See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/23149705 Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease Article  in  Emerging Infectious Diseases · September 2008 DOI: 10.3201/eid1408.071503 · Source: PubMed CITATIONS READS 59 58 10 authors, including: Mark Eric Benbow Heather R Williamson Michigan State University Mississippi State University 208 PUBLICATIONS   2,332 CITATIONS    24 PUBLICATIONS   426 CITATIONS    SEE PROFILE SEE PROFILE Ryan Kimbirauskas Charles Quaye Michigan State University Noguchi Memorial Institute for Medical Research 28 PUBLICATIONS   287 CITATIONS    18 PUBLICATIONS   255 CITATIONS    SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Microbes in Aquatic Ecosystems View project My research mostly focusing on Nosema impacts on Thai Honey bees View project All content following this page was uploaded by Richard W Merritt on 04 June 2014. The user has requested enhancement of the downloaded file. Aquatic Invertebrates as Unlikely Vectors of Buruli Ulcer Disease M. Eric Benbow,*1 Heather Williamson,† Ryan Kimbirauskas,* Mollie D. McIntosh,* Rebecca Kolar,* Charles Quaye,‡ Felix Akpabey,§ D. Boakye,‡ Pam Small,† and Richard W. Merritt* Buruli ulcer is a necrotizing skin disease caused by humans (2–4). Although several water-related risk factors Mycobacterium ulcerans and associated with exposure to have been recognized, none has been consistently reported, aquatic habitats. To assess possible transmission of M. making it diffi cult to identify specifi c water-related risk ulcerans by aquatic biting insects, we conducted a fi eld activities (6–8). Most studies suggest that infection oc- examination of biting water bugs (Hemiptera: Naucoridae, curs through inoculation of M. ulcerans into skin lesions Belostomatidae, Nepidae) in 15 disease-endemic and 12 non–disease-endemic areas of Ghana, Africa. From collec- or insect bites (2,4,9–11). Portaels et al. (11) were the fi rst tions of 22,832 invertebrates, we compared composition, to propose that aquatic insects might serve as vectors of abundance, and associated M. ulcerans positivity among M. ulcerans. This hypothesis maintains that M. ulcerans sites. Biting hemipterans were rare and represented a small is found in biofi lms of aquatic habitats and concentrated percentage (usually <2%) of invertebrate communities. No by grazing or fi lter-feeding invertebrates that are then con- signifi cant differences were found in hemipteran abundance sumed by predators known to bite humans (11). Initial or pathogen positivity between disease-endemic and non– evidence for this hypothesis used PCR detection of the disease-endemic sites, and between abundance of biting insertion sequence IS2404 to document M. ulcerans’ as- hemipterans and M. ulcerans positivity. Therefore, although sociation with biting water bugs (Hemiptera), fi ltered con- infection through insect bites is possible, little fi eld evidence centrates of water, detritus, and aquatic plants (4,12–14). supports the assumption that biting hemipterans are prima- These studies were important for understanding the pos- ry vectors of M. ulcerans. sible environmental reservoirs of M. ulcerans. However, IS2404 is now understood to be not specifi c for M. ulcer- Mycobacterium ulcerans infection is an emerging skin ans because this insertion sequence has been found in a disease often called Buruli ulcer (BU). Infection re- number of other aquatic mycobacterial species, including sults in illness and lasting negative socioeconomic effects in M. marinum (15–17). When more discriminatory methods rural areas of the tropics and subtropics (1). The pathologic based on detection of variable number tandem repeats were changes, clinical signs and symptoms, and treatment have used, many IS2404-positive environmental samples were been reviewed elsewhere (2–5). In this article we evaluate reported to lack M. ulcerans (18). In light of these recent fi eld evidence for the potential of aquatic invertebrates to fi ndings, the relative frequency or abundance of M. ulcer- be vectors of M. ulcerans. ans among aquatic invertebrates or other environmental The exact mode of BU transmission remains unknown; reservoirs, remains tenuous, and thus, the role of aquatic however, past epidemiologic studies have associated BU insect vectors is uncertain. with human activity near, or within, slow-fl owing or stand- A series of laboratory experiments provided initial evi- ing water bodies that have been created or disturbed by dence for biting hemipteran vectors of M. ulcerans (19–23). Marsollier et al. (9,24) demonstrated that a South American *Michigan State University, East Lansing, Michigan, USA; †Uni- isolate of M. ulcerans could survive and multiply within the versity of Tennessee, Knoxville, Tennessee, USA; ‡University of salivary glands of aquatic bugs indigenous to France (Nau- Ghana, East Legon, Ghana; and §Water Resources Research In- coridae: Naucoris cimicoides). Furthermore, N. cimicoides stitute, Accra, Ghana could transmit M. ulcerans by feeding on inoculated prey DOI: 10.3201/eid1408.071503 1Current affi liation: University of Dayton, Dayton, Ohio, USA. Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 1247 RESEARCH and then biting mice, which then exhibited BU (9). Most Ghana (Figure 1). The water bodies were located within recently, exposure to hemipteran insect saliva was reported or very near (<100–200 m) each community of housing to infer protection against lesion development in laboratory structures and were routinely used for daily domestic pur- mouse models (21). That study also reported correlations poses and refl ect habitats of routine human exposure. These between aquatic insect salivary gland antibodies in humans water bodies were chosen after discussions with commu- categorized as exposed or patient, when the former group nity members who directed us to the main water source for had exhibited BU. However, 3 limitations of that study drinking water, recreation, domestic washing, irrigation, or have been noted (25): 1) the antibodies against salivary bathing for that community. Six of these sites were sampled proteins might only be biomarkers of protection; 2) pos- in both years, providing information on annual variation: sible geographically related polymorphisms in the salivary Afuaman, Amasaman, Abbeypanya, Afi enya, Odumse, and proteins among hemipteran taxa could limit the generaliz- Weija. Human BU case data for the years 2003–2005 were ability of protection among distant communities; and 3) the provided by the Ghana Ministry of Health and used to clas- overall relevance of biting aquatic insects infected with M. sify communities into 2 site types: 15 BU–endemic (BU+) ulcerans in the natural environment is unknown. and 12 BU–nonendemic (BU–). A site was classifi ed as a A confounding factor in these experimental studies is BU+ type if at least 1 case of BU had been reported during that they used 1 South American isolate of M. ulcerans. the 3-year period. Recent data support 2 major lineages of M. ulcerans: the ancestral strains that closely resemble M. marinum in chro- Aquatic Invertebrate Sampling and Processing mosomal content, and the classic strains that have under- Within each water body, two 10–20-m transects were gone substantial genome reduction (26). The latter strains measured parallel to the shoreline and positioned through the account for all severe disease and include the African, Ma- dominant macrophyte community. Along each transect, we laysian, and Australian isolates. The aforementioned labo- randomly placed two 1-m2 polyvinyl chloride quadrats and ratory studies have been elegantly performed, but the use collected invertebrates by sweeping within the quadrat with of a French species of Naucoridae and a South American a 500-μm mesh dip net. The quadrats fl oated on top of the isolate of M. ulcerans makes it diffi cult to assess the im- water and delineated 1 m2 of area to be sampled by using an portance of insect transmission in Africa. Thus, although aquatic dip net designed to capture the aquatic life stages of provocative experimental data support a potential role for invertebrates. Three sweeps of the dip net were performed aquatic hemipterans as vectors of M. ulcerans in laboratory from the water surface to the bottom substrate for compre- settings, no supporting evidence has been obtained from hensive sampling of specimens in the water column. All studies conducted in the natural setting. Results from fi eld studies that identify the relative abundance and exposure potential of biting aquatic hemipterans can provide insight into the importance of biting insects in BU transmission. This study had 3 objectives: 1) to describe the aquatic invertebrate samples collected during a large-scale, 2-year standardized fi eld-sampling program of 27 bodies of wa- ter in Ghana, West Africa; 2) to investigate M. ulcerans positivity among the same aquatic invertebrates from those water bodies, directly linking aquatic invertebrate commu- nities with pathogen positivity; and 3) to discuss the role of human-biting hemipterans as primary vectors of M. ulcer- ans. Data on the detection of M. ulcerans within aquatic samples based on the use of variable number tandem re- peats analysis are presented in another article (18). In the current article, we associate presumptive M. ulcerans posi- tivity rates with relative abundance and percentage compo- sition of the same aquatic communities. Methods Study Sites Figure 1. Regional site map of water bodies sampled in Ghana for aquatic invertebrates during 2004, 2005, or both. Small maps In June 2004 and August 2005, we sampled 27 wa- on left show location of Ghana in Africa and location of regions ter bodies associated with human communities in southern sampled within Ghana (boxes). 