Landscape and environmental influences on Mycobacterium ulcerans distribution among aquatic sites in Ghana.

Buruli ulcer, caused by Mycobacterium ulcerans, is highly endemic in West Africa. While the mode of transmission is unknown, many studies associate Buruli ulcer with different types of water exposure. We present results from the largest study to date to test for M. ulcerans in aquatic sites and identify environmental attributes associated with its presence. Environmental samples from 98 aquatic sites in the Greater Accra, Ashanti, and Volta regions of Ghana were tested for the presence of M. ulcerans DNA by polymerase chain reaction. The proportion of aquatic sites positive for M. ulcerans varied by region: Ashanti 66% (N = 39), Greater Accra 34% (N = 29), and Volta 0% (N = 30). We explored the spatial distribution of M. ulcerans positive and negative water bodies and found no significant clusters. We also determined both highly localized water attributes and broad scale remotely sensed land cover and terrain environmental characteristics associated with M. ulcerans presence through logistic regression. Our results concur with published results regarding conditions suitable for M. ulcerans growth and associations with Buruli ulcer disease burden with regards to water characteristics and disturbed environments, but differ from others with regards to spatial associations and topographic effects such as elevation and wetness. While our results suggest M. ulcerans is an environmental organism existing in a specific ecological niche, they also reveal variation in the elements defining this niche across the sites considered. In addition, despite the causal association between Buruli ulcer and M. ulcerans, we observed no significant statistical association between case reports of Buruli ulcer and presence of M. ulcerans in nearby waterbodies.

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.

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.

Identifying the Achilles' heel of multi-host pathogens: The concept of keystone "host" species illustrated by Mycobacterium ulcerans transmission.

Pathogens that use multiple host species are an increasing public health issue due to their complex transmission, which makes them difficult to mitigate. Here, we explore the possibility of using networks of ecological interactions among potential host species to identify the particular disease-source species to target to break down transmission of such pathogens. We fit a mathematical model on prevalence data of Mycobacterium ulcerans in western Africa and we show that removing the most abundant taxa for this category of pathogen is not an optimal strategy to decrease the transmission of the mycobacterium within aquatic ecosystems. On the contrary, we reveal that the removal of some taxa, especially Oligochaeta worms, can clearly reduce rates of pathogen transmission and should be considered as a keystone organism for its transmission because it leads to a substantial reduction in pathogen prevalence regardless of the network topology. Besides its potential application for the understanding of M. ulcerans ecology, we discuss about how networks of species interactions can modulate transmission of multi-host pathogens.

A molecular approach to identifying the natural prey of the African creeping water bug Naucoris, a potential reservoir of Mycobacterium ulcerans.

The extra-oral digestion of creeping water bugs (Naucoridae: Hemiptera) hinders the study of their diet using the standard method of identifying prey body parts in the gut. Genetic methods are available, but rely on PCR tests or similar diagnostics to confirm suspected prey. Where the potential prey is unknown and a broad search for all possible prey is desirable, methods that can potentially capture any prey item are required. Naucoris sp. is known to harbor Mycobacterium ulcerans (Actinomycetales: Mycobacteriaceae), the causative bacterium of Buruli ulcer. Outbreaks of Buruli ulcer have been associated with disturbed freshwater habitats, but the mode of transmission to humans remains unclear. Here we examine the diet of Naucoris sp., a dominant aquatic predator in water bodies in Ghana where the prevalence of Buruli ulcer is high. We cloned and sequenced 576 PCR products (mtDNA rrnL, cox1) isolated from the gut of 60 Naucoris sp. individuals to determining diet composition as broadly as possible. Using phylogenetic analysis of newly sequenced clones and 6 potential prey taxa collected from the site, sequences isolated from Naucoris sp. guts matched locally collected Coleoptera (Hydrophilidae). Blastn queries to GenBank of other clone sequences produced matches to (Anura) (n = 1), Rotifera (n = 5), and fungi (n = 4) as additional components of the diet. Our results suggest that sp. in this Buruli ulcer-endemic area feeds on a wide range of prey and body sizes, and that the approach could be successfully applied to studies of aquatic food webs where morphological identification of prey is impossible and where little or no a priori knowledge is available.

Effectiveness of wound cleansing treatments on maggot (Diptera, Calliphoridae) mortality.

Myiasis is defined as an infestation of the organs and/or tissues of human and other animals by fly maggots. Fly species that normally breed in meat or carrion (Diptera: Calliphoridae, Sarcophagidae) may become involved in cutaneous myiasis by colonizing preexisting wounds. Reports of human wound myiasis contracted in hospitals and nursing homes, especially when patients are chronically ill or bed-ridden, are not uncommon across North America and often result in cases of neglect and civil litigation. Based on a case history dealing with this latter situation and circumstances surrounding the treatment of maggot infestation, we designed an experiment to assess the effectiveness of wound cleansing solutions on maggot mortality. Treatments, consisting of four commonly used cleaning solutions (isopropyl alcohol, Dakin's solution, iodine, and hydrogen peroxide) and a control (deionized water), were applied to experimental units (n=5), with each unit consisting of groups of actively feeding Lucilia sericata maggots (Diptera: Calliphoridae). Every 24h, treatments were applied and mortality was assessed for the duration of the study (14 days). Total mean mortality increased over the duration of the experiment, with an initial large increase (10-25%) after the first treatment application, followed by a gradual increase over the remainder of the study. General differences among treatments indicated greatest mean total mortality for Dakin's solution (sodium hypochlorite) (46%), followed by isopropyl alcohol (42%), Betadine (37%), hydrogen peroxide (33%) and lowest mortality for the control (25%); however, no statistically significant differences were observed among treatments and no treatment resulted in 100% maggot mortality. Traditional wound cleansing solutions may not be sufficient for maggot infestations of pre-existing wounds and supplemental treatments may be necessary to effectively treat cases of wound myiasis.

