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Oocyst shedding in domestic and wild felids is a critical yet understudied topic in Toxoplasma gondii ecology and epidemiology that shapes human and animal disease burden. We synthesized published literature dating from the discovery of felids as the definitive hosts of T. gondii in the 1960s through March 2021 to examine shedding prevalence, oocyst genotypes, and risk factors for shedding. Oocyst shedding prevalence in many geographic regions exceeded the commonly accepted 1% reported for domestic cats; crude prevalence from cross-sectional field studies of domestic cat shedding ranged from 0% in Australia to 18.8% in Africa, with greater variation in reports of oocyst shedding in free-ranging, wild felids. Shedding in wild felid species has primarily been described in captive animals, with attempted detection of oocyst shedding reported in at least 31 species. Differences in lifestyle and diet play an important role in explaining shedding variation between free-ranging unowned domestic cats, owned domestic cats, and wild felids. Additional risk factors for shedding include the route of infection, diet, age, and immune status of the host. It is widely reported that cats only shed oocysts after initial infection with T. gondii, but experimental studies have shown that repeat oocyst shedding can occur. Factors associated with repeat shedding are common amongst free-ranging felids (domestic and wild), which are more likely to eat infected prey, be exposed to diverse T. gondii genotypes, and have coinfections with other parasites. Repeat shedding events could play a significant yet currently ignored role in shaping environmental oocyst loading with implications for human and animal exposure. Oocyst presence in the environment is closely linked to climate variables such as temperature and precipitation, so in quantifying risk of exposure, it is important to consider the burden of T. gondii oocysts that can accumulate over time in diverse environmental matrices and sites, as well as the spatial heterogeneity of free-ranging cat populations. Key directions for future research include investigating oocyst shedding in under-sampled regions, genotyping of oocysts detected in feces, and longitudinal studies of oocyst shedding in free-ranging felids. This article is protected by copyright. All rights reserved.

BackgroundWastewater-based epidemiology (WBE) has been deployed broadly as an early warning tool for emerging COVID-19 outbreaks. WBE can inform targeted interventions and identify communities with high transmission, enabling quick and effective response. As wastewater becomes an increasingly important indicator for COVID-19 transmission, more robust methods and metrics are needed to guide public health decision making.ObjectivesThe aim of this research was to develop and implement a mathematical framework to infer incident cases of COVID-19 from SARS-CoV-2 levels measured in wastewater. We propose a classification scheme to assess the adequacy of model training periods based on clinical testing rates and assess the sensitivity of model predictions to training periods.MethodsWe present a Bayesian deconvolution method and linear regression to estimate COVID-19 cases from wastewater data. We described an approach to characterize adequacy in testing during specific time periods and provided evidence to highlight the importance of model training periods on the projection of cases. We estimated the effective reproductive number (Re) directly from observed cases and from the reconstructed incidence of cases from wastewater. The proposed modeling framework was applied to three Northern California communities served by distinct wastewater treatment plants.ResultsBoth deconvolution and linear regression models consistently projected robust estimates of prevalent cases andRefrom wastewater influent samples when assuming training periods with adequate testing. Case estimates from models that used poorer-quality training periods consistently underestimated observed cases.DiscussionWastewater surveillance data requires robust statistical modeling methods to provide actionable insight for public health decision-making. We propose and validate a modeling framework that can provide estimates of COVID-19 cases andRefrom wastewater data that can be used as tool for disease surveillance including quality assessment for potential training data.

High prevalences of Cryptosporidium and Giardia were recently found in enteric illness patients in the Qikiqtaaluk region of Nunavut, Canada, with a foodborne, waterborne or animal source of parasites suspected. Clams (Mya truncata) are a commonly consumed, culturally important and nutritious country food in Iqaluit; however, shellfish may concentrate protozoan pathogens from contaminated waters. The goal of this work was to investigate clams as a potential source of Cryptosporidium and Giardia infections in residents in Iqaluit, Nunavut. The objectives were to estimate the prevalence and genetically characterize Cryptosporidium and Giardia in locally harvested clams. Clams (n = 404) were collected from Iqaluit harvesters in September 2016. Haemolymph (n = 328) and digestive gland (n = 390) samples were screened for Cryptosporidium and Giardia via PCR, and amplified products were further processed for sequence analyses for definitive confirmation. Giardia DNA was found in haemolymph from 2 clams, while Cryptosporidium was not detected. The two Giardia sequences were identified as zoonotic Giardia enterica assemblage B. The overall prevalence of Giardia in clams near Iqaluit was low (0.6%) compared with other studies in southern Canada and elsewhere. The presence of Giardia DNA in clams suggests human or animal faecal contamination of coastal habitat around Iqaluit in shellfish harvesting waters. Results from this study are intended to inform public health practice and planning in Inuit Nunangat.