1248 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 Buruli Ulcer Disease contents were washed through a 500-μm sieve and preserved Data Analysis in 100% ethanol for laboratory identifi cation and PCR. The 2 Using all invertebrate data, we initially evaluated dif- quadrats were combined into 1 composite sample. ferences between site types (i.e., BU+ vs BU–) by com- paring total abundance and percentage composition. Only M. ulcerans Detection in Invertebrate Samples those taxa that represented >3% of total invertebrates col- Samples were analyzed in a 2-step procedure so that lected from all sites were used for subsequent statistical an initial screening reduced sample numbers. Small inver- analyses because some taxa were so rare that any compari- tebrates were analyzed in pools of 3–15, whereas larger sons would limit meaningful conclusions. However, be- specimens were tested individually. DNA was extracted cause we were interested in evaluating Hemiptera known by using a protocol adapted from Lamour and Finley (27). to bite humans, the families Belostomatidae, Naucoridae, Samples were ground and vortexed in 400 μL of lysis solu- and Nepidae also were included, although each represented tion (100 mmol/L Tris, pH 8.0), 50 mmol/L EDTA, 500 <2% of total collections. mmol/L NaCl, 1.33% sodium dodecyl sulfate, and 0.2 mg/ To compare abundance differences between site types, mL RNase A) and 1 g of 1.0-mm glass beads (Sigma-Al- t tests were used after data were log + 1 transformed to meet drich, St. Louis, MO, USA), then centrifuged. After 150 μL the assumptions of normality and equal variances. For per- of 5 mol/L potassium acetate was added, each sample was centage composition differences, data were arc-sine square incubated overnight at –20° C. After a 30-min centrifuga- root transformed, but they still did not demonstrate a nor- tion, supernatants were transferred to new tubes contain- mal distribution, so the nonparametric Wilcoxon/Kruskal- ing 0.66 mol/L guanidine hydrochloride in a 63.3% ethanol Wallis rank sum test was used. Because multiple tests were solution. The samples were added to a spin fi lter (MO BIO performed, it was necessary to calculate a Bonferroni ad- Laboratories Inc., Carlsbad, CA, USA) in a 2-mL microcen- justed α (and corresponding p value) of 0.006 to assist in trifuge tube (MO BIO Laboratories Inc.). The fl ow-through interpreting statistically signifi cant differences. However, was discarded and the fi lter was rinsed fi rst with 500 μL of to evaluate the biological meaning of these multiple tests, wash solution (10 mmol/L Tris, pH 8, 1 mmol/L EDTA, 50 Cohen d effect size (and 95% confi dence intervals) was cal- mmol/L NaCl, 67% ethanol) and then with 500 μL of 95% culated with Hedges adjustment (28). To compare overall ethanol. The spin fi lters were dried by centrifugation and ER positivity proportions between BU+ and BU– sites, a transferred to new 2-mL microcentrifuge tubes, immersed t test was used after data were arc-sine square root trans- in 200 μL elution solution (10 mmol/L Tris, pH 8), and formed. Lastly, we evaluated correlations between total bit- incubated at room temperature for 15 min. The DNA was ing hemipterans (and each individual family) and ER posi- eluted and stored at –20°C. tivity using Spearman rank correlations with a Bonferroni Presumptive identifi cation of M. ulcerans in inverte- adjusted α = 0.008. This nonparametric test was used after brates was based on detection of the enoyl reduction domain attempts to transform the data for normality and homoge- (ER) in mlsA that encodes the lactone core of the mycolac- neity of variances failed. tone toxin, the major virulence determinant of M. ulcer- ans. All samples were screened for the presence of the ER Results gene, which has been evaluated for M. ulcerans specifi city in a companion study that used a multitiered PCR approach Invertebrate Abundance and Composition (18). Amplifi cation of the ER gene was achieved using a Of 22,832 invertebrates collected, ≈50% came from 50-μL reaction