Assessment of Giardia and Cryptosporidium spp. as a Microbial Source Tracking Tool for Surface Water: Application in a Mixed-Use Watershed

Knowledge of host specificity, combined with genomic sequencing of Giardia and Cryptosporidium spp., has demonstrated a microbial source tracking (MST) utility for these common waterborne microbes. To explore the source attribution potential of these pathogens, water samples were collected in a mixed rural-urban watershed in the Township of Langley, in southwestern British Columbia (BC), Canada, over a 2-year period. Cryptosporidium was detected in 63% of surface water samples at concentrations ranging from no positive detection (NPD) to 20,600 oocysts per 100 liters. Giardia was detected in 86% of surface water samples at concentrations ranging from NPD to 3,800 cysts per 100 liters of water. Sequencing at the 18S rRNA locus revealed that 50% of Cryptosporidium samples and 98% of Giardia samples contained species/genotypes (Cryptosporidium) or assemblages (Giardia) that are capable of infecting humans, based on current knowledge of host specificity and taxonomy. Cryptosporidium genotyping data were more promising for source tracking potential, due to the greater number of host-adapted (i.e., narrow-host-range) species/genotypes compared to Giardia, since 98% of Giardia isolates were zoonotic and the potential host could not be predicted. This report highlights the benefits of parasite genomic sequencing to complement Method 1623 (U.S. Environmental Protection Agency) and shows that Cryptosporidium subtyping for MST purposes is superior to the use of Giardia subtyping, based on better detection limits for Cryptosporidium-positive samples than for Giardia-positive samples and on greater host specificity among Cryptosporidium species. These additional tools could be used for risk assessment in public health and watershed management decisions.     

Benthic phosphorus regeneration in the Potomac River Estuary

The flux of dissolved reactive phosphate from Potomac riverine and estuarine sediments is controlled by processes occurring at the water-sediment interface and within surficial sediment.In situ benthic fluxes (0.1 to 2.0 mmoles m⁻² day⁻¹) are generally five to ten times higher than calculated diffusive fluxes (0.020 to 0.30 mmoles m⁻² day⁻¹). The discrepancy between the two flux estimates is greatest in the transition zone (river mile 50 to 70) and is attributd to macrofaunal irrigation. Bothin situ and diffusive fluxes of dissolved reactive phosphate from Potomac tidal river sediments are low while those from anoxic lower estuarine sediments are high. The net accumulation rate of phosphorus in benthic sediment exhibits an inverse pattern. Thus a large fraction of phosphorus is retained by Potomac tidal river sediments, which contain a surficial oxidized layer and oligochaete worms tolerant of low oxygen conditions, and a large fraction of phosphorus is released from anoxic lower estuary sediments. Tidal river sediment pore waters are in equilibrium with amorphous Fe (OH)₃ while lower estuary pore waters are significantly undersaturated with respect to this phase. Benthic regeneration of dissolved reactive phosphorus is sufficient to supply all the phosphorus requirements for net primary production in the lower tidal river and transition-zone waters of the Potomac River Estuary. Benthic regeneration supplies approximately 25% as much phosphorus as inputs from sewage treatment plants and 10% of all phosphorus inputs to the tidal Potomac River. When all available point source phosphorus data are put into a steady-state conservation of mass model and reasonable coefficients for uptake of dissolved phosphorus, remineralization of particulate phosphorus, and sedimentation of particulate phosphorus are used in the model, a reasonably accurate simulation of dissolved and particulate phosphorus in the water column is obtained for the summer of 1980.     

Distribution of submersed aquatic macrophytes in the tidal Potomac River

Results of a 3-year survey (1978–1980) and review of historic trends have shown a major decline in the number of species and the distribution of submersed aquatic macrophytes in the tidal Potomac River since the early 1900's. The freshwater tidal river is essentially devoid of plants and only very sparse populations remain in the mesohaline section of the estuary. Present plant populations are largely confined to the transition-zone region where salinity instability at the fresh-to-brackish water interface is believed to reduce biotic stress on submersed vegetation. Many factors may be implicated in the loss of vegetation over major regions of the tidal Potomac River; however, long-term conditions of excessive nutrients appear to be primarily responsible for the present distribution.     

