Aquatic Insects as a Vector for Mycobacterium ulcerans

Mycobacterium ulcerans is an emerging environmental pathogen which causes chronic skin ulcers (i.e., Buruli ulcer) in otherwise healthy humans living in tropical countries, particularly those in Africa. In spite of epidemiological and PCR data linking M. ulcerans to water, the mode of transmission of this organism remains elusive. To determine the role of aquatic insects in the transmission of M. ulcerans, we have set up an experimental model with aquariums that mimic aquatic microenvironments. We report that M. ulcerans may be transmitted to laboratory mice by the bite of aquatic bugs (Naucoridae) that are infected with this organism. In addition, M. ulcerans appears to be localized exclusively within salivary glands of these insects, where it can both survive and multiply without causing any observable damage in the insect tissues. Subsequently, we isolated M. ulcerans from wild aquatic insects collected from a zone in the Daloa region of Ivory Coast where Buruli ulcer is endemic. Taken together, these results point to aquatic insects as a possible vector of M. ulcerans.     

Aquatic Plants Stimulate the Growth of and Biofilm Formation by Mycobacterium ulcerans in Axenic Culture and Harbor These Bacteria in the Environment

Mycobacterium ulcerans is the causative agent of Buruli ulcer, one of the most common mycobacterial diseases of humans. Recent studies have implicated aquatic insects in the transmission of this pathogen, but the contributions of other elements of the environment remain largely unknown. We report here that crude extracts from two green algae added to the BACTEC 7H12B culture medium halved the doubling time of M. ulcerans and promoted biofilm formation. Using the 7H12B medium, modified by the addition of the algal extract, and immunomagnetic separation, we also demonstrate that M. ulcerans is associated with aquatic plants in an area of the Ivory Coast where Buruli ulcer is endemic. Genotype analysis showed that plant-associated M. ulcerans had the same profile as isolates recovered in the same region from both aquatic insects and clinical specimens. These observations implicate aquatic plants as a reservoir of M. ulcerans and add a new potential link in the chain of transmission of M. ulcerans to humans.     

Aquatic Snails, Passive Hosts of Mycobacterium ulcerans

Accumulative indirect evidence of the epidemiology of Mycobacterium ulcerans infections causing chronic skin ulcers (i.e., Buruli ulcer disease) suggests that the development of this pathogen and its transmission to humans are related predominantly to aquatic environments. We report that snails could transitorily harbor M. ulcerans without offering favorable conditions for its growth and replication. A novel intermediate link in the transmission chain of M. ulcerans becomes likely with predator aquatic insects in addition to phytophage insects. Water bugs, such as Naucoris cimicoides, a potential vector of M. ulcerans, were shown to be infected specifically by this bacterium after feeding on snails experimentally exposed to M. ulcerans.     

First cultivation and characterization of Mycobacterium ulcerans from the environment

Background

Mycobacterium ulcerans disease, or Buruli ulcer (BU), is an indolent, necrotizing infection of skin, subcutaneous tissue and, occasionally, bones. It is the third most common human mycobacteriosis worldwide, after tuberculosis and leprosy. There is evidence that M. ulcerans is an environmental pathogen transmitted to humans from aquatic niches; however, well-characterized pure cultures of M. ulcerans from the environment have never been reported. Here we present details of the isolation and characterization of an M. ulcerans strain (00-1441) obtained from an aquatic Hemiptera (common name Water Strider, Gerris sp.) from Benin.

Methodology/Principal Findings

One culture from a homogenate of a Gerris sp. in BACTEC became positive for IS2404, an insertion sequence with more than 200 copies in M. ulcerans. A pure culture of M. ulcerans 00-1441 was obtained on Löwenstein-Jensen medium after inoculation of BACTEC culture in mouse footpads followed by two other mouse footpad passages. The phenotypic characteristics of 00-1441 were identical to those of African M. ulcerans, including production of mycolactone A/B. The nucleotide sequence of the 5′ end of 16S rRNA gene of 00-1441 was 100% identical to M. ulcerans and M. marinum, and the sequence of the 3′ end was identical to that of the African type except for a single nucleotide substitution at position 1317. This mutation in M. ulcerans was recently discovered in BU patients living in the same geographic area. Various genotyping methods confirmed that strain 00-1441 has a profile identical to that of the predominant African type. Strain 00-1441 produced severe progressive infection and disease in mouse footpads with involvement of bone.

