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The transmission of Buruli ulcer (BU), caused by  Mycobacterium ulcerans  ( MU ), remains puzzling although a number of hypothesis including through bites of infected aquatic insects have been proposed. We report the results of experiments using ICR mice that give credence to our hypothesis that  Acanthamoeba  species may play a role in BU transmission. We cocultured  MU  N2 and  MU  1615 which expresses red fluorescent protein (RFP) and  Acanthamoeba polyphaga  ( AP ), and confirmed infected  AP  by Ziehl-Neelsen (ZN) staining. We tested for viability of  MU  inside  AP  and observed strong RFP signals inside both trophozoites and cysts after 3 and 42 days of coculturing respectively. ICR mice were topically treated, either on shaved intact or shaved pinpricked rumps, with one of the following;  MU  N2 only (2.25 x 10 6  colony forming units [CFU] / ml),  MU  N2: AP  coculture (2.96 x 10 4  CFU: 1.6 x 10 6  cells/ml),  AP  only (1.6 x 10 6  cells/ml), PYG medium and sterile distilled water. Both  MU  N2 only and  MU  N2: AP  elicited reddening on day (D) 31; edema on D 45 and D 44 respectively, and ulcers on D 49 at pinpricked sites only. To ascertain infectivity and pathogenicity of  MU  N2 only and  MU  N2: AP , and compare their virulence, the standard mouse footpad inoculation method was used.  MU  N2: AP  elicited reddening in footpads by D 3 compared to D 14 with  MU  N2 only of the same dose of  MU  N2 (2.96 x 10 4  CFU). ZN-stained  MU  were observed in both thin sectioned and homogenized lesions, and aspirates from infected sites. Viable  MU  N2 were recovered from cultures of the homogenates and aspirates. This study demonstrates in ICR mice  MU  transmission via passive infection, and shows that punctures in the skin are prerequisite for infection, and that coculturing of  MU  with  AP  enhances pathogenesis.

Experimental demonstration of the possible role of Acanthamoeba polyphaga in the infection and disease progression in Buruli Ulcer (BU) using ICR mice