Detection of Infectious Cryptosporidium parvum Oocysts in Surface and Filter Backwash Water Samples by Immunomagnetic Separation and Integrated Cell Culture-PCR

A new strategy for the detection of infectious Cryptosporidium parvum oocysts in water samples, which combines immunomagnetic separation (IMS) for recovery of oocysts with in vitro cell culturing and PCR (CC-PCR), was field tested with a total of 122 raw source water samples and 121 filter backwash water grab samples obtained from 25 sites in the United States. In addition, samples were processed by Percoll-sucrose flotation and oocysts were detected by an immunofluorescence assay (IFA) as a baseline method. Samples of different water quality were seeded with viable C. parvum to evaluate oocyst recovery efficiencies and the performance of the CC-PCR protocol. Mean method oocyst recoveries, including concentration of seeded 10-liter samples, from raw water were 26.1% for IMS and 16.6% for flotation, while recoveries from seeded filter backwash water were 9.1 and 5.8%, respectively. There was full agreement between IFA oocyst counts of IMS-purified seeded samples and CC-PCR results. In natural samples, CC-PCR detected infectious C. parvum in 4.9% (6) of the raw water samples and 7.4% (9) of the filter backwash water samples, while IFA detected oocysts in 13.1% (16) of the raw water samples and 5.8% (7) of the filter backwash water samples. All CC-PCR products were confirmed by cloning and DNA sequence analysis and were greater than 98% homologous to the C. parvum KSU-1 hsp70 gene product. DNA sequence analysis also revealed reproducible nucleotide substitutions among the hsp70 fragments, suggesting that several different strains of infectious C. parvum were detected.     

A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts

Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed P value (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log₁₀ inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.     

Detection of UV-Induced Thymine Dimers in Individual Cryptosporidium parvum and Cryptosporidium hominis Oocysts by Immunofluorescence Microscopy

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ·cm⁻² doses of UV irradiation. Thymine dimers were detected by immunofluorescence microscopy using a monoclonal antibody against cyclobutyl thymine dimers (anti-TDmAb). Dimer-specific fluorescence within sporozoite nuclei was confirmed by colocalization with the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Oocyst walls were visualized using either commercial fluorescein isothiocyanate-labeled anti-Cryptosporidium oocyst antibodies (FITC-CmAb) or Texas Red-labeled anti-Cryptosporidium oocyst antibodies (TR-CmAb). The use of FITC-CmAb interfered with TD detection at doses below 40 mJ·cm⁻². With the combination of anti-TDmAb, TR-CmAb, and DAPI, dimer-specific fluorescence was detected in sporozoite nuclei within oocysts exposed to 10 to 40 mJ·cm⁻² of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ·cm⁻² of UV light failed to infect neonatal mice, confirming that results of our anti-TD immunofluorescence assay paralleled the outcomes of our neonatal mouse infectivity assay. These results suggest that our immunofluorescence assay is suitable for detecting DNA damage in C. parvum and C. hominis oocysts induced following exposure to UV light.     

Detection of Infectious Adenoviruses in Environmental Waters by Fluorescence-Activated Cell Sorting Assay

