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Facing challenges in water demands and population size, particularly in the water-scarce regions in the United States, the reuse of treated municipal wastewater has become a viable potential to relieve the ever-increasing demands of providing water for (non-)potable use. The objectives of this study were to assess microbial quality of reclaimed water and to investigate treatability of microorganisms during different treatment processes. Raw and final treated effluent samples from three participating utilities were collected monthly for 16 months and analyzed for various microbial pathogens and fecal indicator organisms. Results revealed that the detectable levels of microbial pathogens tested were observed in the treated effluent samples from all participating utilities. Log10 reduction values (LRVs) of Cryptosporidium oocysts and Giardia cysts were at least two orders of magnitude lower than those of human adenovirus and all fecal indicator organisms except for aerobic endospores, which showed the lowest LRVs. The relatively higher LRV of the indicator organisms such as bacteriophages suggested that these microorganisms are not good candidates of viral indicators of human adenovirus during wastewater treatment processes. Overall, this study will assist municipalities considering the use of wastewater effluent as another source of drinking water by providing important data on the prevalence, occurrence, and reduction of waterborne pathogens in wastewater. More importantly, the results from this study will aid in building a richer microbial occurrence database that can be used towards evaluating reuse guidelines and disinfection practices for water reuse practices.

This study developed and evaluated Giardia duodenalis cyst propagation using a dexamethasone immunosuppressed CF-1 mouse model as an alternative to a previously described Mongolian gerbil model. The CF-1 mouse model shed significantly more cysts per animal during a 16–18 h collection period compared to the gerbil (averages: 7.8 × 106 cysts/CF-1 mouse and 2.5 × 106 cysts/gerbil). In addition, the patency period for this model differed from both G. muris in mice and G. duodenalis in gerbils in that cysts were shed continuously for over 20 days. Results further showed that the β-giardin gene sequences from gerbil derived and mouse derived G. duodenalis were identical, after 34 serial passages through the CF-1 mouse model. Overall, the CF-1 mouse model produced higher concentrations of cysts per animal, and were genetically and phenotypically stable based on β-giardin gene sequences.

Surveillance monitoring for microbial water quality typically involves collecting single discrete grab samples for analyzing only one contaminant. While informative, current approaches suffer from poor recoveries and only provide a limited snapshot of the microbial contaminants only at the time of collection. To overcome these limitations, bivalves have been proposed as effective biosentinels of water quality particularly for their ability to efficiently concentrate and retain microbial contaminants for long periods of time. In this study, we examined the use of indigenous blue mussels (Mytilus spp.) as biosentinels to monitor for the presence of Toxoplasma gondii and Cryptosporidium water. An efficient method to extract oocyst DNA from various mussel tissues followed by PCR-based detection of these pathogens was developed, which resulted in the detection down to 10 oocysts. This method was then used to conduct a small survey in Point Lobos and Morro Bay, California to determine prevalence T. gondii and Cryptosporidium. Results revealed that mussels from Morro Bay were contaminated with T. gondii (33 %), while mussels from Point Lobos were contaminated with T. gondii (54 %) and Cryptosporidium (26.9 %) oocysts. Phylogenetic analysis using the SSU rRNA gene identified two novel Cryptosporidium parvum-like genotypes. Overall, this study demonstrated the application of using native California Mytilus spp. as biosentinels for pathogen contamination along the central California shorelines. More importantly, T. gondii and Cryptosporidium were found at higher prevalence rates in Morro Bay and in Point Lobos, an area not previously reported to be contaminated with these pathogens.