Aquatic invertebrates as unlikely vectors of Buruli ulcer disease.

Buruli ulcer is a necrotizing skin disease caused by Mycobacterium ulcerans and associated with exposure to aquatic habitats. To assess possible transmission of M. ulcerans by aquatic biting insects, we conducted a field examination of biting water bugs (Hemiptera: Naucoridae, Belostomatidae, Nepidae) in 15 disease-endemic and 12 non-disease-endemic areas of Ghana, Africa. From collections of 22,832 invertebrates, we compared composition, abundance, and associated M. ulcerans positivity among sites. Biting hemipterans were rare and represented a small percentage (usually <2%) of invertebrate communities. No significant differences were found in hemipteran abundance or pathogen positivity between disease-endemic and non-disease-endemic sites, and between abundance of biting hemipterans and M. ulcerans positivity. Therefore, although infection through insect bites is possible, little field evidence supports the assumption that biting hemipterans are primary vectors of M. ulcerans.

Distribution of Mycobacterium ulcerans in buruli ulcer endemic and non-endemic aquatic sites in Ghana.

Mycobacterium ulcerans, the causative agent of Buruli ulcer, is an emerging environmental bacterium in Australia and West Africa. The primary risk factor associated with Buruli ulcer is proximity to slow moving water. Environmental constraints for disease are shown by the absence of infection in arid regions of infected countries. A particularly mysterious aspect of Buruli ulcer is the fact that endemic and non-endemic villages may be only a few kilometers apart within the same watershed. Recent studies suggest that aquatic invertebrate species may serve as reservoirs for M. ulcerans, although transmission pathways remain unknown. Systematic studies of the distribution of M. ulcerans in the environment using standard ecological methods have not been reported. Here we present results from the first study based on random sampling of endemic and non-endemic sites. In this study PCR-based methods, along with biofilm collections, have been used to map the presence of M. ulcerans within 26 aquatic sites in Ghana. Results suggest that M. ulcerans is present in both endemic and non-endemic sites and that variable number tandem repeat (VNTR) profiling can be used to follow chains of transmission from the environment to humans. Our results suggesting that the distribution of M. ulcerans is far broader than the distribution of human disease is characteristic of environmental pathogens. These findings imply that focal demography, along with patterns of human water contact, may play a major role in transmission of Buruli ulcer.

Caddisflies assist with homicide case: determining a postmortem submersion interval using aquatic insects.

Although few indicators of time since death for corpses found in aquatic ecosystems are comparable in precision to the insect indicators used in terrestrial cases, there are observations that can be useful in suggesting or ruling out an approximate PMSI (postmortem submersion interval). For example, the time intervals required for certain growth phases of aquatic insects, such as caddisflies, that may attach themselves to the submerged remains can be used to estimate a minimum PMSI. Approximately 8 of the 13 orders of insects containing species with aquatic or semi-aquatic stages are likely to be associated with carrion or corpses in aquatic habitats. We present a case study in which portions of a body from an adult male were discovered in a south central Michigan stream. The body was dismembered and portions were recovered from two bags floating and submerged in the stream. Insect specimens collected from mesh and plastic bags consisted of one fly larva belonging to the family Muscidae, and caddisfly larvae belonging to two families: the Limnephilidae. (case-makers) and the Hydropsychidae, (net spinners). We used unique case-building behaviors of the limnephilid caddisflies found on the remains to elucidate a PMSI range consistent with the disappearance of the victim. It is important for forensic investigators to understand that although some precision is lost in estimating a PMSI with aquatic insects, these organisms should not be ignored in gathering evidence from aquatic crime scenes, and in fact, they can provide valuable details in estimating a PMSI.

Collembola of the grave: a cold case history involving arthropods 28 years after death.

This report describes a cold case in which a cadaver of a 28-year-old female was exhumed in February 2005 from a cemetery in Battle Creek, Michigan. She had sustained a gunshot wound to the head and was found dead in her home on November 15, 1977. The body of the victim was subsequently embalmed and then buried at a depth of 1.8 m in an unsealed casket that was placed inside an unsealed cement vault. The exhumation yielded thousands of live specimens of a single species of the order Collembola or spring tails, Sinella (Coecobrya) tenebricosa (Entomobryidae). This species is considered to be a "tramp" species, cosmopolitan in the United States and Canada. Due to the ideal environmental conditions at the site, the population of this species underwent growth and development inside the casket for a number of years. Collected with the Collembola were large numbers of Acarina (mites) of the Family Glycyphagidae, and fly puparia, Conicera tibialis Schmitz (Order: Diptera, Family: Phoridae), also known as coffin flies. These invertebrates are sometimes mentioned by forensic investigators as occurring on corpses in graves, but aspects of their life history are rarely described. The species of Collembola that was found surviving and reproducing on this corpse in a casket exhumed after 28 years was the oldest reported grave site occurrence for any collembolan species based on a survey of the literature back to 1898.