High occurrence of viable forms of Cryptosporidium and Giardia in domestic sewage from an agricultural region of Brazil.

Cryptosporidium and Giardia are the main etiologies of waterborne outbreaks caused by protozoa. These parasites are commonly detected in wastewater; however, there is little knowledge about the concentration of viable forms in treated sewage, mainly in small communities. To understand more about the presence of viable oocysts and cysts in domestic sewage, we monitored the affluent and effluent of a wastewater treatment plant (WWTP) in inner-city Brazil. Ten samplings and seven follow-ups were performed in 2020. Samples were concentrated by centrifugation, filtration and purified by fluctuation. Viability was accessed by propidium-monoazide (PMA) associated with nPCR and qPCR. Both viable protozoa were detected in all raw sewage samples (average: 438.5 viable oocysts/L). Regarding treated sewage, Cryptosporidium was detected in all of the samples (average: 92.8 viable oocysts/L) and Giardia was detected in 70% with viable cysts in 30%. Considering the follow-ups, 31.17% of Cryptosporidium viable oocysts remained in the effluent after the treatment. High amounts of Cryptosporidium and a high frequency of Giardia were detected, therefore both arrived at WWTP and were discharged into the river. These alert the presence of agro-industrial effluents into domestic sewage and demonstrated the effectiveness of the concentration technique for monitoring protozoa in wastewater.

Parasitic threat in commercial organic fertilizers.

The use of fertilizers based on sewage sludge is common practice. Due to the possible presence of pathogens and eggs of intestinal parasites like Ascaris sp., Toxocara sp., and Trichuris sp. in these products, it is necessary to control them. The aim of the study was to determine the presence of parasite eggs in commercial organic fertilizers available on the market. Selected commercial products were tested using the Quinn flotation method and a method dedicated to the study of dewatered sewage sludge. Assessment of the viability of helminth eggs was carried out on the basis of staining with calcein and propidium iodide. In 57% of the tested samples, the presence of live eggs of the abovementioned parasites was detected, and in 21% of samples, the eggs with live larvae were detected. Eggs of Trichuris sp. (50%) and Ascaris sp. (36%) were the most common. The obtained results clearly indicate that the process of hygienization of the sewage sludge before the production of fertilizers was not effective enough and it is necessary to standardize the prophylaxis against the spread of parasitic nematodes in commercially available products.

Performance of sewage treatment technologies for the removal of Cryptosporidium sp. and Giardia sp.: Toward water circularity.

Cryptosporidium sp. and Giardia sp. are parasites that cause diseases in the population. Most of parasite diseases regarding the consumption of drinking water polluted with sewage are caused by Cryptosporidium sp. or Giardia sp. it is because of the incomplete disinfection of the wastewater treatment. Therefore, in this work the removal or inactivation efficiency of different treatment technologies presented by around 40 scientific studies was evaluated, with a view to water circularity. For Cryptosporidium sp., we conclude that the most efficient secondary technologies are aerobic technologies, which remove between 0.00 and 2.17 log units (Ulog), with activated sludge presenting the greatest efficiency, and that the tertiary technologies with the greatest removal are those that use ultrasound, which reach removal values of 3.17 Ulog. In the case of Giardia sp., the secondary technologies with the greatest removal are anaerobic technologies, with values between 0.00 and 3.80 Ulog, and the tertiary technologies with the greatest removal are those that combine filtration with UV or a chemical disinfection agent. Despite the removal values obtained, the greatest concern remains detecting and quantifying the infectious forms of both parasites in effluents; therefore, although the technologies perform adequately, discharge effluents must be monitored with more sensitive techniques, above all aiming for circularity of the treated water in a context of the water scarcity that affects some parts of the world.

Pathogenic parasites in sewage irrigated crops and soil: pattern of occurrence and health implications.