mixture containing 1 μL each of forward each group of BU+ and BU– site types (online and reverse primer (15,18), 10 μL 5× Go Taq reaction buf- Technical Appendix, available from www.cdc.gov/EID/ fer (Promega, Madison, WI, USA), 1 μL 10 mmol/L PCR content/14/8/1247-Techapp.pdf). A total of 85 taxa were nucleotide mix (Promega), 31.7 μL double-distilled water, represented among all sites: 80 taxa were collected from 1.6 units Go Taq polymerase enzyme (Promega), and 5 μL BU+ sites compared with 71 from BU– sites. The abun- DNA template. Cycling conditions began with an initial de- dance of specifi c taxa was not consistent between site naturation at 94°C for 5 min, 35 cycles of 94°C for 1 min, types, indicating that the invertebrate communities were 58°C for 45 seconds, 72°C for 1 min, and a fi nal 10-min highly variable. This variability was confi rmed in statis- extension at 72°C. The amplifi ed DNA was subjected to tical analyses comparing the most abundant taxa (>3%) gel electrophoresis by using a 1.5% agarose gel, and band with substantial effect size variation within and among sizes were compared by using a 1-kb DNA ladder (Invit- taxa (online Technical Appendix). The invertebrates found rogen, Carlsbad, CA, USA). PCR products of appropriate in greatest abundance were 2 families of Diptera (i.e., size were cloned into the pCR2.1 Topo vector (Invitrogen) Chironomidae and Culicidae), 1 family of Ephemeroptera and sequenced by using an ABI 3100 automated genetic (Baetidae), and several Crustacea. More than 300 indi- analyzer (Applied Biosystems, Foster City, CA, USA). viduals of some families of Hemiptera, Coleoptera, and Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 1249 RESEARCH Odonata were encountered (online Technical Appendix). The biting Hemiptera were usually rare. For instance, 55 Naucoridae in total were collected, which was about 0.2% of all invertebrates sampled (online Technical Appendix). Insects made up the greatest percentage of the inverte- brates collected from BU+ sites but were nearly equivalent to the Crustacea in BU– sites. In BU– sites, Anura made up a relatively higher percentage, but most (1,231 of 1,303 individuals) were from a single site (Figure 2; online Tech- nical Appendix). The Crustacea were most often represent- ed by copepods, ostracods, and shrimp (Atyidae); fewer shrimp were collected from BU+ sites. Most shrimp were from BU– sites Adumanya (197) and Keedmos (120). Fur- ther, in BU– sites the large copepod abundance occurred primarily at Odumse, where 1,723 were collected from a total 1,884 (online Technical Appendix). Insects were reduced by 40% in BU– sites compared with BU+ sites (Figures 2, 3). When individual insect orders were com- pared, the Ephemeroptera (mayfl ies) and Diptera (true fl ies) made up the greatest percentages of insects in both BU+ and BU– site types (Figure 3; online Technical Appendix). When the abundance and percent composition of dom- inant taxa were statistically compared between BU+ and BU– site types, there were no signifi cant differences for any taxa (online Technical Appendix). However, the ef- fect size varied greatly, refl ecting a need to collect from more sites in future studies. On average, the Chironomi- dae (midges) made up the greatest percentage of the in- vertebrate communities, representing 9%–20% of the to- tal, while the Baetidae (mayfl ies) ranged from 6% to 15% and the Culicidae (mosquitoes) from 2% to 5%. The biting Hemiptera made up a very small percentage of the domi- Figure 2. Higher level classifi cation (e.g., class, phylum) taxa nant invertebrate communities, with Naucoridae <0.5%, percentage composition between A) Buruli ulcer–endemic (n = 15) and B) Buruli ulcer–nonendemic (n = 12) site types, Ghana. Belostomatidae <2%, and Nepidae <0.3% (online Techni- cal Appendix). (online Technical Appendix). There were 26 taxa positive Presumptive Identifi cation of M. ulcerans from BU+ site types compared with only 18 from BU– sites. from Invertebrates Only 2 taxa were positive in BU– and not in BU+ sites, and Presumptive identifi cation of M. ulcerans from a total for those taxa, <5 samples were tested from the BU+ type. of 1,032 invertebrate sample pools tested found no signifi - When only those taxa with >5 samples tested were com- cant difference between BU+ and BU– site types (online pared, no observable pattern in ER positivity was apparent Technical Appendix). Furthermore, there was no detect- among sites or taxa. The most abundant taxa did not always able pattern of invertebrate taxa ER positivity among sites, have the greatest ER positivity. For