Wastewater surveillance is a useful complement to clinical testing for managing COVID-19. While good agreement has been found between community-scale wastewater and clinical data, little is known about sub-community relationships between the two data types. Moreover, effects of non-detects in qPCR wastewater data have been largely overlooked. We used data collected from September 2020–June 2021 in Davis, California (USA) to address these gaps. By applying a predictive probability model to spatially disaggregate clinical results, we compared wastewater and clinical data at the community scale, in 16 sampling zones isolating city sub-regions, and in seven zones isolating high-priority building complexes or neighborhoods. We found reasonable agreement between wastewater and clinical data at all scales. Greater activity (i.e., more frequent detections) in clinical data tended to be mirrored in wastewater data. Small, isolated clinical-data spikes were often matched as well. We also developed a method for handling such non-detects using multiple imputation and compared results to (i) single imputation using half the qPCR limit of detection, (ii) single imputation using maximum qPCR cycle number, and (iii) non-detect censoring. Apparent wastewater trends were significantly influenced by non-detect handling. Multiple imputation improved correlation relative to single imputation, though not necessarily relative to censoring.

Cyclospora cayetanensis is an emerging food- and waterborne pathogen that causes cyclosporiasis, a gastrointestinal disease in humans. The parasite is endemic in tropical and subtropical regions; however, its prevalence is largely dependent on environmental factors, such as climate and rainfall patterns. The objective of this paper was to conduct a systematic review and meta-analysis to determine the prevalence of C. cayetanensis in water and to determine if geography, water source and other variables influence this prevalence. A literature search was performed using search terms relating to water and C. cayetanensis in MEDLINE®, CAB Direct, Food Science and Technology Abstracts, Agricola databases and Environmental Science Index. Observational studies published in English after 1979 were eligible. Screening, data extraction and risk-of-bias assessment were performed independently by two reviewers. A multi-level random-effects meta-analysis was completed to determine the prevalence of C. cayetanensis in water and subgroup meta-analyses were performed to explore between-study heterogeneity. The search identified 828 unique articles, and after the screening, 33 articles were included in the review. The pooled prevalence of C. cayetanensis in water was 6.90% [95% confidence interval (CI) 2.25%–13.05%, I2 = 84.38%]. Subgroup meta-analyses revealed significant differences in the prevalence between continents. Additionally, laboratory methods between studies were highly variable and these findings highlight the need for further environmental research on C. cayetanensis in water using detection methods that include PCR and sequencing to accurately identify the organism. The results of this study can be used to help assess the risk of waterborne cyclosporiasis.

Plastics are widely recognized as a pervasive marine pollutant. Microplastics have been garnering increasing attention due to reports documenting their ingestion by animals, including those intended for human consumption. Their accumulation in the marine food chain may also pose a threat to wildlife that consume species that can accumulate microplastic particles. Microplastic contamination in marine ecosystems has thus raised concerns for both human and wildlife health. Our study addresses an unexplored area of research targeting the interaction between plastic and pathogen pollution of coastal waters. We investigated the association of the zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum, and Giardia enterica with polyethylene microbeads and polyester microfibers. These pathogens were chosen because they have been recognized by the World Health Organization as underestimated causes of illness from shellfish consumption, and due to their persistence in the marine environment. We show that pathogens are capable of associating with microplastics in contaminated seawater, with more parasites adhering to microfiber surfaces as compared with microbeads. Given the global presence of microplastics in fish and shellfish, this study demonstrates a novel pathway by which anthropogenic pollutants may be mediating pathogen transmission in the marine environment, with important ramifications for wildlife and human health.

Plastics are widely recognized as a pervasive marine pollutant. Microplastics have been garnering increasing attention due to reports documenting their ingestion by animals, including those intended for human consumption. Their accumulation in the marine food chain may also pose a threat to wildlife that consume species that can accumulate microplastic particles. Microplastic contamination in marine ecosystems has thus raised concerns for both human and wildlife health. Our study addresses an unexplored area of research targeting the interaction between plastic and pathogen pollution of coastal waters. We investigated the association of the zoonotic protozoan parasites Toxoplasma gondii, Cryptosporidium parvum, and Giardia enterica with polyethylene microbeads and polyester microfibers. These pathogens were chosen because they have been recognized by the World Health Organization as underestimated causes of illness from shellfish consumption, and due to their persistence in the marine environment. We show that pathogens are capable of associating with microplastics in contaminated seawater, with more parasites adhering to microfiber surfaces as compared with microbeads. Given the global presence of microplastics in fish and shellfish, this study demonstrates a novel pathway by which anthropogenic pollutants may be mediating pathogen transmission in the marine environment, with important ramifications for wildlife and human health.

