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Ecology and Transmission of Buruli Ulcer Disease: A Systematic Review
Article  in  PLoS Neglected Tropical Diseases · December 2010
DOI: 10.1371/journal.pntd.0000911 · Source: PubMed
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Ecology and Transmission of Buruli Ulcer Disease:
A Systematic Review
Richard W. Merritt1*, Edward D. Walker2, Pamela L. C. Small3, John R. Wallace4, Paul D. R. Johnson5, M.
Eric Benbow6, Daniel A. Boakye7
1 Department of Entomology, Michigan State University, East Lansing, Michigan, United States of America, 2 Department of Entomology and Microbiology and Molecular
Genetics, Michigan State University, East Lansing, Michigan, United States of America, 3 Department of Microbiology, University of Tennessee, Knoxville, Tennessee,
United States of America, 4 Department of Biology, Millersville University, Millersville, Pennsylvania, United States of America, 5 Austin Health, Melbourne, Australia,
6 Department of Biology, University of Dayton, Dayton, Ohio, United States of America, 7 University of Ghana, East Legon, Ghana
Abstract
Buruli ulcer is a neglected emerging disease that has recently been reported in some countries as the second most frequent
mycobacterial disease in humans after tuberculosis. Cases have been reported from at least 32 countries in Africa (mainly
west), Australia, Southeast Asia, China, Central and South America, and the Western Pacific. Large lesions often result in
scarring, contractual deformities, amputations, and disabilities, and in Africa, most cases of the disease occur in children
between the ages of 4–15 years. This environmental mycobacterium, Mycobacterium ulcerans, is found in communities
associated with rivers, swamps, wetlands, and human-linked changes in the aquatic environment, particularly those created
as a result of environmental disturbance such as deforestation, dam construction, and agriculture. Buruli ulcer disease is
often referred to as the ‘‘mysterious disease’’ because the mode of transmission remains unclear, although several
hypotheses have been proposed. The above review reveals that various routes of transmission may occur, varying amongst
epidemiological setting and geographic region, and that there may be some role for living agents as reservoirs and as
vectors of M. ulcerans, in particular aquatic insects, adult mosquitoes or other biting arthropods. We discuss traditional and
non-traditional methods for indicting the roles of living agents as biologically significant reservoirs and/or vectors of
pathogens, and suggest an intellectual framework for establishing criteria for transmission. The application of these criteria
to the transmission of M. ulcerans presents a significant challenge.
Citation: Merritt RW, Walker ED, Small PLC, Wallace JR, Johnson PDR, et al. (2010) Ecology and Transmission of Buruli Ulcer Disease: A Systematic Review. PLoS
Negl Trop Dis 4(12): e911. doi:10.1371/journal.pntd.0000911
Editor: Richard O. Phillips, Kwame Nkrumah University of Science and Technology (KNUST) School of Medical Sciences, Ghana
Received July 21, 2010; Accepted November 11, 2010; Published December 14, 2010
This is an open-access article distributed under the terms of the Creative Commons Public Domain declaration which stipulates that, once placed in the public
domain, this work may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose.
Funding: The authors acknowledge the following research support from: grant no. R01TW007550 from the Fogarty International Center through the National
Institutes of Health/National Science Foundation Ecology of Infectious Diseases Program; McCord Research Buruli Ulcer Foundation, Iowa City, Iowa; UBS Optimus
Foundation, Zurich, Switzerland; PASSHE Faculty Professional Development Council and Millersville University Faculty Grants, Millersville, PA, and the World Health
Organization, Geneva, Switzerland. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: merrittr@msu.edu
Introduction disease found in rural areas located near wetlands (ponds, swamps,
marshes, impoundments, backwaters) and slow-moving rivers,
Buruli ulcer (BU) is a serious necrotizing cutaneous infection especially in areas prone to flooding [3,4,23,27,29,31–36] (Fig. 2).
caused by Mycobacterium ulcerans [1–7]. Before the causative agent Cases have been reported from at least 32 countries in Africa
was specifically identified, it was clinically given geographic (mainly west), Australia, Southeast Asia, China, Central and South
designations such as Bairnsdale, Searles, and Kumasi ulcer, America, and the Western Pacific [3,6,20,28,37,38] (Fig. 3). A
depending on the country [8–11]. BU is a neglected emerging number of cases have been reported in non-endemic areas of North
disease that has recently been reported in some countries as the America and Europe as a sequel to international travel [20,39–42].
second most frequent mycobacterial disease in humans after Buruli ulcer disease is often referred to as the ‘‘mysterious disease’’
tuberculosis (TB) [12–14]. Large lesions often result in scarring, because the mode of transmission remains unclear, although several
contractual deformities, amputations, and disabilities [2–4,7,14– hypotheses have been proposed. The objectives of this article are to:
22] (Fig. 1). Approximately 80% of the ulcers are located on the 1) review the current state of knowledge on the ecology and
limbs, most commonly on the lower extremities yet some variation transmission of M. ulcerans, 2) discuss traditional and non-traditional
exists [3,13,23,24]. In Africa, all ages and sexes are affected, but methods for investigating transmission, and 3) suggest an intellectual
most cases of the disease occur in children between the ages of 4– framework for establishing criteria for transmission.
15 years [5,13,17,25–28].
BU is a poorly understood disease that has emerged dramatically Methods
since the 1980’s, reportedly coupled with rapid environmental
change to the landscape including deforestation, eutrophication, Data Sources and Search Strategy
dam construction, irrigation, farming (agricultural and aquacul- Selection of the publications cited was based on the following
ture), mining, and habitat fragmentation [3–7,29,30]. BU is a approaches: 1) Direct knowledge of the authors of this manuscript
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Buruli Ulcer Disease Review
Author Summary Centers for Disease Control (CDC); 3) Knowledge in the field of
Buruli Ulcer research in that three of the authors (Merritt, Small,
Buruli ulcer (BU) is a serious necrotizing cutaneous Johnson) are on the WHO Technical Advisory Committee for
infection caused by Mycobacterium ulcerans. It is a Buruli Ulcer in Geneva, Switzerland; 4) Review of the following
neglected emerging disease that has recently been websites: Buruli ulcer disease maintained by WHO in Geneva,
reported in some countries as the second most frequent Switzerland (http://www.who.int/buruli/en), The Buruli Ulcer
mycobacterial disease in humans after tuberculosis (TB). Disease Ecology Research Consortium (BUDERC) (https://www.
Cases have been reported from at least 32 countries in msu.edu/,budiseco/index.html); and UBS Optimus Foundation
Africa (mainly west), Australia, Southeast Asia, China, (http://www.stopburuli.org).
