Non-resident definitive host presence is sufficient to sustain avian schistosome populations.

To control swimmer's itch in northern Michigan inland lakes, USA, one species of bird, the common merganser (Mergus merganser), has been relocated from several lakes since 2015. Relocation efforts are driven by a desire to reduce the prevalence of the swimmer's itch-causing parasite Trichobilharzia stagnicolae. The intention of this state-sponsored control effort was to interrupt the life cycle of T. stagnicolae and reduce parasite egg contribution into the environment from summer resident mergansers such that infections of the intermediate snail host Stagnicola emarginata declined. Reduced snail infection prevalence was expected to substantially reduce the abundance of the swimmer's itch-causing cercarial stage of the parasite in water. With no official programme in place to assess the success of this relocation effort, we sought to study the effectiveness and impact of the removal of a single definitive host from a location with high definitive host and parasite diversity. This was assessed through a comprehensive, lake-wide monitoring study measuring longitudinal changes in the abundance of three species of avian schistosome cercariae in four inland Michigan lakes. Environmental measurements were also taken at these lakes to understand how they can affect swimmer's itch incidence. This study demonstrates that the diversity of avian schistosomes at the study lakes would likely make targeting a single species of swimmer's itch-causing parasite meaningless from a swimmer's itch control perspective. Our data also suggest that removing the common merganser is not an effective control strategy for the T. stagnicolae parasite, likely due to contributions of the parasite made by non-resident birds, possibly migrants, in the autumn and spring. It appears likely that only minimal contact time between the definitive host and the lake ecosystem is required to contribute sufficient parasite numbers to maintain a thriving population of parasite species with high host specificity.

Pathogen performance testing of a natural swimming pool using a cocktail of microbiological surrogates and QMRA-derived management goals.

In recent decades, natural swimming pools (NSPs) have gained popularity in Europe, especially in Germany and Austria. NSPs differ from swimming pools in that they utilize biological treatment processes based on wetland processes with no disinfection residual. However, data are missing on the specific log-reduction performance of NSPs to address enteric virus, bacteria, and parasitic protozoa removal considered necessary to meet the North American risk-based benchmark (<35 illnesses per 1,000 swimming events) set by the USEPA for voluntary swimming. In this study, we examined Canada's first NSP at Borden Park, Edmonton, Canada, to address the following three questions: (1) Given normal faecal shedding rates by bathers, what is the total log reduction (TLR) theoretically needed to meet the EPA benchmark? (2) what is the in-situ performance of the NSP based on spiking suitable microbial surrogates (MS2 coliphage, Enterococcus faecalis, and Saccharomyces cerevisiae [Baker's yeast])? and (3) how much time is required to reach acceptable bather risk levels under different representative volume-turnover rates? A reverse-quantitative microbial risk assessment (QMRA) revealed that of the four reference pathogens selected (Norovirus, Campylobacter, Cryptosporidium, and Giardia), only Norovirus was estimated to exceed the risk benchmark at the 50th, 75th, and 95th percentiles, while Campylobacter was the only other reference pathogen to exceed at the 95th percentile. Log-reduction values (LRVs) were similar to previous reports for bacterial indicators, and novel LRVs were estimated for the other two surrogates. A key finding was that more than 24 h treatment time would be necessary to provide acceptable bather protection following heavy bather use (378 bathers/day for main pool and 26 bathers/day for children's pool), due to the mixing dynamics of the treated water diluting out possible residual pool faecal contamination. The theoretical maximum number of people in the pool per day to be below USEPA's 35 gastro cases in 1,000 swimming events was 113, 47, and 8, at the 50th, 75th, and 95th percentiles. Further, the use of ultra-violet disinfection to the pool return flow had little effect on reducing the treatment time required.

Evidence of a Putative Novel Species of Avian Schistosome Infecting Planorbella trivolvis.

Freshwater gastropods of the genera Lymnaea Lamarck, 1799, Physa Draparnaud, 1801, Gyraulus Charpentier, 1837, Radix Montfort, 1810, and Stagnicola Jeffreys, 1830 are considered suitable intermediate hosts for avian schistosomes. A large trematode biodiversity survey performed across 3 yr on 6 lakes in Alberta confirmed 3 already-reported snail hosts for 7 North American avian schistosomes; however, the cytochrome c oxidase subunit 1 (COI) nucleotide sequence from 1 cercarial sample (from a single specimen of Planorbella trivolvis) was distinct from all other COI schistosome sequences. As part of a simultaneous, comparable study of P. trivolvis by us in Michigan, we collected another cercarial type from 6 lakes that was 99% similar (COI) to the aforementioned cercarial type. Phylogenetic analyses of the COI and 28S rDNA genes recovered the former cercaria in a clade of avian schistosomes. In Michigan, the feces of a Canada goose (Branta canadensis Linnaeus, 1758) had a miracidium with an identical COI nucleotide sequence. Preliminary swimmer's itch and cercarial emergence studies were performed to determine if the cercariae could cause swimmer's itch and to study the emergence pattern as compared with species of Trichobilharzia Skrjabin and Zakharow, 1920.

