Characteristics of the case-patients and control participants are shown in Table 1. Both sexes were equally affected by BU. Most case-patients were elderly adults, and their median age was higher than that of the control participants (p = 0.01). Among case-patients, ≈69% reported that they were either permanent residents of Point Lonsdale or had visited the town ≈3 months before onset of symptoms. Personal health factors such as cancer, immunosuppressive medication taken within the previous year, or having had an M. bovis BCG vaccination did not alter the odds of having BU.

Table 2 displays the participants’ behavioral and lifestyle choices on the Bellarine Peninsula. Wearing insect repellent (OR 0.38, 95% CI 0.20–0.71) or wearing long trousers when outdoors (OR 0.51, 95% CI 0.27–0.97) were each found to reduce the odds of having BU. Immediately washing a wound sustained outdoors was also found to decrease the odds for disease (OR 0.47, 95% CI 0.24–0.94). Owning a household pet or using gardening products such as fertilizer, potting mix, and topsoil was not found to be associated with BU. Participants were also assessed on whether they frequented any of 8 prominent lakes or marshes in Point Lonsdale or St Leonards. Case-patients were more likely than control participants to have visited a small ornamental lake at the western edge of Point Lonsdale during autumn (p = 0.04), but associations with all the other water areas surveyed were not statistically significant.

The extent of insect exposure on the Bellarine Peninsula is shown in Table 3. The odds more than doubled if participants reported that they had received mosquito bites on commonly exposed sites of the body, i.e., lower arms (OR 2.56, 95% CI 1.23–5.33) and legs (OR 2.51, 95% CI 1.18–5.31). The same biting pattern was not noted in response to the same questions regarding bites from March flies and sandflies.

A variety of outdoor activities were surveyed, including beach activities, freshwater or salt water swimming and fishing, surfing, sailing, bushwalking, lawn bowling, golf, bird watching, cycling, and gardening. None of these activities was associated with BU.

A backward elimination technique was applied to further investigate all significant factors. In a multivariate model, after adjusting for age and location, insect repellent use (OR 0.37, 95% CI 0.19–0.69) and being bitten by mosquitoes on the lower legs (OR 2.60, 95% CI 1.22–5.53) were found to be independently associated with BU.

BU occurs in focal outbreaks in >30 countries worldwide. Although the disease has been long neglected (21), substantial progress has been recently made and includes the development of rapid diagnosis by PCR (19), the discovery of the lipid toxin that explains the destructive nature of the disease (22), and the identification of the virulence plasmid that harbors the genes that produce the toxin (23). Also, increasing evidence supports the use of drug therapy for BU (24–26), which until recently has been regarded as a disease that would respond only to surgery. However, the ecologic factors driving the increasing incidence of BU in different regions of the world and the mode of transmission to humans who live in BU-endemic areas remain to be determined.

One of the major differences between BU in southeastern Australia and BU in sub-Saharan Africa is the proportion of older persons affected. Of the total number of cases linked to the Bellarine Peninsula, 33 (67%) case-patients were >60 years of age, whereas many studies from Africa report that most BU cases occur in children <15 years of age (3,6,7,27). A high proportion of the permanent residents of the Bellarine Peninsula are older Australians who have relocated to the coast for their retirement. However, especially during summer, young families visit the area and increase the local population considerably. Although at least 9 children or teenagers have acquired BU linked to the Bellarine Peninsula, advanced age may be an independent risk factor for BU, as was reported recently in Benin (28,29).

Our results suggest that protecting the body from environmental exposure appears to reduce the odds for disease. These measures include wearing long trousers and immediately washing wounds after sustaining minor skin trauma; such measures have also been identified as protective factors against BU in west Africa (3,13,30). These findings support the established hypothesis of M. ulcerans transmission, which postulates that direct contact with the skin is required for transmission to occur. Negative findings with regard to animal exposure factors (domestic pets and wildlife) also make it unlikely that contact with these animals is a risk factor for infection with M. ulcerans on the Bellarine Peninsula, although the bacterium has been isolated from skin ulcers in several wild animals near the original Bairnsdale region and other BU-endemic areas in Victoria (31,32).

Only 1 case-patient in this study reported having had direct physical contact with environmental water (other than the ocean) from a BU-endemic town, and only 14 (2%) of control participants reported having swum in fresh water on the Bellarine Peninsula (data not shown). Case-patients were more likely than control participants to have visited only 1 of our surveyed water areas, a small park reserve (Table 2), although the low numbers of positive responses to this question limited our ability to draw further insight from these data. A small percentage of participants reported having spent time near marshy areas on the Peninsula, which suggests that transmission is most likely to occur indirectly or that M. ulcerans has spread to areas outside of its presumed aquatic environment. Previous studies conducted in BU-endemic African countries found that case-patients were more likely to report that they regularly immersed themselves in, or worked along, rivers or marshy areas (12). On the Bellarine Peninsula, contact with lakes and rivers is not a large part of the lifestyle because recreational swimming and boating activities are largely based around coastal saltwater areas. This difference in lifestyle could also possibly explain the lower incidence rate of BU in Australia than in the highly affected African regions.

More case-patients than control-participants reported having been bitten by mosquitoes on the distal areas of their arms and legs, the sites most commonly exposed to the environment. Although the responses to our insect-exposure questions suggest an association between mosquito bites and M. ulcerans, this association may be just a nonspecific marker of environmental exposure. However, use of insect repellent remained an independent predictor of reduced risk, which suggests that insects themselves may play a role in transmission. Although no case-control study can establish causation, our findings are consistent with the hypothesis that mosquitoes play a role in transmission of M. ulcerans to humans, at least in southeastern Australia. In a previous outbreak of BU in Australia in the 1990s, contaminated aerosolized water droplets that could be inhaled or ingested were suggested as a means of infection for persons who did not have direct contact with the likely point source. Although mosquitoes and other insects were not tested during that outbreak, they also could have played a role in transmission (1,33).

We acknowledge several limitations in our study. Reliability of patient recall may be biased because some BU cases date back to 1998. However, the median time between the date of completing the questionnaire and the time of diagnosis of BU was 1.51 years (interquartile range 0.90–3.29 years). Avoidance of recall bias is impossible because the incubation period varies by several months, and delay between first noticing a skin lesion and the diagnosis of BU is often substantial (34,35). Studies with more cases would help validate and refine the protective and risk factors identified in this study.

Despite many differences between the BU-endemic areas in southeastern Australia and western Africa, case-control studies in these diverse environments produce surprisingly concordant results. Direct contact with the environment appears to be a universal risk factor for acquisition of BU, and use of protective clothing appears to reduce this risk. In this study we also established a new risk factor—exposure to mosquitoes. Our data support an association between mosquitoes and M. ulcerans on the Bellarine Peninsula during the outbreak and demonstrate that BU case-patients were likely to have been bitten by mosquitoes. We hypothesize that in this BU-endemic region, mosquitoes become colonized or passively contaminated when they come into contact with M. ulcerans in the environment and then transmit it to humans living nearby. However, before introducing specific public health interventions, such as mosquito control and campaigns to encourage the use of repellents and protective clothing for those who live in BU-endemic areas, the presence of M. ulcerans in mosquitoes (36–38) from the BU-endemic area should be confirmed and transmissibility under laboratory conditions should be demonstrated.