Central and South America, and the Western Pacific. BU
is a disease found in rural areas located near wetlands
(ponds, swamps, marshes, impoundments, backwaters) Results and Discussion
and slow-moving rivers, especially in areas prone to
The Pathogen
human-made disturbance and flooding. Despite consider-
able research on this disease in recent years, the mode of M. ulcerans is a slow-growing environmental mycobacterium that
transmission remains unclear, although several hypotheses can be isolated from primary lesions after a 5–8 week incubation
have been proposed. In this article we review the current period, although up to 6 months may be required [43,44]. M.
state of knowledge on the ecology and transmission of M. ulcerans falls into a group of closely related mycobacterial
ulcerans in Africa and Australia, discuss traditional and pathogens which comprise the M. marinum complex. The M.
non-traditional methods for investigating transmission, marinum complex contains mycobacterial species pathogenic for
and suggest an intellectual framework for establishing aquatic vertebrates and includes M. marinum (fish), M. pseudoschottsii
criteria for transmission. (fish) and M. liflandii (frogs) [45–48]. All of these species are
characterized by slow growth rates and low optimal growth
regarding their background in the field of Buruli Ulcer research temperatures [49]. From a genomic standpoint, the species in the
and knowledge of key papers and unpublished data; 2) Online M. marinum complex can be considered a single species based on
search engines for Buruli Ulcer and Mycobacterium ulcerans the fact that they share over 97% identity in the 16sRNA gene
(predominantly PubMed, ISI Web of Knowledge, Web of Science, sequence [50]. However, practical considerations have led to the
Figure 1. Buruli ulcer on leg and contractual deformity on wrist and hand. (Photo by R. Kimbirauskas).
doi:10.1371/journal.pntd.0000911.g001
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Buruli Ulcer Disease Review
Figure 2. Typical Buruli ulcer riverine endemic sites in Ghana and Benin, respectively. (Photos by M. E. Benbow and M. McIntosh,
respectively).
doi:10.1371/journal.pntd.0000911.g002
establishment of separate names based on differences in host virulence plasmid and accumulation of multiple copies of insertion
tropism and pathogenesis analogous to other mycobacterial sequences, IS2404 and IS2606. The genome has undergone
groupings, such as the M. avium and M. tuberculosis complexes. considerable reductive evolution through a number of mutational
Genomic analysis suggests that M. ulcerans evolved from an M. events including transposon insertion. As a result, the genome has
marinum-like ancestor [21,51] through the acquisition of a large accumulated over 700 pseudogenes [21,52]. Although it has been
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Buruli Ulcer Disease Review
Figure 3. A global map representing countries that have reported cases of Buruli ulcer disease as of 2009 (WHO).
doi:10.1371/journal.pntd.0000911.g003
reported that micro-aerophilic conditions enhance the growth of 30–33uC [56]. The restricted growth temperature of M. ulcerans is
M. ulcerans in the BACTEC system [53], the M. ulcerans genome thought to play a substantial role in the pathogenesis of BU by
strain lacks both nitrate and fumarate reductase systems, limiting infection to the skin. The organism has never been isolated
suggesting that M. ulcerans is considerably handicapped in the from internal organs of human patients or from bone in cases of
ability to grow under low oxygen conditions compared with M. osteomylelitis, or from the internal organs or blood of experimen-
marinum. The reported discrepancy in the oxygen requirements of tally infected animals [51,57–59]. It has been recently reported that
M. ulcerans may be due to strain differences and requires closer many isolates of M. ulcerans survive at 37uC for 13 days, although
investigation. A mutation in crtI, a key gene in the pathway for numbers decline after the first few days. No one has isolated or
carotinoid biosynthesis, is suggested to compromise the ability of derived a strain capable of growth at 37uC [60].
M. ulcerans to survive in direct sunlight [52]. A number of genes in The characteristic pathology of BU is mediated by a polyketide-
ion transport and lipid biosynthesis have been lost and the derived macrolide exotoxin called mycolactone, which is cytotoxic
repertoire of PE, PPE genes are considerably reduced compared and immunosuppressive [51,61,62]. Because of the large meta-
with M. tuberculosis or M. marinum. Taken together, these results bolic cost of producing mycolactone, it is likely that mycolactone
suggest that M. ulcerans is undergoing adaptation to a different and plays an important role in the survival and growth of M. ulcerans in
narrower niche than M. marinum. This idea has recently gained its environmental niche.
support from experimental work in which Medaka fish were
infected with M. marinum and M. ulcerans. In these studies, M. Ecology and Distribution of the Pathogen and Disease
marinum produced a lethal infection in Medaka, whereas M. ulcerans Detecting M. ulcerans in the environment. The slow
was not pathogenic and declined over a 23-week infection period growth rate of M. ulcerans and the complex mix of many faster
(L. Mosi, unpubl. data). growing bacteria and fungi in environmental samples have
The most important phenotypic characteristic of M. ulcerans is the prevented direct culture on artificial media of M. ulcerans from
low optimal growth temperature and the extremely restricted the environment. A major breakthrough in environmental studies
growth temperature range. M. marinum exhibits growth between 25– occurred with the development of the first PCR probes for M.
35uC, although the optimal growth temperature is 30–35uC [54,55] ulcerans based on detection of IS2404 by Ross et al. [63]. This
and many M. marinum isolates are capable of growth at 37uC. In technique was rapidly adopted by a number of laboratories leading
contrast, growth of M. ulcerans strains under laboratory conditions is to identification of M. ulcerans DNA in environmental samples
characterized by a remarkably narrow temperature range bet- including detritus, soil, biofilms, water filtrates, fish, frogs, snails,
ween 28–34uC and optimal growth of most strains is found between insects and other invertebrates [18,35,64–75].
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Buruli Ulcer Disease Review
Although IS2404 PCR has become the gold standard for sedimentation (e.g., turbidity) also would provide ultraviolet light
clinical diagnosis of Buruli ulcer, there are several caveats in (UV) attenuation and protection for M. ulcerans biofilm near the
applying these methods to environmental samples. First, PCR bottom substrates and on submerged plant surfaces as proposed by
detects DNA, not intact organisms. The death of infected Merritt et al. [30]. It has been documented that UV lowers M.
organisms will lead to the release of M. ulcerans DNA into the ulcerans cell viability [52], and thus deforestation and high-impact
environment where it may stick to a number of substrates. agriculture may promote increased nutrients, higher temperatures,
Although in two different countries in Africa, Williamson et al. UV attenuation and lower dissolved oxygen – environmental
[67] found M. ulcerans DNA in 9.7% (8/82) of water filtrant conditions that facilitate M. ulcerans growth.
samples and Vandelannoote et al. [59] found 7.7% (1/13) water Because of the association with freshwater habitats, Eddyani et
samples positive for M. ulcerans, the significance of these small al. [83] hypothesized that freshwater plankton, specifically
quantities of M. ulcerans in an environmental sample is difficult to protozoans, may act as reservoirs for M. ulcerans, or may even
evaluate. In southeastern Australia, M. ulcerans also has been facilitate the multiplication of the bacteria [18]. Although the
detected in a range of environmental samples. Recently, Fyfe et al. former authors did not detect M. ulcerans DNA in free-living
[76], reported that 30% of selected samples including detritus, amoebae collected BU endemic areas in Benin, this area of
plant material, suspended solids, and soil collected from one research definitely warrants further investigation.
highly-endemic area were weakly positive by quantitative PCR. Landscape ecology of the disease. Buruli ulcer has been
However, in a low endemicity area, only 4/156 (3%) of samples (2 widely associated with proximity to aquatic habitats. The disease is
soil, 2 terrestrial plant) were positive. Interpretation of results from rare in the savanna regions of West Africa and drier areas of
environmental PCR is complex. PCR methodology detects DNA, Australia. Its presence in Australia is notably costal however,
but it does not provide definitive proof for the presence of intact where water is often saline. This association between ecosystem
bacteria in a matrix. DNA bound to the surface of potential ecology and disease has not been quantified. Rather, the
vectors in the water column also will be detected. However, the association is most often anecdotal or related to specific human
successful culture of M. ulcerans from an aquatic water bug risk factors (e.g., wading, swimming, fishing, bathing, washing,
collected in Benin [71] provides definitive evidence for the farming, mining, etc.) in different countries and/or regional
presence of M. ulcerans in an aquatic invertebrate. This districts (see review below). To date, there have been few
considerable achievement was based on earlier observations using ecological studies focused on statistically determining why
IS2404 PCR that implicated aquatic water bugs as possible residence near certain water bodies is associated with BU,
reservoirs or vectors of M. ulcerans [70]. whereas the disease is absent along others [30,67,68]. For
Ecological associations with disturbed water bodies. example, BU is highly associated with residence along several
Until recently, a systematic and/or quantitative approach to the major river systems in both Benin and Ghana [12,14,20,84,85],
ecology of M. ulcerans in the environment has received little whereas disease is essentially non-existent in communities within a
attention, despite the fact that nearly all epidemiological studies few kilometers of Lake Volta, the largest water system in Ghana, as
have associated disease outbreaks with villages in close proximity well as along the Mono River in Benin. Williamson et al. [67]
to human-disturbed aquatic habitats, including both standing and recently found that in Ghana, PCR results suggesting that M.