instance, positivity of indicating that no single taxon was more often likely to be Chironomidae (19.5% of all invertebrates) was only about positive at a particular site. The number of ER positive taxa 7%, even though positivity of Caenidae (<2% of all inver- that were detected at any site ranged from 0 to 15 and 0 to tebrates) ranged from 6% to 17% (online Technical Appen- 6 in BU+ and BU– sites, respectively (Figure 4). Clearly, dix). For taxa with >5samples tested from either BU+ or not all BU+ or BU– sites had ER positive invertebrates. BU– sites, the ER positivity was >20% for 5 taxa and from There were 6/15 BU+ sites without a single taxon positive 10% to 20% for 12 taxa (online Technical Appendix). The compared with only 3/12 BU– sites (Figure 4). biting Hemiptera had neither the highest nor consistently Taxon-specifi c ER positivity was highly variable, and higher ER positivity compared with more abundant taxa percentage positivity ranged from 0% to 100% among taxa (online Technical Appendix). Fifteen taxa with >5 samples 1250 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 Buruli Ulcer Disease tions or evaluated associated M. ulcerans positivity rates for specifi c invertebrate communities. Understanding the relative abundance and composition of the invertebrate taxa is a useful initial approach for assessing exposure risk of populations that use waterbodies for domestic needs. If biting water bugs are primary vectors of M. ulcerans, then the minimum (but not only) supporting evidence should confi rm at least 1 of the following characteristics: 1) biting water bugs should be relatively more abundant at sites with BU cases compared with those without BU, indicating increased exposure potential to the vector in disease-endemic communities; 2) biting water bugs should have relatively higher M. ulcerans positivity rates within disease-endemic sites compared with disease-nonendemic sites; 3) M. ulcerans positivity rates should be higher in biting water bugs than in other invertebrates in the same sites, demonstrating increased potential pathogen expo- sure in the vector compared with background exposure; or 4) a correlation should exist between M. ulcerans positiv- ity and vector abundance. This study addressed each of these characteristics and did not fi nd strong confi rming evidence that biting water bugs were any more important in the transmission of M. ulcerans than passive contact ex- posure to the environment. This fi nding is consistent with reports that few infected persons remember being bitten by water bugs (30). Although our results do not prove that infection could never occur from biting water bugs, they suggest that such an event would be rare. In a companion study, Williamson et al. (18) reported M. ulcerans ER positivity from a broad spectrum of envi- Figure 3. Insect order percentage composition between Buruli ronmental samples, including animals, water fi ltrate, and ulcer–endemic (n = 15) and Buruli ulcer–nonendemic site types (n biofi lm on glass slides. They found that M. ulcerans DNA = 12), Ghana. was detectable, not only at sites with or without a history of BU cases, but also in the environment, independent of invertebrates; positive results were detected for all sample tested had 0 positivity. These taxa represented all inverte- types. Although M. ulcerans has been detected on the exo- brate functional feeding groups (e.g., predators, shredders, skeleston of experimentally infected Naucoridae (9), the scrapers, collector-gatherers, and fi lterers). possibility that invertebrates could serve as substrates for M. ulcerans in a natural environment has not been addressed, Biting Hemiptera Correlations but it is certainly possible and may explain the wide range No signifi cant correlation was found between mean of taxa that were found positive in this study. ER positivity and total biting Hemiptera (r = 0.25; p = The invertebrate communities in this study demon- 0.218) or any individual family: Belostomatidae (r = 0.31; strated high intersite variation (online Technical Appen- p = 0.118), Naucoridae (r = -0.03; p = 0.850), and Nepidae dix), a fi nding similar to those of other studies of lentic (r = 0.37; p = 0.060). These results confi rmed that biting invertebrate habitats (31,32). This variation suggests that Hemiptera were not signifi cantly associated with the patho- additional collection sites should be included for a more gen in the environment. comprehensive evaluation of invertebrate communities; an expanded study is under way. Hydrologic and physical/ Discussion chemical attributes regulate the structure and abundance