Effect of Glycosaminoglycans on Cryptosporidium Oocyst Attachment and Excystation.

Cryptosporidium causes severe gastrointestinal disease resulting from the ingestion of oocysts, followed by oocyst excystation in the small intestine and the release of infective sporozoites. An understudied strategy for Cryptosporidium inactivation is purposeful oocyst excystation, as sporozoites do not survive long in the environment. This study showed that C. parvum oocyst excystation was induced by direct contact with various glycosaminoglycans (GAGs), including heparin (Hep), chondroitin sulfate A (CSA), and hyaluronan (HA), assembled on polydopamine (PD)-functionalized surfaces. PD surfaces elicited 97.9 ± 3.6% oocyst attachment, with some of the attached oocysts partially (7.3 ± 1.3%) or fully (4.0 ± 0.6%) excysted after 4 days. The PD-GAG surfaces (GAG concentration = 2 mg/mL) elicited similarly high attachment (>97%) and higher oocyst excystation efficiencies after 4 days. The PD-Hep surfaces elicited the highest number of attached excysted oocysts (11.8 ± 0.63% partially excysted; 11.9 ± 0.49% fully excysted), and the PD-HA surfaces elicited the lowest (8.8 ± 2.1% partially excysted; 7.8 ± 1.2% fully excysted). Surface characterization revealed that the addition of GAGs to the PD surface changed both the surface roughness as well as the surface wettability. Treatment of oocysts with an enzyme that degraded the surface glycocalyx markedly reduced excystation (to <2%) of the oocysts attached to the PD and PD-GAG surfaces. These findings suggest that GAGs provide an important local signal for the excystation of C. parvum oocysts and that certain surface-expressed oocyst receptors are necessary for efficient excystation. These oocyst-receptor relationships may be useful in the design of functionalized surfaces for the purposeful inactivation of oocysts in the environment or in water treatment systems. IMPORTANCE Polydopamine surfaces functionalized with glycosaminoglycans were shown to facilitate the attachment and excystation of Cryptosporidium parvum oocysts. Our findings suggest that a surface-expressed receptor on the oocyst wall plays a key role in excystation, with glycosaminoglycans serving as ligands that trigger the initiation of the process. Future technologies and treatment strategies designed to promote premature excystation of oocysts will minimize the ingestion of sporozoites that initiate infection. Therefore, the results from this study have important implications for the protection of public health from waterborne cryptosporidiosis and may serve as a foundation for engineered surfaces designed to remove oocysts from surface waters or inactivate oocysts in water treatment systems.

Biofilm Sampling for Detection of Cryptosporidium Oocysts in a Southeastern Pennsylvania Watershed.

This study investigated the use of biofilms to monitor Cryptosporidium in water. Benthic rock and submersible slide biofilms were sampled upstream and downstream of point sources in a suburban watershed in southeastern Pennsylvania. More oocysts were detected in biofilms scraped from rocks downstream than upstream of a wastewater treatment plant (WWTP) (19 versus 5, respectively; n = 1). Although not statistically significant, Cryptosporidium oocysts were detected more frequently, and in greater numbers, in biofilms grown on slides downstream than upstream of this same WWTP (83.3% positive samples [n = 12] versus 45.5% positive samples [n = 11], respectively; P = 0.0567). Similarly, Cryptosporidium oocysts were detected more frequently, and in greater numbers, in rock biofilms collected downstream than upstream of a stormwater outfall impacted by defective sewer laterals (50% positive samples downstream and 17% positive samples upstream; n = 6; P = 0.2207). While oocyst detection data obtained by slide biofilms versus filters did not necessarily agree on a given day, there was no seasonal difference in the frequency of oocyst detection (P > 0.05) or numbers of oocysts detected (P > 0.05) whether the water was monitored by filtration or slide biofilm sampling. Within any given season, there was no difference in the frequency of oocyst detection (P > 0.05) or the numbers of oocysts detected (P > 0.05) whether the water was monitored by filtration or slide biofilm sampling. These data show that oocyst detection in biofilms is comparable to oocyst detection in filtered water samples. Biofilm sampling offers significant cost savings compared to the filtration-based EPA Method 1623.1 and could be used to identify watershed locations at potential risk for increased oocyst loads.IMPORTANCE Monitoring Cryptosporidium occurrence in watersheds that provide drinking water is necessary to determine where limited resources should most effectively be directed to protect consumers from waterborne exposure to pathogenic oocysts. Biofilms are a useful tool to monitor complex watersheds and identify point sources of Cryptosporidium oocyst contamination that need to be managed to protect public health. Compared to EPA Method 1623.1, the cost benefit of using biofilms to monitor for Cryptosporidium contamination will enable utilities to sample water supplies more frequently, and at more locations, than is currently possible given limited operating budgets. Biofilm sampling could be used to identify high-risk regions within a large, complex watershed and the associated water treatment plants at potential risk for increased oocyst loads in the water supply; this information could then be used to select the locations within the watershed where the more expensive EPA Method 1623.1 is warranted.