Methods for rapid detection and quantification of infectious viruses in the environment are urgently needed for public health protection. A fluorescence-activated cell-sorting (FACS) assay was developed to detect infectious adenoviruses (Ads) based on the expression of viral protein during replication in cells. The assay was first developed using recombinant Ad serotype 5 (rAd5) with the E1A gene replaced by a green fluorescent protein (GFP) gene. Cells infected with rAd5 express GFP, which is captured and quantified by FACS. The results showed that rAd5 can be detected at concentrations of 1 to 10⁴ PFU per assay within 3 days, demonstrating a linear correlation between the viral concentration and the number of GFP-positive cells with an r² value of >0.9. Following the same concept, FACS assays using fluorescently labeled antibodies specific to the E1A and hexon proteins, respectively, were developed. Assays targeting hexon showed greater sensitivity than assays targeting E1A. The results demonstrated that as little as 1 PFU Ads was detected by FACS within 3 days based on hexon protein, with an r² value greater than 0.9 over a 4-log concentration range. Application of this method to environmental samples indicated positive detection of infectious Ads in 50% of primary sewage samples and 33% of secondary treated sewage samples, but none were found in 12 seawater samples. The infectious Ads ranged in quantity between 10 and 165 PFU/100 ml of sewage samples. The results indicate that the FACS assay is a rapid quantification tool for detecting infectious Ads in environmental samples and also represents a considerable advancement for rapid environmental monitoring of infectious viruses.Waterborne viral infection is one of the most important causes of human morbidity in the world. There are hundreds of different types of human viruses present in human sewage, which, if improperly treated, may become the source of contamination in drinking and recreational waters (6, 12, 19). Furthermore, as water scarcity intensifies in the nation, so has consideration of wastewater reuse as a valid and essential alternative for resolving water shortages (31).Currently, routine viral monitoring is not required for drinking or recreational waters, nor is it required for wastewater that is discharged into the environment. This lack of a monitoring effort is due largely to the lack of methods that can rapidly and sensitively detect infectious viruses in environmental samples. In the past 20 years, tremendous progress has been made in detection of viruses in the environment based on molecular technology (32, 33, 35). PCR and quantitative real-time PCR (qPCR) methods have improved both the speed and sensitivity of viral detection compared with detection by the traditional tissue culture method (2, 11, 17, 18). However, they provide little information on viral infectivity, which is crucial for human health risk assessment (22-24, 35). Our previous work using a real-time PCR assay to detect human adenoviruses (Ads) in sewage could not differentiate the infectious viruses in the secondary treated sewage from those killed by chlorination disinfection (15). In this research, we pursued an innovative approach to detecting infectious viruses in water using fluorescence-activated cell sorting (FACS). This method is rapid and sensitive, with an established record in microbiological research (29, 34, 39).FACS is a specialized type of flow cytometry which provides a method for counting and sorting a heterogeneous mixture of biological cells into two or more kinds, one cell at a time, based upon the specific light-scattering and fluorescent characteristics of each cell (4, 25, 34, 38). It is a useful method since it provides fast and quantitative recording of fluorescent signals from individual cells (14, 16, 34, 47). The FACS viral assay is based on the expression of viral protein inside the recipient cell during viral replication (16). Specific antibody labeled with fluorescence is bound to the target viral protein, which results in fluorescence emission from infected cells. Viral particles outside the cell will not be captured, because the size of virus is below the detection limit of flow cytometry. Therefore, detection of cells, which can be captured with fluorescently labeled viral antibody, is a definitive indication of the presence of infectious virus.This research used human Ads as the target for development of the FACS method. The rationale for this choice is as follows. (i) Ads are important human pathogens that may be transmitted by water consumption and water spray (aerosols) (26, 32). The health hazard associated with exposure to Ads has been demonstrated by epidemiological data and clinical research (1, 7, 9, 35, 40, 43). (ii) Ads are among the most prevalent human viruses identified in human sewage and are frequently detected in marine waters and the Great Lakes (17, 32, 33, 35). (iii) Ads are more resistant to UV disinfection than any other bacteria or viruses (3, 5, 10, 24, 41, 42, 44). Thus, they may survive wastewater treatment as increasing numbers of wastewater treatment facilities switch from chlorination to UV to avoid disinfection by-products. (iv) Some serotypes of Ads, including enteric Ad 40 and 41, are fastidious. They are difficult to detect by plaque assay, and a routine assay of infectivity takes 7 to 14 days (8, 20).In this study, recombinant Ad serotype 5 (rAd5) with the E1A gene (the first transcribed gene after infection) replaced by a green fluorescent protein (GFP) gene was first used to test for sensitivity and speed of the assay. Two other viral proteins were then used as targets for development of FACS assays using Ad serotype 2 (Ad2) and Ad41. This study demonstrated the feasibility, sensitivity, and reliability of the assay for detection of infectious Ads in environmental samples.     