Conclusion

Strain 00-1441 represents the first genetically and phenotypically identified strain of M. ulcerans isolated in pure culture from the environment. This isolation supports the concept that the agent of BU is a human pathogen with an environmental niche.     

Associations Between Mycobacterium ulcerans and Aquatic Plant Communities of West Africa: Implications for Buruli Ulcer Disease

Numerous studies have associated Buruli ulcer (BU) disease with disturbed aquatic habitats; however, the natural reservoir, distribution, and transmission of the pathogen, Mycobacterium ulcerans, remain unknown. To better understand the role of aquatic plants in the ecology of this disease, a large-scale survey was conducted in waterbodies of variable flow throughout three regions of Ghana, Africa. Our objectives were to characterize plant communities and identify potential relationships with M. ulcerans and other mycolactone-producing mycobacteria (MPM). Waterbodies with M. ulcerans had significantly different aquatic plant communities, with submerged terrestrial plants identified as indicators of M. ulcerans presence. Mycobacterium ulcerans and MPM were detected on 14 plant taxa in emergent zones from both lotic and lentic waterbodies in endemic regions; however, M. ulcerans was not detected in the non-endemic Volta region. These findings support the hypothesis that plants provide substrate for M. ulcerans colonization and could act as potential indicators for disease risk. These findings also suggest that M. ulcerans is a widespread environmental bacteria species, but that it is absent or reduced in regions of low disease incidence. A better understanding is needed regarding the mechanistic associations among aquatic plants and M. ulcerans for identifying the mode of transmission of BU disease.     

A Major Role for Mammals in the Ecology of Mycobacterium ulcerans

Background

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU), a destructive skin disease found predominantly in sub-Saharan Africa and south-eastern Australia. The precise mode(s) of transmission and environmental reservoir(s) remain unknown, but several studies have explored the role of aquatic invertebrate species. The purpose of this study was to investigate the environmental distribution of M. ulcerans in south-eastern Australia.

Methodology/Principal Findings

A range of environmental samples was collected from Point Lonsdale (a small coastal town southwest of Melbourne, Australia, endemic for BU) and from areas with fewer or no reported incident cases of BU. Mycobacterium ulcerans DNA was detected at low levels by real-time PCR in soil, sediment, water residue, aquatic plant biofilm and terrestrial vegetation collected in Point Lonsdale. Higher levels of M. ulcerans DNA were detected in the faeces of common ringtail (Pseudocheirus peregrinus) and common brushtail (Trichosurus vulpecula) possums. Systematic testing of possum faeces revealed that M. ulcerans DNA could be detected in 41% of faecal samples collected in Point Lonsdale compared with less than 1% of faecal samples collected from non-endemic areas (p<0.0001). Capture and clinical examination of live possums in Point Lonsdale validated the accuracy of the predictive value of the faecal surveys by revealing that 38% of ringtail possums and 24% of brushtail possums had laboratory-confirmed M. ulcerans skin lesions and/or M. ulcerans PCR positive faeces. Whole genome sequencing revealed an extremely close genetic relationship between human and possum M. ulcerans isolates.

Conclusions/Significance

The prevailing wisdom is that M. ulcerans is an aquatic pathogen and that BU is acquired by contact with certain aquatic environments (swamps, slow-flowing water). Now, after 70 years of research, we propose a transmission model for BU in which terrestrial mammals are implicated as reservoirs for M. ulcerans.     

