Mycobacterium ulcerans in Mosquitoes Captured during Outbreak of Buruli Ulcer, Southeastern Australia

Mosquitoes positive for M. ulcerans were linked to outbreaks of Buruli ulcer in humans.

Buruli ulcer (BU) occurs in >30 countries. The causative organism, Mycobacterium ulcerans, is acquired from the environment, but the exact mode of transmission is unknown. We investigated an outbreak of BU in a small coastal town in southeastern Australia and screened by PCR mosquitoes caught there. All cases of BU were confi rmed by culture or PCR. Mosquitoes were trapped in multiple locations during a 26-month period. BU developed in 48 residents of Point Lonsdale/Queenscliff and 31 visitors from January 2001 through April 2007. We tested 11,504 mosquitoes trapped at Point Lonsdale (predominantly Aedes camptorhynchus). Forty-eight pools (5 species) were positive for insertion sequence IS2404 (maximum likelihood estimate 4.3/1,000), and we confi rmed the presence of M. ulcerans in a subset of pools by detection of 3 additional PCR targets.
B uruli ulcer (BU), also known as Bairnsdale ulcer (1), Daintree ulcer (2), and Mossman ulcer in Australia, is an emerging disease of skin and soft tissue with potential to cause scarring and disability (3). It is caused by Mycobacterium ulcerans (4), an environmental pathogen that produces a destructive polyketide toxin, mycolactone (5); the genes for the production of this toxin are encoded on newly described plasmid pMUM001 (6). BU occurs in >30 countries worldwide, but it affects mainly children in sub-Saharan Africa, where it is now more common than tuberculosis and leprosy in some regions (7). This disease occurs in people of all ages and races who live in or visit BU-endemic areas, but the precise mode of transmission remains unknown.
Analysis of the recently sequenced M. ulcerans genome has shown that in addition to pMUM001, there are unusually high copy numbers of 2 independent insertion sequences (IS2404 and IS2606) and a high incidence of pseudogene formation (8). These data suggest that M. ulcerans is unlikely to be free-living in the environment but is instead undergoing adaptation to a specifi c ecologic niche in which the products of some ancestral genes are no longer essential. One such niche may be in aquatic insects because M. ulcerans has recently been reported to colonize the salivary glands of carnivorous water bugs (Naucoridae) under laboratory conditions (9), and mycolactone production appears to be necessary for this colonization (10). Studies from disease-endemic areas in Africa have reported that farming activities near rivers (11) and swimming in rivers or marshes (12) may be risk factors for BU; bites from contaminated water bugs may transmit the infection.
In temperate southeastern Australia, outbreaks of M. ulcerans infection occur in localized areas, but few patients report direct contact with environmental water other than the ocean, which led to the proposal that aerosols from contaminated water may cause human infections (13). However, these low-lying disease-endemic areas also harbor large populations of mosquitoes, and some patients have reported that BU fi rst appeared at the site of what may have been a mosquito bite ( Figure 1). These observations, and

Mycobacterium ulcerans in Mosquitoes Captured during
Outbreak of Buruli Ulcer, Southeastern Australia knowledge from fi eld studies in Africa implicating insects as either a reservoir or mode of transmission, led us to capture and screen mosquitoes during our investigation of a large outbreak of BU in humans in a small coastal town in southeastern Australia (Point Lonsdale), ≈60 km south of Melbourne ( Figure 2).

Outbreak Investigation
M. ulcerans infection has become increasingly common in the southern Australian state of Victoria since the early 1990s (14,15) and characteristically causes localized outbreaks (16). In 1995, a research group at the Royal Children's Hospital in Melbourne developed an IS2404 PCR to improve speed and accuracy of diagnosis of BU (17). This method has now become the initial diagnostic method of choice in Australia and elsewhere (18). All PCR-and culture-positive cases of M. ulcerans infection in Victoria have been unoffi cially reported to the Victorian Department of Human Services (DHS) since the 1990s, and investigators from DHS began to routinely contact and interview all new reported case-patients in 2000. All new cases of M. ulcerans infection were made legally reportable in Victoria in January 2004 (19).