The current study aimed to determine the contamination level by human parasite eggs and cysts of sewage-irrigated soil, food crops and fodder crops. Field trials were conducted on spray-irrigated lettuce and flood-irrigated crops using raw wastewater and fresh water. Findings showed that irrigation by raw wastewater led to crops contamination with parasite eggs and cysts in varying levels. Lettuce samples were positive (27.7%) for one or more helminth eggs including the pathogens Ascaris and Trichuris with an average of 4.7 eggs/kg. Wastewater application caused an increase in lettuce contamination, followed by a decreasing phase. Cessation of irrigation resulted in helminth eggs disappearance within a week. Flood-irrigated crops were contaminated with helminth eggs in levels ranging from 1.5 eggs/kg in alfalfa to 4.75 eggs/kg in coriander. The pathogenic Giardia,Entamoeba histolytica/dispar and commensal amoeba cysts were highly detected reaching 9.97x103 cysts/kg in alfalfa. Parasites highly concentrated in soil, with levels up to 159 eggs/100gdw and 1.3x103 cysts/gdw. Ascaris and Trichuris eggs developed in soil and crops attaining the infective stage. Crops and soil irrigated with fresh water were free from parasite eggs and cysts. The occurrence of pathogens in soil and crops implies health risks for farmers, produce handlers and consumers.

Human health risk assessment for (re)emerging protozoan parasites in surface water used for public supply and recreational activities.

Gastrointestinal diseases caused by protozoan parasites remain a major challenge in developing countries and ingestion of contaminated surface water represents one of the main sources by which these diseases are contracted. This study assessed the risk of infection and diseases caused by Cryptosporidium spp. and Giardia sp. due to ingestion of surface water used for public supply and recreational activities, focusing on the southeastern Brazilian Pardo River and applying the USEPA 1623 method to quantify (oo)cyst concentrations. Infection and disease probabilities due to ingestion of drinking water or during recreational activities were estimated using the Quantitative Microbial Risk Assessment (QMRA) approach. Mean concentrations of Cryptosporidium spp. and Giardia sp. in surface water ranged from 0.2 to 0.4 oocysts L-1 and 0.2 to 4.4 cysts L-1, respectively. Considering public water supply, annual infection probabilities were higher for adults than children and exceeded the USEPA limit; also, disease probabilities were higher for adults than children. For recreational activities, annual infection and disease probabilities were higher for children, followed by men and women. The occurrence of both parasites likely reflects raw sewage discharge, effluent from sewage treatment plants, and diffuse sources of pollution, such as runoff from pasture lands and deforested riparian forest corridors. Our results highlight substantial infection risks by both parasite types after conventional treatment of water used for public supply and also call for careful monitoring of water bodies used for recreational purposes.

Helminth eggs inactivation efficiency by sludge co-composting under arid climates.

Primary sludge (PS) from the wastewater treatment plants is constituted of organic matter and other nutrients that could be reused as soil fertilizers. However, primary sludge amendment is hampered due to the presence of pathogens that could be responsible for several infections in various ways depending on the prevalence of the human sources. This study investigates helminth eggs (HE) removal and inactivation efficiency by co-composting. So, PS was mixed with date palm waste as a bulking material at a 1:1 volume ratio. The C/N ratio decreased from 25:1 to 12:1 and temperatures above 50ºC were observed since 33 days. The initial concentration of HE eggs in the PS is 135.9 per 10 g DR. Microscopic examination of the PS, according to the Bailenger method, showed the presence of nematodes and Cestodes represented by: Ascaris lombricoïde, Ancylostomes duodenale, Trichuris trichiura, Capilaria spp, Hymenolepis nana, Taenia saginata, and Ascaris lombricoïde. After 60 days of co-composting, the charge of parasites was reduced to 18 eggs per 10 g per DR. The viability of ascaris eggs was examined using a light microscope and the percentage of the embryonated eggs was determined. No viable eggs were observed in the final compost. The result corroborates the WHO guidelines for the application of the composted sewage sludge safely for agriculture purposes.

Helminth Egg Automatic Detector (HEAD): Improvements in development for digital identification and quantification of helminth eggs and their application online.