Mycobacterium ulcerans Fails to Infect through Skin Abrasions in a Guinea Pig Infection Model: Implications for Transmission

Transmission of M. ulcerans, the etiological agent of Buruli ulcer, from the environment to humans remains an enigma despite decades of research. Major transmission hypotheses propose 1) that M. ulcerans is acquired through an insect bite or 2) that bacteria enter an existing wound through exposure to a contaminated environment. In studies reported here, a guinea pig infection model was developed to determine whether Buruli ulcer could be produced through passive inoculation of M. ulcerans onto a superficial abrasion. The choice of an abrasion model was based on the fact that most bacterial pathogens infecting the skin are able to infect an open lesion, and that abrasions are extremely common in children. Our studies show that after a 90d infection period, an ulcer was present at intra-dermal injection sites of all seven animals infected, whereas topical application of M. ulcerans failed to establish an infection. Mycobacterium ulcerans was cultured from all injection sites whereas infected abrasion sites healed and were culture negative. A 14d experiment was conducted to determine how long organisms persisted after inoculation. Mycobacterium ulcerans was isolated from abrasions at one hour and 24 hours post infection, but cultures from later time points were negative. Abrasion sites were qPCR positive up to seven days post infection, but negative at later timepoints. In contrast, M. ulcerans DNA was detected at intra-dermal injection sites throughout the study. M. ulcerans was cultured from injection sites at each time point. These results suggest that injection of M. ulcerans into the skin greatly facilitates infection and lends support for the role of an invertebrate vector or other route of entry such as a puncture wound or deep laceration where bacteria would be contained within the lesion. Infection through passive inoculation into an existing abrasion appears a less likely route of entry.     

Detection of Mycobacterium ulcerans in the environment predicts prevalence of Buruli ulcer in Benin

Background

Mycobacterium ulcerans is the causative agent of Buruli ulcer (BU). In West Africa there is an association between BU and residence in low-lying rural villages where aquatic sources are plentiful. Infection occurs through unknown environmental exposure; human-to-human infection is rare. Molecular evidence for M. ulcerans in environmental samples is well documented, but the association of M. ulcerans in the environment with Buruli ulcer has not been studied in West Africa in an area with accurate case data.

Methodology/Principal Finding

Environmental samples were collected from twenty-five villages in three communes of Benin. Sites sampled included 12 BU endemic villages within the Ouheme and Couffo River drainages and 13 villages near the Mono River and along the coast or ridge where BU has never been identified. Triplicate water filtrand samples from major water sources and samples from three dominant aquatic plant species were collected. Detection of M. ulcerans was based on quantitative polymerase chain reaction. Results show a significant association between M. ulcerans in environmental samples and Buruli ulcer cases in a village (p = 0.0001). A “dose response” was observed in that increasing numbers of M. ulceran- positive environmental samples were associated with increasing prevalence of BU cases (R² = 0.586).

Conclusions/Significance

This study provides the first spatial data on the overlap of M. ulcerans in the environment and BU cases in Benin where case data are based on active surveillance. The study also provides the first evidence on M. ulcerans in well-defined non-endemic sites. Most environmental pathogens are more broadly distributed in the environment than in human populations. The congruence of M. ulcerans in the environment and human infection raises the possibility that humans play a role in the ecology of M. ulcerans. Methods developed could be useful for identifying new areas where humans may be at high risk for BU.     

Seasonal and Regional Dynamics of M. ulcerans Transmission in Environmental Context: Deciphering the Role of Water Bugs as Hosts and Vectors

Background

Buruli ulcer, the third mycobacterial disease after tuberculosis and leprosy, is caused by the environmental mycobacterium M. ulcerans. Various modes of transmission have been suspected for this disease, with no general consensus acceptance for any of them up to now. Since laboratory models demonstrated the ability of water bugs to transmit M. ulcerans, a particular attention is focused on the transmission of the bacilli by water bugs as hosts and vectors. However, it is only through detailed knowledge of the biodiversity and ecology of water bugs that the importance of this mode of transmission can be fully assessed. It is the objective of the work here to decipher the role of water bugs in M. ulcerans ecology and transmission, based on large-scale field studies.

