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.

Fig 1. Schematic representations of the two BU transmission models tested in this study.

(A) Model-1 tests transmission of M. ulcerans present on a skin surface following a puncturing injury created by mosquito blood-feeding or needle stick. (B) Visualization of bioluminescent M. ulcerans JKD8049 (harbouring plasmid pMV306 hsp:luxG13) [29, 30] on the mouse-tail in model-1, showing the distribution of bacteria immediately after coating for two mice, versus an uncoated animal. M. ulcerans culture concentration used for tail coating was 8.3x10⁵ CFU/mL.

Fig 2. Mechanical transmission of M. ulcerans.

(A) An example of the development of Buruli ulcer following mosquito blood-feeding through a skin surface (mouse-tail) contaminated with M. ulcerans. (B) Composite histological cross-section with Ziehl–Neelsen staining through the infected tail showing the focus of acid-fast bacilli (arrow) within the subcutaneous tissue. (C) Higher magnification view of the focus of infection, with the yield of viable M. ulcerans obtained from the infected tissue. Panels (D)–(F) show the same analyses as for the mosquito-bitten mouse #182, but for a mouse developing a lesion following sterile needle-stick puncture through a contaminated skin surface (mouse #201).

Fig 3. Summary of M. ulcerans burden on mosquitoes post-feeding on contaminated mouse-tails.

(A) Visualization of the mean number of M. ulcerans detected per dissected mosquito segment, as assessed by IS2404 qPCR and expressed as genome equivalents (GE). ‘N’ indicates the total number of mosquitoes tested. Red-shaded mosquitoes transmitted M. ulcerans, leading to mouse-tail lesions. Green-shaded mosquitoes blood-fed on mouse-tails but lesions did not develop. (B, C, D) Plots of the individual qPCR results for each mosquito segment, listed by experiment. Red dots correspond to qPCR bacterial load for mosquitoes that transmitted M. ulcerans infection. Null hypothesis (no difference in means) was rejected (p<0.05)* (Kruskal-Wallis test). Horizontal bar indicates the mean bacterial load per mosquito and error bars depict standard deviation. The y-axis is GE and x-axis is experiment. The qPCR data for individual insects is contained in S1 Table.

Fig 4. M. ulcerans incubation period and infectious dose₅₀.

(A) Incubation period of M. ulcerans based on the time between sterile-needle puncture of an M. ulcerans contaminated mouse-tail and first observation of a lesion. (B) Estimated M. ulcerans ID50 for contaminated skin surface transmission model. Dashed lines indicate 95% confidence intervals. Dotted lines depicts ID50.