moving water bodies [7,9–11,19,20,25,33,77–80]. Increased BU ulcerans and/or other mycolactone producing mycobacteria are
incidence has been reported in association with: 1) unprecedented widely distributed in water bodies in endemic and non-endemic
flooding of lakes and rivers during heavy rainfall [9,16,30,37,81]; villages. In these studies, however, the identification of endemic
2) the damming of streams and rivers to create impoundments and versus non-endemic sites was based on passive surveillance. A
wetlands [4,9,30,37]; 3) resorts that modify wetlands [16,30]; 4) community was considered endemic if a case had been identified
deforestation practices and increased agriculture leading to in the public health center in the past three years. A community
increased flooding [4,9,18,30,37]; 5) construction of agricultural that is not listed in the health center records, in association with a
irrigation systems [4,30,81]; 6) rice cultivation [4,9]; 7); alluvial, pit case of Buruli ulcer, was considered non-endemic. A preliminary
and sand mining operations [30,37,82]; and 8) population survey to validate the non-endemic status of several communities
expansion, resettlement and migration closer to water bodies in the GA district of Ghana through active surveillance showed
[9,16,18,27,30,37]. that Buruli ulcer cases could be indentified in nearly all of the
Indeed, many water bodies associated with increased sedimen- villages visited along the Densu River in the GA district (P. C.
tation and eutrophication have low dissolved oxygen concentra- Small, unpubl. data). In areas where much of the disease is not
tions that may enhance the growth of M. ulcerans [53]. Hayman [9] reported, this can lead to significant error in the designation of
speculated that in Australia M. ulcerans enters surface waters ‘‘non-endemic.’’
through deforestation, erosion and run-off contamination. He There have been case control studies and observational reports
suggested that populations of M. ulcerans were washed into aquatic of disturbed landscape associations with BU disease [29,30,86];
habitats where environmental conditions facilitated growth and however, there have only been a few recent studies to statistically
proliferation, much like an algal bloom. Because most infectious quantify landscape characteristics and relationships with disease
diseases have a strong correlation between infective dose and [36,79,81,87]. Duker et al. [79] found that arsenic levels in soil
incubation period for disease, Hayman [9] speculated that slow and gold mining were significant covariates related to increased
growth of M. ulcerans might be required for the bacteria to achieve disease risk in the Amansie West district of Ghana, while Wagner
population numbers sufficient to produce infection and the et al. [36,81] addressed larger scale land use/land cover
appearance of disease. The way in which M. ulcerans could be relationships using satellite imagery, GIS, and country wide BU
washed down into these habitats has never been explained, but is data from Benin. In the latter studies, Wagner et al. [36,81]
consistent with other reports of increased BU outbreaks associated reported highest disease in communities surrounded by an
with deforested and heavily flooded African lands [20,33]. agriculture matrix, and thus deforestation, with abundant wetlands
Further, deforestation leads to lost riparian cover, resulting in and other habitats that experience frequent flooding. These were
increased water temperatures that may facilitate M. ulcerans growth low-lying areas with complex topography far removed from urban
at optimal temperatures of 30–33uC [11,18,20]. Associated settings [36,81]. In another country-wide study using GIS, Brou
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Buruli Ulcer Disease Review
et al. [88] found that in Côte d’Ivoire, communities near factor in the analysis. Several of the commonly reported risk factors
landscapes of irrigated rice and other agriculture near dams used showed few consistent associations depending on the country, type
for irrigation were related to increased risk of BU. These studies of analysis conducted, use of different case definitions, and based on
confirm previous epidemiological studies and indicate that there the control populations used [89]. For instance, in a case-control
are quantifiable relationships between landscape features and land study from Ghana, Aiga et al. [25] found that swimming in rivers on
use that are related to BU disease. It is also clear that communities a habitual basis was a significant risk factor, whereas drinking,
involved with these activities are at high risk for disease, yet how cooking, washing clothing and bathing were not. However, in
specific activities are associated with transmission remains another Ghanaian study, wading, bathing, and swimming were all
unresolved. confirmed to be significant risk factors for BU [77]. Two studies
Risk factors associated with Buruli ulcer disease. found a decreased risk of infection with mosquito net use, while
Recently, Jacobson and Padgett [89] systematically reviewed the another study found no association between bed net use and
risk factors associated with M. ulcerans infection throughout the infection (Table 1). However, in a case control study performed in
world and concluded that poor wound care, failure to wear southeastern Australia, use of insect repellent was associated with
protective clothing, and living or working near water bodies were reduced risk and the reporting of mosquito bites on the forearms
commonly identified risk factors in most studies. However, a and lower legs was associated with increased risk [90]. Despite the
number of epidemiological studies have identified other potential association with water contact, fishermen were not found to be at
risk factors associated with M. ulcerans infection and these are high risk for the disease (Table 1). Although a review of these
summarized in Table 1. For each specific risk factor investigated, it potential risk factors suggests that transmission of M. ulcerans might
is stated as to whether or not there was an increased or decreased occur through direct inoculation of bacteria into the skin via contact
risk of infection reported, or if the factor was not considered a risk with environmental sources, insect bites or trauma, it was clear that
Table 1. A summary of reported risk factors associated with infection Mycobacterium ulcerans.
Increased Risk Decreased Risk Not Considered
Country Risk Factor(s) of Infection of Infection a Risk Factor Citation
Ghana 1) Arsenic-enriched drinking water (from mining) X Duker et al. (2004)
Ghana 1) Exposed skin X Raghunathan et al.