Testing surfaces in school classrooms for the presence of SARS-CoV-2, the virus that causes COVID-19, can provide public-health information that complements clinical testing. We monitored the presence of SARS-CoV-2 RNA in five schools (96 classrooms) in Davis, California (USA) by collecting weekly surface-swab samples from classroom floors and/or portable high-efficiency particulate air (HEPA) units (n = 2,341 swabs). Twenty-two surfaces tested positive, with qPCR cycle threshold (Ct) values ranging from 36.07–38.01. Intermittent repeated positives in a single room were observed for both floor and HEPA filter samples for up to 52 days, even following regular cleaning and HEPA filter replacement after a positive result. We compared the two environmental sampling strategies by testing one floor and two HEPA filter samples in 57 classrooms at Schools D and E. HEPA filter sampling yielded 3.02% and 0.41% positivity rates per filter sample collected for Schools D and E, respectively, while floor sampling yielded 0.48% and 0% positivity rates. Our results indicate that HEPA filter swabs are more sensitive than floor swabs at detecting SARS-CoV-2 RNA in interior spaces. During the study, all schools were offered weekly free COVID-19 clinical testing through Healthy Davis Together (HDT). HDT also offered on-site clinical testing in Schools D and E, and upticks in testing participation were observed following a confirmed positive environmental sample. However, no confirmed COVID-19 cases were identified among students associated with classrooms yielding positive environmental samples. The positive samples detected in this study appeared to contain relic viral RNA from individuals infected before the monitoring program started and/or RNA transported into classrooms via fomites. High-Ct positive results from environmental swabs detected in the absence of known active infections supports this conclusion. Additional research is needed to differentiate between fresh and relic SARS-CoV-2 RNA in environmental samples and to determine what types of results should trigger interventions.

Author(s): Gomez Marin, Jorge; de-la-Torre, Alejandra; Shapiro, Karen; Dumetre, Aurelien | Abstract: In this TrendsTalk article, the organizers of the 15th International Congress on Toxoplasma Biology and Toxoplasmosis, Professors Jorge Gomez Marin and Alejandra de-la-Torre, bring the highlights of this event and the key outcome from the inaugural workshop on the environmental transmission of Toxoplasma gondii organized by Doctors Aurelien Dumetre and Karen Shapiro.

Food and waterborne protozoan pathogens including Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii are a global concern for human public health. While all three pathogens have been detected in commercial shellfish, there is currently no standard approach for detecting protozoan parasites in shellfish. Common molecular and microscopic methods are limited in the number of pathogens they can simultaneously detect and are often targeted at one or two of these pathogens. Previously, we developed and validated a novel 18S amplicon-based next-generation sequencing assay for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii in shellfish. In this study, we applied the assay for protozoan pathogen detection in wild oysters from Prince Edward Island (PEI). Oysters were harvested from restricted and prohibited areas, classified by the Canadian government according to fecal coliform counts in surrounding waters, and different fractions (whole tissue homogenate and hemolymph) were analyzed. Protozoan DNA was detected using metabarcoding in 28%, of oysters tested (N = 128), and the pathogen read counts in oyster homogenate were considerably higher than those in hemolymph. Protozoan read count thresholds were established for classifying probable oyster contamination with pathogens to account for low levels of background protozoan reads detected in negative controls. Assay results showed protozoan contamination was not associated with harvesting site classifications, suggesting that using fecal indicators for ensuring food safety may be insufficient. Due to the complex matrix, an oyster DNA reduction step may further improve the pathogen detection sensitivity of the assay. Results from this study affirm that novel metabarcoding is a promising screening tool for detection of protozoan pathogens in shellfish.

Increasing reports of marine mammal deaths have been attributed to the parasite Sarcocystis neurona. Infected opossums, the only known definitive hosts, shed S. neurona sporocysts in their feces. Sporocysts can contaminate the marine environment via overland runoff, and subsequent ingestion by marine mammals can lead to fatal encephalitis. Our aim was to determine the prevalence of S. neurona in opossums from coastal areas of Washington State (USA) and to compare genetic markers between S. neurona in opossums and marine mammals. Thirty-two road-kill opossums and tissue samples from 30 stranded marine mammals meeting inclusion criteria were included in analyses. Three opossums (9.4%) and twelve marine mammals (40%) were confirmed positive for S. neurona via DNA amplification at the ITS1 locus. Genetic identity at microsatellites (sn3, sn7, sn9) and the snSAG3 gene of S. neurona was demonstrated among one harbor porpoise and two opossums. Watershed mapping further demonstrated plausible sporocyst transport pathways from one of these opossums to the location where an infected harbor porpoise carcass was recovered. Our results provide the first reported link between S. neurona genotypes on land and sea in the Pacific Northwest, and further demonstrate how terrestrial pathogen pollution can impact the health of marine wildlife.