Calcium-Mediated Biophysical Binding of Cryptosporidium parvum Oocysts to Surfaces Is Sensitive to Oocyst Age.

Cryptosporidium parvum causes potentially life-threatening gastrointestinal disease in humans and may not be effectively removed from drinking water via conventional methods. Prior research has shown that environmental biofilms immobilize oocysts from the water column, but the biophysical mechanisms driving this attraction are still under investigation. This study investigates the affinity of C. parvum oocysts to silanized surfaces. Surfaces were prepared with hydroxyl, amine, and carboxyl moieties. Binding forces between the oocysts and these engineered substrates were analyzed, with and without divalent ions, using atomic force microscopy. Binding forces were measured over several weeks to investigate the influence of age on adhesion. C. parvum oocysts bind most strongly to carboxylic acid functional groups, with rupture forces greater than that required to break noncovalent molecular bonds, regardless of oocyst age. This adhesion is shown to be due to divalent cation bridging mechanisms. In addition, the binding strength increases over a 5-week period as the oocysts age, followed by a decrease in the binding strength, which may be related to structural or biochemical changes in the outer wall-bound glycosylated proteins. This study sheds new light on the biochemical parameters that influence C. parvum oocyst binding to surfaces. Increased understanding of how age and water chemistry influence the binding strength of oocysts may inform future developments in environmental detection and drinking water treatment, such as with the development of oocyst-specific sensors that allow for more frequent tracking of oocysts in the environment.IMPORTANCE The mechanisms by which pathogens bind to surfaces are of interest to a wide variety of scientific communities, as these mechanisms drive infectivity, fate, and transport of the pathogenic organisms. This study begins to reveal the mechanism of direct binding of Cryptosporidium parvum to surfaces containing both carboxylic acid and amine moieties, in an attempt to understand how much of the binding ability is due to long-range electrostatic forces versus other mechanisms (specific or nonspecific) of bonding. In addition to improving the scientific understanding of fate and transport of oocysts, an expanded understanding of the binding mechanisms may aid in the development of new tools and sensors designed to detect and track oocysts in waterways. Furthermore, the methods used to examine binding in this study could be translated to other waterborne pathogens of interest.

Pseudo-Second-Order Calcium-Mediated Cryptosporidium parvum Oocyst Attachment to Environmental Biofilms.