2) Bednet and mosquito coils use X 2005
3) Insect bites, cuts, scratches, and other wounds X
4) Exposure to riverine areas (wading and swimming) X
5) Association between BCG and vaccination or HIV infection X
6) Not wearing protective clothing
7) Fishing X
X
Ghana 1) Age 2–14 years of age X Aiga et al. 2004
2) Use of water for drinking, cooking, bathing, washing X
3) Association with agricultural activities X
4) Swimming in rivers X
Benin 1) 5–14 years of age X Debacker et al. 2004,
2) Unprotected water from swamps X 2006
3) BCG-vacinated patients .5 years old X
4) Participated in agricultural activities X
5) Sex X
Benin 1) Mosquito bed net use X Nackers et al. 2007
2) Association with agricultural activities X
3) Improper wound care X
Cameroon 1) Living near cocoa plantation or woods X Pouillot et al. 2007
2) Wading in swamps X
3) Wearing protective clothing while farming X
4) Association with agricultural activities X
5) Improper wound care X
6) Bed nets X
7) Mosquito coils X
8) Unprotected water sources X
9) Fishing X
Cote d9 Ivoire 1) Age group X Marston et al. 1995
2) Wearing protective clothing during farming activities X
3) Washing clothes X
4) Swimming X
5) Fishing X
Australia 1) Wearing protective clothing X Quek et al. 2007
2) Use of insect repellent X
3) Most patients . 60 years old X
4) Washing wounds after sustaining minor skin trauma X
5) Exposure to mosquitoes X
doi:10.1371/journal.pntd.0000911.t001
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additional comparative studies are required to clarify the potential natural infection model in which M. ulcerans-infected mosquito
modes of transmission of M. ulcerans [89]. larvae served as prey that were then fed to the predacious bugs,
Although there have been reports of a seasonal distribution in Mosi and colleagues confirmed Marsollier’s finding that infected
BU cases related to rainfall-influenced patterns of village water- belostomatid bugs could become infected with M. ulcerans via
body usage [32], and by season in southeastern Australia [91], feeding. However, they concluded that transfer of bacteria through
other studies have not shown this relationship [12]. Recording feeding was most likely to have occurred through contact with the
monthly trends for BU cases over a 3-year period in Benin, Sopoh heavily colonized raptorial arms and other external parts of the
et al. [12] found consistent average monthly BU case occurrence, belostomatid, rather than through saliva or contact with other
without an apparent seasonal trend. However, country-wide data internal organs as originally reported [66]. Together, these
can obscure local variation in climate and the issue of seasonal experiments indeed support the hypothesis that predaceous
trends needs to be more closely investigated at the local level. The aquatic insects may play an important role in maintaining M.
unknown incubation period for Buruli ulcer, which may vary from ulcerans within food webs in the aquatic environment [1,30,68,70]
2 weeks to 7 months [92,93], also makes it difficult to analyze but, as detailed below, their role in actual transmission to humans
seasonal factors with Buruli ulcer occurrence. Duker et al. [4], and remains unclear.
more recently Marion et al. [94], discussed seasonal variations and The role of other non-insect aquatic invertebrates as interme-
M. ulcerans infections reported from different countries and diate hosts or environmental reservoirs for M. ulcerans has been
concluded that there may be a temporal relationship between suggested by several authors [30,66,70,73,99], and recently
BU incidences and relatively dry periods; however, it also has been confirmed in more field research [67,68]. It was experimentally
reported that M. ulcerans infections occurred mainly after flooding confirmed that aquatic snails could be transiently colonized by M.
events [9,16,33,34,95]. ulcerans after feeding on M. ulcerans-containing aquatic plant
biofilms [64]. Aquatic plant extracts stimulated biofilm formation,
Environmental Reservoirs and Transmission and increased the uptake of labeled metabolites by M. ulcerans in
Africa. Unlike leprosy and tuberculosis, which are laboratory experiments [65]. In the field, Kotlowski et al. [73]
characterized by person-to-person transmission, it is hypothesized recorded M. ulcerans DNA in aquatic snails from endemic regions
that M. ulcerans is acquired through environmental contact. Direct of Ghana and Benin, and other studies have found that average
human to human transmission of M. ulcerans is extremely rare. The estimates of M. ulcerans increased by two orders of magnitude in
one reported case occurred following a human bite [96]. In this detritus compared to water [72]. More recently, Marsollier et al.
instance it was hypothesized that the patient’s skin surface was [104] described an extracellular matrix associated with the biofilm
contaminated with M. ulcerans from an environmental source (e.g. of M. ulcerans that may confer selective advantages to the
swamps) and driven into the skin by the playmate’s bite. Non- mycobacteria in colonizing various microhabitats in the environ-
human mammals and reptiles have been tested in the environment ment. Based on these studies and extensive environmental studies
without positive findings [95], and several arthropods (i.e., bedbugs, by Williamson et al. [67], it is evident that M. ulcerans DNA can be
black flies, mosquitoes) in Africa associated with vectoring other detected within biofilm on the plant surface, and as part of
disease agents tested negative in early studies [18,32]. However, few decaying organic matter (detritus) both of which serve as food for
organisms of each taxonomic group were tested in these studies, and certain aquatic invertebrates and fish, suggesting reservoirs and
insect sampling methods were neither systematically employed nor movement throughout the aquatic food web.
standardized. Buruli ulcer cases in wild and domesticated animals in A conceptual model, expanded and modified from Portaels
Africa have not been reported [97]. et al. [70], illustrating the potential reservoirs and movement of M.
Portaels and colleagues [70] were first to suggest that aquatic ulcerans within and among aquatic environments was detailed by
bugs (Hemiptera) might be reservoirs of M. ulcerans in nature, and Merritt et al. [30] and more recently by Marion et al. [94].
recently they described the first isolation in pure culture of M. Basically, M. ulcerans has been reported from mud, detritus, water
ulcerans from a water strider (Hemiptera: Gerridae, Gerris sp.) from filtrants, and plant biofilms, thereby allowing grazing or filtering
Benin [71]. A survey study [18] based on detection of M. ulcerans aquatic insects (e.g., midges and mosquito larvae) or other
DNA in aquatic insects (Hemiptera, water bugs; Odonata, invertebrates (snails, crustaceans, plankton) to concentrate myco-
dragonfly larvae; Coleoptera, beetle larvae) collected from African bacteria through their feeding activities. Then, predatory aquatic
BU endemic swamps confirmed their earlier findings, and vertebrates (i.e., some fish) and invertebrates (e.g., true bugs,
suggested that small fish might also contain M. ulcerans [66,98– beetles and dragonfly larvae) feed on other invertebrate prey or
100]. Marsollier et al. [64,66,98–100] conducted a series of small fish, serving to move M. ulcerans from prey to biting insects.
laboratory studies and demonstrated that M. ulcerans could survive Lastly, aquatic insects capable of flight, and birds that prey on fish
and show limited replication within the salivary glands of biting and/or aquatic invertebrates may potentially disseminate M.
aquatic bugs (Naucoridae: Naucoris cimicoides). In their experimental ulcerans to other aquatic environments [30].
model they demonstrated that M. ulcerans could be acquired from Although the potential for different aquatic invertebrates in
feeding on inoculated insect prey (a blow fly maggot), transmitted Africa to serve as environmental reservoirs for M. ulcerans has been
to mice via biting; and that the infected mice subsequently clearly demonstrated, direct transmission by biting water bugs,
developed clinical BU [66]. Although there has been some other than by purely accidental means appears very unlikely for
controversy regarding the interpretation of this work [68,101,102] the following reasons. First, in Africa M. ulcerans DNA has only
and subsequent follow-up studies on tracing the pathogen through been detected in invertebrates that are not hematophagous.
the bug [103,104], Marsollier and colleagues concluded that biting Predatory semi-aquatic Hemiptera (i.e., Naucoridae, Belostoma-
water bugs belonging to the families Naucoridae (creeping water tidae, Notonectidae) mainly feed on invertebrates (aquatic insects,
bugs) and Belostomatidae (giant water bugs) could be considered Crustacea, snails) by inserting their piercing mouth parts into their
reservoirs, and most importantly could serve as vectors in the prey, injecting saliva containing proteolytic enzymes, and then
transmission of M. ulcerans to humans in nature. More recently, imbibing the liquefied prey tissues [105,106]. Most employ an
Mosi et al. [101] investigated the ability of M. ulcerans to colonize ambush strategy, waiting motionless clinging to vegetation for
aquatic bugs (Belostomatidae) collected from Africa. Using a unsuspecting prey (Belostomatidae), while others may actively
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swim and pursue their prey (Naucoridae, Notonectidae) [107,108]. In attempting to understand possible modes of transmission, two
Adults of most species of semi-aquatic Hemiptera possess the competing models have been proposed to explain this pattern of
ability to disperse by flight, but mainly at night, and end up being limited environmental contact, brief exposure, and high attack
attracted to electric lights during the breeding season, often rates. Hayman [9] proposed that transmission by aerosol could
correlated with the lunar cycle. Because of this, they often find partially explain outbreaks of M. ulcerans disease and an
their way into houses by accident [107,108]. However, the very opportunity arose to test this hypothesis during a three year
low disease prevalence among children less than three years of age period when a large cluster of Buruli ulcer cases occurred in East
suggests that infection does not occur in the house. When humans Cowes, Phillip Island. This outbreak was significant in that only
accidently come into contact with the bugs in the water, on aquatic part of the town was affected, and there was a newly created
vegetation, or away from water, they can be bitten [109]. wetland and a golf course at the center of the affected area. The
However, these bugs do not actively search for humans, they do golf course used a mixture of ground water and recycled water for
not require a blood meal or protein source to mature their eggs, irrigation and run-off from the golf course was likely to have
nor is there any evolutionary history suggesting or supporting a drained towards the new wetland, connecting the two systems.