Biosolids are nutrient-rich organic residuals that are currently used to amend soils for food production. Treatment requirements to inactivate pathogens for production of Class A biosolids are energy intensive. One less energy intensive alternative is to treat biosolids to Class B standards, but it could result in higher pathogen loads. Quantitative microbial risk assessments models have been developed on land application of Class B biosolids but contain many uncertainties because of limited data on specific pathogen densities and the use of fecal indicator organisms as accurate surrogates of pathogen loads. To address this gap, a 12-mo study of the levels and relationships between Cryptosporidium, Giardia, and human adenovirus (HAdV) with fecal coliform, somatic, and F-RNA coliphage levels in Class B biosolids from nine wastewater treatment plants throughout the United States was conducted. Results revealed that fecal coliform, somatic, and F-RNA coliphage densities were consistent throughout the year. More important, results revealed that HAdV ( x = 2.5 × 10 3 genome copies dry g -1 ) and Giardia ( x = 4.14 × 10 3 cysts dry g -1 ) were in all biosolids samples regardless of treatment processes, location, or season. Cryptosporidium oocysts were also detected (38% positive; range: 0-1.9 × 10 3 oocysts dry g -1 ), albeit sporadically. Positive correlations among three fecal indicator organisms and HAdV, but not protozoa, were also observed. Overall, this study reveals that high concentrations of enteric pathogens (e.g., Cryptosporidium, Giardia, and HAdV) are present in biosolids throughout the United States. Microbial densities found can further assist management and policymakers in establishing more accurate risk assessment models associated with land application of Class B biosolids.

Aim USEPA Method 1623, or its equivalent, is currently used to monitor for protozoan contamination of surface drinking water sources worldwide. At least three approved staining kits used for detecting Cryptosporidium and Giardia are commercially available. This study focuses on understanding the differences among staining kits used for Method 1623. Methods and Results Merifluor and EasyStain labeling kits were used to monitor Cryptosporidium oocyst and Giardia cyst densities in New York City's raw surface water sources. In the year following a change to the approved staining kits for use with Method 1623, an anomaly was noted in the occurrence of Giardia cysts in New York City's raw surface water. Specifically, Merifluor-stained samples had higher Giardia cyst densities as compared with those stained with EasyStain. Side by side comparison revealed significantly lower fluorescence intensities of Giardia muris as compared with G. duodenalis cysts when labeled with EasyStain. Conclusions This study showed very poor fluorescence intensity signals by EasyStain on G. muris cysts results in lower cyst counts, while Merifluor, with its broader Giardia cyst staining specificity, results in higher cyst counts, when using Methods 1623. Significance and Impact of Study These results suggest that detected Giardia cyst concentrations are dependent on the staining kits used, which can result in a more or less conservative estimation of occurrences and densities of zoonotic Giardia cysts by detecting a broader range of Giardia species/Assemblages. This article is protected by copyright. All rights reserved.

Aims This study developed and systematically evaluated performance and limit of detection of an off-the-slide genotyping procedure for both Cryptosporidium oocysts and Giardia cysts. Methods and Results Slide standards containing flow-sorted (oo)cysts were used to evaluate the off-the-slide genotyping procedure by microscopy and PCR. Results show approximately 20% of cysts and oocysts are lost during staining. Although transfer efficiency from the slide to the PCR tube could not be determined by microscopy, it was observed that the transfer process aided in the physical lysis of the (oo)cysts likely releasing DNA. PCR detection rates for a single event on a slide were 44% for Giardia and 27% for Cryptosporidium, and a minimum of five cysts and 20 oocysts are required to achieve a 90% PCR detection rate. A Poisson distribution analysis estimated the relative PCR target densities and limits of detection, it showed that 18 Cryptosporidium and five Giardia replicates are required for a 95% probability of detecting a single (oo)cyst on a slide. Conclusions This study successfully developed and evaluated recovery rates and limits of detection of an off-the-slide genotyping procedure for both Cryptosporidium and Giardia (oo)cysts from the same slide. Significance and Impact of the Study This off-the-slide genotyping technique is a simple and low cost tool that expands the applications of US EPA Method 1623 results by identifying the genotypes and assemblages of the enumerated Cryptosporidium and Giardia. This additional information will be useful for microbial risk assessment models and watershed management decisions.

