Mycobacterium ulcerans low infectious dose and mechanical transmission support insect bites and puncturing injuries in the spread of Buruli ulcer

Abstract

Addressing the transmission enigma of the neglected disease Buruli ulcer (BU) is a World Health Organization priority. In Australia, we have observed an association between mosquitoes harboring the causative agent, Mycobacterium ulcerans, and BU. Here we tested a contaminated skin model of BU transmission by dipping the tails from healthy mice in cultures of the causative agent, Mycobacterium ulcerans. Tails were exposed to mosquito (Aedes notoscriptus and Aedes aegypti) blood feeding or punctured with sterile needles. Two of 12 of mice with M. ulcerans contaminated tails exposed to feeding A. notoscriptus mosquitoes developed BU. There were no mice exposed to A. aegypti that developed BU. Eighty-eight percent of mice (21/24) subjected to contaminated tail needle puncture developed BU. Mouse tails coated only in bacteria did not develop disease. A median incubation time of 12 weeks, consistent with data from human infections, was noted. We then specifically tested the M. ulcerans infectious dose-50 (ID50) in this contaminated skin surface infection model with needle puncture and observed an ID50 of 2.6 colony-forming units. We have uncovered a biologically plausible mechanical transmission mode of BU via natural or anthropogenic skin punctures.

Vaccine-specific immune responses againstMycobacterium ulceransinfection in a low-dose murine challenge model

“…We have previously described the use of a low-dose tail infection model for studying insect-mediated transmission of M. ulcerans (10). We reasoned that because BU patients were likely to be initially infected with a low bacterial inoculum (10, 11, 59) we could use this model to test the protective efficacy of responses induced by the ER + R 4 Pam 2 Cys vaccine. This model features the use of a bioluminescent strain of M. ulcerans (10, 60) and its infectious characteristics are summarised in Fig.…”

“…A key feature of this model also allows the use of a significantly lower bacterial challenge dose compared to other studies (approximately 14-20 CFU compared to 10 4 -10 6 CFU) (25, 32-37, 49, 52-54) (Table 1) and enables measuring bacterial growth in the same animal over time. This lower dose is likely to be more physiologically relevant, in terms of reflecting the bacterial inoculum that occurs during M. ulcerans transmission to humans (10, 11, 59). Sporadic healing of BU disease was also seen in this model, an observation that has been noted in humans and other animals (29, 66-68).…”

“…Escherichia coli ClearColi©BL21 (DE3) containing the plasmid, pJexpress-ER (strain TPS847) was grown at 37°C in Luria-Bertani (LB) broth (Difco, Becton Dickinson, MD, USA) supplemented with 100 µg/ml ampicillin (Sigma-Aldrich, USA) to express the enoyl reductase (ER) protein (57). Log-phase bioluminescent Mycobacterium ulcerans (strain JKD8049 containing integrated plasmid pMV306 hsp:lux G13) (10, 60) was grown at 30°C in 7H9 broth or 7H10 agar (Middlebrook, Becton Dickinson, MD, USA) supplemented with oleic acid, albumin, dextrose and catalase growth supplement (OADC) (Middlebrook, Becton Dickinson, MD, USA), 0.5% glycerol (v/v) and 25 µg/ml kanamycin (Sigma-Aldrich, USA). M. bovis BCG (strain ‘Danish 1331’) used for vaccinations was grown at 37°C in 7H9 broth or 7H10 agar supplemented with OADC.…”

“…This can occur if a region of contaminated skin surface is punctured or by insertion of an object contaminated with the bacteria into subcutaneous tissue ( e.g. via an insect bite) (10-12). BU-endemic areas include certain regions of West and Sub-Saharan Africa and south eastern Australia (13, 14).…”

“…Previous research has shown that as few as three colony forming units (CFU) of bacteria are required to initiate infection (10), however most animal models challenge with >10 4 CFU (see Table 1) (25, 32-37, 49, 52-54). This dose is likely to be far higher than the dose of bacteria that leads to natural infection in humans and other animals (10). Such an unrealistic high dose may overwhelm immune responses and underestimate the true efficacy of potential M. ulcerans vaccines.…”