Protozoan contamination in produce is of growing importance due to their capacity to cause illnesses in consumers of fresh leafy greens. Viability assays are essential to accurately estimate health risk caused by viable parasites that contaminate food. We evaluated the efficacy of reverse transcription quantitative PCR (RT-qPCR), propidium monoazide coupled with (q)PCR, and viability staining using propidium iodide through systematic laboratory spiking experiments for selective detection of viable Cryptosporidium parvum, Giardia enterica, and Toxoplasma gondii. In the presence of only viable protozoa, the RT-qPCR assays could accurately detect two to nine (oo)cysts/g spinach (in 10 g processed). When different proportions of viable and inactivated parasite were spiked, mRNA concentrations correlated with increasing proportions of viable (oo)cysts, although low levels of false-positive mRNA signals were detectable in the presence of high amounts of inactivated protozoa. Our study demonstrated that among the methods tested, RT-qPCR performed more effectively to discriminate viable from inactivated C. parvum, G. enterica and T. gondii on spinach. This application of viability methods on leafy greens can be adopted by the produce industry and regulatory agencies charged with protection of human public health to screen leafy greens for the presence of viable protozoan pathogen contamination. Submitted/Accepted version This work was supported by the Center for Produce Safety (California Department of Food and Agriculture grant# SBC15064CPS).

Sarcocystis spp. are protozoan parasites that cause a spectrum of lesions in various hosts. Hepatic sarcocystosis and encephalitis have been described in captive American black bears (Ursus americanus) and polar bears (Ursus maritimus), and in a free-ranging grizzly bear (Ursus arctos horribilis), but have not previously been reported in free-ranging American black bears. This study aimed to characterize the presence and lesions associated with Sarcocystis spp. in free-ranging bears in British Columbia, Canada from samples submitted to the provincial diagnostic laboratory. From 2007 to 2019, 102 free-ranging American black bear and grizzly bear tissues were examined postmortem for sarcocystosis using histopathology and follow-up molecular diagnostics. Sarcocystosis was confirmed in 41 (40%) free-ranging bears including 39 American black bears and two grizzly bears. Microscopic lesions included multifocal necrotizing hepatitis, nonsuppurative encephalitis, and/or intramuscular sarcocysts with or without associated inflammation. Sarcocystosis was considered the cause of death in eight (20%) of these bears, exclusively in cubs of the year (

Food and waterborne protozoan pathogens can cause serious disease in people. Three common species Cryptosporidium parvum, Giardia enterica and Toxoplasma gondii can contaminate diverse shellfish species, including commercial oysters. Current methods of protozoan detection in shellfish are not standardized, and few are able to simultaneously identify multiple species. Here, we present a novel metabarcoding assay targeting the 18S rRNA gene followed by next generation sequencing (NGS) for simultaneous detection of Cryptosporidium spp., Giardia spp. and T. gondii spiked into oyster samples. We further developed a bioinformatic pipeline to process and analyze 18S rRNA data for protozoa classification. The ability of the NGS assay to detect protozoa was later compared with conventional PCR. Results demonstrated that background amplification of oyster and other eukaryotic DNA competed with that of protozoa for obtained sequence reads. Sequences of target protozoans were obtained across all spiking levels; however, low numbers of target sequences in negative controls imply that a threshold for true positives must be defined for assay interpretation. While this study focused on three target parasites, the ability of this approach to detect numerous known and potentially unknown protozoan pathogens make it a promising screening tool for monitoring protozoan contamination in food and water.

Pollution of marine ecosystems with the protozoan parasites Toxoplasma gondii, Cryptosporidium spp. and Giardia duodenalis can be studied using bivalve shellfish as biosentinels. Although evidence suggests that these parasites are present in New Zealand coastal waters, the extent of protozoal pollution has not been investigated. This study used optimised molecular methods to detect the presence of Cryptosporidium spp., G. duodenalis and T. gondii in commercially sourced green-lipped mussel (Perna canaliculus), an endemic species found throughout coastal New Zealand. A nested polymerase chain reaction was validated for detection of T. gondii DNA and applied to 104 commercially sourced mussels. Thirteen mussels were positive for T. gondii DNA with an estimated true prevalence of 16.4% using Bayesian statistics, and the presence of T. gondii in mussels was significantly associated with collection during the summer compared with that in the winter (P = 0.003). Consumption of contaminated shellfish may also pose a health risk for humans and marine wildlife. As only sporulated T. gondii oocysts can be infectious, a reverse transcriptase-polymerase chain reaction was used to confirm presence of a sporozoite-specific marker (SporoSAG), detected in four mussels. G. duodenalis assemblage B, known to be pathogenic in humans, was also discovered in 1% mussels, tested by polymerase chain reaction (n = 90). Cryptosporidium spp. was not detected in the sampled mussel haemolymph. Results suggest that New Zealand may have high levels of coastal contamination with T. gondii, particularly in summer months, and that naturally exposed mussels can ingest and retain sporulated oocysts, further establishing shellfish consumption as a health concern.

