A refined low-dose murine model of <i>Mycobacterium ulcerans</i> infection to assess integrated immune networks in Buruli ulcer pathogenesis

<i>Mycobacterium ulcerans,</i> the causative agent of Buruli ulcer (BU), has a low infectious dose (<10 colony-forming units [CFU]). BU animal infection models typically use challenge doses orders of magnitude higher than natural infection. These doses are unrealistic for vaccine trials and studies of immunity. Here, we address this issue and describe a murine tail infection model in two genetically distinct mouse strains (BALB/c and C57BL/6) using quality-controlled <i>M. ulcerans</i> challenge doses (10-20 CFU and 100 CFU). Over 24 weeks, we measured >70 clinical, immunological, and microbiological parameters. Principal findings included a 100% infection rate even at the lowest inoculum, but with a dose-dependent delay in lesion onset and disease progression for both mouse strains. Bacterial growth kinetics were similar between mouse strains, but there was a difference in immune profiles between mouse strains and between "low" (10 CFU) versus "high" (100 CFU) bacterial challenges. C57BL/6 mice exhibited more robust systemic cellular responses and more rapid lesion onset compared to BALB/c mice. Activated CD8⁺ T cells and dendritic cells were dominant in responses to low-dose infection in C57BL/6 mice. Murine low-dose <i>M. ulcerans</i> infection models provide confidence for future human Buruli ulcer challenge trials and will inform the development of effective vaccines and therapeutics.IMPORTANCEBuruli ulcer (BU) is an infection of subcutaneous tissue caused by <i>Mycobacterium ulcerans</i>. This bacterial infection affects the lives of thousands of people each year across West and Central Africa and Australia. Recent research showed that as few as 2-3 <i>M. ulcerans</i> are sufficient to cause infection. Unfortunately, earlier laboratory studies have used unrealistically high bacterial doses to test new vaccines or to understand host responses, compromising subsequent conclusions. This research is significant because it takes a fresh approach to develop an experimental animal infection model for BU that uses a carefully calibrated and realistic infectious dose. The findings from assessing this new infection model are the foundation for an ambitious new program to develop a controlled human infection model for <i>M. ulcerans</i>, a platform for developing new therapies to prevent and treat BU.