Helminths are parasitic worms that constitute a major public health problem. Conventional analytical techniques to evaluate helminth eggs in environmental samples rely on different steps, namely sedimentation, filtration, centrifugation, and flotation, to separate the eggs from a variety of particles and concentrate them in a pellet for direct observation under an optical microscope. To improve this process, a new approach was implemented in which various image processing algorithms were developed and implemented by a Helminth Egg Automatic Detector (HEAD). This allowed identification and quantification of pathogenic helminth eggs of global medical importance and it was found to be useful for relatively clean wastewater samples. After the initial version, two improvements were developed: first, a texture verification process that reduced the number of false positive results; and second, the establishment of the optimal thresholds (morphology and texture) for each helminth egg species. This second implementation, which was found to improve on the results of the former, was developed with the objective of using free software as a platform for the system. This does not require the purchase of a license, unlike the previous version that required a Mathworks® license to run. After an internal statistical verification of the system was carried out, trials in internationally recognized microbiology laboratories were performed with the aim of reinforcing software training and developing a web-based system able to receive images and perform the analysis throughout a web service. Once completed, these improvements represented a useful and cheap tool that could be used by environmental monitoring facilities and laboratories throughout the world; this tool is capable of identifying and quantifying different species of helminth eggs in otherwise difficult environmental samples: wastewater, soil, biosolids, excreta, and sludge, with a sensitivity and specificity for the TensorFlow (TF) model in the web service values of 96.82% and 97.96% respectively. Additionally, in the case of Ascaris, it may even differentiate between fertile and non-fertile eggs.

Bacteria and microeukaryotes are differentially segregated in sympatric wastewater microhabitats.

Wastewater purification is mostly performed in activated sludge reactors by bacterial and microeukaryotic communities, populating organic flocs and a watery liquor. While there are numerous molecular community studies of the bacterial fraction, those on microeukaryotes are rare. We performed a year-long parallel 16S rRNA gene and 18S rRNA-gene based analysis of the bacterial and of the microeukaryote communities, respectively, of physically separated flocs and particle-free liquor samples from three WWTPs. This uncovered a hitherto unknown large diversity of microeukaryotes largely composed of potential phagotrophs preferentially feeding on either bacteria or other microeukaryotes. We further explored whether colonization of the microhabitats was selective, showing that for both microbial communities, different but often closely taxonomically and functionally related populations exhibiting different dynamic patterns populated the microhabitats. An analysis of their between plants-shared core populations showed the microeukaryotes to be dispersal limited in comparison to bacteria. Finally, a detailed analysis of a weather-caused operational disruption in one of the plants suggested that the absence of populations common to the floc and liquor habitat may negatively affect resilience and stability.

Detection of Cryptosporidium and Giardia in the slaughterhouse, sewage and river waters of the Qinghai Tibetan plateau area (QTPA), China.

The occurrence of Cryptosporidium and Giardia species in slaughter, sewage and river waters of the Qinghai Tibetan Plateau Area (QTPA), China, was investigated. A total of 456 samples were collected from different locations in the QTPA to study the contamination rates of Cryptosporidium spp. and Giardia via PCR and subsequent sequence analysis. Ten samples were Cryptosporidium positive, and 97 were Giardia positive, as confirmed by PCR amplification of the SSU rRNA gene. The percentages of positive Cryptosporidium and Giardia detection were 2.2% (10/456) and 21.3% (97/456), respectively. Cryptosporidium was detected in only sewage and river waters. Six species of Cryptosporidium were identified: Cryptosporidium hominis (n = 5), C. andersoni (n = 1), C. environmental (n = 1), C. struthionis (n = 1), C. canis (n = 1), and C. parvum (n = 1). G. duodenalis assemblage A was identified in almost all positive samples (n = 96), and one sample harboured G. duodenalis assemblage E. The results suggest that Cryptosporidium and Giardia species circulate through the aqueous environment and different hosts. Therefore, we strongly recommend that the local government and health authorities in China undertake control measures to reduce the contamination of water sources by these protozoa to protect the health of humans and animals.

Comparative assessment of DNA extraction procedures for Ascaris spp. eggs.

A central and critical step in the molecular detection of soil-transmitted helminths from environmental sources is the extraction of DNA from the eggs. In this study, we investigated the yield of DNA extracted from known quantities (500, 100, 50, 20, 10 and 5) of Ascaris suum eggs, as well as directly from wastewater and sludge samples containing Ascaris spp. eggs, using six commercial DNA extraction kits. The amount of DNA extracted was quantified with NanoDrop, Qubit and Ct values from quantitative polymerase chain reaction (qPCR) assay using CFX96 Touch™ real-time PCR equipment. The PowerLyzer Ultraclean Microbial DNA isolation kit and PowerSoil DNA isolation kit gave the highest yield of DNA based on the NanoDrop, Qubit and Ct values. However, the qPCR results indicate that in some of the kits, PCR inhibitors may have been carried over to the PCR reaction. DNA extraction kits that incorporate a bead-beating step as well as other mechanical eggshell disruption steps were superior in extracting DNA from Ascaris spp. eggs. Additionally, for the accurate quantification of extracted DNA, the use of Ct values from qPCR and Qubit readings gives better results compared to the NanoDrop readings. For efficient downstream applications, the use of DNA extraction kits with superior inhibitor removal technology is essential, in addition to a high yield of DNA.