Cryptosporidium Genotypes in Wildlife from a New York Watershed

To identify the animal sources for Cryptosporidium contamination, we genotyped Cryptosporidium spp. in wildlife from the watershed of the New York City drinking water supply, using a small-subunit rRNA gene-based PCR-restriction fragment length polymorphism analysis and DNA sequencing. A total of 541 specimens from 38 species of wildlife were analyzed. One hundred and eleven (20.5%) of the wildlife specimens were PCR positive. Altogether, 21 Cryptosporidium genotypes were found in wildlife samples, 11 of which were previously found in storm runoff in the watershed, and six of these 11 were from storm water genotypes of unknown animal origin. Four new genotypes were found, and the animal hosts for four storm water genotypes were expanded. With the exception of the cervine genotype, most genotypes were found in a limited number of animal species and have no major public health significance.     

Sources and Species of Cryptosporidium Oocysts in the Wachusett Reservoir Watershed

Understanding the behavior of Cryptosporidium oocysts in the environment is critical to developing improved watershed management practices for protection of the public from waterborne cryptosporidiosis. Analytical methods of improved specificity and sensitivity are essential to this task. We developed a nested PCR-restriction fragment length polymorphism assay that allows detection of a single oocyst in environmental samples and differentiates the human pathogen Cryptosporidium parvum from other Cryptosporidium species. We tested our method on surface water and animal fecal samples from the Wachusett Reservoir watershed in central Massachusetts. We also directly compared results from our method with those from the immunofluorescence microscopy assay recommended in the Information Collection Rule. Our results suggest that immunofluorescence microscopy may not be a reliable indicator of public health risk for waterborne cryptosporidiosis. Molecular and environmental data identify both wildlife and dairy farms as sources of oocysts in the watershed, implicate times of cold water temperatures as high-risk periods for oocyst contamination of surface waters, and suggest that not all oocysts in the environment pose a threat to public health.     

Effects of increasing salinity on a cyanobacteria bloom in the Potomac River estuary

From June through August, 1985, a bloom of the cyanobacteriumMicrocyslis aeruginosa was observed in the upper Potomac Riverreaching densities of 193 x 10⁶ cells 1^–1 and 83% of totalcells in the surface mixed layer. However, in regions typifiedby salinities of 1–2 p.p.t. immediately down-river, thealga disappeared from the phytoplankton assemblage, never contributing>17% of total phytoplankton numbers. In an attempt to determinethe effect of increasing salinity on limiting horizontal distributionsof cyanobacteria blooms in the Potomac River estuary, threelaboratory experiments were conducted. Bloom samples collectedfrom the river were exposed to daily salinity increases of 1–2p.p.t. through the addition of NaCl or a complement of fullsea salts. Relative to samples receiving no salt supplement,densities of Microcyslis spp. declined in salinity-stressedsamples. However, total cyanobacteria remained constant or increaseddue to rapid growth of Aphanizomenon. The addition of a mixtureof full sea salts favored aggregation of cyanobacteria, yieldingan average of 132 cells colony^–1; 36 cells colony^–1were observed in populations maintained in river water and NaCl-supplementedsamples. Chlorophyll concentrations declined slightly relativeto assemblages receiving no salt additions while carbon fixationwas reduced in salinity-stressed assemblages. These resultssuggest that salinities from 0.5 to 7 p.p.t., typical of theoligohaline region of the Potomac River, could limit the distributionof Microcystis-dominated blooms down-estuary. In addition, increasingsalinities should result in the aggregation of photosyntheticallyinhibited cells and, through prolonged exposure to increasingosmotic stress, produce large, rapidly sinking detrital particlessupporting microbial decomposition in oligohaline and mesohalineregions of the lower Potomac River estuary, perhaps perpetuatinghigh microbial oxygen demand and anoxia in this portion of theriver/estuary.     

Randomly Amplified Polymorphic DNA PCR Analysis of Bovine Cryptosporidium parvum Strains Isolated from the Watershed of the Red River of the North

Cryptosporidium parvum is a protozoan parasite that causes the disease cryptosporidiosis in a variety of mammals, including neonatal calves and humans. Millions of oocysts are shed during acute cryptosporidiosis, and zoonotic transmission is inferred, though not proven, to be a general phenomenon. Very little is known about the degree of strain variation exhibited by bovine and human isolates, though such knowledge would enable the amount of bovine-to-human transmission to be more precisely analyzed. This research was initiated to determine whether variations exist among bovine strains isolated from a localized geographic area, the watershed of the Red River of the North. Sixteen strains were isolated and compared to each other and to two human and two calf strains from Australia by randomly amplified polymorphic DNA PCR. A statistical analysis of the data indicated that the isolates belonged to four different groups of strains.     