Cryptosporidium parvum oocysts are able to infect a wide range of mammals, including humans, via fecal-oral transmission. The remobilization of biofilm-associated C. parvum oocysts back into the water column by biofilm sloughing or bulk erosion poses a threat to public health and may be responsible for waterborne outbreaks; thus, the investigation of C. parvum attachment mechanisms to biofilms, particularly the physical and chemical factors controlling oocyst attachment to biofilms, is essential to predict the behavior of oocysts in the environment. In our study, biofilms were grown in rotating annular bioreactors using prefiltered stream water (0.2-µm retention) and rock biofilms (6-µm retention) until the mean biofilm thickness reached steady state. Oocyst deposition followed a calcium-mediated pseudo-second-order kinetic model. Kinetic parameters (i.e., initial oocyst deposition rate constant and total number of oocysts adhered to biofilms at equilibrium) from the model were then used to evaluate the impact of water conductivity on the attachment of oocysts to biofilms. Oocyst deposition was independent of solution ionic strength; instead, the presence of calcium enhanced oocyst attachment, as demonstrated by deposition tests. Calcium was identified as the predominant factor that bridges the carboxylic functional groups on biofilm and oocyst surfaces to cause attachment. The pseudo-second-order kinetic profile fit all experimental conditions, regardless of water chemistry and/or lighting conditions. IMPORTANCE: The cation bridging model in our study provides new insights into the impact of calcium on the attachment of C. parvum oocysts to environmental biofilms. The kinetic parameters derived from the model could be further analyzed to elucidate the behavior of oocysts in commonly encountered complex aquatic systems, which will enable future innovations in parasite detection and treatment technologies to protect public health.

Role of Wall Shear Stress in Cryptosporidium parvum Oocyst Attachment to Environmental Biofilms.

This study investigated Cryptosporidium parvum oocyst deposition onto biofilms as a function of shear stress under laminar or turbulent flow. Annular rotating bioreactors were used to grow stabilized stream biofilms at shear stresses ranging from 0.038 to 0.46 Pa. These steady-state biofilms were then used to assess the impact of hydrodynamic conditions on C. parvum oocyst attachment. C. parvum deposition onto biofilms followed a pseudo-second-order model under both laminar (after a lag phase) and turbulent flows. The total number of oocysts attached to the biofilm at steady state decreased as the hydrodynamic wall shear stress increased. The oocyst deposition rate constant increased with shear stress but decreased at high shear, suggesting that increasing wall shear stress results in faster attachment of Cryptosporidium due to higher mass transport until the shear forces exceed a critical limit that prevents oocyst attachment. These data show that oocyst attachment in the short and long term are impacted differently by shear: higher shear (to a certain limit) may be associated with faster initial oocyst attachment, but lower shear is associated with greater numbers of oocysts attached at equilibrium.IMPORTANCE This research provides experimental evidence to demonstrate that shear stress plays a critical role in protozoan-pathogen transport and deposition in environmental waters. The data presented in this work expand scientific understanding of Cryptosporidium attachment and fate, which will further influence the development of timely and accurate sampling strategies, as well as advanced water treatment technologies, to target protozoan pathogens in surface waters that serve as municipal drinking water sources.

Effect of Sand Bed Depth and Medium Age on Escherichia coli and Turbidity Removal in Biosand Filters.

The main objective of this study was to build several full-scale biosand filters (BSFs) and assess the long-term (9 month) efficacy for particulate and Escherichia coli removal under simulated real-world usage. Four replicates of three different filter designs were built: the traditional concrete BSF and two scaled-down versions that use a 5 or 2 gal bucket as the casing material. The smaller sand bed depths in the bucket-sized filters did not impact filter performance with respect to (i) turbidity and E. coli removal or (ii) effluent levels of turbidity and E. coli. All filters produced effluents with a mean turbidity of <0.6 nephelometric turbidity unit. In addition, 78, 74, and 72% of effluent samples for the concrete, 5 gal, and 2 gal filters, respectively, had E. coli concentrations of <1 colony-forming unit/100 mL. The bucket-sized filters were found to be a potential alternative to the concrete BSFs for the removal of E. coli and turbidity from drinking water. Because smaller BSFs must be filled more frequently than larger BSFs to produce comparable water volumes, the effect of shorter pause periods on BSF performance should be investigated.

Emergency water treatment with bleach in the United States: the need to revise EPA recommendations.