Predictive models, based on environmental and water quality variables, have been used to improve the timeliness and accuracy of recreational water quality assessments, but their effectiveness has not been studied in inland waters. Sampling at eight inland recreational lakes in Ohio was done in order to investigate using predictive models for Escherichia coli and to understand the links between E. coli concentrations, predictive variables, and pathogens. Based upon results from 21 beach sites, models were developed for 13 sites, and the most predictive variables were rainfall, wind direction and speed, turbidity, and water temperature. Models were not developed at sites where the E. coli standard was seldom exceeded. Models were validated at nine sites during an independent year. At three sites, the model resulted in increased correct responses, sensitivities, and specificities compared to use of the previous day's E. coli concentration (the current method). Drought conditions during the validation year precluded being able to adequately assess model performance at most of the other sites. Cryptosporidium , adenovirus, eaeA ( E. coli ), ipaH ( Shigella ), and spvC ( Salmonella ) were found in at least 20% of samples collected for pathogens at five sites. The presence or absence of the three bacterial genes was related to some of the model variables but was not consistently related to E. coli concentrations. Predictive models were not effective at all inland lake sites; however, their use at two lakes with high swimmer densities will provide better estimates of public health risk than current methods and will be a valuable resource for beach managers and the public.

Bacterial indicators are used to indicate increased health risk from pathogens and to make beach closure and advisory decisions; however, beaches are seldom monitored for the pathogens themselves. Studies of sources and types of pathogens at beaches are needed to improve estimates of swimming-associated health risks. It would be advantageous and cost-effective, especially for studies conducted on a regional scale, to use a method that can simultaneously filter and concentrate all classes of pathogens from the large volumes of water needed to detect pathogens. In seven recovery experiments, stock cultures of viruses and protozoa were seeded into 10-liter lake water samples, and concentrations of naturally occurring bacterial indicators were used to determine recoveries. For the five filtration methods tested, the highest median recoveries were as follows: glass wool for adenovirus (4.7%); NanoCeram for enterovirus (14.5%) and MS2 coliphage (84%); continuous-flow centrifugation (CFC) plus Virocap (CFC+ViroCap) for Escherichia coli (68.3%) and Cryptosporidium (54%); automatic ultrafiltration (UF) for norovirus GII (2.4%); and dead-end UF for Enterococcus faecalis (80.5%), avian influenza virus (0.02%), and Giardia (57%). In evaluating filter performance in terms of both recovery and variability, the automatic UF resulted in the highest recovery while maintaining low variability for all nine microorganisms. The automatic UF was used to demonstrate that filtration can be scaled up to field deployment and the collection of 200-liter lake water samples.

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay. The results from the animal bioassay show that 1- and 3-log 10 inactivation is achieved with 4 mJ/cm 2 UV and 10 mJ/cm 2 low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with bioassay results. In conclusion, a 3-log 10 inactivation of T. gondii oocysts is achieved by 10-mJ/cm 2 low-pressure UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.

Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne(R), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox(R) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne(R) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox(R) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.

From bottlenose dolphins, to walruses, to sea otters, the parasitic protozoan Toxoplasma gondii is infecting marine mammals around the world. Whereas the terrestrial transmission pathways of T. gondii are well-described, the transmission pathway by which marine mammals are being infected is unknown. We hypothesize that migratory filter feeders, specifically northern anchovies (Engraulis mordax) and Pacific sardines (Sardinops sagax), are serving as biotic vectors for T. gondii within the marine environment. By filtering oocysts from seawater, these fishes could be transporting the oocysts from nearshore to pelagic environments. In this study, we experimentally exposed northern anchovies and Pacific sardines to T. gondii oocysts under laboratory conditions. Following exposure, the fishes' alimentary canals were harvested and assayed for the presence of T. gondii by PCR. Fish exposed to as few as 1197 oocysts/L seawater tested positive for T. gondii by PCR. In total, the PCR assay detected T. gondii DNA in 66% (40/61) of the exposed fishes. Oocyst infectivity was confirmed by mouse bioassay: 30% (7/23) of mice developed toxoplasmosis when fed fish exposed to 100,000 oocysts/L. This study demonstrates that both northern anchovies and Pacific sardines can filter T. gondii oocysts out of seawater under experimental conditions. Our experiments with anchovies demonstrated that the oocysts persisted in the fish for at least 8h post-exposure and our experiments with sardines demonstrated that the oocysts remained infectious inside the fish's alimentary canals.