vectorborne/pathogen transmission or co-evolving host/parasite Many of the case-patients lived close to the wetland or the golf
relationship in the semi-aquatic Hemiptera [107,110]. Therefore, course, supporting the concept of transmission by drifting aerosols
based on the biology and behavior of predaceous aquatic insects, from contaminated irrigation water [116–119].
biting humans appears to be a rare event associated with a purely Initially, no method existed for detection of M. ulcerans in
defensive reaction of these bugs [109,111]. It should be noted, environmental samples. However, as part of the outbreak
however, that the causative agent of Chagas disease (Trypanosoma investigation, Ross et al. [63] discovered IS2404, a high copy
cruzi) in humans is transmitted by a terrestrial hemipteran number insertion sequence in M. ulcerans. A PCR method using
(Reduviidae), but it is through fecal contamination and not by IS2404 as a target sequence has rapidly become the diagnostic
the bite of the bug. Also, in this case the habitat of the vector (bug) method of choice for Buruli ulcer due to its high sensitivity,
is closely tied to that of its host [112]. specificity, and its speed compared with traditional culture
In general, field studies on the prevalence of biting aquatic methods. IS2404 PCR was then adapted for application to
invertebrates do not support the hypothesis that biting aquatic environmental samples, and positive results were obtained from
bugs are vectors of M. ulcerans in nature; however, a recent study the wetland and golf course irrigation system-the first direct
by Marion et al. [94] in Cameroon identified several water bug evidence that M. ulcerans DNA is present in environmental
families as hosts of M. ulcerans in a Buruli ulcer endemic area. samples.
However, in Marion et al. [93], only one endemic area and one IS2404 PCR also can be used as a preliminary test for the
non-endemic area were evaluated, suggesting no replication, and presence of M. ulcerans in Africa, but aquatic mycobacteria
thus, a limitation to testing how variable M. ulcerans is among associated with disease in fish and West African clawed frogs
endemic versus non-endemic areas/villages. This makes it (Xenopus tropicalis) also contain IS2404. For this reason, IS2404 lacks
difficult to compare to studies by Williamson et al. [67] and sufficient specificity for use as sole criteria for M. ulcerans in Africa.
Benbow et al. [68] where multiple replicate sites were evaluated To date, there is no evidence from Australia of the presence of
to test for M. ulcerans variability in standardized ecological IS2404 in any other environmental mycobacterium.
samples. Benbow et al. [68] conducted the largest field study to The above findings supported the hypothesis that the golf
date that examined biting water bugs in 15 disease-endemic and course irrigation system and nearby wetland at Phillip Island had
12 non-disease-endemic areas of Ghana, Africa. From collections become contaminated with M. ulcerans, although transmission by
of over 22,000 invertebrates, they compared composition, aerosol itself was not directly assessed [72,120]. Drainage of the
abundance and invertebrate-associated M. ulcerans positivity wetland, reduction in recycled water use, cleaning of the irrigation
among sites, and concluded that biting hemipterans were rare equipment at the golf course, and subsequent separation of ground
and represented a very small percentage of invertebrate water from recycled water were collectively associated with fewer
communities. When endemic and non-endemic areas were cases in the following years. Buruli ulcer linked to Phillip Island is
compared, there were no significant differences in hemipteran now rare; however, disease activity in at least one other Victorian
abundance or invertebrate-M.ulcerans positivity rates (by PCR) endemic area also declined over a similar time frame without a
between the areas, and there were no significant associations specific intervention, making it difficult to conclude that the
between hemipteran abundance and overall invertebrate-M.ulcer- environmental alterations made at Phillip Island were directly
ans positivity. Thus, there is little field evidence to support the responsible for the decline in cases. During the same period several
assertion that biting bugs are major vectors of M. ulcerans in possums (Australian native tree-dwelling marsupials) with Buruli
nature. However, as concluded by Marion et al. [94], the ulcer were identified at Phillip Island [18], the significance of
detection of M. ulcerans in water bugs in a specific area could which will be discussed further below.
possibly be used as an environmental indicator of the risk of M. In 2002, a new outbreak commenced in a small town on the
ulcerans transmission to humans. Bellarine Peninsula about 60 km to the west of Phillip Island, also
Australia. In Australia, infection with M. ulcerans occurs at in coastal Victoria, southeastern Australia. More than 100 people
low-levels in the wet tropical north where the climate is similar to who either live in or have visited Point Lonsdale have now been
sub-Saharan Africa [113–115]. However, more than 80% of diagnosed with Buruli ulcer [35]. Several other towns on the
Australia’s cases of Buruli ulcer in the past 15 years have been in Bellarine Peninsula have been linked to cases, but in lower
the temperate southeastern state of Victoria [93]. In comparison to numbers thus far. Although Point Lonsdale also has a golf course,
Africa, people in Victoria have less direct contact with the it is not centrally located, and does not use recycled water. In
environment, yet in two well-described outbreaks, 1.2–6.0% of the 2004, intense local mosquito activity seemed to be associated in
entire resident population in the outbreak areas developed Buruli time with new cases of BU and Buruli lesions were observed on
ulcer [35,116]. Visitors may also be at risk, and in one case, ankles and elbows, and on the back where gaps in clothing could
contact with an endemic town for just one day appeared to be allow access for mosquitoes. In one case, Buruli ulcer developed on
sufficient to develop Buruli ulcer up to 7 months later [35]. the ear of a child who was only briefly present in the outbreak
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Buruli Ulcer Disease Review
area. The child’s mother suspected a mosquito bite as the initiating but direct or indirect mosquito transmission from a possum
event [35]. reservoir presents a parallel model with aerosol transmission from
These observations led to a series of studies aimed at assessing a contaminated environmental water sources. Neither the aerosol
possible role for mosquitoes in the transmission of M. ulcerans. nor mosquito transmission hypothesis in temperate Australia is
Using an improved real-time quantitative IS2404 PCR environ- incompatible with transmission by direct contact with the
mental screening method [74], more than 11,000 adult mosquitoes environment or by other vectors not yet examined. Future
captured at Point Lonsdale were tested, and M. ulcerans DNA was research on the biological relationships within each model will
identified in or on an estimated 4.3/1,000 mosquitoes. Most PCR help to resolve the relative probability and plausibility of either
positive mosquito pools were Aedes camptorhynchus (Thomson), the mode.
most common species on the Bellarine peninsula; however, M.
ulcerans DNA also was detected in one or more pools of four other Criteria for Establishing the Role of Insect Vectors of M.