Democratizing water monitoring: Implementation of a community-based qPCR monitoring program for recreational water hazards.

Recreational water monitoring can be challenging due to the highly variable nature of pathogens and indicator concentrations, the myriad of potential biological hazards to measure for, and numerous access points, both official and unofficial, that are used for recreation. The aim of this study was to develop, deploy, and assess the effectiveness of a quantitative polymerase chain reaction (qPCR) community-based monitoring (CBM) program for the assessment of bacterial and parasitic hazards in recreational water. This study developed methodologies for performing qPCR 'in the field,' then engaged with water management and monitoring groups and tested the method in a real-world implementation study to evaluate the accuracy of CBM using qPCR both quantitatively and qualitatively. This study found high reproducibility between qPCR results performed by non-expert field users and expert laboratory results, suggesting that qPCR as a methodology could be amenable to a CBM program.

Species-specific qPCR assays allow for high-resolution population assessment of four species avian schistosome that cause swimmer's itch in recreational lakes.

Swimmer's itch is an allergic condition that occurs when the motile and infectious stage of avian schistosomes penetrate the skin of an individual. Flatworm parasites that cause swimmer's itch belong to the family Schistosomatidae. They utilize a variety of different species of bird and mammal as definitive hosts, and rely on different species of snail, in which they complete their larval development to culminate in a motile, aquatic, infectious stage called a cercaria. Recently, qPCR-based assays have been developed to monitor for swimmer's itch-causing trematodes in recreational water. This environmental DNA approach has been useful for quantifying the abundance of the free-living cercaria, the causative agent of swimmer's itch. However, the existing qPCR test amplifies from all known schistosome species, making it excellent for assessing a site for swimmer's itch potential, but not useful in determining the specific species contributing to swimmer's itch or the likely hosts (snail and bird) of the swimmer's itch-causing parasites. Thus, species-specific resolution built into a qPCR test would be useful in answering ecological questions about swimmer's itch cause, and efficacy of control efforts. This paper details bird, snail, and cercaria surveys conducted in the summer of 2018, that culminated in the development and deployment of four species-specific qPCR assays, capable of detecting Trichobilharzia stagnicolae, Trichobilharzia szidati, Trichobilharzia physellae, and Anserobilharzia brantae in recreational water. These assays were used to assess the relative abundance of each parasite in water samples collected from lakes in Northern Michigan.

Evaluation of targeted copper sulfate (CuSO4) application for controlling swimmer's itch at a freshwater recreation site in Michigan.

Swimmer's itch has historically been controlled by applying copper sulfate (CuSO4) to lakes as a way to eliminate snails that serve as the intermediate hosts for swimmer's itch-causing parasites. CuSO4 is still sometimes applied specifically to areas of lakes where swimmer's itch severity is high. It is unclear whether targeted application of chemical molluscicides like CuSO4 is effective for controlling swimmer's itch. Previous research has found that the larval stage of the parasites responsible for swimmer's itch are released from infected snails and are concentrated by onshore and alongshore winds, and thus, may not be affected by such focused applications. In this study, we evaluated the impact of targeted CuSO4 application to a specific recreational swimming area in a lake in Michigan. We measured the effect on snail populations, as well as on the presence/abundance of swimmer's itch-causing parasites using qPCR. Ultimately, while CuSO4 was confirmed to significantly reduce populations of snails within the treatment area, it was found to have no significant impact on swimmer's itch-causing parasites in the water, likely due to the free-swimming larval stages (cercariae) moving into the treatment area from surrounding regions.

Use of qPCR-Based Cercariometry to Assess Swimmer's Itch in Recreational Lakes.

Swimmer's itch (cercarial dermatitis) is a nuisance encountered by bathers and recreational water users worldwide. The condition is caused by the penetration of larval digenean trematodes (cercariae) of the family Schistosomatidae, into the skin, following their release into freshwater from pulmonate snails that serve as the intermediate hosts for these parasites. This study utilizes qPCR-based cercariometry to monitor and quantify cercariae from water samples collected at 5 lakes in northern Michigan. The resolution provided by qPCR facilitated assessment of the environmental and biological drivers of swimmer's itch-causing cercariae concentrations, allowing us to demonstrate that cercarial abundance is greatest at the top of the water column, in locations with prevailing on- and alongshore winds.

Enterobius vermicularis as a Novel Surrogate for the Presence of Helminth Ova in Tertiary Wastewater Treatment Plants.