During emergencies in the United States, the Environmental Protection Agency (EPA) currently recommends using bottled water, or boiling or treating water by adding 1/8 teaspoon (or 8 drops) of bleach to 1 gal of water. This bleach recommendation is internally inconsistent, a relatively high chlorine dose (5.55-8.67 mg/L), and unsupported by evidence. In this study, bleach was added in three different dosages to six waters available to emergency-affected populations in each of six states; free chlorine residual (FCR) and Escherichia coli/total coliforms were measured 1-24 h after treatment. Data were analyzed using four efficacy criteria. Results indicated the dosages in the current EPA recommendation are unnecessarily high to ensure (1) maintenance of FCR for 24 h after treatment, (2) absence of E. coli/total coliforms, and (3) establishment of a CT-factor sufficient to inactivate Giardia lamblia and enteric viruses 1 h after treatment. Additionally, emergency-prone populations did not have the materials to complete treatment with bleach in their household. Therefore, we recommend EPA review and revise the current recommendation to establish an internally consistent, criteria-based recommendation that is usable by emergency-affected populations. We also recommend investigating the use of new or commercially available water treatment products for emergency response in the United States.

Transport effects on hydraulic loading rate and microbial removal performance in biosand filters.

Biosand filters (BSFs) are increasingly designed using smaller and/or lighter casing material in an effort to reduce logistical requirements and implementation costs. The increased portability of a smaller, lighter design presents a potential negative consequence: the ability to move the installed/operational filter by the homeowner and potentially disturb the system. This study investigated the effects of moving and agitation on filter performance, using mature BSFs which had been in use for over nine months prior to the move. Data were analyzed for four replicate filters of three different filter types: the traditional concrete BSF and two plastic bucket (5-gal and 2-gal, respectively; 5-gal bucket = 18.9-L bucket, 2-gal bucket = 7.6-L bucket) BSFs. Filters were moved approximately 1 km and monitored for hydraulic loading rates (HLRs) and Escherichia coli removal for 8 weeks following the move. Moving the filters resulted in reduced HLRs, likely due to sand compaction, but E. coli removal remained high (log10 removal ≥2.8 for all sizes) and increased significantly as compared to data collected prior to the move. The resulting operational implications of moving BSFs are discussed.

Development of cell-imprinted polymer surfaces for Cryptosporidium capture and detection.

Cryptosporidium parvum is waterborne parasite that can cause potentially life-threatening gastrointestinal disease and is resistant to conventional water treatment processes, including chlorine disinfection. The current Environmental Protection Agency-approved method for oocyst detection and quantification is expensive, limiting the ability of water utilities to monitor complex watersheds thoroughly to understand the fate and transport of C. parvum oocysts. In this work, whole cell imprinting was used to create selective and sensitive surfaces for the capture of C. parvum oocysts in water. Cell-imprinted Polydimethylsiloxane (PDMS) was manufactured using a modified stamping approach, and sensitivity and selectivity were analyzed using different water chemistries and different surrogate biological and non-biological particles. The overall binding affinity was determined to be less than that of highly specific antibodies, but on par with standard antibodies and immune-enabled technologies. These initial results demonstrate the potential for developing devices using cell-imprinting for use in waterborne pathogen analysis.

Influence of sand depth and pause period on microbial removal in traditional and modified biosand filters.

Three different-sized biosand filters (i.e., the center for Affordable Water and Sanitation Technology v10 concrete filter, a 5-gal bucket filter, and a 2-gal bucket filter with fine sand depths of 54, 15, and 10 cm, respectively), configured with and without the addition of iron nails to the diffuser basin, were evaluated for removal of bacteria, protozoa, and viruses over pause periods ranging from 1 to 72 hrs. Biosand filtration proved effective at all pause periods tested, and log10 removal of bacteria and protozoan cysts for all filter sizes and configurations ranged from 3 to 4. The addition of nails resulted in significantly better (p<0.05) bacteria removal for all filter sizes and significantly better (p<0.02) protozoan removal for the bucket-sized filters. Log10 virus removal for all filter types and sizes ranged from <1 to 6. Both the pause period and filter type (size/configuration) influenced virus removal, and the addition of nails to the filter improved virus removal at the shorter pause periods. Scaled-down biosand filters provide a viable household water treatment option for some of the millions of people that still lack access to an improved water source.