species [35]. PCR amplification and sequence analysis of one ulcerans
variable number tandem repeat (VNTR) locus confirmed that Stringent criteria exist in biomedical research for indicting the
mosquitoes were carrying M. ulcerans DNA, indistinguishable from roles of living agents as biologically significant reservoirs and/or
that of the human outbreak strain [74,121]. vectors of pathogens. The application of these criteria to the
A review of notifiable diseases in Victoria in the period 2002-8, transmission of M. ulcerans presents a significant challenge. The
demonstrated a statistically significant correlation between notifi- above review reveals that various routes of transmission may
cations of Buruli ulcer and Ross River Virus/Barmah Forest Virus occur, varying amongst epidemiological setting and geographic
infections (RRV/BFV) – both of which are transmitted by region, and that there may be some role for living agents as
mosquitoes – but there was no correlation with any other non- reservoirs and as vectors of M. ulcerans, in particular aquatic
mosquito borne notifiable disease [122]. insects, adult mosquitoes or other biting arthropods. It is also clear
A case-control study, conducted on the Bellarine Peninsula that the exact mode of transmission, if indeed there is a single
including Point Lonsdale, showed that the odds of being diagnosed mode, remains unknown. We briefly discuss the process by which
with Buruli ulcer were at least halved in respondents who a vector is incriminated to the point of as much certainty as is
frequently used insect repellent, wore long trousers outdoors, possible, and then discuss the application of this process to
and immediately washed minor skin wounds, and were at least indictment of insect vectors for transmission of M. ulcerans. If Buruli
doubled for those who received mosquito bites on the lower legs or ulcer is a vectored disease, intervention might be designed to
lower arms. In a multivariate model, after adjusting for age and reduce the possibility of transmission since there are possibilities
location, use of insect repellent and being bitten by mosquitoes on other than suppressing vector populations.
the lower legs were found to be independently associated with Vector incrimination traditionally involves satisfying a set of
Buruli ulcer risk [90]. criteria analogous to Koch’s postulates, summarized by Barnett
In laboratory experiments using a green fluorescent protein [127] as follows: (1) the vector must be shown to acquire the
(GFP) labeled M. ulcerans mutant, in which GFP was linked to the pathogen from an identified source such as an infected vertebrate
mycolactone toxin polyketide synthase promoter, it was shown host or other reservoir, and thereafter become infected with the
that when fed as a single pulse to live mosquito larvae, M. ulcerans- pathogen; (2) the vector must be shown convincingly to have close
GFP was able to persist through 4 larval instars in the mouth parts associations with infected hosts, including humans, in time and
and midgut of the insect. This was not observed with a closely space; (3) individual vectors collected in endemic settings must
related M. marinum-GFP mutant that did not produce mycolactone repeatedly be found infected with the pathogen; and (4) efficient
[123]. This permissive effect of mycolactone on allowing M. transmission to competent vertebrate hosts must be demonstrated
ulcerans to selectively colonize aquatic insects also was observed in experimentally, under well controlled conditions, by individual
experiments using aquatic water bugs [66,100,104]. However, vectors, such as by bite or other means of direct contact. These
other investigators found equal colonization with mycolactone criteria accommodate mechanical transmission if infection in-
negative and wild type strains [101], and this earlier selective effect cludes recovery of the pathogen from the vector’s body, without
was not observed in a study on M. ulcerans colonization of making any assumptions about replication of the pathogen on or
mosquitoes conducted by Wallace et al. [124].The latter study in the vector. Further, they do not preclude the possibility of
found a nearly 100% infection rate was obtained when wild type parallel modes of transmission other than vectors. For example,
M. ulcerans, an isogenic mycolactone-negative M. ulcerans, and M. the causative agent of plague, Yersinia pestis, has a flea vector and
marinum (a non-toxin producing potential progenitor of M. ulcerans) during sporadic outbreaks is transmitted by flea bites; but these
were used to infect mosquito larva. These findings are in line with bacteria also are transmitted during epidemics in aerosols
the fact that mosquito larvae do not discriminately feed on specific generated by sneezing of pneumonically-infected humans or
bacteria or other foods unless ingestion is mediated by particle size animals such as cats, which is probably the predominant mode
[125,126]. Differences in experimental conditions and bacterial of transmission in epidemics [128]. Similarly, human infection
strains used may help to explain these conflicting findings. with the causative agent of tularemia, Franciscella tularensis, may
Collectively, the above transmission research conducted in occur through direct contact with contaminated water, by
southeastern Australia lends support to mosquitoes as being a aerosols, by contact with blood or infected tissues of animals, or
possible vector of the pathogen for Buruli Ulcer disease in this by bites of infected ticks, deer flies, or mosquitoes [129,130]. The
region of the country (see Bradford Hill guidelines for a critical causative agent of Rift Valley fever, a Phlebovirus in the family
assessment, below). More recently, it also has been discovered that Bunyaviridae, is transmitted amongst infected vertebrate reservoirs
that 38% of ringtail possums (Pseudocheirus peregrinus (Boddaert)) and (mainly ungulates) by mosquitoes; however, many human
24% of brushtail possums (Trichosurus vulpecula Flannery) captured infections occur upon exposure to infected animal blood at the
at Point Lonsdale had laboratory-confirmed M. ulcerans skin lesions time of slaughter, by aerosolization, as well as by mosquito bites
and/or M. ulcerans PCR positive feces (Fyfe et al. [76]). The exact [131]. Another useful illustration is that of Chlamydia trachomatis, the
sequence of events linking mosquitoes, humans, contaminated causative agent of trachoma, where the transmission to human
possum excreta and infected possums has yet to be determined, eyes has been definitively associated with contact by Musca sorbens
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Buruli Ulcer Disease Review
flies (Diptera: Muscidae) that breed in human feces in various parts between presence of M. ulcerans in aquatic environments in which
of Africa [132]. Despite this observation, other mechanisms of larval mosquitoes are found and adult mosquito infection with M.
transmission for this disease are known, such as person-to-person ulcerans, was not confirmed experimentally. However, these studies
contact with contaminated fingers and wash towels [133,134]. In did show that M. ulcerans DNA could be detected on surface
two of the above examples (plague and Rift Valley fever), the components of some adult mosquitoes. This brings up an
pathogen has a close biological relationship with, and dependency important issue regarding experimental design and suggests that
upon, insect vectors; neither pathogen could persist in nature interpretation of PCR results obtained from whole insect lysates
without infecting their respective vectors. For tularemia and must be cautiously interpreted. These findings suggest that further
trachoma, vectors are not essential to pathogen persistence in research is required to confirm the association between mosquito
nature, even though fly control in the latter case was shown to bites, adult mosquito infection, and incidence of Buruli ulcer in
reduce incidence of disease in humans [135]. However, it is humans in Australia (reviewed above), where a link between
unlikely in the case of tularemia and trachoma that even highly mosquito feeding on infected possums and transmission of the
effective fly control could eliminate human infection in endemic agent via the same species of mosquitoes was proposed (Fyfe et
areas owing to other modes of transmission [133]. Therefore, al.[76]). An analysis of blood host choice by mosquitoes,
using a critical approach to address the issue of insect vector documenting blood feeding on both possums and humans in the
incrimination for M. ulcerans, one must be cognizant of the relative area where human cases of Buruli ulcer are occurring, would be
biological dependency of this bacterium on an insect vector, and required as one element of satisfying Barnett’s criterion #2. At
the potential for facultative and facilitative relationships between best, Barnett’s criteria for vector incrimination have not been
these bacteria and various insect ‘‘hosts’’ to exist which may be completely satisfied for a mosquito vector role, but more
ancillary or even spurious to the essential and normal transmission compelling data may be forthcoming on this matter in the future.
modes. A second approach to vector incrimination involves application
The most thorough examination of the role of an insect vector of the Bradford Hill guidelines for establishing causation of
for transmission of M. ulcerans stems from investigations of aquatic, infection and disease in epidemiological/ecological contexts [136].
predaceous Hemiptera (true bugs) as reviewed above, which go far Rather than rely upon experimental evidence, the Bradford Hill
in addressing and meeting Barnett’s criteria. It is important to guidelines emphasize epidemiological/ecological association and
recognize that the vast number of studies of M. ulcerans in use of logical inference to build up support and evidence for a
environmental samples provide qualitative, indirect evidence of M. strong conclusion of cause and effect, where A represents the
ulcerans based on very sensitive methods for detecting M. ulcerans ‘‘cause’’ and B the ‘‘effect’’ in the relationships under study [137].