Case Defi nition
For this study, a case of BU was defi ned as a patient with a suggestive clinical lesion from which M. ulcerans was identifi ed by PCR or culture from a swab or tissue biopsy specimen from January 2002 through April 2007; the patient must have been either a resident of, or a visitor to, Point Lonsdale or Queenscliff (adjacent coastal towns on the Bellarine Peninsula) who did not report a recent history of contact with another known BU-endemic area. Australian Bureau of Statistics data derived from the 2001 Australian Census for Point Lonsdale/Queenscliff (postcode 3225) were used to obtain the resident population numbers and age distribution in the outbreak area (20).

Mosquito Trapping
A total of 8-13 overnight mosquito traps were placed at Point Lonsdale on 22 occasions from December 2004 through January 2007. Adult mosquito sampling was conducted with CO 2 -baited miniature light traps (21). Traps were 2-L, cylindrical, insulated containers designed to hold CO 2 pellets that continuously produce CO 2 , which then diffuses through holes in the bottom of the container. A small electric light and fan at the base of the container defl ected attracted mosquitoes into a holding container. The traps were set before dusk and then retrieved several hours after dawn the next morning. The catches were transported to Primary Industries Research in Attwood, Victoria, where they were counted, sorted, and pooled by sex and species. Mosquito species were identifi ed by using the key of Russell (22). All captured mosquitoes were tested except in  sex and species by using the FastDNA Kit (revision no. 6540-999-1D04) and the FastPrep Instrument (Qbiogene Inc., Irvine, CA, USA) according to the manufacturer's instructions. We adapted fl uorescence-based real-time PCR technology to screen mosquitoes for 3 M. ulcerans-specifi c DNA sequences as described (23). Briefl y, oligonucleotide primers and TaqMan MGB probes (Applied Biosystems, Foster City, CA, USA) labeled with fl uorescent dyes 6FAM or VIC were designed that targeted 3 independent highcopy number repetitive sequences (IS2404 and IS2606 [24] and the ketoreductase B domain [KR] from pMUM001 [6]). The copy number of these targets per bacterial cell in the published sequenced of M. ulcerans, to which the outbreak strain is phylogenetically closely related, is 213 for IS2404, 91 for IS2606, and 30 for KR (8). Assays were conducted with an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).
Each pool was fi rst tested for IS2404 with an internal positive control to test for PCR inhibition and separate negative and positive controls. Samples were considered positive for a given target when they had a result above a previously determined critical threshold (23). Pools that were positive for IS2404 were then screened in duplicate with confi rmatory assays to detect IS2606 and KR. For pools with suffi ciently high signal strength, amplifi cation and sequencing of variable number tandem repeat (VNTR) locus 9 were conducted by using a nested PCR. The fi rst round PCR used 2 primers, MUVNTR9NF (5′-ACTGCCCAGA-CATGGCGA-3′) and MUVNTR9NR (5′-ACGCGAG-GTGGAACAAAGC-3′), designed to fl ank the published VNTR locus 9 primer. First-round PCR products were used as template for a second-round PCR performed as described by Ablordey et al. (25). PCR products of the expected size were sequenced by using the BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems) according to the manufacturer's instructions. Precipitated reaction products were tested in a 3730S Genetic Analyzer (Applied Biosystems) (23). The maximum likelihood estimate (MLE) per 1,000 mosquitoes tested (bias corrected MLE) was calculated by using software recommended for this purpose by the Centers for Disease Control and Prevention (Atlanta, GA, USA) (26).

Description of Outbreak
The climate in Point Lonsdale is temperate with a mean daily maximum temperature of 12.8°C in July (winter) and 22.4°C in January (summer). Average annual rainfall is 660 mm and is spread throughout the year (e.g., average 41.3 mm in January and 59.1 mm in July) (27). Most of the town is low-lying and close to sea level, and there are several natural and human-made swamps and water features in the vicinity ( Most case-patients were adults and many were elderly ( Figure 5), although 14 of the 79 were children <18 years of age. Among visitors, there was a bimodal age distribution, with relatively low numbers of adults 20-50 years of age. An estimate of the age-specifi c attack rate for residents of Point Lonsdale/Queenscliff was obtained with reference to the 2001 Australian census. Because census data were not available for the 2 towns separately, the calculation assumes that the age distribution of Point Lonsdale and Queenscliff is similar. A similar analysis for visitors was not performed because appropriate denominators could not be determined. The risk appeared to increase strongly with age and was ≈7× higher for those >55 years of age than in those <55 years of age (p<0.001) ( Figure 6).
The incubation period for residents and for most visitors could not be assessed because exposure to the BU-en- Her case was diagnosed and reported 7 months after this exposure.