Making Waves: Collaboration in the time of SARS-CoV-2 - rapid development of an international co-operation and wastewater surveillance database to support public health decision-making.

The presence of SARS-CoV-2 RNA in wastewater was first reported in March 2020. Over the subsequent months, the potential for wastewater surveillance to contribute to COVID-19 mitigation programmes has been the focus of intense national and international research activities, gaining the attention of policy makers and the public. As a new application of an established methodology, focused collaboration between public health practitioners and wastewater researchers is essential to developing a common understanding on how, when and where the outputs of this non-invasive community-level approach can deliver actionable outcomes for public health authorities. Within this context, the NORMAN SCORE "SARS-CoV-2 in sewage" database provides a platform for rapid, open access data sharing, validated by the uploading of 276 data sets from nine countries to-date. Through offering direct access to underpinning meta-data sets (and describing its use in data interpretation), the NORMAN SCORE database is a resource for the development of recommendations on minimum data requirements for wastewater pathogen surveillance. It is also a tool to engage public health practitioners in discussions on use of the approach, providing an opportunity to build mutual understanding of the demand and supply for data and facilitate the translation of this promising research application into public health practice.

Turbidity and chlorine demand reduction using alum and moringa flocculation before household chlorination in developing countries.

Over 1.1 billion people in the world lack access to improved drinking water. Diarrhoeal and other waterborne diseases cause an estimated 1.87 million deaths per year. The Safe Water System (SWS) is a household water treatment intervention that reduces diarrhoeal disease incidence among users in developing countries. Turbid waters pose a particular challenge to implementation of SWS programmes; although research shows that a 3.75 mg l(-1) sodium hypochlorite dose effectively treats turbid waters, users sometimes object to the strong chlorine taste and prefer to drink water that is more aesthetically pleasing. This study investigated the efficacy of two locally available chemical water treatments-alum and Moringa oleifera flocculation-to reduce turbidity and chlorine demand at turbidities of 10, 30, 70, 100 and 300 NTU. Both treatments effectively reduced turbidity (alum flocculation 23.0-91.4%; moringa flocculation 14.2-96.2%). Alum flocculation effectively reduced chlorine demand compared with controls at 30, 70, 100 and 300 NTU (p=0.01-0.06). Moringa flocculation increased chlorine demand to the point where adequate free chlorine residual was not maintained for 24 hours after treatment. Alum pretreatment is recommended in waters>or=30 NTU for optimum water disinfection. Moringa flocculation is not recommended before chlorination.

Effect of production variables on microbiological removal in locally-produced ceramic filters for household water treatment.

Diarrhoeal diseases cause an estimated 1.87 million child deaths per year. Point-of-use filtration using locally made ceramic filters improves microbiological quality of stored drinking water and prevents diarrhoeal disease. Scaling-up ceramic filtration is inhibited by lack of universal quality control standards. We investigated filter production variables to determine their affect on microbiological removal during 5-6 weeks of simulated normal use. Decreases in the clay:sawdust ratio and changes in the burnable decreased effectiveness of the filter. Method of silver application and shape of filter did not impact filter effectiveness. A maximum flow rate of 1.7 l(-hr) was established as a potential quality control measure for one particular filter to ensure 99% (2- log(10)) removal of total coliforms. Further research is indicated to determine additional production variables associated with filter effectiveness and develop standardized filter production procedures prior to scaling-up.

Evaluation of consistent use, barriers to use, and microbiological effectiveness of three prototype household water treatment technologies in Haiti, Kenya, and Nicaragua.