Significant effort has gone into assessing the fate and removal of viruses, bacteria, and protozoan parasites during wastewater treatment to provide data addressing potential health risks associated with reuse options. Comparatively less is known about the fate of parasitic worm species ova in these complex systems. It is largely assumed that these helminths settle, are removed with the sludge, and consequently represent a relatively low risk for wastewater reuse applications. However, helminths are a highly diverse group of organisms that display a wide range of physical properties that complicate the application of a single treatment for helminth reduction during wastewater treatment. Moreover, their diverse biological and physical properties make some ova highly resistant to both disinfection (i.e., with chlorine or UV treatment) and physical removal (settling) through the wastewater treatment train, indicating that there may be reason to broaden the scope of our investigations into whether parasitic worm eggs can be identified in treated wastewater. The ubiquitous human parasitic nematode Enterobius vermicularis (pinworm) produces small, buoyant ova. Utilizing a novel diagnostic quantitative PCR (qPCR), this study monitored E. vermicularis presence at two full-scale wastewater treatment plants over the course of 8 months and demonstrated incomplete physical removal of E. vermicularis ova through tertiary treatment, with removal efficiencies approximating only 0.5 and 1.6 log10 at the two wastewater treatment plants based on qPCR. These findings demonstrate the need for more-diverse surrogates of helminthic ova to fully assess treatment performance with respect to reclaimed wastewaters.IMPORTANCE Helminths, despite being a diverse and environmentally resistant class of pathogens, are often underestimated and ignored when treatment performance at modern wastewater treatment plants is considered. A one-size-fits-all surrogate for removal of helminth ova may be inappropriate to adequately assess risk and ensure public safety when treated and partially treated wastewaters are encountered. This study argues for the use of human pinworm as a conservative indicator of the presence of helminth ova due to its small size, buoyancy, prevalence in humans, and environmental resistance.

Endogenous growth factor stimulation of hemocyte proliferation induces resistance to Schistosoma mansoni challenge in the snail host.

Digenean trematodes are a large, complex group of parasitic flatworms that infect an incredible diversity of organisms, including humans. Larval development of most digeneans takes place within a snail (Gastropoda). Compatibility between snails and digeneans is often very specific, such that suitable snail hosts define the geographical ranges of diseases caused by these worms. The immune cells (hemocytes) of a snail are sentinels that act as a crucial barrier to infection by larval digeneans. Hemocytes coordinate a robust and specific immunological response, participating directly in parasite killing by encapsulating and clearing the infection. Hemocyte proliferation and differentiation are influenced by unknown digenean-specific exogenous factors. However, we know nothing about the endogenous control of hemocyte development in any gastropod model. Here, we identify and functionally characterize a progranulin [Biomphalaria glabrata granulin (BgGRN)] from the snail B. glabrata, a natural host for the human blood fluke Schistosoma mansoni Granulins are growth factors that drive proliferation of immune cells in organisms, spanning the animal kingdom. We demonstrate that BgGRN induces proliferation of B. glabrata hemocytes, and specifically drives the production of an adherent hemocyte subset that participates centrally in the anti-digenean defense response. Additionally, we demonstrate that susceptible B. glabrata snails can be made resistant to infection with S. mansoni by first inducing hemocyte proliferation with BgGRN. This marks the functional characterization of an endogenous growth factor of a gastropod mollusc, and provides direct evidence of gain of resistance in a snail-digenean infection model using a defined factor to induce snail resistance to infection.

Dual expression profile of type VI secretion system immunity genes protects pandemic Vibrio cholerae.

The Vibrio cholerae type VI secretion system (T6SS) assembles as a molecular syringe that injects toxic protein effectors into both eukaryotic and prokaryotic cells. We previously reported that the V. cholerae O37 serogroup strain V52 maintains a constitutively active T6SS to kill other Gram-negative bacteria while being immune to attack by kin bacteria. The pandemic O1 El Tor V. cholerae strain C6706 is T6SS-silent under laboratory conditions as it does not produce T6SS structural components and effectors, and fails to kill Escherichia coli prey. Yet, C6706 exhibits full resistance when approached by T6SS-active V52. These findings suggested that an active T6SS is not required for immunity against T6SS-mediated virulence. Here, we describe a dual expression profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 that provides pandemic V. cholerae strains with T6SS immunity and allows T6SS-silent strains to maintain immunity against attacks by T6SS-active bacterial neighbors. The dual expression profile allows transcription of the three genes encoding immunity proteins independently of other T6SS proteins encoded within the same operon. One of these immunity proteins, TsiV2, protects against the T6SS effector VasX which is encoded immediately upstream of tsiV2. VasX is a secreted, lipid-binding protein that we previously characterized with respect to T6SS-mediated virulence towards the social amoeba Dictyostelium discoideum. Our data suggest the presence of an internal promoter in the open reading frame of vasX that drives expression of the downstream gene tsiV2. Furthermore, VasX is shown to act in conjunction with VasW, an accessory protein to VasX, to compromise the inner membrane of prokaryotic target cells. The dual regulatory profile of the T6SS immunity protein-encoding genes tsiV1, tsiV2, and tsiV3 permits V. cholerae to tightly control T6SS gene expression while maintaining immunity to T6SS activity.