD] controls) (P = 0.0002; odds ratio = 3.47). M. avium subsp. paratuberculosis was detected by IS900[L/AV] PCR in MGIT cultures after 14 to 88 weeks of incubation in 14 of 33 (42%) CD patients and 3 of 33 (9%) nIBD controls (P = 0.0019; odds ratio = 4.66). Nine of 15 (60%) MGIT cultures of specimens from CD patients incubated for more than 38 weeks were positive for M. avium subsp. paratuberculosis. In each case the identity of IS900 from M. avium subsp. paratuberculosis was verified by amplicon sequencing. The rate of detection of M. avium subsp. paratuberculosis in individuals with CD is highly significant and implicates this chronic enteric pathogen in disease causation.

* Corresponding author. Mailing address: Department of Surgery, St. George's Hospital Medical School, London SW17 0RE, United Kingdom. Phone: 44 (0)208 767 7631. Fax: 44(0)208 725 3594. E-mail: jhermon{at}sghms.ac.uk.

Journal of Clinical Microbiology, July 2003, p. 2915-2923, Vol. 41, No. 7
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.7.2915-2923.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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