Pathogen contamination of fresh produce presents a health risk for consumers; however, the produce industry still lacks adequate tools for simultaneous detection of protozoan parasites. Here, a simple multiplex PCR (mPCR) assay was developed for detection of protozoan (oo)cysts and compared with previously published real-time PCR assays and microscopy methods. The assay was evaluated for simultaneous detection of Cryptosporidium, Giardia, Cyclospora cayetanensis, and Toxoplasma gondii followed by parasite differentiation via either a nested specific PCR or a restriction fragment length polymorphism (RFLP) assay. Spiking experiments using spinach as a model leafy green were performed for assay validation. Leaf-washing yielded higher recoveries and more consistent detection of parasites as compared with stomacher processing. Lowest limits of detection using the nested mPCR assay were 1–10 (oo)cysts/g spinach (in 10 g samples processed), and this method proved more sensitive than qPCR for parasite detection. Microscopy methods were more reliable for visual detection of parasites in lower spiking concentrations, but are more costly and laborious, require additional expertise, and lack molecular confirmation essential for accurate risk assessment. Overall, the nested mPCR assay provides a rapid (

Toxoplasma gondii is a zoonotic protozoan parasite that can cause morbidity and mortality in humans, domestic animals, and terrestrial and aquatic wildlife. The environmentally robust oocyst stage of T. gondii is fundamentally critical to the parasite's success, both in terms of its worldwide distribution as well as the extensive range of infected intermediate hosts. Despite the limited definitive host species (domestic and wild felids), infections have been reported on every continent, and in terrestrial as well as aquatic environments. The remarkable resistance of the oocyst wall enables dissemination of T. gondii through watersheds and ecosystems, and long-term persistence in diverse foods such as shellfish and fresh produce. Here, we review the key attributes of oocyst biophysical properties that confer their ability to disseminate and survive in the environment, as well as the epidemiological dynamics of oocyst sources including domestic and wild felids. This manuscript further provides a comprehensive review of the pathways by which T. gondii oocysts can infect animals and people through the environment, including in contaminated foods, water or soil. We conclude by identifying critical control points for reducing risk of exposure to oocysts as well as opportunities for future synergies and new directions for research aimed at reducing the burden of oocyst-borne toxoplasmosis in humans, domestic animals, and wildlife. Keywords: Toxoplasma gondii, Oocyst, Transmission, Water, Soil, Food

There are few epidemiologic studies on the role of dogs in zoonotic parasitic transmission in the Circumpolar North. The objectives of this study were to: (a) estimate the faecal prevalence of Giardia spp. and Cryptosporidium spp. in dogs; (b) investigate potential associations between the type of dog population and the faecal presence of Giardia spp. and Cryptosporidium spp.; and (c) describe the molecular characteristics of Giardia spp. and Cryptosporidium spp. in dogs in Iqaluit, Nunavut. We conducted two cross-sectional studies in July and September 2016. In July, the team collected daily faecal samples for 3 days from each of 20 sled dogs. In September, the team collected three faecal samples from each of 59 sled dogs, 111 samples from shelter dogs and 104 from community dogs. We analysed faecal samples for the presence of Giardia spp. and Cryptosporidium spp. using rapid immunoassay and flotation techniques. Polymerase chain reaction (PCR) and sequencing of target genes were performed on positive faecal samples. Overall, the faecal prevalence of at least one of the target parasites, when one faecal sample was chosen at random for all dogs, was 8.16% (CI: 5.52-11.92), and for Giardia spp. and Cryptosporidium spp., prevalence was 4.42% (CI: 2.58-7.49) and 6.12% (CI: 3.88-9.53), respectively. The odds of faecal Giardia spp. in sled dogs were significantly higher than those in shelter and community dogs (OR 10.19 [CI: 1.16-89.35]). Sequence analysis revealed that 6 faecal samples were Giardia intestinalis, zoonotic assemblage B (n = 2) and species-specific assemblages D (n = 3) and E (n = 1), and five faecal samples were Cryptosporidium canis. Giardia intestinalis is zoonotic; however, Cryptosporidium canis is rare in humans and, when present, usually occurs in immunosuppressed individuals. Dogs may be a potential source of zoonotic Giardia intestinalis assemblage B infections in residents in Iqaluit, Nunavut, Canada; however, the direction of transmission is unclear.