Aims: To evaluate the effectiveness of continuous separation channel centrifugation for concentrating Toxoplasma gondii and Cyclospora cayetanensis from drinking water and environmental waters. Methods and Results: Ready-to-seed vials with known quantities of T. gondii and C. cayetanensis oocysts were prepared by flow cytometry. Oocysts were seeded at densities ranging from 1 to 1000 oocysts l−1 into 10 to 100 l test volumes of finished drinking water, water with manipulated turbidity, and the source waters from nine drinking water utilities. Oocysts were recovered using continuous separation channel centrifugation and counted on membrane filters using epifluorescent microscopy. Recovery efficiencies of both parasites were ≥84% in 10 l volumes of drinking water. In source waters, recoveries ranged from 64% to 100%, with the lowest recoveries in the most turbid waters. Method precision was between 10% and 20% coefficient of variation. Conclusion: Toxoplasma gondii and C. cayetanensis are effectively concentrated from various water matrices by continuous separation channel centrifugation. Significance and Impact of the Study: Waterborne transmission of T. gondii and C. cayetanensis presents another challenge in producing clean drinking water and protecting public health. Detection of these parasites relies on effectively concentrating oocysts from ambient water, otherwise false negatives may result. Validation data specific to T. gondii and C. cayetanensis concentration methods are limited. Continuous separation channel centrifugation recovers oocysts with high efficiency and precision, the method attributes required to accurately assess the risk of waterborne transmission.

This study describes fluorogenic 5′ nuclease PCR assays suitable for rapid, sensitive, high-throughput detection of the human-pathogenic microsporidial species Encephalitozoon hellem, E. cuniculi and E.intestinalis . The assays utilize species-specific primer sets and a genus-specific dual fluorescent-labeled probe that anneals to a region within the Encephalitozoon 16S rRNA gene. The assay design theoretically permits the probe to be used either with one set of primers for species-level determination or with a combination of all three primer sets for a genus-level screening of samples. The linear range of all three species-specific calibration curves that were developed using serial ten-fold dilutions of genomic DNA isolated from hemacytometer counted spores was determined to be between 10 4 and 10 −1 spores per PCR sample. The coefficients of variation were ≤5·2% over the entire 5-log span of each calibration curve. When DNA isolated from flow cytometric enumerated spores from each of the three Encephalitozoon species was used to evaluate the quantitative capability of the species' respective calibration curves, the results from 34 out of 36 (94%) samples were within 2 standard deviations. The species-specificity of each assay was confirmed using DNA isolated from 10 4 spores from each of the other two Encephalitozoon species as well as DNA extracted from numerous other protozoa, algae and bacteria.

Cryptosporidiosis has been traced to drinking contaminated surface water, which was either not treated or was ineffectively treated. Testing to detect Cryptosporidium parvum in surface water has been suggested to help prevent future outbreaks. In the present study, the same sample collection and filtration methods were used to compared sample processing and detection steps from 4 testing methods: a modified information collection rule (ICR) method and method 1623 (both developed by the U.S. Environmental Protection Agency), a flow cytometric method, and a solid-phase cytometric method. All of these methods use fluorescent antibody staining, which is only a presumptive indication of the presence of this parasite. Confirmation requires another assay. Methods were evaluated for both presumptive and confirmed detection. Solid-phase cytometry had the highest presumptive and confirmed detection rates. Flow cytometry had the next highest presumptive detection rate in reagent water but was third in spiked surface and tap waters, with no confirmation procedure. The ICR method had the third highest presumptive detection rate in reagent water and the second highest in spiked surface and tap waters but failed to confirm any oocysts. Method 1623 had significantly lower presumptive detection than any other method and a significantly lower confirmation rate than the solid-phase cytometry method.