Household water treatment (HWT) can improve drinking water quality and reduce diarrheal disease. New HWT technologies are typically evaluated under ideal conditions; however, health gains depend on consistent, effective household use, which is less often evaluated. We conducted four evaluations of three prototype HWT technologies: two filters and one electrochlorinator. Evaluations consisted of a baseline survey, HWT distribution to households (ranging from 60 to 82), and four visits (ranging from 1 week-14 months after distribution). Each visit included a survey, observation of treated water presence (confirmed use), and microbiological analysis of treated and untreated samples for E. coli. Consistent use was defined as the proportion of total visits with confirmed use. Overall, confirmed use declined 2.54% per month on average, and 2-72% of households demonstrated 100% consistent use. Consistent use was positively associated with baseline HWT knowledge and practice and belief that drinking water was unsafe, and negatively associated with technological problems. Reported barriers to use were behavioral, such as forgetting or when outside the home, and technological failures. Technologies demonstrated 68-96% E. coli reductions, with 18-70% of treated samples having detectable E. coli. Results highlight the importance of household use evaluations within prototype HWT technology design cycles, the need for standard evaluation metrics, and difficulties in achieving both consistent use and microbiological effectiveness with HWT technologies.

Turbidity and chlorine demand reduction using locally available physical water clarification mechanisms before household chlorination in developing countries.

Over 1.1 billion people in the world lack access to improved drinking water. Diarrhoeal and other waterborne diseases cause an estimated 1.9 million deaths per year. The Safe Water System (SWS) is a proven household water treatment intervention that reduces diarrhoeal disease incidence among users in developing countries. Turbid waters pose a particular challenge to implementation of SWS programmes; although research shows that a 3.75 mg l(-1) sodium hypochlorite dose effectively treats turbid waters, users sometimes object to the strong chlorine taste and prefer to drink water that is more aesthetically pleasing. This study investigated the efficacy of three locally available water clarification mechanisms-cloth filtration, settling/decanting and sand filtration-to reduce turbidity and chlorine demand at turbidities of 10, 30, 70, 100 and 300 NTU. All three mechanisms reduced turbidity (cloth filtration -1-60%, settling/decanting 78-88% and sand filtration 57-99%). Sand filtration (P=0.002) and settling/decanting (P=0.004), but not cloth filtration (P=0.30), were effective at reducing chlorine demand compared with controls. Recommendations for implementing organizations based on these results are discussed.

Impact of Bioreactor Environment and Recovery Method on the Profile of Bacterial Populations from Water Distribution Systems.

Multiple rotating annular reactors were seeded with biofilms flushed from water distribution systems to assess (1) whether biofilms grown in bioreactors are representative of biofilms flushed from the water distribution system in terms of bacterial composition and diversity, and (2) whether the biofilm sampling method affects the population profile of the attached bacterial community. Biofilms were grown in bioreactors until thickness stabilized (9 to 11 weeks) and harvested from reactor coupons by sonication, stomaching, bead-beating, and manual scraping. High-throughput sequencing of 16S rRNA amplicons was used to profile bacterial populations from flushed biofilms seeded into bioreactors as well as biofilms recovered from bioreactor coupons by different methods. ß diversity between flushed and reactor biofilms was compared to ß diversity between (i) biofilms harvested from different reactors and (ii) biofilms harvested by different methods from the same reactor. These analyses showed that average diversity between flushed and bioreactor biofilms was double the diversity between biofilms from different reactors operated in parallel. The diversity between bioreactors was larger than the diversity associated with different biofilm recovery methods. Compared to other experimental variables, the method used to recover biofilms had a negligible impact on the outcome of water biofilm analyses based on 16S amplicon sequencing. Results from this study show that biofilms grown in reactors over 9 to 11 weeks are not representative models of the microbial populations flushed from a distribution system. Furthermore, the bacterial population profile of biofilms grown in replicate reactors from the same flushed water are likely to diverge. However, four common sampling protocols, which differ with respect to disruption of bacterial cells, provide similar information with respect to the 16S rRNA population profile of the biofilm community.

Source tracking identifies deer and geese as vectors of human-infectious Cryptosporidium genotypes in an urban/suburban watershed.