Detection of Infectious Virus from Field-collected Mosquitoes by Vero Cell Culture Assay

Mosquitoes transmit a number of distinct viruses including important human pathogens such as West Nile virus, dengue virus, and chickungunya virus. Many of these viruses have intensified in their endemic ranges and expanded to new territories, necessitating effective surveillance and control programs to respond to these threats. One strategy to monitor virus activity involves collecting large numbers of mosquitoes from endemic sites and testing them for viral infection. In this article, we describe how to handle, process, and screen field-collected mosquitoes for infectious virus by Vero cell culture assay. Mosquitoes are sorted by trap location and species, and grouped into pools containing ≤50 individuals. Pooled specimens are homogenized in buffered saline using a mixer-mill and the aqueous phase is inoculated onto confluent Vero cell cultures (Clone E6). Cell cultures are monitored for cytopathic effect from days 3-7 post-inoculation and any viruses grown in cell culture are identified by the appropriate diagnostic assays. By utilizing this approach, we have isolated 9 different viruses from mosquitoes collected in Connecticut, USA, and among these, 5 are known to cause human disease. Three of these viruses (West Nile virus, Potosi virus, and La Crosse virus) represent new records for North America or the New England region since 1999. The ability to detect a wide diversity of viruses is critical to monitoring both established and newly emerging viruses in the mosquito population.Download video file.^{(46M, mov)}     

Method Detection Limits of PCR and Immunofluorescence Assay for Cryptosporidium parvum in Soil

We determined and compared the method detection limits (MDL_α) of a PCR and an immunofluorescence assay (IFA) for detection of Cryptosporidium parvum oocysts in soils. Based on the MDL_α and the quantitative nature and stability of the IFA, PCR analysis is not a useful screening step for soil studies of oocyst transport.     

Assessment of Methods for Detection of Infectious Cryptosporidium Oocysts and Giardia Cysts in Reclaimed Effluents

This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.     

Concentration and Detection of Cryptosporidium Oocysts in Surface Water Samples by Method 1622 Using Ultrafiltration and Capsule Filtration

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.     

Detection of Cryptosporidium Oocysts in Water: Effect of the Number of Samples and Analytic Replicates on Test Results

Due to the small number of Cryptosporidium oocysts in water, the number of samples taken and the analyses performed can affect the results of detection. In this study, 42 water samples were collected from one watershed during 20 storm events over 1 year, including duplicate or quadruplicate samples from 16 storm events. Ten samples from four events had three to eight subsamples. They were processed by EPA method 1623, and Cryptosporidium oocysts present were detected by immunofluorescent microscopy or PCR. Altogether, 24 of 39 samples (47 of 67 samples and subsamples) analyzed by microscopy were positive for Cryptosporidium. In contrast, 36 of 42 samples (62 of 76 samples and subsamples) were positive by PCR, including 10 microscopy-negative samples (13 microscopy-negative samples and subsamples). Six of the 24 microscopy-positive samples were negative by PCR, and all samples had one or less oocyst in a 0.5-ml packed pellet volume calculated. Discordant results were obtained by microscopy and PCR from six and three of the storm events, respectively, with multiple samples. Discordant microscopy or PCR results were also obtained among subsamples. Most of the 14 Cryptosporidium genotypes were found over a brief period. Cryptosporidium-positive samples had a mean of 1.9 genotypes per sample, with 39 of the 62 positive samples/subsamples having more than one genotype. Samples/subsamples with more than one genotype had an overall PCR-positive rate of 73%, compared to 34% for those with one genotype. The PCR amplification rate of samples was affected by the volume of DNA used in PCR.     