Methodology/Principal Findings

The distribution of M. ulcerans-hosting water bugs was monitored on previously unprecedented time and space scales: a total of 7,407 water bugs, belonging to large number of different families, were collected over one year, in Buruli ulcer endemic and non endemic areas in central Cameroon. This study demonstrated the presence of M. ulcerans in insect saliva. In addition, the field results provided a full picture of the ecology of transmission in terms of biodiversity and detailed specification of seasonal and regional dynamics, with large temporal heterogeneity in the insect tissue colonization rate and detection of M. ulcerans only in water bug tissues collected in Buruli ulcer endemic areas.

Conclusion/Significance

The large-scale detection of bacilli in saliva of biting water bugs gives enhanced weight to their role in M. ulcerans transmission. On practical grounds, beyond the ecological interest, the results concerning seasonal and regional dynamics can provide an efficient tool in the hands of sanitary authorities to monitor environmental risks associated with Buruli ulcer.     

Potential Wildlife Sentinels for Monitoring the Endemic Spread of Human Buruli Ulcer in South-East Australia

The last 20 years has seen a significant series of outbreaks of Buruli/Bairnsdale Ulcer (BU), caused by Mycobacterium ulcerans, in temperate south-eastern Australia (state of Victoria). Here, the prevailing view of M. ulcerans as an aquatic pathogen has been questioned by recent research identifying native wildlife as potential terrestrial reservoirs of infection; specifically, tree-dwelling common ringtail and brushtail possums. In that previous work, sampling of environmental possum faeces detected a high prevalence of M. ulcerans DNA in established endemic areas for human BU on the Bellarine Peninsula, compared with non-endemic areas. Here, we report research from an emergent BU focus recently identified on the Mornington Peninsula, confirming associations between human BU and the presence of the aetiological agent in possum faeces, detected by real-time PCR targeting M. ulcerans IS2404, IS2606 and KR. Mycobacterium ulcerans DNA was detected in 20/216 (9.3%) ground collected ringtail possum faecal samples and 4/6 (66.6%) brushtail possum faecal samples. The distribution of the PCR positive possum faecal samples and human BU cases was highly focal: there was a significant non-random cluster of 16 M. ulcerans positive possum faecal sample points detected by spatial scan statistics (P<0.0001) within a circle of radius 0.42 km, within which were located the addresses of 6/12 human cases reported from the area to date; moreover, the highest sample PCR signal strength (equivalent to ≥10⁶ organisms per gram of faeces) was found in a sample point located within this cluster radius. Corresponding faecal samples collected from closely adjacent BU-free areas were predominantly negative. Possums may be useful sentinels to predict endemic spread of human BU in Victoria, for public health planning. Further research is needed to establish whether spatial associations represent evidence of direct or indirect transmission between possums and humans, and the mechanism by which this may occur.     

Amoebae as Potential Environmental Hosts for Mycobacterium ulcerans and Other Mycobacteria, but Doubtful Actors in Buruli Ulcer Epidemiology

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, remain unknown. Ecological, genetic and epidemiological information nonetheless suggests that M. ulcerans may reside in aquatic protozoa.

Methodology/Principal Findings

We experimentally infected Acanthamoeba polyphaga with M. ulcerans and found that the bacilli were phagocytised, not digested and remained viable for the duration of the experiment. Furthermore, we collected 13 water, 90 biofilm and 45 detritus samples in both Buruli ulcer endemic and non-endemic communities in Ghana, from which we cultivated amoeboid protozoa and mycobacteria. M. ulcerans was not isolated, but other mycobacteria were as frequently isolated from intracellular as from extracellular sources, suggesting that they commonly infect amoebae in nature. We screened the samples as well as the amoeba cultures for the M. ulcerans markers IS2404, IS2606 and KR-B. IS2404 was detected in 2% of the environmental samples and in 4% of the amoeba cultures. The IS2404 positive amoeba cultures included up to 5 different protozoan species, and originated both from Buruli ulcer endemic and non-endemic communities.