Background Toxoplasma gondii is a zoonotic parasite that can have severe implications for human health. Acutely infected cats shed environmentally resistant T. gondii oocysts in their faeces that contaminate soil, and soil can serve as a reservoir of infection for humans. Free-roaming domestic cats are thought to play an important role in environmental contamination with T. gondii, but few studies have directly measured the direct contribution of free-roaming cats to T. gondii in soil. Methods Our goals were to determine whether T. gondii soil contamination occurs in public areas with free-roaming cat colonies in central California and examine spatial and temporal variation in soil contamination. We initially performed spiking experiments to compare the limit of T. gondii detection in soil using three conventional nested PCR assays and one real-time quantitative PCR. The nested PCR targeting the internal transcribed spacer (ITS-1) of the small subunit ribosomal RNA was the most sensitive assay, with a limit of detection between 20 and 200 oocysts per gram of soil. We applied the ITS1 PCR assay on soil from sites in city and state parks, public playgrounds and community gardens in central California, USA. Samples were collected during spring, summer and fall and in sites located along the coast and inland. Results We detected and sequence-confirmed T. gondii in 5.6% of all of our soil sub-samples, but with large seasonal and spatial variation in soil contamination: we only detected T. gondii during fall and only in coastal sites (44.3% soil prevalence), despite similar sampling intensity across space and time. Conclusions Our results suggest that free-roaming cat colonies are an important source of T. gondii in spaces where people recreate and grow food and that soil contamination is highly seasonal and spatially variable. Management of free-roaming cats could prevent T. gondii infections by reducing environmental contamination with this zoonotic pathogen.

Toxoplasma gondii is recognised as an important pathogen in the marine environment, with oocysts carried to coastal waters in overland runoff. Currently, there are no standardised methods to detect T. gondii directly in seawater to assess the extent of marine ecosystem contamination, but filter-feeding shellfish may serve as biosentinels. A variety of PCR-based methods have been used to confirm presence of T. gondii DNA in marine shellfish; however, systematic investigations comparing molecular methods are scarce. The primary objective of this study was to evaluate analytical sensitivity and specificity of two nested-PCR (nPCR) assays targeting dhps and B1 genes and two real-time (qPCR) assays targeting the B1 gene and a 529-bp repetitive element (rep529), for detection of T. gondii. These assays were subsequently validated for T. gondii detection in green-lipped mussel (Perna canaliculus) haemolymph using oocyst spiking experiments. All assays could reliably detect 50 oocysts spiked into mussel haemolymph. The lowest limit of detection was 5 oocysts using qPCR assays, with the rep529 primers performing best, with good correlation between oocyst concentrations and Cq values, and acceptable efficiency. Assay specificity was evaluated by testing DNA from closely related protozoans, Hammondia hammondi, Neospora caninum, and Sarcocystis spp. Both nPCR assays were specific to T. gondii. Both qPCR assays cross-reacted with Sarcocystis spp. DNA, and the rep529 primers also cross-reacted with N. caninum DNA. These studies suggest that the rep529 qPCR assay may be preferable for future mussel studies, but direct sequencing is required for definitive confirmation of T. gondii DNA detection.

SUMMARYSmall marine snails and abalone have been identified as high- and low-risk prey items, respectively, for exposure of threatened southern sea otters to Toxoplasma gondii, a zoonotic parasite that can cause fatal encephalitis in animals and humans. While recent work has characterized snails as paratenic hosts for T. gondii, the ability of abalone to vector the parasite has not been evaluated. To further elucidate why abalone predation may be protective against T. gondii exposure, this study aimed to determine whether: (1) abalone are physiologically capable of acquiring T. gondii; and (2) abalone and snails differ in their ability to concentrate and retain the parasite. Abalone were exposed to T. gondii surrogate microspheres for 24 h, and fecal samples were examined for 2 weeks following exposure. Concentration of surrogates was 2–3 orders of magnitude greater in abalone feces than in the spiked seawater, and excretion of surrogates continued for 14 days post-exposure. These results indicate that, physiologically, abalone and snails can equally vector T. gondii as paratenic hosts. Reduced risk of T. gondii infection in abalone-specializing otters may therefore result from abalone's high nutritional value, which implies otters must consume fewer animals to meet their caloric needs.