This study evaluates Cryptosporidium parvum oocyst production in dexamethasone suppressed CF-1 and C57BL/6 mice. Both models can yield 1 × 10 9 total oocysts over a 20-day production period; however, only 20 CF-1 mice are required to reliably achieve this goal compared to 40 C57BL/6 mice. Although oocyst yields per mouse are similar for both mouse strains, the survival rate for CF-1 mice is higher, resulting in reduced lost production time per study when compared to the C57BL/6 mice. This study presents a more efficient and cost effective dexamethasone suppressed murine model of propagating high concentrations of C. parvum oocysts.

In this study, we examined the potential for detecting fecal bacteria and microbial source tracking markers in samples discarded during the concentration of Cryptosporidium and Giardia using USEPA Method 1623. Recovery rates for different fecal bacteria were determined in sewage spiked samples and environmental waters using different group-specific and host-specific PCR assays. Bacteroidales DNA recovery ranged from 59 to 71% for aliquots of supernatant collected after the elution step. The recovery of human-specific Bacteroidales DNA from sewage spiked samples was 54% in the elution step. An additional 1-7% Bacteroidales DNA was recovered after the immunomagnetic separation step, while recovery from the pellet left after the immunomagnetic separation of protozoa parasites was substantially lower. Comparison of Bacteroidales 16S rRNA gene sequences from elution and immunomagnetic separation discarded samples indicated that the distribution of clones was not statistically different, suggesting that there were no recovery biases introduced by these steps. Human- and cow-specific Bacteroidales and fecal indicator bacteria (i.e., enterococci,) were also detected in the discarded fractions of environmental samples collected from different geographic locations. Overall, the results of this study demonstrated the potential application of leftover sample fractions that are currently discarded for the PCR detection of fecal bacterial indicators and molecular source tracking.

Riverbank filtration has been shown to be effective for removing viable Cryptosporidium parvum oocysts. Drinking water systems that employ riverbank filtration may receive additional treatment credits beyond that which they can obtain using traditional engineering approaches. In order to develop guidance for removal effectiveness, screening level predictive modeling by colloid filtration theory combined with advection and dispersion modeling is potentially useful. Currently, only few studies have measured basic effective colloid filtration parameters for Cryptosporidium oocysts with naturally occurring riverbank sediments. In the focus of this study we conducted flow column experiments in triplicate and measured effective attachment rate coefficients for sandy river sediments of the Southern Great Plains which are low in organic matter. We found that for sediment sampled from these high-energy rivers there was no apparent dependency of C. parvum removal with carbon content, bacterial colony forming units, or with gross texture properties of the sands. The differences in particle size distribution for the sediments suggested that straining did not play a role in removal efficiency. First-order colloid attachment rate coefficients followed lognormal distribution functions. The coefficients also appeared to be unrelated to the differences in particle size distributions of the sediments, bacterial counts, or levels of total carbon or total organic carbon. Using Monte Carlo analyses, the lowest observed 5th percentile was 8.0 × 10 −6 min −1 and the highest observed 95th percentile was 1.6 × 10 −3 . Total log 10 removals ranged from 23 to 200 m −1 . These results have application for screening level colloid filtration modeling of riverbank filtration in these systems.

Cryptosporidium is an important waterborne protozoan parasite that can cause severe diarrhea and death in the immunocompromised. The current methods used to monitor for Cryptosporidium oocysts in water are the microscopy-based USEPA methods 1622 and 1623. These methods assess total levels of oocysts in source waters, but do not determine oocyst viability or genotype. Recently, propidium monoazide (PMA) has been used in conjunction with molecular diagnostic tools to identify species and assess the viability of bacteria. The goal of this study was the development of a Cryptosporidium PMA-PCR (CryptoPMA-PCR) assay that includes PMA treatment prior to PCR analysis in order to prevent the amplification of DNA from dead oocysts. The results demonstrated that PMA penetrates only dead oocysts and blocks amplification of their DNA. The CryptoPMA-PCR assay can also specifically detect live oocysts within a mixed population of live and dead oocysts. More importantly, live oocysts, not dead oocysts, were detected in raw waste or surface water samples spiked with Cryptosporidium oocysts. This proof-of-concept study is the first to demonstrate the use of PMA for pre-PCR treatment of Cryptosporidium oocysts. The CryptoPMA-PCR assay is an attractive approach to specifically detect and genotype viable Cryptosporidium oocysts in the water, which is critical for human health risk assessment.