Growth and Reproductive Responses of Daphnia to Cyanobacterial Blooms on the Potomac River

We examined reproductive responses of the cladoceran Daphnia parvula to algal assemblages from the Potomac River, during and after Cyanobacterial blooms. Algal assemblages were manipulated by filtration, dilution, or additions of nutritious algae, in order to determine causes for growth inhibition. There was no evidence for lethal toxic effects or food limitation of population growth rates during the algal bloom, although growth was food limited later in the year. However, growth rates were lower in the experiment conducted during the Cyanobacteria bloom than in later experiments, and the results suggest an inhibitory factor associated with the particulate material. Results from filtered treatments indicate that picoplankton present in hypereutrophic waters can support Daphnia growth and reproduction.     

Life history and status of shortnose sturgeon (Acipenser brevirostrum) in the Potomac River

We collected the first life history information on shortnose sturgeon ( Acipenser brevirostrum ) in any of the rivers to Chesapeake Bay, the geographic center of the species range. In the Potomac River, two telemetry-tagged adult females used 124 km of river: a saltwater/freshwater reach at river km (rkm) 63−141 was the foraging−wintering concentration area, and one female migrated to spawn at rkm 187 in Washington, DC. The spawning migration explained the life history context of an adult captured 122 years ago in Washington, DC, supporting the idea that a natal population once lived in the river. Repeated homing migrations to foraging and wintering areas suggested the adults were residents, not transient coastal migrants. All habitats that adults need to complete life history are present in the river. The Potomac River shortnose sturgeon offers a rare opportunity to learn about the natural rebuilding of a sturgeon population.     

Host Specificity and Source of Enterocytozoon bieneusi Genotypes in a Drinking Source Watershed

To assess the host specificity of Enterocytozoon bieneusi and to track the sources of E. bieneusi contamination, we genotyped E. bieneusi in wildlife and stormwater from the watershed of New York City''s source water, using ribosomal internal transcribed spacer (ITS)-based PCR and sequence analyses. A total of 255 specimens from 23 species of wild mammals and 67 samples from stormwater were analyzed. Seventy-four (29.0%) of the wildlife specimens and 39 (58.2%) of the stormwater samples from streams were PCR positive. Altogether, 20 E. bieneusi genotypes were found, including 8 known genotypes and 12 new ones. Sixteen and five of the genotypes were seen in animals and stormwater from the watershed, respectively, with WL4 being the most common genotype in both animals (35 samples) and stormwater (23 samples). The 20 E. bieneusi genotypes belonged to five genogroups (groups 1, 3, 4, and 7 and an outlier), with only 23/113 (20.4%) E. bieneusi-positive samples belonging to zoonotic genogroup 1 and 3/20 genotypes ever being detected in humans. The two genogroups previously considered host specific, groups 3 and 4, were both detected in multiple groups of mammals. Thus, with the exception of the type IV, Peru11, and D genotypes, which were detected in only 7, 5, and 2 animals, respectively, most E. bieneusi strains in most wildlife samples and all stormwater samples in the watershed had no known public health significance, as these types have not previously been detected in humans. The role of different species of wild mammals in the contribution of E. bieneusi contamination in stormwater was supported by determinations of host-adapted Cryptosporidium species/genotypes in the same water samples. Data from this study indicate that the host specificity of E. bieneusi group 3 is broader than originally thought, and wildlife is the main source of E. bieneusi in stormwater in the watershed.     