SUMMARYSarcocystis neurona is a terrestrial parasite that can cause fatal encephalitis in the endangered Southern sea otter (Enhydra lutris nereis). To date, neither risk factors associated with marine contamination nor the route of S. neurona infection to marine mammals has been described. This study evaluated coastal S. neurona contamination using California mussels (Mytilus californianus) as sentinels for pathogen pollution. A field investigation was designed to test the hypotheses that (1) mussels can serve as sentinels for S. neurona contamination, and (2) S. neurona contamination in mussels would be highest during the rainy season and in mussels collected near freshwater. Initial validation of molecular assays through sporocyst spiking experiments revealed the ITS-1500 assay to be most sensitive for detection of S. neurona, consistently yielding parasite amplification at concentrations ⩾5 sporocysts/1 mL mussel haemolymph. Assays were then applied on 959 wild-caught mussels, with detection of S. neurona confirmed using sequence analysis in three mussels. Validated molecular assays for S. neurona detection in mussels provide a novel toolset for investigating marine contamination with this parasite, while confirmation of S. neurona in wild mussels suggests that uptake by invertebrates may serve as a route of transmission to susceptible marine animals.

Toxoplasma gondii is a zoonotic protozoan parasite that can cause morbidity and mortality in humans, domestic animals, and terrestrial and aquatic wildlife. The environmentally robust oocyst stage of T. gondii is fundamentally critical to the parasite's success, both in terms of its worldwide distribution as well as the extensive range of infected intermediate hosts. Despite the limited definitive host species (domestic and wild felids), infections have been reported on every continent, and in terrestrial as well as aquatic environments. The remarkable resistance of the oocyst wall enables dissemination of T. gondii through watersheds and ecosystems, and long-term persistence in diverse foods such as shellfish and fresh produce. Here, we review the key attributes of oocyst biophysical properties that confer their ability to disseminate and survive in the environment, as well as the epidemiological dynamics of oocyst sources including domestic and wild felids. This manuscript further provides a comprehensive review of the pathways by which T. gondii oocysts can infect animals and people through the environment, including in contaminated foods, water or soil. We conclude by identifying critical control points for reducing risk of exposure to oocysts as well as opportunities for future synergies and new directions for research aimed at reducing the burden of oocyst-borne toxoplasmosis in humans, domestic animals, and wildlife., Highlights • Oocyst ingestion constitutes a significant proportion of T. gondii infections. • Oocyst attributes facilitate T. gondii transmission in terrestrial and aquatic habitats. • Oocysts are persistent and prevalent in water, soil and foods. • Mitigating oocyst-borne infections requires transdisciplinary international efforts.

Toxoplasma gondii is a coccidian parasite with the cat as its definitive host but any warm-blooded animal, including humans, may act as intermediate hosts. It has a worldwide distribution where it may cause acute and chronic toxoplasmosis. Infection can result from ingestion either of tissue cysts in infected meat of intermediate hosts or oocysts found in cat faeces via contaminated water or food. In this review, we highlight how the oocyst and sporocyst walls sustain the persistence and transmission of infective T. gondii parasites from terrestrial and aquatic environments to the host. We further discuss why targeting the oocyst wall structure and molecules may reduce the burden of foodborne and waterborne T. gondii infections. Keywords: Oocyst, Sporocyst, Wall structure, Molecules, Environment

Pollution of nearshore waters with disease-causing microorganisms impacts ecosystems health through illness and deaths in people and wildlife, as well as negative socioeconomic consequences of impaired marine resources. Insight on pathogen ecology in coastal habitats is crucial for accurately mitigating inputs and impacts of microbial pollution. Three objectives were addressed to (i) compare fecal pollution in proximity to (a) freshwater runoff, and (b) endemic marine wildlife; (ii) evaluate presence and magnitude of fecal microorganisms in marine snow and mussels and (iii) determine if pathogens in mussels and FIB levels in seawater or mussels are correlated. Sampling during the wet season, proximity to freshwater, and FIB levels in mussel homogenates (but not seawater) were associated with pathogen presence in mussels. Pathogens and FIB were enriched in aggregate-rich fractions, further supporting an important role of marine snow in pathogen transmission. The lack of association between FIB in surrounding waters and presence of pathogens in mussels calls into question current regulations for insuring safe seafood to consumers in the United States, and alternative monitoring approaches such as direct testing for select pathogens should be further evaluated.

Freeze-thaw DNA extraction methods and PCR primers were compared to optimize detection of Cryptosporidium parvum and Toxoplasma gondii oocysts in different matrices. Increasing FT cycles did not increase parasite DNA detection, and primers targeting the 18S ssrRNA gene yielded the most sensitive detection of C. parvum oocysts.

