Muhi, S.; Foo, I. J. H.; Kedzierski, L.; Porter, J. L.; McQuilten, H. A.; Howden, B.; Kedzierska, K.; Buultjens, A. H.; Chua, B. Y.; Stinear, T. P.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is a slow-growing zoonotic pathogen with distinctive pathogenesis linked primarily to its toxin mycolactone. Recent research has shown that the M. ulcerans infectious dose is very low (<10 colony forming units [CFU]). Buruli ulcer animal infection models traditionally use bacterial challenge doses in the range 10^4 - 10^6 CFU; a range orders of magnitude higher than natural infection. These large doses represent an unrealistic challenge 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, M. ulcerans challenge doses (10 - 20 CFU and 100 CFU). Over 24-weeks, we assessed host responses to infection by measuring >70 clinical, immunological and microbiological parameters. Principal findings included a 100% infection rate even at the lowest bacterial challenge, but with a dose-dependent delay in lesion onset and disease progression for both mouse strains. Bacterial growth kinetics were similar between mouse strains. There was a difference in immune profiles between mouse strains and between \"low\" (10 CFU) versus \"high\" (100 CFU) bacterial challenge doses. C57BL/6 mice exhibited more robust systemic cellular responses and more rapid lesion onset compared to BALB/c mice. There were dose-dependent cytokine and chemokine differences in C57BL/6 mice, while BALB/c mice displayed similar responses across both doses. Antibody responses were only detected late in the infection and were associated with the high-dose inoculum in both strains. Machine learning and other statistical analyses highlighted the importance of activated CD8+ T cells and dendritic cells in the immune response to low-dose infection in C57BL/6 mice. Murine low-dose M. ulcerans infection models provide confidence for future human Buruli ulcer challenge trials and will inform the development of effective vaccines and therapeutics.