Efficacy of Common Laboratory Disinfectants on the Infectivity of Cryptosporidium parvum Oocysts in Cell Culture

Nine liquid disinfectants were tested for their ability to reduce infectivity of Cryptosporidium parvum oocysts in cell culture. A 4-min exposure to 6% hydrogen peroxide and a 13-min exposure to ammonium hydroxide-amended windshield washer fluid reduced infectivity 1,000-fold. Other disinfectants tested (70% ethanol, 37% methanol, 6% sodium hypochlorite, 70% isopropanol, and three commercial disinfectants) did not reduce the infectivity after a 33-min exposure. The results indicate that hydrogen peroxide and windshield washer fluid or ammonium hydroxide disinfectant may be suitable laboratory disinfectants against C. parvum oocysts.     

Maximizing Recovery and Detection of Cryptosporidium parvum Oocysts from Spiked Eastern Oyster (Crassostrea virginica) Tissue Samples

Numerous studies have documented the presence of Cryptosporidium parvum, an anthropozoonotic enteric parasite, in molluscan shellfish harvested for commercial purposes. Getting accurate estimates of Cryptosporidium contamination levels in molluscan shellfish is difficult because recovery efficiencies are dependent on the isolation method used. Such estimates are important for determining the human health risks posed by consumption of contaminated shellfish. In the present study, oocyst recovery was compared for multiple methods used to isolate Cryptosporidium parvum oocysts from oysters (Crassostrea virginica) after exposure to contaminated water for 24 h. The immunomagnetic separation (IMS) and immunofluorescent antibody procedures from Environmental Protection Agency method 1623 were adapted for these purposes. Recovery efficiencies for the different methods were also determined using oyster tissue homogenate and hemolymph spiked with oocysts. There were significant differences in recovery efficiency among the different treatment groups (P < 0.05). We observed the highest recovery efficiency (i.e., 51%) from spiked samples when hemolymph was kept separate during the homogenization of the whole oyster meat but was then added to the pellet following diethyl ether extraction of the homogenate, prior to IMS. Using this processing method, as few as 10 oocysts could be detected in a spiked homogenate sample by nested PCR. In the absence of water quality indicators that correlate with Cryptosporidium contamination levels, assessment of shellfish safety may rely on accurate quantification of oocyst loads, necessitating the use of processing methods that maximize oocyst recovery. The results from this study have important implications for regulatory agencies charged with determining the safety of molluscan shellfish for human consumption.     

猴泡沫病毒间接免疫荧光检测方法的建立及初步应用

目的建立检测血清中猴泡沫病毒（SFV）抗体的间接免疫荧光方法,为检测实验用猴群中SFV的感染情况提供参考依据。方法用SFV-1病毒感染BHK-21细胞,待50%细胞出现病变时,用胰蛋白酶消化细胞后以2×107/mL浓度40μL的细胞滴到10孔镀膜的玻片上,丙酮固定。利用制备的抗原片通过间接免疫荧光法对34份猴血清标本进行检测。结果建立了检测SFV抗体的间接免疫荧光染色方法,SFV抗体阳性19例,15例血清检测为阴性。结论本方法具有良好的特异性,可作为SFV检测的可靠方法。     

Detection of Infectious Cryptosporidium parvum Oocysts in Mussels (Mytilus galloprovincialis) and Cockles (Cerastoderma edule)

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10³ oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.     