of The role of aquatic invertebrates in the transmission invertebrate communities (31), while biotic factors such as of BU has been proposed several times (3,4,29). How- macrophytes and fi sh can also infl uence communities (33). ever, to date, no large-scale fi eld studies have assessed Few basic ecologic studies have been conducted on non– aquatic invertebrate communities from multiple loca- disease-related aquatic invertebrates in West Africa. The Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 1251 RESEARCH Figure 4. Number of enoyl-reduction- domain–positive taxa detected for each A) Buruli ulcer–endemic site (n = 15) and B) Buruli ulcer–nonendemic site (n = 12), Ghana. most comprehensive articles on ecology have come from subtropical regions, or that any seasonal pattern in BU studies of small, fast-fl owing streams or large lakes (34,35), cases is related to seasonal population changes of biting which are different habitats than those in this study. hemipterans. Season may also play a role in invertebrate abundance Various researchers have proposed that biting water patterns; however, in many tropical and subtropical regions, bugs could be vectors for M. ulcerans, and laboratory stud- most invertebrate taxa show minimal seasonally based abun- ies have provided evidence for this possibility. However, dance patterns (36–38). Most tropical species have multi- no complementary fi eld studies had tested these laboratory voltine (multiple generations) and asynchronous (overlap- results. Results from this fi eld study do not support the hy- ping) life cycles throughout the year (39). For instance, all pothesis that biting aquatic insects are primary vectors of life stages of tropical naucorids have been reported through M. ulcerans. The results do not rule out the possibility of both wet and dry seasons over 2 years (38), and the same has biting Hemiptera or other invertebrates as vectors or pos- been documented for other aquatic invertebrates in Kenya sible reservoirs for M. ulcerans, but rather, they suggest (36) and Lake Tanganyika (37). Therefore, although season caution in describing their role in transmission. These fi eld might have had a small effect on the abundance variation data on biting hemipteran abundance and M. ulcerans posi- of biting hemipterans and other invertebrates, this infl uence tivity suggest a need to reevaluate future research direc- was unlikely to have limited our potential for detecting dif- tions for understanding BU transmission. ferences between BU+ and BU– sites. If season affects biting Hemiptera populations, and Acknowledgements these insects are important vectors, then human BU case We are grateful to E. Ampadu for providing the BU case data data should refl ect seasonal patterns, but this is not gen- and Kingsley Asiedu for continued support of our research. We erally reported (4). In a recent study, no seasonal pattern also thank Todd White and Lydia Mosi for fi eld assistance and in- was shown in monthly BU cases for 2003, 2004, and 2005 vertebrate collections and identifi cations, and Lindsay Campbell (40). In the current study, sampling each site throughout the and Jenni van Ravensway for generating the site map. year was not logistically feasible. In other ongoing studies, we have sampled an additional 55 sites, including 22 sites This work was funded by the World Health Organization and from 2004 to 2005 that have been sampled at least twice the National Institutes of Health (NIH). The project described was and 6 sites sampled 3 times over 3 years. The abundance supported by grant no. R01TW007550 from the Fogarty Interna- of biting Hemiptera and other invertebrates from these ad- tional Center through the NIH/National Science Foundation Ecol- ditional sites are similar to what is reported here. There- ogy of Infectious Diseases Program and grant no. R03AI062719. fore, although season may have infl uenced our invertebrate Dr Benbow is an assistant professor in the Department of Bi- community abundances, little evidence suggests that BU+ ology at the University of Dayton. His research interests include and BU– sites would be differentially affected, that Ghana- the role of human-mediated environmental changes in aquatic ian invertebrate communities should respond differently to ecosystem structure and function, with emphasis on the ecology season compared with communities in other tropical and of M. ulcerans infection in West Africa. 1252 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 14, No. 8, August 2008 Buruli Ulcer Disease References 18. 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