Conclusions/Significance

This is the first report of experimental infection of amoebae with M. ulcerans and of the detection of the marker IS2404 in amoeba cultures isolated from the environment. We conclude that amoeba are potential natural hosts for M. ulcerans, yet remain sceptical about their implication in the transmission of M. ulcerans to humans and their importance in the epidemiology of Buruli ulcer.     

Interaction of Mycobacterium ulcerans with Mosquito Species: Implications for Transmission and Trophic Relationships

Mycobacterium ulcerans is the causative agent of Buruli ulcer, a severe necrotizing skin disease that causes significant morbidity in Africa and Australia. Person-to-person transmission of Buruli ulcer is rare. Throughout Africa and Australia infection is associated with residence near slow-moving or stagnant water bodies. Although M. ulcerans DNA has been detected in over 30 taxa of invertebrates, fish, water filtrate, and plant materials and one environmental isolate cultured from a water strider (Gerridae), the invertebrate taxa identified are not adapted to feed on humans, and the mode of transmission for Buruli ulcer remains an enigma. Recent epidemiological reports from Australia describing the presence of M. ulcerans DNA in adult mosquitoes have led to the hypothesis that mosquitoes play an important role in the transmission of M. ulcerans. In this study we have investigated the potential of mosquitoes to serve as biological or mechanical vectors or as environmental reservoirs for M. ulcerans. Here we show that Aedes aegypti, A. albopictus, Ochlerotatus triseriatus, and Culex restuans larvae readily ingest wild-type M. ulcerans, isogenic toxin-negative mutants, and Mycobacterium marinum isolates and remain infected throughout larval development. However, the infections are not carried over into the pupae or adult mosquitoes, suggesting an unlikely role for mosquitoes as biological vectors. By following M. ulcerans through a food chain consisting of primary (mosquito larvae), secondary (predatory mosquito larva from Toxorhynchites rutilus septentrionalis), and tertiary (Belostoma species) consumers, we have shown that M. ulcerans can be productively maintained in an aquatic food web.Infection with Mycobacterium ulcerans, the causative agent of Buruli ulcer (BU) disease, is associated with residence near stagnant and slow-moving water bodies in areas in which the disease is endemic (5, 36, 40, 45, 50). A plasmid-encoded macrolide toxin, mycolactone, is the primary virulence determinant of M. ulcerans (8, 41). Biting aquatic insects, such as several taxa in the Belostomatidae and Naucoridae families (Hemiptera), have been suggested as possible vectors of M. ulcerans in several laboratory experiments (16, 19, 20, 24, 31, 32); however, there is little empirical evidence from field studies to support the contention that these biting insects vector M. ulcerans to humans (2). In Melbourne, Australia, recent epidemiological evidence suggests that mosquitoes may serve as vectors in the transmission of BU disease (10, 11, 12, 34, 35). In this study, 957 pools consisting of over 11,000 mosquitoes of four different species were collected and tested by quantitative PCR (qPCR) for the presence of M. ulcerans DNA, and positive results were obtained from 48 of 957 pools tested (10). Of the 48 positive pools, 13 were positive for PCR directed against two insertion sequences (IS2404 and IS2606) as well as against sequence based on the ketoreductase domain of the mycolactone toxin genes. Because all of these target sequences are present multiple times in the genome, it was difficult to assign genome equivalents to these results. However, data from laboratory experiments suggested that 10 to 100 M. ulcerans isolates per mosquito were present in the positive pools. Epidemiological work also suggested a seasonal relationship between Buruli ulcer and mosquito-vectored diseases in Australia (12). These studies are extremely provocative and raise a number of questions for further work. What is the prevalence of M. ulcerans in other invertebrate taxa in the same environment? What is the infection rate in equal numbers of mosquitoes collected from areas in which the disease is not endemic? Is it possible to obtain physical evidence for the presence of M. ulcerans through