DNA. Such studies revealed repeatedly that natural infection by The result is an ‘‘evidence hierarchy’’ that can be used in formal
M. ulcerans in field-collected bugs occurred, but it was tempered by deduction [138], and represents an interdisciplinary approach to
detection of M. ulcerans in many other aquatic insects [18,67]. causal investigation in disease ecology. Here, ‘‘A’’ would be
Thus, definitive incrimination of a single species or group of contact between an insect vector infected with M. ulcerans, and ‘‘B’’
closely-related aquatic and semi-aquatic Hemiptera to the would be human infection with M. ulcerans. The guidelines are
exclusion of other insects was not initially established. Other qualitative in nature and do not require the clear endpoints of
studies suggested natural contamination of the surfaces of these Barnett’s criteria, yet represent a logical approach to the problem
insects with M. ulcerans and suggested that M. ulcerans growth could of cause and effect under epidemiological circumstances [139].
occur as biofilms on the external appendages of such ‘bugs’ [101]. They are as follows (Table 2):
Thus, although aquatic and semi-aquatic Hemiptera and other (1) Plausibility. The cause and effect association of A and B must
insects found to harbor M. ulcerans in nature might provide habitat be plausible, that is, rational and lacking in speciousness. By this is
for the bacteria, along with numerous other living and non-living meant that the association reflects the common understanding of the
surfaces where biofilms could form [104], this is insufficient normal behavior and other attributes of both A and B, bringing the
evidence for indicating an obligatory or even facultative vectorial appropriate factors together in such a way that abnormally
role to these insects. Although the experiments reported by implausible (i.e., irrational) explanations must be discounted. In
Marsollier et al. [64,66,98–100] suggested modest bacterial formal philosophy, plausibility must be demonstrated by sets of
replication in internal tissues of bugs, acquisition of bacterial binary outcomes whose relationships are clearly defined proposi-
infection from a live source (infected fly maggots meant to simulate tions which can be resolved by the application of logical discourse
an infected prey item), and transmission to mice, this evidence [140]. Although plausibility can be formulated axiomatically, it
does not establish natural infection coupled with transmission to cannot be analyzed statistically. It is important, therefore, not to
humans. Finally, there has been no epidemiological association confuse ‘‘plausible’’ with ‘‘probable’’ as the latter allows for rare and
established between spatial and temporal distribution of contacts unusual circumstances and events to be explanatory under the right
with aquatic Hemiptera, or bites by them, and development of circumstances, whereas the former involves a rigorous, but non-
Buruli ulcer in humans [68]. As reviewed above, the common probabilistic analytical process. Put more simply, plausibility
understanding of the feeding habitats of aquatic and semi-aquatic addresses qualitatively how likely or unlikely it is that A results in
Hemiptera does not include feeding on humans. More likely, B. A common problem in epidemiological scenarios that confronts
infection in aquatic insects is associated with exposure to M. plausibility is the issue of clusters of cases of infection (e.g., [134]),
ulcerans in detritus and on biofilms formed on submerged materials, which may or may not have spatial associations with other nearby
leading to a generalized distribution of M. ulcerans and M. ulcerans cases or with the landscape qualities near those cases [136]. In the
DNA in aquatic environments. In this particular scenario, despite case of Buruli ulcer and vector transmission of M. ulcerans, it is not
the body of research on the topic, Barnett’s criteria have not yet implausible that Hemiptera and human cases are associated in time
been fulfilled satisfactorily. and space, but it is not plausible that there is a direct, causal
The recent research by Wallace et al. [124], whilst firmly relationship between the pair except in rare, accidental circum-
documenting growth of M. ulcerans in mosquito larvae and stances. Hence, there is insufficient evidence to conclude that biting
transtadial infection after the molt, showed that infection did not hemipterans are a significant vector of M. ulcerans, although they
persist upon metamorphosis to the adult stage. Thus, the link may act as environmental reservoirs.
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Table 2. Listing of Hill’s guidelines (Bradford Hill guidelines, Hill 1965) for associating a role of insect vectors of pathogens causing
human disease.
Term Descriptor/Qualifier
1. Plausibility Plausible, rational given knowledge of the biology of the putative vector, biology of the pathogen, and epidemiology of the
disease. Specious associations would contraindicate a positive association.
2. Temporality The insect vector must show a temporal association with infection in humans; in particular, infected vectors should be found in
endemic areas immediately before human cases occur.
3. Strength The association of the putative insect vector with human cases must be strong in time and space and in an epidemiological
context. Correlation analysis supports the conclusion of strength if the correlation is positive.
4. Biological Gradient Prevalence of human cases should co-vary with prevalence of infection in the insect population.
5. Consistency Confirmed human cases should consistently be associated with infected insect vectors in time and space.
6. Alternate Explanations Explanations other than those related to a role of an insect vector should be considered and ruled out, or validated.
7. Experimentation Role of an insect species as a vector should be validated through experimental analysis with adequate controls and with realism
in experimental design.
8. Specificity Infection with M. ulcerans in humans occurs when, and only when, a bite by an infected insect occurs first.
9. Coherence The association of human infection with insect transmission must cohere to knowledge of similar relationships in other similar
associations.
doi:10.1371/journal.pntd.0000911.t002
(2) Temporality. If A results in B, then A must consistently insects are the predominant vector in most geographic regions.
precede B in temporal sequence. For Buruli ulcer, there is no Consistent data are lacking for the ubiquitous role of vectors in the
evidence that bites of particular insects consistently precede M. ulcerans transmission system.
development of patent M. ulcerans infection in humans, although (6) Consideration of alternate explanations and analogous
there is evidence that mosquito bites are associated with increased situations. Explanations other than causation due to A must be
risk [90]. The problem with this guideline is the prolonged period carefully weighed as alternatives. Causation may be inferred by
of time between exposure and development of symptoms in Buruli analogous correspondence with other scenarios. For Buruli ulcer, a
ulcer disease. However, if bites from true bugs always preceded wide range of alternate explanations for transmission exists, such
disease, patients are likely to remember these due to the painful as human behavior linkages involving activities that increase direct
nature of a naucorid or belostomatid bite, in contrast to bites by skin contacts with contaminated water and inoculation with
mosquitoes that often go unnoticed. infective doses of M. ulcerans through lesions. However, as we have
(3) Strength. Is the ‘‘strength’’ of the association great? For seen, several diseases with insect vector associations have
example, is there a statistically significant correlation between A alternative transmission modes, such as tularemia, plague, Rift
and B in space and or time? The association between contact with Valley fever, and trachoma. Thus, it is plausible that there are
water sources and M. ulcerans infection in humans is reasonably multiple modes of transmission in Buruli ulcer, with certain modes
strong, but between insect bites and infection it is not for more likely given specific environmental and socio-cultural
hemipterans, nor yet firmly established for mosquitoes in Australia contexts.
