Abstract. The reservoir of Mycobacterium ulcerans causing Buruli ulcer (BU) remains unknown. Here, sterilized watery soil was mixed with 2 × 10 6 colony-forming units (CFU)/g of M. ulcerans Agy99 or M. ulcerans ATCC 33728 and incubated in a microaerophilic atmosphere in the presence of negative controls. Both M. ulcerans strains survived in soil for 4 months with a final inoculum of 300-440 CFU/g. Further, three groups of five mice with and without footpad scarification were exposed to control soil or M. ulcerans-inoculated soil. Although no specific clinical and histopathological lesions were observed in control animals, red spots observed on 8/20 scarified feet in 8/10 challenged mice yielded inflammatory infiltrates and positive real-time polymerase chain reaction detection of M. ulcerans DNA in five mice. BU can be acquired as an inoculation infection with watery soil as a transient source of infection. These experimental observations warrant additional field observations. Moreover, the only environmental M. ulcerans isolate was recovered from a water strider in Benin. 7 These observations suggest a link between BU and stagnant and slow-flowing aquatic environments in West Africa. However, freshwater itself may not be the ultimate source of M. ulcerans. In particular, fishermen who are presumably in frequent contact with water have a lower prevalence of BU than other groups in endemic regions in Africa. 8,9 In addition, the M. ulcerans inoculum has been estimated to be 100-1,000 mycobacteria/mL in water, based on real-time polymerase chain reaction (RT-PCR) targeting IS2404.3 This estimated inoculum level is three to four times lower than the levels reported for major waterborne human pathogens such as Vibrio. 10We have recently shown that Mycobacterium tuberculosis, a Mycobacterium that is phylogenetically closely related to M. ulcerans, retains its infectivity after an extended stay in soil. 11 We therefore hypothesized that watery soil could be a source of M. ulcerans.To test the hypothesis, M. ulcerans ATCC 19423 TMT (strain Agy99) and M. ulcerans ATCC 33728 (both obtained from the American Type Culture Collection, Masassas, VA) were cultured in an incubator at 32°C on Middlebrook 7H10 Agar (Becton Dickinson, Le Pont de Claix, France), supplemented with OADC (oleic acid, bovine albumin, dextrose, and catalase) (Becton Dickinson). Natural soil was steam sterilized at 134°C for 15 minutes, and the sterility was then assessed by inoculating 2 g of steam-sterilized soil onto Middlebrook 7H10 Agar incubated at 32°C for 4 weeks. A 20-g sample of sterilized soil was inoculated and mixed with 2 × 10 6 colonyforming units (CFU)/g of each of the two M. ulcerans isolates, placed into 25-cm 2 cell culture flasks (Corning, Corning, NY) and thoroughly mixed. Three flasks were prepared for each M. ulcerans strain. The flasks were placed inside a transparent sterile plastic container where they were exposed to a microaerophilic atmosphere created using a CampyGen atmosphere generation system (Oxoid Ltd., Basingstoke, Un...