microscopy or culture of mosquitoes in areas in which the disease is endemic, and, finally, what can we learn from laboratory studies concerning the interaction between mosquitoes and M. ulcerans?The recent work from Australia suggesting that M. ulcerans is spread by mosquitoes is particularly significant because adult mosquitoes are the most important group of insects in the spread of human disease. They may serve as biological vectors that provide a major environment for pathogen replication, as in malaria or yellow fever, or as mechanical vectors that carry organisms between hosts without serving as a site of replication (1, 4, 7, 9, 38). Larval mosquitoes are common in habitats associated with BU disease, most notably lentic or standing water habitats, and feed by filtering particles in the water using labral head fans (21). Members of some genera (i.e., Anopheles) aggregate at the air-water interface in microlayers near plant stems and algal mats (27, 28, 46), where they feed on microorganisms such as bacteria and algae (47). Because of their collecting-filtering feeding mode, there is potential for larvae to consume M. ulcerans and concentrate mycobacteria through their feeding activities (22, 23).In Ghana, the occurrence of M. ulcerans among invertebrate communities in lentic habitats has been documented from regions in Ga West and Ga East Districts in which the disease is endemic as well as those in which it is not endemic (2, 49) but not in geographically distinct areas in which the disease is not endemic such as the Volta region (49). M. ulcerans has been identified in a suite of environmental samples such as filtered water, biofilms, and algae as well as among a broad spectrum of invertebrate taxa, including both larval and adult mosquitoes (2, 11, 17, 49). However, the replication and trophic movement of M. ulcerans within these environmental samples and invertebrate communities have not been experimentally investigated. Conceptual models have been proposed that assume that the primary consumers of M. ulcerans (e.g., mosquito larvae, cladocerans, and chironomid larvae) may feed on bacteria and algae in biofilms, filter suspended matter from the water column, and then initiate the passage of M. ulcerans through an aquatic food web (2, 22, 31). This model predicts the movement of M. ulcerans through secondary and tertiary consumers and implies a complex trophic relationship in the ecology of M. ulcerans as well as an important role of aquatic invertebrates in the disease ecology of M. ulcerans.In the studies reported here, we have explored the role of mosquitoes as biological or mechanical vectors of M. ulcerans, as well as the potential of mosquito larvae to play a central role in the movement of M. ulcerans through an aquatic food web. In order to investigate the ability of mosquito larvae to ingest and maintain M. ulcerans within their digestive tract as well as to persist throughout the mosquito development cycle, we took advantage of the fact that mosquito larvae naturally feed upon bacteria. Results presented here show that strains of M. ulcerans from Africa and Australia, as well as Mycobacterium marinum, were maintained at high levels in the larval mosquito gut for 6 days. However, neither M. ulcerans nor M. marinum was detected in adult mosquitoes that were infected in the larval stage. These results suggest that mosquitoes are unlikely to serve as biological vectors of M. ulcerans.We further developed a model for following the passage of M. ulcerans through a series of consumers to determine whether M. ulcerans could be passed up a trophic chain from primary to tertiary consumers. In this model, we conducted similar experiments using four species of nonpredatory mosquito larvae, Aedes aegypti (Linnaeus), Aedes albopictus (Skuse), Ochlerotatus triseriatus (Theobald), and Culex restuans (Theobald), as primary consumers. These larvae were infected with isogenic wild-type (WT) and toxin-negative isolates of M. ulcerans and of M. marinum, the closest relative to M. ulcerans (13, 14, 51). We have shown that M. ulcerans in mosquito larvae survive passage through secondary and tertiary consumers, thus providing the first laboratory evidence that M. ulcerans has the potential to move between and be maintained within different species in an aquatic food web.     