An optimized cell culture immunofluorescence (IFA) procedure, using the HCT-8 cell line, was evaluated in blind trials to determine the sensitivity and reproducibility of measuring the infectivity of flow-cytometry-prepared inocula of Cryptosporidium parvum oocysts. In separate trials, suspensions consisting of between 0% and 100% viable oocysts were prepared at the US Environmental Protection Agency, shipped to the American Water Laboratory, and analyzed blindly by cell culture IFA. Data indicated the control (100% live) oocyst suspensions yielded statistically similar results to cell culture dose–response curve data developed previously at the American Water Laboratory. For test samples containing oocyst suspensions of unknown infectivity, cell culture IFA analyses indicated a high degree of correlation (r2= 0.89; n = 26) with the values expected by the US Environmental Protection Agency. Cell culture infectivity correlates well with neonatal mouse infectivity assays, and these blind validation trials provide credibility for the cell culture IFA procedure as a cost-effective and expedient alternative to mouse infectivity assays for determining in vitro infectivity of C. parvum oocysts.

IARDIA is the protozoan parasite that is the etiologic agentof giardiasis. This illness is the most common parasitic dis-ease and is estimated to infect at least 100,000 people each year inthe United States (Furness, Beach, and Roberts 2000). Symptomsof giardiasis range from asymptomatic to severe abdominal pain,chronic diarrhea, andin rarecases,death, withyoung children andimmunocompromised individuals being at the greatest risk of ser-ious illness (Adam 1991; Upcroft and Upcroft 2001). Infectiontypically occurs through the fecal-oral route and has been docu-mented to be associated with many waterborne disease outbreaksworldwide. A majority of these outbreaks occurred due to con-tamination of the drinking water supplies with untreated sewage.There are at least six species of Giardia including Giardia lam-blia (also known as Giardia duodenalis or Giardia intestinalis),which can infect a wide range of hosts including humans (Caccioetal.2005).Thecurrentdetectionmethodemployedtomonitorthepresence of Giardia cysts in surface and drinking water relies pri-marily on microscopic techniquesthat detect thepresenceof Giar-dia cysts in the sample, but the method is not species specific nordoes it determine cyst viability (U.S. Environmental ProtectionAgency 1999). More recently, a PCR-based genotyping tool hasbeen developed and used to identify the different Giardia spp.present in environmental water (Sulaiman et al. 2004). This tech-nique however can be prone to contamination and thus other al-ternative approaches are currently being explored. In particular,matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF MS) has been used to identify andclassify bacteria (Donohue et al. 2006) and parasites (Magnuson,Owens,andKelty2000;Mouraetal.2003),althoughthisapproachhas not been applied to study Giardia spp.This manuscript describes a MALDI-TOF MS-based approachthat is used to characterize the mass spectral fingerprints of intactG. lambliaandGiardiamuriscysts.Thisstudy identified commonmass spectral peaks shared by the two species as well as peaksspecific to G. lamblia and others specific to G. muris, which areuseful in differentiating the two organisms. Additional analysesrevealed that the mass spectral profiles of intact cysts consistedpartly of peaks representing trophozoite-derived proteins, basedon comparison with purified trophozoites. These results suggestthe potential application of intact cell MALDI-TOF MS as an al-ternative high throughput approach for species identification ofGiardia spp.MATERIALS AND METHODSCyst propagation and purification. Giardia lamblia (H3strain; assemblage B) and G. muris (obtained from Drs. ErikHewlett and John Andrews, Case Western Reserve School ofMedicine, Cleveland, OH) cysts were propagated using Mongo-lian gerbils or CF-1 mice, respectively (Jackson Laboratories, BarHarbor, ME). Feces from infected rodents were collected andcysts were harvested and purified using a sucrose/percoll (Sigma,St. Louis, MO) gradient (Hayes et al. 2003). Purified cysts used inall experiments were 7 days from the time of collection.In vitro excystation and trophozoite purification. The invitro excystation of cysts was performed as described (Rice andSchaefer 1981) and efficiency of excystation was determinedmicroscopically to routinely be 90% excysted. Following ex-cystation, trophozoite/cyst mixtures were labeled with FITC-con-jugated anti-Giardia antibody (Giardi-a-Glo