This study identified Cryptosporidium genotypes in the Wissahickon watershed from May 2005 to April 2008. We analyzed 129 samples from Wissahickon Creek, 83 effluent samples from wastewater treatment plants (WWTPs), and 240 fecal droppings. Genotyping was based on the hypervariable region of the 18S rRNA gene. Oocysts were detected year-round, independent of wet weather events, in 22% of Wissahickon Creek samples, 5% of WWTP effluents, and 7% of fecal samples. Of the genotypes detected, 67% were human-infectious: 30% C. hominis or C. hominis-like, 12% C. parvum, 14% cervine genotype, 9% skunk genotype, and 1% chipmunk I genotype. Similar genotype profiles were detected in Wissahickon Creek each year, and human-infectious genotypes were detected year-round. Unusual genotypes detected in a deer (a C. hominis-like genotype) and geese (C. hominis-like genotypes, C. parvum, and muskrat genotype I) show that these animals are vectors of human-infectious genotypes in this watershed. Results suggest that deer, geese, and WWTPs are appropriate targets for source water protection in the Wissahickon watershed.

Phylogenetic analysis implicates birds as a source of Cryptosporidium spp. oocysts in agricultural watersheds.

Cryptosporidium parvum and C. hominis are protozoan parasites responsible for cryptosporidiosis, an acute gastrointestinal illness that can be life-threatening for immunocompromised persons. Sources and genotypes of Cryptosporidium oocysts were investigated in two agricultural areas within the Wachusett Reservoir watershed, a drinking water source for Boston, Massachusetts. Two brooks (denoted Brook SF and Brook JF, respectively), each downgradient from a dairy farm, were chosen as sample sites. For one year, Brooks SF and JF were sampled monthly; oocysts were detected in 6 (50%) out of 12 samples from Brook JF, and no oocysts were detected in Brook SF. Oocyst genotypes from agricultural surface waters were compared to oocyst genotypes from Genbank, as well as fecal samples of cattle and birds, using phylogenetic analysis of a hypervariable region of the 18S rRNA gene by both neighbor-joining and parsimony methods. Results show extensive heterogeneity among Cryptosporidium spp. 18S rRNA sequences, and also suggest that birds are an oocyst source in this watershed. Principal components analysis showed oocyst presence correlating strongly with seasonal factors, and oocysts in surface waters were only detected in the summer through late fall, co-incident with the presence of migratory birds in this watershed. If birds are confirmed to be an important source of oocysts infectious to humans, the data suggest that protection of raw drinking water supplies in some agricultural areas may depend upon management and control of resident and migratory bird populations.

Artificial UV-B and solar radiation reduce in vitro infectivity of the human pathogen Cryptosporidium parvum.

The potential for solar ultraviolet (UV) radiation to act as a significant abiotic control of Cryptosporidium parvum oocysts in nature is unknown. Infectivity of C. parvum following exposure to artificial UV-B and natural solar radiation, with and without UV wavelengths, was tested under controlled pH and temperature conditions. Percent infectivity of exposed oocysts was determined by in vitro cell culture. Artificial UV-B exposures of 32 and 66 kJ/m2 significantly decreased oocyst infectivity by an average of 58 and 98%, respectively. Exposure of oocysts to approximately half and full intensity of full solar spectrum (all wavelengths) for a period of less than 1 day (10 h) in mid-summer reduced mean infectivity by an average of 67% and >99.99%, respectively. Exposure of the C. parvum oocysts to UV-shielded solar radiation (>404 nm) in early autumn reduced mean infectivity by 52%, while full spectrum solar radiation (exposure at all wavelengths) reduced mean infectivity by 97%. The data provide strong evidence that exposure to natural solar radiation can significantly reduce C. parvum infectivity. Direct effects of solar radiation on oocysts in nature will depend on the depth distribution of the oocysts, water transparency, mixing conditions, and perhaps other environmental factors such as temperature, pH, and stress.

Sources and species of cryptosporidium oocysts in the Wachusett Reservoir watershed.

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