Mosquito Testing
A total of 23,692 mosquitoes were captured in Point Lonsdale during a 25-month period; 96% were Aedes camptorhynchus (Thomson). Twelve other species comprised the remaining 4% (Table 1). Of 11,504 mosquitoes tested, 48 pools were positive for IS2404; of these, 13 pools were also positive for KR and IS2606. Forty-one of 48 pools were female Ae. camptorhynchus, 4 were positive pools of Coquillettidia linealis (Skuse), and 1 each were Anopheles annulipes (Walker s.l.), Culex australicus (Dobrotworsky and Drummond), and Ae. notoscriptus (Skuse). For 2 positive pools with particularly high M. ulcerans DNA concentrations, VNTR locus 9 was amplifi ed and the sequence was identical to that of the local outbreak strain cultured from case-patients (23).
Thirty-fi ve IS2404-positive pools did not contain IS2606 and KR. However, the cycle threshold (Ct) values for IS2404 were lower for those pools that did have IS2606 and KR, which suggested that failure to detect KR and IS2606 in some pools was caused by low DNA concentration, rather than lack of specifi city for M. ulcerans. This fi nding is consistent with known differences in copy number per cell of targets used for PCR screening and confi rmation (23). A total of 124 pools of mosquitoes that were negative for IS2404 by PCR were screened with probes for KR and IS2606. None were positive, which indicated that these 2 loci are consistently linked to IS2404 and do not occur independently.
The MLE (bias corrected) for all mosquitoes over the entire testing period at Point Lonsdale was 4.

Mosquito Numbers, Proportion PCR Positive, and Reporting of BU
Trapping was conducted at Point Lonsdale between December 2004 and January 2007. Mosquito numbers varied during the period, and traps were not set when local reports suggested low mosquito numbers (online Appendix Figure, available from www.cdc.gov/EID/content/13/11/1653-appG.htm). There appeared to be a qualitative relationship between PCR-positive mosquitoes in spring and summer (September-February) and reporting of new cases of human disease in autumn and winter (March-August). The exposure-to-reporting interval is typically longer than the actual incubation period because patients do not always seek medical assistance immediately and doctors do not always diagnose BU when a patient is fi rst seen (28).

Mosquitoes Caught at Other Locations in Victoria
To test that the observed association between M. ulcerans and mosquitoes only occurs in outbreak areas, we tested 3,385 mosquitoes from several inhabited areas with lower BU endemicity than Point Lonsdale. Ae. camptorhynchus (48%), but all pools were negative for IS2404. When analyzed together, an association was observed between degree of endemicity and probability of trapping mosquitoes that are positive by PCR for M. ulcerans (Table 2), but this association did not show statistical signifi cance (p = 0.07).