Insertion Sequence Element Single Nucleotide Polymorphism Typing Provides Insights into the Population Structure and Evolution of Mycobacterium ulcerans across Africa

Buruli ulcer is an indolent, slowly progressing necrotizing disease of the skin caused by infection with Mycobacterium ulcerans. In the present study, we applied a redesigned technique to a vast panel of M. ulcerans disease isolates and clinical samples originating from multiple African disease foci in order to (i) gain fundamental insights into the population structure and evolutionary history of the pathogen and (ii) disentangle the phylogeographic relationships within the genetically conserved cluster of African M. ulcerans. Our analyses identified 23 different African insertion sequence element single nucleotide polymorphism (ISE-SNP) types that dominate in different areas where Buruli ulcer is endemic. These ISE-SNP types appear to be the initial stages of clonal diversification from a common, possibly ancestral ISE-SNP type. ISE-SNP types were found unevenly distributed over the greater West African hydrological drainage basins. Our findings suggest that geographical barriers bordering the basins to some extent prevented bacterial gene flow between basins and that this resulted in independent focal transmission clusters associated with the hydrological drainage areas. Different phylogenetic methods yielded two well-supported sister clades within the African ISE-SNP types. The ISE-SNP types from the “pan-African clade” were found to be widespread throughout Africa, while the ISE-SNP types of the “Gabonese/Cameroonian clade” were much rarer and found in a more restricted area, which suggested that the latter clade evolved more recently. Additionally, the Gabonese/Cameroonian clade was found to form a strongly supported monophyletic group with Papua New Guinean ISE-SNP type 8, which is unrelated to other Southeast Asian ISE-SNP types.     

Investigating the Role of Free-living Amoebae as a Reservoir for Mycobacterium ulcerans

Background

The reservoir and mode of transmission of Mycobacterium ulcerans, the causative agent of Buruli ulcer, still remain a mystery. It has been suggested that M. ulcerans persists with difficulty as a free-living organism due to its natural fragility and inability to withstand exposure to direct sunlight, and thus probably persists within a protective host environment.

Methodology/Principal Findings

We investigated the role of free-living amoebae as a reservoir of M. ulcerans by screening the bacterium in free-living amoebae (FLA) cultures isolated from environmental specimens using real-time PCR. We also followed the survival of M. ulcerans expressing green fluorescence protein (GFP) in Acanthameoba castellanii by flow cytometry and observed the infected cells using confocal and transmission electron microscopy for four weeks in vitro. IS2404 was detected by quantitative PCR in 4.64% of FLA cultures isolated from water, biofilms, detritus and aerosols. While we could not isolate M. ulcerans, 23 other species of mycobacteria were cultivated from inside FLA and/or other phagocytic microorganisms. Laboratory experiments with GFP-expressing M. ulcerans in A. castellani trophozoites for 28 days indicated the bacteria did not replicate inside amoebae, but they could remain viable at low levels in cysts. Transmission electron microscopy of infected A. castellani confirmed the presence of bacteria within both trophozoite vacuoles and cysts. There was no correlation of BU notification rate with detection of the IS2404 in FLA (r = 0.07, n = 539, p = 0.127).

Conclusion/Significance

This study shows that FLA in the environment are positive for the M. ulcerans insertion sequence IS2404. However, the detection frequency and signal strength of IS2404 positive amoabae was low and no link with the occurrence of BU was observed. We conclude that FLA may host M. ulcerans at low levels in the environment without being directly involved in the transmission to humans.     

Aquatic Macroinvertebrate Assemblages of Ghana,West Africa: Understanding the Ecology of a Neglected Tropical Disease

Buruli ulcer (BU) is an emerging, but neglected tropical disease, where there has been a reported association with disturbed aquatic habitats and proposed aquatic macroinvertebrate vectors such as biting Hemiptera. An initial step in understanding the potential role of macroinvertebrates in the ecology of BU is to better understand the entire community, not just one or two taxa, in relation to the pathogen, Mycobacterium ulcerans, at a large spatial scale. For the first time at a country-wide scale this research documents that M. ulcerans was frequently detected from environmental samples taken from BU endemic regions, but was not present in 30 waterbodies of a non-endemic region. There were significant differences in macroinvertebrate community structure and identified potential indicator taxa in relation to pathogen presence. These results suggest that specific macroinvertebrate taxa or functional metrics may potentially be used as aquatic biological indicators of M. ulcerans. Developing ecological indicators of this pathogen is a first step for understanding the disease ecology of BU and should assist future studies of transmission.     

