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Applied and Environmental Microbiology, January 2006, p. 854-859, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.854-859.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Replication and Long-Term Persistence of Bovine and Human Strains of Mycobacterium avium subsp. paratuberculosis within Acanthamoeba polyphaga

Manuela Mura,1 Tim J. Bull,1* Hugh Evans,3 Karim Sidi-Boumedine,1 Liz McMinn,1 Glenn Rhodes,2 Roger Pickup,2 and John Hermon-Taylor1

Department of Cardiac and Vascular Sciences (Surgery), St. George's University of London, London SW17 0RE, United Kingdom,1 Centre for Ecology and Hydrology, Lancaster Environment Centre, Library Avenue, Bailrigg, Lancaster LA1 4AP, United Kingdom,2 Department of Surgery, Prince Philip Hospital, Llanelli, South Wales SA14 8QF, United Kingdom3

Received 29 July 2005/ Accepted 4 October 2005

Free-living protists are ubiquitous in the environment and form a potential reservoir for the persistence of animal and human pathogens. Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease, a systemic infection accompanied by chronic inflammation of the intestine that affects many animals, including primates. Most humans with Crohn's disease are infected with this chronic enteric pathogen. Subclinical infection with M. avium subsp. paratuberculosis is widespread in domestic livestock. Infected animals excrete large numbers of robust organisms into the environment, but little is known about their ability to replicate and persist in protists. In the present study we fed laboratory cultures of Acanthamoeba polyphaga with bovine and human strains of M. avium subsp. paratuberculosis. Real-time PCR showed that the numbers of the pathogens fell over the first 4 to 8 days and recovered by 12 to 16 days. Encystment of the amoebic cultures after 4 weeks resulted in a 2-log reduction in the level of M. avium subsp. paratuberculosis, which returned to the original level by 24 weeks. Extracts of resection samples of human gut from 39 patients undergoing abdominal surgery were fed to cultures of A. polyphaga. M. avium subsp. paratuberculosis detected by nested IS900 PCR with amplicon sequencing and visualized by IS900 in situ hybridization and auramine-rhodamine staining was found in cultures derived from 13 of the patients and was still present in the cultures after almost 4 years of incubation. Control cultures were negative. M. avium subsp. paratuberculosis has the potential for long-term persistence in environmental protists.

* Corresponding author. Mailing address: Department of Cardiac and Vascular Sciences (Surgery), St. George's University of London, London SW17 0RE, United Kingdom. Phone: 44 (0)208 725 5580. Fax: 44 (0)208 725 3594. E-mail: tim.bull{at}sgul.ac.uk

Applied and Environmental Microbiology, January 2006, p. 854-859, Vol. 72, No. 1
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.1.854-859.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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