Microbial Source Tracking in Adjacent Karst Springs

Modern man-made environments, including urban, agricultural, and industrial environments, have complex ecological interactions among themselves and with the natural surroundings. Microbial source tracking (MST) offers advanced tools to resolve the host source of fecal contamination beyond indicator monitoring. This study was intended to assess karst spring susceptibilities to different fecal sources using MST quantitative PCR (qPCR) assays targeting human, bovine, and swine markers. It involved a dual-time monitoring frame: (i) monthly throughout the calendar year and (ii) daily during a rainfall event. Data integration was taken from both monthly and daily MST profile monitoring and improved identification of spring susceptibility to host fecal contamination; three springs located in close geographic proximity revealed different MST profiles. The Giach spring showed moderate fluctuations of MST marker quantities amid wet and dry samplings, while the Zuf spring had the highest rise of the GenBac3 marker during the wet event, which was mirrored in other markers as well. The revelation of human fecal contamination during the dry season not connected to incidents of raining leachates suggests a continuous and direct exposure to septic systems. Pigpens were identified in the watersheds of Zuf, Shefa, and Giach springs and on the border of the Gaaton spring watershed. Their impact was correlated with partial detection of the Pig-2-Bac marker in Gaaton spring, which was lower than detection levels in all three of the other springs. Ruminant and swine markers were detected intermittently, and their contamination potential during the wet samplings was exposed. These results emphasized the importance of sampling design to utilize the MST approach to delineate subtleties of fecal contamination in the environment.     

Cluster analysis of occurrence and distribution of insect species in a portion of the Potomac River

Summary Limnological surveys of a portion of the Potomac River were made from 1956 to 1965 to determine the effects of operation of the PEPCO Dickerson Power Station on the aquatic biota. Samples were collected at three stations on each of four high-water and six-low-water surveys. Cluster analyses were made of various combinations of Jaccard coefficients relating 50 aggregations of 370 insect species.Similarities of aggregations of species with a survey were in all cases greater than similarities among aggregations from different surveys, indicating relatively strong within-year or along-stream environmental influences. Clusters of aggregations from middle-year survey show a greater similarity to each other than to the earlier or later surveys. This may indicate environmental change at all stations, including the control. The marked dissimilarity of aggregations from the last survey to all others suggests biotic change in the river. One explanation is that increased urbanization upstream from the power station after 1956 caused some environmental change. No changes in aquatic biota can be attributed to thermal loading as a direct result of operation of the electric power generating station.The clustering method used provides a convenient quantitative analysis of limnological survey data and a comparison of environmental information derived from the study of distribution and ocurrence of different groups or organisms. The significance of this is that similarities and differences between communities can be expressed numerically and similar communities will cluster. If the control station clusters with the stations receiving heated waste water this indicates that no significant biological changes were caused by the waste heat.

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Zusammenfassung Um festzustellen ob die Lebensgemeinschaften des Potomacflusses irgendwie beeinflut oder durch thermischer höhte Abwassern vom PEPCO Dickerson Power Station geändert wurde, wurden mehrere limnologische Untersuchungen (1956–1965) im Flu durchgeführt.Proben wurden 10 Male von 3 Stationen entnommen (4 Male-Hochwasser, 6 Male-Niedrigwasser). Verschiedene Jaccard Coefficient Cluster Analyses wurden geprüft in Beziehung auf 50 Ansammlungen von 370 Arten von Insecten.Ansammlungen innerhalb einzelnen Untersuchungen waren ähnlicher als diejenigen zwischen verschiedenen Untersuchungen. Das zeigt starke Beeinflüsung von Umweltfaktoren in einzelnen Jahren oder in einzelnen Strom-Orten.Ansammlung-Klumpen von den mitteljährigen Untersuchungen älnelt sich mehr als die Früh-oder Spätjährigen. Das könnte auf Umweltsänderungen (alle Stationen) hinweisen. Die letzte Untersuchung war ganz verschieden; wohl ein Zeichen von biotischer Änderung. Die Ursache dafür könnte sich wohl als eine Folge der stromaufwärts des Untersuchungsgebietes neuzeitlichen Verstädterung. Keine von den festgestellten biotischen Änderungen sind auf den thermisch-erhöhten Abwässern des PEPCO elektrizitätswerk zurückzuführen.Diese Klump-Methode wird eine Beihilfe sein um Ähnichkeiten und Verschiedenheiten zwischen Lebensgemeinschaften mathematisch auszudrücken. Ähnlichen Gemeinschaften werden klumpen.Wenn die Kontrolle Biota und die Abwasser Biota klumpen, können wir vermuten da es sich um keinen bedeutsamen biotischen Veränderungen handelt; ferner, da die erhöhte Abwasser-Temperature keine bedeustame Wirkung auf die Biota veranstaltete.