Inactivation of Cryptosporidium parvum Oocysts by Ammonia

The survival of Cryptosporidium parvum oocysts in soil and water microhabitats may be affected by the environmental production and release of free ammonia. The objective of this study was to determine the effects of increasing free ammonia concentrations and times of exposure on oocyst viability. Wild-type oocysts were obtained from naturally infected calf feces by chemical (continuous-flow) centrifugation and sucrose gradients. Ammonia (NH₃) from a commercial solution was applied in concentrations ranging from 0.007 to 0.148 M. Exposure times ranged from 10 min to 24 h at a constant temperature of 24 ± 1°C. Viability of oocysts was determined with a dye permeability assay and an in vitro excystation assay (M. B. Jenkins, L. J. Anguish, D. D. Bowman, M. J. Walker, and W. C. Ghiorse, Appl. Environ. Microbiol. 63:3844–3850, 1997). Even the lowest concentration of ammonia decreased significantly the viability of oocysts after 24 h of exposure. Increasing concentrations of ammonia increased inactivation rates, which ranged from 0.014 to 0.066 h⁻¹. At the highest concentration of ammonia, a small fraction of viable oocysts still remained. Exposure to pH levels corresponding to those associated with the ammonia concentrations showed minimal effects of alkaline pH alone on oocyst viability. This study shows that environmentally relevant concentrations of free ammonia may significantly increase the inactivation of oocysts in ammonia-containing environments.     

Molecular Characterization of Cryptosporidium Oocysts in Samples of Raw Surface Water and Wastewater

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.     

Detection of Cryptosporidium parvum Oocysts on Fresh Produce Using DNA Aptamers

There are currently no standard methods for the detection of Cryptosporidium spp., or other protozoan parasites, in foods, and existing methods are often inadequate, with low and variable recovery efficiencies. Food testing is difficult due to the low concentrations of parasites, the difficulty in eluting parasites from some foods, the lack of enrichment methods, and the presence of PCR inhibitors. The main objectives of the present study were to obtain DNA aptamers binding to the oocyst wall of C. parvum, and to use the aptamers to detect the presence of this parasite in foods. DNA aptamers were selected against C. parvum oocysts using SELEX (Systematic Evolution of Ligands by EXponential enrichment). Ten rounds of selection led to the discovery of 14 aptamer clones with high affinities for C. parvum oocysts. For detecting parasite-bound aptamers, a simple electrochemical sensor was employed, which used a gold nanoparticle-modified screen-printed carbon electrode. This aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and the anti- C. parvum aptamer. Square wave voltammetry was employed to quantitate C. parvum in the range of 150 to 800 oocysts, with a detection limit of approximately 100 oocysts. The high sensitivity and specificity of the developed aptasensor suggests that this novel method is very promising for the detection and identification of C. parvum oocysts on spiked fresh fruits, as compared to conventional methods such as microscopy and PCR.     

Seasonal Retention and Release of Cryptosporidium parvum Oocysts by Environmental Biofilms in the Laboratory