The prevailing paradigm is that immunosuppressed individuals are more susceptible to infection and are at higher risk of infection from Cryptosporidium oocysts if present in drinking water. To test this hypothesis, three immune conditions were examined: genetically immunocompromised T cell deficient CD-1 nude mice, B and T cell deficient Fox Chase CB-17/IcrClB SCID mice, and chemically immunosuppressed C57Bl/6 mice. Chemical immunosuppression was induced with a single subcutaneous injection of methylprednisolone acetate (MPA) at 600 mg/kg. The MPA immunosuppressed C57Bl/6 mice were characterized by a sustained decrease in circulating CD3, CD4 and CD8 T-lymphocytes of greater than 80% and a similar decrease in B-lymphocytes. A sharp rise in circulating mature segmented neutrophils followed MPA injection, dropping sharply after 10-14 days, mirroring the decrease in lymphocytes. The cessation of oocyst production after MPA was not accompanied by a radical rise in circulating CD3 or CD4 T-lymphocytes, but rather a rise in CD8 T-lymphocytes. The ID50 for the MPA immunosuppressed C57Bl/6 mice was 122 oocysts, whereas the ID50 for the C57Bl/6 immunocompetent group was 44. The genetically immunocompromised mice showed similar differences. The ID50 for CD-1 nude mice was 166 oocysts compared to 64 in CD-1 immunocompetent mice. For Fox Chase CB-17/IcrClB SCID and the immunocompetent CB-17 mice, the ID50's were 83 and 60 oocysts, respectively. These results suggest that the lack of an immune response does not increase the ability of C. muris to establish a productive infection and produce oocysts.

Accurately comparing filtration methods is indeed difficult. Our method ([1][1]) and the method described by [Borchardt et al.][2] for determining recoveries are both acceptable approaches; however, each is designed to achieve a different research goal. Our study was designed to compare recoveries

U.S. Environmental Protection Agency method 1623 is widely used to monitor source waters and drinking water supplies for Cryptosporidium oocysts. Matrix spikes, used to determine the effect of the environmental matrix on the method's recovery efficiency for the target organism, require the collection and analysis of two environmental samples, one for analysis of endemic oocysts and the other for analysis of recovery efficiency. A new product, ColorSeed, enables the analyst to determine recovery efficiency by using modified seeded oocysts that can be differentiated from endemic organisms in a single sample. Twenty-nine stream water samples and one untreated effluent sample from a cattle feedlot were collected in triplicate to compare modified seeding procedures to conventional seeding procedures that use viable, unmodified oocysts. Significant negative correlations were found between the average oocyst recovery and turbidity or suspended sediment; this was especially apparent in samples with turbidities greater than 100 nephelometric turbidity units and suspended sediment concentrations greater than 100 mg/liter. Cryptosporidium oocysts were found in 16.7% of the unseeded environmental samples, and concentrations, adjusted for recoveries, ranged from 4 to 80 oocysts per 10 liters. Determining recovery efficiency also provided data to calculate detection limits; these ranged from r = 0.802) and were not significantly different. Recoveries by using modified oocysts, therefore, were comparable to recoveries by using conventional seeding procedures.