     

The application of the ecotone concept in defining nutrient management requirements for the upper Potomac River basin

The utility of the ecotone concept in nutrient management studies of the Potomac River basin, a large USA watershed, is examined. The MAB hypothesis suggesting that the influence of an ecotone or adjacent system is proportional to the length and scope of the interfaces (Naiman et al., 1989) is the major focus of this paper.The land-riverine ecotone appears to have a major influence on the total nitrogen balance and river export flux of nitrogen for the upper Potomac River basin. A method of converting conventional areal flux units into linear flux rates is suggested.     

Data synthesis and bioindicator development for nontidal streams in the interstate Potomac River basin, USA

Water resource agencies are increasingly confronted with issues of methods and data comparability when assessing inter-jurisdictional waters. The Interstate Commission on the Potomac River Basin (ICPRB) is developing an assessment framework for the diverse nontidal monitoring data collected by Potomac basin jurisdictions. The ICPRB's goal is to augment the jurisdictions’ water quality assessments with uniform, basin-wide evaluations of habitat and biological integrity. Disparate datasets were integrated and used to select and calibrate a common suite of biological indicators of human disturbance. Aggregated physiographic regions were most effective at grouping streams into site classes with similar attributes. Common elements from states’ habitat and water quality assessments were used to define regional reference and impairment criteria. Seven macroinvertebrate metrics distinguished reference from impaired sites in most regions: EPT richness, Hilsenhoff Family Biotic Index, percent clingers, percent collectors, percent dominance, percent EPT, and taxonomic richness. Reference communities in the Piedmont region of the basin were dissimilar from those in Highland and Valley regions, suggesting a need for different reference conditions and metric thresholds in these regions. Results confirm that useful bioindicators of aquatic health can be generated from multiple datasets if the synthesis is done with care.     

Availability of Ferric Iron for Microbial Reduction in Bottom Sediments of the Freshwater Tidal Potomac River

The distribution of Fe(III), its availability for microbial reduction, and factors controlling Fe(III) availability were investigated in sediments from a freshwater site in the Potomac River Estuary. Fe(III) reduction in sediments incubated under anaerobic conditions and depth profiles of oxalate-extractable Fe(III) indicated that Fe(III) reduction was limited to depths of 4 cm or less, with the most intense Fe(III) reduction in the top 1 cm. In incubations of the upper 4 cm of the sediments, Fe(III) reduction was as important as methane production as a pathway for anaerobic electron flow because of the high rates of Fe(III) reduction in the 0- to 0.5-cm interval. Most of the oxalate-extractable Fe(III) in the sediments was not reduced and persisted to a depth of at least 20 cm. The incomplete reduction was not the result of a lack of suitable electron donors. The oxalate-extractable Fe(III) that was preserved in the sediments was considered to be in a form other than amorphous Fe(III) oxyhydroxide, since synthetic amorphous Fe(III) oxyhydroxide, amorphous Fe(III) oxyhydroxide adsorbed onto clay, and amorphous Fe(III) oxyhydroxide saturated with adsorbed phosphate or fulvic acids were all readily reduced. Fe₃O₄ and the mixed Fe(III)-Fe(II) compound(s) that were produced during the reduction of amorphous Fe(III) oxyhydroxide in an enrichment culture were oxalate extractable but were not reduced, suggesting that mixed Fe(III)-Fe(II) compounds might account for the persistence of oxalate-extractable Fe(III) in the sediments. The availability of microbially reducible Fe(III) in surficial sediments demonstrates that microbial Fe(III) reduction can be important to organic matter decomposition and iron geochemistry. However, the overall extent of microbial Fe(III) reduction is governed by the inability of microorganisms to reduce most of the Fe(III) in the sediment.     