Cryptosporidium is a genus of waterborne protozoan parasites that causes significant gastrointestinal disease in humans. These parasites can accumulate in environmental biofilms and be subsequently released to contaminate water supplies. Natural microbial assemblages were collected each season from an eastern Pennsylvania stream and used to grow biofilms in laboratory microcosms in which influx, efflux, and biofilm retention were determined from daily oocyst counts. For each seasonal biofilm, oocysts attached to the biofilm quickly during oocyst dosing. Upon termination of oocyst dosing, the percentage of oocysts retained within the biofilm decreased to a new steady state within 5 days. Seasonal differences in biofilm retention of oocysts were observed. The spring biofilm retained the greatest percentage of oocysts, followed (in decreasing order) by the winter, summer, and fall biofilms. There was no statistically significant correlation between the percentage of oocysts attached to the biofilm and (i) any measured stream water quality parameter (including temperature, pH, conductivity, and dissolved organic carbon concentration) or (ii) experimental temperature. Seasonal differences in oocyst retention persisted when biofilms were tested with stream water from a different season. These data suggest that seasonal variation in the microbial community and resulting biofilm architecture may be more important to oocyst transport in this stream site than water quality. The biofilm attachment and detachment dynamics of C. parvum oocysts have implications for public health, and the drinking water industry should recognize that the potential exists for pathogen-free water to become contaminated during the distribution process as a result of biofilm dynamics.Cryptosporidium is a genus of waterborne protozoan parasites that cause a gastrointestinal disease in humans (cryptosporidiosis) that can be prolonged and life-threatening for people with compromised immune systems. Recent advances in medical treatment for cryptosporidiosis exist but are not entirely effective for immunocompromised patients (1). In addition, conventional water treatment does not effectively target Cryptosporidium oocysts because the oocysts'' small size (4 to 8 μm) limits the ability of filters to remove them and, more importantly, oocysts are resistant to chlorine (25). Therefore, environmental control of Cryptosporidium is important to protect public health. To determine the risk of human exposure and infection, the fate and transport of Cryptosporidium oocysts in the environment, including biofilms, should be examined.Within the past two decades, biofilms have been recognized as ubiquitous habitats found on most surfaces exposed to water (20, 24). Environmental biofilms can rapidly accumulate pathogens at densities much higher than water column densities, and the potential for gradual or sudden pathogen loss from the biofilm exists long after entrapment (8, 22). Biofilm sloughing events are commonplace, occurring when a biofilm detaches from the substrate to be resuspended as large particles in the water column, and may result in the release of pathogen reservoirs from the biofilm into the water column (8).Biofilms have been identified as a possible contamination source for drinking water supplies, which may lead to infections for which the source cannot be identified (7, 9). An example of the impact of biofilm sloughing events on human health is seen in the cryptosporidiosis outbreak that occurred in Lancashire County, England, in March 2000 (10). After the outbreak, the oocyst source was identified as cattle feces from adjacent farmland that contaminated the drinking water after abnormally heavy rainfall. The water source was subsequently changed to two upland impounding reservoirs containing filtered surface water. However, oocysts persisted in the water distribution system for 19 days, with large peaks associated with major water main disturbance events, including the initial flushing of the system and a burst in the main supply pipe. This persistence of oocysts in the water supply was attributed to the release of oocysts trapped in biofilms on the interior surface of the distribution pipes and may have contributed to additional infections.Several studies have examined pathogen transport dynamics in biofilms using glass or latex beads of various sizes as surrogates for pathogens (5, 8, 16, 17). A few studies examined the attachment of C. parvum oocysts to biofilms but did not use natural microbial assemblages to make the biofilms (3, 23) or quantify how many oocysts attached or sloughed (9, 22). Rogers and Keevil (22) showed that oocysts attached to a biofilm composed of a natural microbial assemblage collected from a reservoir at a concentration of 1,400 oocysts/cm² after the addition of 10⁸ oocysts in 10 ml of sterile water. Dai and Hozalski (3) and Searcy et al. (23) used pure culture biofilms to demonstrate oocyst attachment; however, only Searcy et al. (23) accounted for sloughing, although no oocyst release from the biofilm was seen during the course of their experiments. Helmi et al. (9) noted attachment and detachment of oocysts from a natural biofilm but did not include a quantitative analysis to account for all oocysts in the flow system over time. None of these studies examined pathogen attachment seasonally over the course of a year. Seasonal changes in temperature, precipitation, and water quality (including nutrient availability) may have significant impacts on the microbial composition and functional structure of a biofilm (14). These changes include structural changes in the biofilm thickness and morphology, as well as changes in the water composition and suspended matter. In addition, seasonal changes in stream flow dynamics may alter biofilm composition and morphology, as well as oocyst attachment and release patterns.This study provides novel information about C. parvum oocyst attachment to biofilms grown in the laboratory from natural microbial assemblages collected seasonally (i.e., in January, April, July, and October) from Monocacy Creek in Bethlehem, PA. Previous work (26) showed that (i) a significant fraction of C. parvum oocysts adhered to the surface of experimental biofilms during a 3-day oocyst dosing period, (ii) a portion of the adhered oocysts immediately released from the biofilm, and (iii) a portion of the oocysts remained attached to the biofilm for a period of days after termination of oocyst dosing. Here, we test the hypotheses that (i) oocyst retention by biofilms varies seasonally and (ii) seasonal changes in water quality influence oocyst retention.