U.S. EPA Methods 1622 and 1623 are used to detect and quantify Cryptosporidium oocysts in water. The protocol consists of filtration, immunomagnetic separation (IMS), staining with a fluorescent antibody, and microscopic analysis. Microscopic analysis includes detection by fluorescent antibody and confirmation by the demonstration of 1-4 sporozoites or nuclei after staining with 4',6-diamidino-2-phenyl indole dihydrochloride (DAPI). The purpose of this study was to evaluate a new IMS dissociation, a 10-min incubation at 80 degrees C. Heat dissociation improved the average oocyst recovery from 41% to 71% in seeded reagent water, and from 10% to 51% in seeded river samples. The average DAPI confirmation rate improved from 49% to 93% in reagent water, and from 48% to 73% in river samples. This modification improved both oocyst recovery and confirmation.

This is the first report of blue autofluorescence as a useful characteristic in the microscopic detection of Toxoplasma gondii, Hammondia hammondi, Hammondia heydorni, Neospora caninum, Besnoitia darlingi, and Sarcocystis neurona oocysts or sporocysts. This autofluorescence is of sufficient intensity and duration to allow identification of these oocysts from complex microscopic sample backgrounds. As with the autofluorescence of related coccidia, the oocysts glow pale blue when illuminated with an ultraviolet (UV) light source and viewed with the correct UV excitation and emission filter set.

Cyclospora cayetanensis, a coccidian parasite, with a fecal-oral life cycle, has become recognized worldwide as an emerging human pathogen. Clinical manifestations include prolonged gastroenteritis. While most cases of infection with C. cayetanensis in the United States have been associated with foodborne transmission, waterborne transmission has also been implicated. We report on the development and application of a real-time, quantitative polymerase chain reaction assay for the detection of C. cayetanensis oocysts, which is the first reported use of this technique for this organism. Both a species-specific primer set and dual fluorescent-labeled C. cayetanensis hybridization probe were designed using the inherent genetic uniqueness of the 18S ribosomal gene sequence of C. cayetanensis. The real-time polymerase chain reaction assay has been optimized to specifically detect the DNA from as few as 1 oocyst of C. cayetanensis per 5 microl reaction volume.

Aims: The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low-pressure u.v. irradiation. Methods and Results: Cysts of Giardia muris were inactivated using a low-pressure u.v. light source. Giardia muris was employed as a surrogate for the human pathogen Giardia lamblia. Cyst viability was determined by both in vitro excystation and animal infectivity. Cyst doses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ cm−2 did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, u.v. fluences of 1·4, 1·9 and 2·3 mJ cm−2 yielded log10 reductions ranging from 0·3 to ≥ 4·4. Conclusions: Results indicate that in vitro excystation is not a reliable indicator of G. muris cyst viability after u.v. disinfection. Very low doses of u.v. light rendered G. muris cysts non-infective in the mouse model employed. Significance and Impact of the Study: Data presented represent the only complete u.v. inactivation curve for G. muris. This research provides evidence that u.v. can be an effective barrier against Giardia spp. in the treatment of drinking water supplies.

Toxoplasma gondii oocysts are environmentally resistant and can infect virtually all warm-blooded hosts, including humans and livestock. Little is known about the biochemical basis for this resistance of oocysts, and mechanism for excystation of T. gondii sporozoites. The objective of the present study was to evaluate different methods (mechanical fragmentation, gradients, flow cytometry) to separate and purify T. gondii oocyst walls and sporocysts. Oocyst walls were successfully separated and purified using iodixanol gradients. Sporocysts were successfully separated and purified using iodixanol and Percoll gradients. Purification was also achieved by flow cytometry. Flow cytometry with fluorescence-activated cell sorting (FACS) yielded analytical quantities of oocyst walls and intact sporocysts. Flow cytometry with FACS also proved useful for quantitation of purity obtained following iodixanol gradient fractionation. Methods reported in this paper will be useful for analytical purposes, such as proteomic analysis of components unique to this life cycle stage, development of detection methods, or excystation studies.