Discussion
To our knowledge, the outbreak of BU in Point Lonsdale is the largest ever recorded in Australia and has now caused more than twice as many cases as the well-described outbreak at Phillip Island a decade earlier (16,29). A striking feature of both outbreaks is their intensely localized nature. We identifi ed 79 cases that were epidemiologically linked to Point Lonsdale and the western edges of Queenscliff, but the town of Queenscliff, only 4 km to the east along the same beach, has so far remained disease free. The cumulative attack rate for both towns is estimated to be 1.2% of the resident population, but it could be up to twice as high if only the population of Point Lonsdale, where all transmission appears to have occurred, were considered. Although Queenscliff remains unaffected, the nearby towns of Barwon Heads and Ocean Grove, ≈12 km west of Point Lonsdale, began reporting their fi rst cases in 2005.
The fi rst case at Point Lonsdale was reported in January 2002. In 2004, the outbreak increased in intensity and began to involve visitors as well as residents, which suggested that environmental contamination with M. ulcerans has steadily increased over 5 years. Among local residents, we found a higher attack rate in the elderly, with 3.7% of residents of Point Lonsdale/Queenscliff >75 years of age with BU. The reasons for this age distribution are not known, but increasing risk with age could be caused by an age-related immune defect or an unrecognized behavioral factor. Among visitors, there was a pronounced bimodal age distribution, which probably represents a skewing of the exposed population (e.g., young children going to stay with their retired grandparents over the summer while their parents stayed at work) but may also refl ect increased susceptibility in young persons. This bimodal pattern, which included increased incidence in young persons and the elderly, has also been reported in Africa (30).
During our investigations at Point Lonsdale, we focused initially on several marshy areas and obtained positive PCR results for plant material from 2 small ornamental lakes and soil from storm water drains (23). However, case-patients did not report direct contact with these lakes or drains (these sources of water are not used for swimming or wading). Thus, how people were exposed is not clear. In Figure 6. Estimated age-specifi c attack rates of Buruli ulcer for residents of Point Lonsdale/Queenscliff, Australia (postcode 3225). Values above the bars are cases per total no. residents in each age group. an outbreak in Phillip Island, many cases were clustered around a newly formed wetland and a golf course irrigation system, and we proposed transmission from these sites by aerosol (16,29). However, this hypothesis may not be supported by our new evidence, which suggests that M. ulcerans may not be free-living in the environment but may have adapted to specifi c niches within aquatic environments, including salivary glands of some insects. Thus, we investigated whether M. ulcerans could be detected in mosquitoes, which had been reported in higher than usual numbers at Point Lonsdale. We also investigated behavior in a case-control study (the subject of a separate report), which found that being bitten by mosquitoes increased the odds of having BU (31). A total of 14,889 mosquitoes obtained over a 25month period (11,504 from Point Lonsdale) were tested for M. ulcerans by using a highly sensitive and specifi c realtime PCR (23). We used PCR because direct culture of M. ulcerans from the environment is extremely diffi cult and was only achieved when IS2404 PCR screening of environmental samples accurately directed researchers to specifi c microenvironments that include water insects and aquatic plants (32). Although IS2404, IS2606, and the mycolactone-producing virulence plasmid have been detected in mycobacteria other than M. ulcerans (33)(34)(35), identifi cation of these targets in expected relative proportions and the VNTR locus 9 sequence identical to that of the outbreak strain in a subset of mosquito pools with suffi ciently high DNA concentrations confi rms that we identifi ed the outbreak strain (23).
We also demonstrated that over a 2-year cycle at Point Lonsdale absolute numbers of mosquitoes and PCR-positive mosquitoes increased in spring and summer followed by a cluster of new human cases in autumn and winter. This pattern is consistent with recent point estimates that suggest the incubation period for BU in Australia is 3-7 months (2 cases) (36) and 1-4 months (3 cases) (28), and that an additional 1-6 weeks may elapse before cases are diagnosed and reported (28).
The predominant species trapped was Ae. camptorhynchus; however, identifi cation of M. ulcerans in 4 other spe-cies suggests that M. ulcerans contamination of mosquitoes is not species specifi c. Ae. camptorhynchus is a salt marsh species, an aggressive biter, and a major pest in coastal areas of southeastern Australia that has been linked to transmission of Ross River virus. The mosquito appears in large numbers after rain as minimum temperatures begin to increase, with a lag time of ≈1 month (37). Of the other species from which at least 1 PCR-positive pool was identifi ed, An. annulipes and Cq. linealis are fresh water species (38). Ae. notoscriptus is a peridomestic species that breeds in containers (e.g., in roof gutters) (39), can transmit dog hookworm, and has a limited fl ight range (e.g., <200 m) (40). In contrast, Cx. australicus may have a fl ight range of many kilometers (41). A limited number of other biting or aquatic insects were also tested and none were positive for M. ulcerans. However, larger numbers must be screened before it can be concluded that they do not transmit M. ulcerans.
Our results do not demonstrate viability or transmissibility of M. ulcerans at the time mosquitoes were captured, and the method we used does not answer questions about location of M. ulcerans within the insect. Because M. ulcerans is an environmental pathogen, PCR-positive mosquitoes may only be indicators of its presence in the environment and not linked to transmission. The Ct values obtained for mosquito pools suggest that only 10-100 organisms were present per positive pool, which is more consistent with organisms being acquired on outer surfaces of mosquitoes when resting or feeding in storm water drains (23), rather than mosquitoes being a true productive reservoir and vector. However, if some bacterial cells were present on the proboscis, they could have been injected beneath the keratin layer during feeding. Although the inoculum size required to cause a human infection is unknown, the long incubation period suggests a low initial inoculum. Our fi ndings do not demonstrate that mosquitoes are responsible for transmission, but this possibility should be investigated. Studies are underway to artifi cially infect mosquito larvae with M. ulcerans and initiate infection in a mouse model, as has been conducted with naucorids (9).
Although our fi ndings may not apply to the situation in Africa, the close genetic relationship of Australian isolates