and virtually non-existent for mosquitoes in Africa. (7) Experimentation. If experimental manipulations are feasible
(4) Biological gradient or dose-response relationship. Infection and can be structured realistically, then outcomes of the treatment
in B should increase proportionately as A increases. This principle regime conferred upon B (such as exposure to the effects of A)
can operate at the dose-response level, as in a toxicological series; must reflect the association in a positive way. Often, however,
or at the population level, as when, e.g., more dengue virus Bradford Hill guidelines are utilized because experiments are
infected mosquitoes results in more human cases of infection with either not possible, or not sufficiently rigorous or realistic.
that virus in space and time. The relationship may not be linear, Experimental data on insect-M. ulcerans relationships have been
thus confounding the interpretation of the relationship. There is reviewed above. There seems to be a sufficient body of work with
no evidence that higher infection rate of M. ulcerans in aquatic sufficient variation in outcomes that the treatment manipulations
insects results in higher incidence of infection in humans, although do not lead to easily generalized conclusions on the association.
there is evidence that adult mosquitoes caught in highly endemic Furthermore, it is often difficult to find true replication for large-
area in southeastern Australia are more likely to be PCR positive scale experiments (e.g., treating replicate ponds with a specific
than those caught in areas with lower endemicity [35]. chemical agent to test of changes in M. ulcerans), making it difficult
(5) Consistency. Episodes and research data where A and B to rigorously evaluate and experimentally test complex dynamics
show spatial and temporal associations commensurate with the related to multiple modes of transmission of M. ulcerans within the
other Bradford Hill guidelines must consistently reveal the environment.
association to be a positive one. Consistency could be revealed (8) Specificity. In this guideline, B follows A, but B does not
by meta-analysis of many data sets or through replicated, follow when other plausible explanatory factors and events occur
longitudinal studies across time and space. If scenarios emerge in in temporal or spatial association. It is one of the most difficult of
which B occurs, but A does not in space and time, then doubt the guidelines to satisfy and comes closest to a strict criterion,
emerges regarding the veracity of the association. Although there usually because of incomplete information, multiple causes of B,
are vignettes, correlations, and observations regarding insect random effects, and systematic errors of measurement. The review
vectors of M. ulcerans, there is no clear consistency among of the literature on cause and effect between insects and Buruli
epidemiological scenarios to currently support the notion that ulcer cases indicates a paucity of data to prove specificity.
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Furthermore, there are few studies relating disease incidence and Conclusions
insect abundance in time and space especially in Africa, and none Recommended research directions on Buruli ulcer
of the alternate explanations for transmission reviewed above, such disease. As stated in the beginning of this review, Buruli ulcer
as through aerosols (9), have been discounted. The current disease has been referred to as the ‘‘mysterious disease’’ because
available data points to a multiple transmission model for Buruli the exact mode(s) of transmission, in the strictest sense, remain
ulcer, indicating that the Buruli ulcer disease system lacks unclear, although several hypotheses have been proposed. We
specificity with regard to vector insects, with the possible exception have reviewed the hypotheses and reported on studies that provide
of southeastern Australia. Therefore, more complete and rigorous good evidence of probable reservoirs for the disease, particularly in
qualitative assessments of data are critical to provide evidence for Australia. An intellectual framework for establishing criteria for
consistency and specificity with regard to the role of vectors and transmission followed this. Finally, we recommend that the
reservoirs in transmission of M. ulcerans. following research studies be conducted to help better
(9) Coherence. The association of B with A must cohere to understand transmission of M. ulcerans in nature: 1) in depth
knowledge of similar relationships in other similar associations. For studies of human behavior patterns in African endemic villages to
M. ulcerans, insect transmission is quite unusual, as the remainder better understand exposure to the pathogen in the environment; 2)
of the M. marinum group does not depend upon invertebrate a search for mammalian and/or other animal reservoirs and
vectors for transmission and infection in fish hosts. Furthermore, potential arthropod vectors in Africa; 3) understanding the
there is no scientific precedent for transmission of any disease relationship between mosquitoes, humans and infected possums
agent from the direct bites of hemipteran bugs, nor is there who frequently share the same habitats in Australia; 4) laboratory
precedent for biological transmission of any bacterial pathogen by competency studies with Australian mosquitoes using local strains
mosquitoes known. Thus, coherence is overall not strong. of MU to determine whether transmission could occur vertically
However, although closely related to M. marinum, M. ulcerans is a (larvae to adult) or horizontally (adult feeds on possum and then on
distinct species with a genomic signature indicating it has diverged humans); 5) further field and laboratory experiments on vector
from its free-living ancestor and now occupies a specialized niche transmission and vector competence to confirm current
environment. Either a vertebrate gastrointestinal tract (e.g. hypotheses and experimental evidence on arthropod
possums) or insects may provide this unknown microenvironment. transmission; and 6) the development of new and innovative
In summary, neither the application of Barnett’s strict criteria studies aimed at satisfying Hill’s Criteria to provide strong and
nor the Bradford Hill guidelines support conclusively that bites by logically defendable evidence about the true mode, or modes, of
M. ulcerans-infected insects’ result in human infection with M. Buruli ulcer transmission in nature.
ulcerans. However, further research will reveal if any associations
might result in higher risk of infection under certain circumstanc- Supporting Information
es. Infection with anthrax bacteria, Bacillus anthracis, provides a
useful comparison, not as a directly transferable model, but rather Checklist S1 PRISMA checklist.
as a model for conceptualization of how insects, like mosquitoes, Found at: doi:10.1371/journal.pntd.0000911.s001 (0.07 MB
may have ancillary roles in bacterial transmission when other DOC)
transmission modes also exist [141]. In that system, infection
occurs in animals endemically and sporadically. When they are Acknowledgments
stressed (as in a drought), they become susceptible to low dosages
of bacterial spores in soil. As animals die, colonization of We would like to thank Dr. K. Asiedu of the World Health Organization
for his continued encouragement and support of our research on Buruli
necrophilic flies during decomposition results in infection locally
Ulcer. We also would like to thank E. Campbell for assistance with final
and increased bacterial sporulation and more animal cases occur copy editing.
as a result (the so-called ‘‘case multipliers’’ effect of insects). As
more animals become infected, an insect-mediated dispersal of
Author Contributions
bacteria occurs by biting flies such as deer flies and horse flies,
whose mouthparts can become contaminated with bacteria during Conceived and designed the experiments: RWM EDW PLCS JRW
blood feeding (the so-called ‘‘space multiplier’’ effect of insects). PDRJ MEB. Analyzed the data: RWM EDW DAB. Contributed
reagents/materials/analysis tools: PLCS JRW PDRJ MEB DAB. Wrote
The role of flies in both modes furthers epizootics of anthrax.
the paper: RWM. Wrote part of the criteria section of the paper: EDW.
Although these two processes are unlikely to occur for Buruli ulcer,
Wrote part of the pathogen section: PLCS. Contributed to analyses of
which appears to be mainly an endemic disease, the scenario for particular studies and wrote one section: JRW. Wrote part of the paper
anthrax establishes a model by which insects might be envisioned on Australia transmission: PDRJ. Wrote part of the landscape
to have ancillary roles in transmission for M. ulcerans as well. epidemiology section: MEB.
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www.plosntds.org 15 December 2010 | Volume 4 | Issue 12 | e911
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