Human noroviruses (NoVs) are responsible for 50% of food-related disease outbreaks and are notably associated with shellfish consumption. Despite the detrimental health impacts of human NoV-contaminated seafood to public health, there is a lack of knowledge on the physicochemical conditions that govern NoV transmission in aquatic ecosystems. In the present study, we investigated the propensity for NoVs to associate with aquatic aggregates, which have been shown to efficiently deliver nano-sized particles to shellfish. Specific physicochemical conditions characteristic of shellfish cultivation waters, specifically salinity and transparent exopolymer particles (TEP), were targeted in this study for investigating aggregate formation and NoV association dynamics. Murine norovirus (MNV) was used in aggregation experiments as a model surrogate for NoVs. Results demonstrate increased aggregate formation as a function of increasing salinity and TEP concentrations, as well as greater numbers of MNV genomes incorporated into aggregates under conditions that favor aggregation. As aggregate formation was enhanced in waters representing optimal conditions for shellfish production, specifically saline and high TEP waters, the implications to virus transport and shellfish food safety are profound: more aggregates implies increased scavenging of virus particles from surrounding waters and therefor greater risk for bivalve contamination with nano-sized pathogens. These novel data provide insight into where and when NoVs are most likely to be ingested by shellfish via contaminated aggregates, thereby informing best management and water quality monitoring practices aimed at providing safe seafood to consumers.

To evaluate the impact of treated wastewater (TWW) irrigation for produce safety, field experiments were conducted to compare secondary and tertiary TWW with potable water using tomatoes as a model crop. Human pathogens including a suite of obligate and opportunistic bacterial pathogens (Campylobacter, Pseudomonas, Salmonella, Shigella, Staphylococcus), protozoa (Cryptosporidium and Giardia), and viruses (Adenovirus and Enterovirus) were monitored in two field trials using a combination of microscopic, cultivation-based, and molecular (qPCR) techniques. Results indicate that microbial contamination on the surface of tomatoes was not associated with the source of irrigation waters; fecal indicator bacteria (FIB) contamination was not statistically different on tomatoes irrigated with TWW or potable water. In fact, indicator bacteria testing did not predict the presence of pathogens in any of the matrices tested. Indicator bacteria and the opportunistic pathogens were detected in water, soil and on tomato surfaces from all irrigation treatment schemes, and were positively correlated with duration of time in the field (p

Summary The parasite Toxoplasma gondii is an environmentally persistent pathogen that can cause fatal disease in humans, terrestrial warm-blooded animals and aquatic mammals. Although an association between T. gondii exposure and prey specialization on marine snails was identified in threatened California sea otters, the ability of kelp-dwelling snails to transmit terrestrially derived pathogens has not been previously investigated. The objective of this study was to measure concentration and retention of T. gondii by marine snails in laboratory aquaria, and to test for natural T. gondii contamination in field-collected snails. Following exposure to T. gondii-containing seawater, oocysts were detected by microscopy in snail faeces and tissues for 10 and 3 days respectively. Nested polymerase chain reaction was also applied as a method for confirming putative T. gondii oocysts detected in snail faeces and tissues by microscopy. Toxoplasma gondii was not detected in field-collected snails. Results suggest that turban snails are competent transport hosts for T. gondii. By concentrating oocysts in faecal pellets, snails may facilitate entry of T. gondii into the nearshore marine food web. This novel mechanism also represents a general pathway by which marine transmission of terrestrially derived microorganisms can be mediated via pathogen concentration and retention by benthic invertebrates.

Summary Coastal habitat contamination with Toxoplasma gondii is a health risk to humans and marine wildlife, with infections documented in both nearshore and pelagic marine mammals. Due to lack of sensitive methods for detection of T. gondii in water, this study utilized an alternative surveillance approach for evaluating marine habitat contamination using wild mussels. The objectives of this study were to (i) validate sensitive molecular tools for T. gondii detection in mussels and (ii) apply optimized methods in a surveillance study to determine the prevalence and genotype(s) of T. gondii in mussels. Simplex polymerase chain reaction screening and multiplex genotyping assays were validated and then applied on 959 wild-caught mussels collected from central California. Thirteen mussels (1.4%) had detectable T. gondii DNA and the presence of T. gondii in mussels was significantly associated with proximity to freshwater run-off and collection during the wet season. Molecular characterization revealed alleles from T. gondii types I, II/III, X at the B1 locus, and a novel atypical B1 allele that was recently documented in T. gondii-infected carnivores from California. Findings demonstrate higher than previously reported T. gondii contamination of California coastlines, and describe novel strains of the parasite that further link terrestrial sources with marine contamination.