Risk of Buruli ulcer and detection of Mycobacterium ulcerans in mosquitoes in southeastern Australia

Background

Buruli ulcer (BU) is a destructive skin condition caused by infection with the environmental bacterium, Mycobacterium ulcerans. The mode of transmission of M. ulcerans is not completely understood, but several studies have explored the role of biting insects. In this study, we tested for an association between the detection of M. ulcerans in mosquitoes and the risk of BU disease in humans in an endemic area of southeastern Australia.

Methodology/Principal Findings

Adult mosquitoes were trapped in seven towns on the Bellarine Peninsula in Victoria, Australia, from December 2004 to December 2009 and screened for M. ulcerans by real-time PCR. The number of laboratory-confirmed cases of BU in permanent residents of these towns diagnosed during the same period was tallied to determine the average cumulative incidence of BU in each location. Pearson''s correlation coefficient (r) was calculated for the proportion of M. ulcerans-positive mosquitoes per town correlated with the incidence of BU per town. We found a strong dose-response relationship between the detection of M. ulcerans in mosquitoes and the risk of human disease (r, 0.99; 95% CI, 0.92–0.99; p<0.001).

Conclusions/Significance

The results of this study strengthen the hypothesis that mosquitoes are involved in the transmission of M. ulcerans in southeastern Australia. This has implications for the development of intervention strategies to control and prevent BU.     

Potential Role for Fish in Transmission of Mycobacterium ulcerans Disease (Buruli Ulcer): an Environmental Study

This study reports a potential role that fish may play in the transmission of Mycobacterium ulcerans disease (Buruli ulcer). Fish found positive for M. ulcerans DNA all appear to feed on insects or plankton and are believed to concentrate M. ulcerans from this usual food source. These observations provide additional data supporting our previous hypothesis on sources of M. ulcerans and modes of transmission.     

Mycobacterium ulcerans Persistence at a Village Water Source of Buruli Ulcer Patients

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.     

Identification of the Mycobacterium ulcerans Protein MUL_3720 as a Promising Target for the Development of a Diagnostic Test for Buruli Ulcer

Buruli ulcer (BU) caused by Mycobacterium ulcerans is a devastating skin disease, occurring mainly in remote West African communities with poor access to health care. Early case detection and subsequent antibiotic treatment are essential to counteract the progression of the characteristic chronic ulcerative lesions. Since the accuracy of clinical BU diagnosis is limited, laboratory reconfirmation is crucial. However, currently available diagnostic techniques with sufficient sensitivity and specificity require infrastructure and resources only accessible at a few reference centres in the African endemic countries. Hence, the development of a simple, rapid, sensitive and specific point-of-care diagnostic tool is one of the major research priorities for BU. In this study, we have identified a previously unknown M. ulcerans protein, MUL_3720, as a promising target for antigen capture-based detection assays. We show that MUL_3720 is highly expressed by M. ulcerans and has no orthologs in other prevalent pathogenic mycobacteria. We generated a panel of anti-MUL_3720 antibodies and used them to confirm a cell wall location for MUL_3720. These antibodies could also specifically detect M. ulcerans in infected human tissue samples as well as in lysates of infected mouse footpads. A bacterial 2-hybrid screen suggested a potential role for MUL_3720 in cell wall biosynthesis pathways. Finally, we demonstrate that a combination of MUL_3720 specific antibody reagents in a sandwich-ELISA format has sufficient sensitivity to make them suitable for the development of antigen capture-based diagnostic tests for BU.