Soil-transmitted helminth infections associated with wastewater and sludge reuse: a review of current evidence.

OBJECTIVE: To review current evidence on infections related to the concentration of soil-transmitted helminth (STH) eggs in wastewater, sludge and vegetables irrigated with wastewater or grown on sludge-amended soils. METHOD: Search of Web of Science, Science Direct, PubMed and Google Scholar databases for publications reporting on STH egg concentration in wastewater, sludge and vegetables and for epidemiological studies on wastewater/sludge reuse and STH infections. RESULTS: STH egg concentrations were variable but high in wastewater and sludge especially in developing countries. They ranged from 6 to 16 000 eggs/L in wastewater and from 0 to 23 000 eggs/g in sludge and far exceed limits set in the WHO guideline for wastewater/sludge reuse. Numbers of STH eggs on vegetables ranged from 0 to 100 eggs/g. The concentration of STH eggs in wastewater, sludge and vegetables therefore relates to risks of infection through different exposure routes. CONCLUSION: Epidemiological evidence reveals an increased prevalence of STH infections associated with direct exposure to wastewater or sludge (farmers) and consumption of vegetables grown on soil treated with it. This calls for increased efforts to reduce the adverse health impact of wastewater and sludge reuse in line with the WHO multi-barrier approach.

Molecular surveillance of Cryptosporidium and Giardia duodenalis in sludge and spent filter backwash water of a water treatment plant.

The purpose of this study was to monitor the presence of Cryptosporidium spp. and Giardia duodenalis in a water treatment plant (WTP) using settling sludge and backwash water (BW) samples in previous and post the deflocculation of polyaluminium chloride (PAC) flacks. Eleven collections were performed. BW and settling sludge (SSF) were concentrated by calcium carbonate flocculation, and another aliquot of settling sludge (SSC) by centrifugation. The samples were divided as follows: Group A, containing 33 samples without degradation of PAC flakes, and Group B, with degradation by alkalinization with 10 M NaOH. Sample DNA was extracted with a commercial kit, and nested polymerase chain reaction (PCR) was used to detect Cryptosporidium and G. duodenalis. All samples from Group A were negative for Cryptosporidium spp., and 6.1% (2/33) were positive for G. duodenalis in SSC samples. While the absence of Cryptosporidium may be due to a low contamination level of the water resource, the presence of G. duodenalis indicates contamination of the raw water. The detection of G. duodenalis in SSC samples indicates that this detection method was the most effective. The 33 samples from Group B were negative for both protozoa, probably due to the presence of aluminium and humic substances.

Cryptosporidium parvum decay during air drying and stockpiling of mesophilic anaerobically digested sewage sludge in a simulation experiment and oocyst counts in sludge collected from operational treatment lagoons in Victoria, Australia.

The inactivation of Cryptosporidium species oocysts during sewage sludge treatment is important to protect human health when the residual biosolids are applied to agricultural land. Quantifying the decay of Cryptosporidium species during sludge treatment for microbiological assurance purposes is difficult if low numbers are present in wastewater. The rate of decay of Cryptosporidium parvum oocysts during solar/air drying treatment and in sludge stockpiles in temperate environment conditions was simulated in laboratory inoculation experiments using sludge sampled from a mesophilic anaerobic digester. Oocyst numbers were also determined in settled lagoon sludge samples collected from three operational rural wastewater treatment plants (WWTPs). C. parvum oocysts were enumerated by immunomagnetic separation followed by staining with vital dyes and examination by confocal laser scanning microscopy. An air-drying/storage period equivalent to 11 weeks was required for a 1 log10 reduction of viable oocysts inoculated into digested sludge. Oocyst viability in air-dried and stored digested sludge decreased with time, but was independent of sludge desiccation and dry solids (DS) content. No oocysts were detected in sludge samples collected from the anaerobic digester, and the average concentration of oocysts found in settled lagoon sludge from the rural WWTP was 4.6 × 102 oocysts/g DS.