Reproduction rates and secondary production of three species of the rotifer genus Synchaeta in the estuarine Potomac River

Rotifers are a relatively well-studied component of lacustrinesystems but their role is only poorly understood in estuaries.Three species of the genus Synchaeta—S. baltica, S. triophthalmaand S.cecilia—dominate the cold-water assemblage of rotifersin Chesapeake Bay. Laboratory experiments were conducted todetermine the temperature dependence of egg development time(EDT) for each species; EDT varied over an approximate rangeof 90–9 h as temperature (T) varied from 2 to 22°C.The EDT/temperature relationships could be closely fitted bya simple polynomial equation of the form log(EDT) = a + b(logT) + c(log T)² for each species. Natural populations of thesethree rotifers were sampled during a cruise in the Potomac River(7–11 March 1983). Estimates of specific reproductiverates (b) were calculated based on the previously defined EDT/temperaturerelationship and the observed ratio of eggs/rotifers for eachspecies. The two most abundant species, S.triophthalma and S.cecilia,showed a clear dependence of b on the observed chlorophyll aconcentrations. Maximum reproductive rates ({small tilde}0.015h^–1) were attained only at relatively high phytoplanktondensities within a bloom of Heterocapsa triquetra where thechlorophyll a concentrations exceeded 10 µg l^–1.Estimates of secondary production suggest that Synchaeta spp.may contribute to the trophic flow of carbon in this systemwith a significance at least similar to that of the planktoniccopepods.     

Patterns of Cryptosporidium Oocyst Shedding by Eastern Grey Kangaroos Inhabiting an Australian Watershed

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 × 10⁶ oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum “bovine” genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium “marsupial” genotype I or “marsupial” genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.     

Biotic Interactions and Sunlight Affect Persistence of Fecal Indicator Bacteria and Microbial Source Tracking Genetic Markers in the Upper Mississippi River

The sanitary quality of recreational waters that may be impacted by sewage is assessed by enumerating fecal indicator bacteria (FIB) (Escherichia coli and enterococci); these organisms are found in the gastrointestinal tracts of humans and many other animals, and hence their presence provides no information about the pollution source. Microbial source tracking (MST) methods can discriminate between different pollution sources, providing critical information to water quality managers, but relatively little is known about factors influencing the decay of FIB and MST genetic markers following release into aquatic environments. An in situ mesocosm was deployed at a temperate recreational beach in the Mississippi River to evaluate the effects of ambient sunlight and biotic interactions (predation, competition, and viral lysis) on the decay of culture-based FIB, as well as molecularly based FIB (Entero1a and GenBac3) and human-associated MST genetic markers (HF183 and HumM2) measured by quantitative real-time PCR (qPCR). In general, culturable FIB decayed the fastest, while molecularly based FIB and human-associated genetic markers decayed more slowly. There was a strong correlation between the decay of molecularly based FIB and that of human-associated genetic markers (r², 0.96 to 0.98; P < 0.0001) but not between culturable FIB and any qPCR measurement. Overall, exposure to ambient sunlight may be an important factor in the early-stage decay dynamics but generally was not after continued exposure (i.e., after 120 h), when biotic interactions tended to be the only/major influential determinant of persistence.     

Source Tracking Mycobacterium ulcerans Infections in the Ashanti Region,Ghana

Although several studies have associated Mycobacterium ulcerans (MU) infection, Buruli ulcer (BU), with slow moving water bodies, there is still no definite mode of transmission. Ecological and transmission studies suggest Variable Number Tandem Repeat (VNTR) typing as a useful tool to differentiate MU strains from other Mycolactone Producing Mycobacteria (MPM). Deciphering the genetic relatedness of clinical and environmental isolates is seminal to determining reservoirs, vectors and transmission routes. In this study, we attempted to source-track MU infections to specific water bodies by matching VNTR profiles of MU in human samples to those in the environment. Environmental samples were collected from 10 water bodies in four BU endemic communities in the Ashanti region, Ghana. Four VNTR loci in MU Agy99 genome, were used to genotype environmental MU ecovars, and those from 14 confirmed BU patients within the same study area. Length polymorphism was confirmed with sequencing. MU was present in the 3 different types of water bodies, but significantly higher in biofilm samples. Four MU genotypes, designated W, X, Y and Z, were typed in both human and environmental samples. Other reported genotypes were only found in water bodies. Animal trapping identified 1 mouse with lesion characteristic of BU, which was confirmed as MU infection. Our findings suggest that patients may have been infected from community associated water bodies. Further, we present evidence that small mammals within endemic communities could be susceptible to MU infections. M. ulcerans transmission could involve several routes where humans have contact with risk environments, which may be further compounded by water bodies acting as vehicles for disseminating strains.