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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.

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.

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.

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.

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.

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.

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.

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.

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.