Sewage sludge amendment and inoculation with plant-parasitic nematodes do not facilitate the internalization of Salmonella Typhimurium LT2 in lettuce plants.

Contamination of fruits and vegetables with Salmonella is a serious threat to human health. In order to prevent possible contaminations of fresh produce it is necessary to identify the contributing ecological factors. In this study we investigated whether the addition of sewage sludge or the presence of plant-parasitic nematodes foster the internalization of Salmonella enterica serovar Typhimurium LT2 into lettuce plants, posing a potential threat for human health. Greenhouse experiments were conducted to investigate whether the amendment of sewage sludge to soil or the presence of plant-parasitic nematodes Meloidogyne hapla or Pratylenchus crenatus promote the internalization of S. Typhimurium LT2 from soil into the edible part of lettuce plants. Unexpectedly, numbers of cultivable S. Typhimurium LT2 decreased faster in soil with sewage sludge than in control soil but not in root samples. Denaturing gradient gel electrophoresis analysis revealed shifts of the soil bacterial communities in response to sewage sludge amendment and time. Infection and proliferation of nematodes inside plant roots were observed but did not influence the number of cultivable S. Typhimurium LT2 in the root samples or in soil. S. Typhimurium LT2 was not detected in the leaf samples 21 and 49 days after inoculation. The results indicate that addition of sewage sludge, M. hapla or P. crenatus to soil inoculated with S. Typhimurium LT2 did not result in an improved survival in soil or internalization of lettuce plants.

Pit Latrines: A Noninvasive Sampling Strategy to Assess Fecal Pathogen Occurrence in Low Resource Communities.

Limited understanding of disease in low resource communities continues to hamper improvements in health. We evaluated household pit latrine sampling as a non-invasive approach to investigate important fecal pathogens such as Giardia lamblia and Cryptosporidium spp. in impoverished communities where health-seeking behavior limits the sensitivity of health facility-based surveillance. Fecal samples were collected from pit latrines in randomly selected households and from patients presenting to the only hospital in the region during the same time periods. Samples were tested with a commercially available ELISA. Giardia household prevalence was 28.7% in 2016 and 48.4% in 2017, while individual samples from hospital submission had a Giardia prevalence of 2.4% in 2016 and 8.0% in 2017. Cryptosporidium was only found in one household. Results suggest that pit latrine surveillance for fecal-borne infections provide course estimates of community infection levels that are unbiased by health seeking behaviors and allow surveillance of vulnerable sectors of a population.

Performance comparison of three methods for detection of Giardia spp. cysts and Cryptosporidium spp. oocysts in drinking-water treatment sludge.

Detecting pathogenic protozoa in drinking-water treatment sludge is a challenge as existing methods are complex, and unfortunately, there are no specific technical standards to follow. Selecting an efficient analytical method is imperative in developing countries, such as Brazil, in order to evaluate the risk of parasite infection. In this context, three methods to detect Giardia spp. cysts and Cryptosporidium spp. oocysts were tested in sludge generated when water with protozoa and high turbidity was treated. Jar testing was carried out using polyaluminium chloride as a coagulant to generate the residue to be analyzed. The results showed that calcium carbonate flocculation with reduced centrifugation and immunomagnetic separation obtained the highest recoveries in the tested matrix showing 60.2% ± 26.2 for oocysts and 46.1% ± 5 for cysts. The other two methods, the first using the ICN 7× cleaning solution and the second considering the acidification of the sample, both followed by the immunomagnetic separation step, also presented high recoveries showing 41.2% ± 43.3 and 37.9% ± 52.9 for oocysts and 11.5% ± 85.5 and 26% ± 16.3 for cysts, respectively. Evidently, these methods and others should be studied in order to make it possible to detect protozoa in settled residue.

Environmental Transport of Emerging Human-Pathogenic Cryptosporidium Species and Subtypes through Combined Sewer Overflow and Wastewater.

The environmental transport of Cryptosporidium spp. through combined sewer overflow (CSO) and the occurrence of several emerging human-pathogenic Cryptosporidium species in developing countries remain unclear. In this study, we collected 40 CSO samples and 40 raw wastewater samples from Shanghai, China, and examined them by PCR and DNA sequencing for Cryptosporidium species (targeting the small subunit rRNA gene) and Giardia duodenalis (targeting the triosephosphate isomerase, ß-giardin, and glutamate dehydrogenase genes) and Enterocytozoon bieneusi (targeting the ribosomal internal transcribed spacer) genotypes. Human-pathogenic Cryptosporidium species were further subtyped by sequence analysis of the 60-kDa glycoprotein gene, with additional multilocus sequence typing on the emerging zoonotic pathogen Cryptosporidium ubiquitum. Cryptosporidium spp., G. duodenalis, and E. bieneusi were detected in 12 and 15, 33 and 32, and 37 and 40 CSO and wastewater samples, respectively, including 10 Cryptosporidium species, 3 G. duodenalis assemblages, and 8 E. bieneusi genotypes. In addition to Cryptosporidium hominis and Cryptosporidium parvum, two new pathogens identified in industrialized nations, C. ubiquitum and Cryptosporidium viatorum, were frequently detected. The two novel C. ubiquitum subtype families identified appeared to be genetic recombinants of known subtype families. Similarly, the dominant group 1 E. bieneusi genotypes and G. duodenalis subassemblage AII are known human pathogens. The similar distribution of human-pathogenic Cryptosporidium species and E. bieneusi and G. duodenalis genotypes between wastewater and CSO samples reaffirms that storm overflow is potentially a significant contamination source of pathogens in surface water. The frequent identification of C. ubiquitum and C. viatorum in urban wastewater suggests that these newly identified human pathogens may be endemic in China.IMPORTANCECryptosporidium spp., Giardia duodenalis, and Enterocytozoon bieneusi are major waterborne pathogens. Their transport into surface water through combined sewer overflow, which remains largely untreated in developing countries, has not been examined. In addition, the identification of these pathogens to genotypes and subtypes in urban storm overflow and wastewater is necessary for rapid and accurate assessment of pathogen transmission in humans and transport in the environment. Data from this study suggest that, like untreated urban wastewater, combined sewer overflow is commonly contaminated with human-pathogenic Cryptosporidium, G. duodenalis, and E. bieneusi genotypes and subtypes, and urban storm overflow potentially plays a significant role in the contamination of drinking source water and recreational water with human pathogens. They also indicate that Cryptosporidium ubiquitum and Cryptosporidium viatorum, two newly identified human pathogens, may be common in China, and genetic recombination can lead to the emergence of novel C. ubiquitum subtype families.

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.

An 18S rRNA Workflow for Characterizing Protists in Sewage, with a Focus on Zoonotic Trichomonads.

Microbial eukaryotes (protists) are important components of terrestrial and aquatic environments, as well as animal and human microbiomes. Their relationships with metazoa range from mutualistic to parasitic and zoonotic (i.e., transmissible between humans and animals). Despite their ecological importance, our knowledge of protists in urban environments lags behind that of bacteria, largely due to a lack of experimentally validated high-throughput protocols that produce accurate estimates of protist diversity while minimizing non-protist DNA representation. We optimized protocols for detecting zoonotic protists in raw sewage samples, with a focus on trichomonad taxa. First, we investigated the utility of two commonly used variable regions of the 18S rRNA marker gene, V4 and V9, by amplifying and Sanger sequencing 23 different eukaryotic species, including 16 protist species such as Cryptosporidium parvum, Giardia intestinalis, Toxoplasma gondii, and species of trichomonad. Next, we optimized wet-lab methods for sample processing and Illumina sequencing of both regions from raw sewage collected from a private apartment building in New York City. Our results show that both regions are effective at identifying several zoonotic protists that may be present in sewage. A combination of small extractions (1 mL volumes) performed on the same day as sample collection, and the incorporation of a vertebrate blocking primer, is ideal to detect protist taxa of interest and combat the effects of metazoan DNA. We expect that the robust, standardized methods presented in our workflow will be applicable to investigations of protists in other environmental samples, and will help facilitate large-scale investigations of protistan diversity.