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Ultraviolet light-emitting diodes (UV-LEDs) have demonstrated the ability to inactivate microorganisms in water, offering an environmentally safer alternative to the conventional mercury lamp, in UV applications. While several studies have explored the microbiological effect of UVC-LEDs (200nm-280nm), limited information exists regarding their effects on waters with critical qualities. These critical qualities encompass bacteria, viruses, and protozoa - drinking water quality indicators defined by the World Health Organization for small water systems. For the first time, this work reports on the Escherichia coli, PhiX-174, MS2, and Cryptosporidium oocysts inactivation using a bench-scale UVC-LED (280 nm) water disinfection system. UV doses at a wavelength of 280 nm (UV 280 ) of up to 143.4 mJ/cm 2 were delivered under two quality-critical water conditions: filtered water (UV transmittance at 280 nm - UVT 280 90.2 %) and WHO challenge water (UVT 15.7 %). Results revealed microbiological reductions dependent on exposure time and UVT. For UV 280 dose of 16.1 mJ/cm 2 , 2.93-3.70 log E. coli reductions were observed in UVT 90.2 % and 15.7 %, 3.49-4.21 log for PhiX-174, 0.63-0.78 log for MS2, and 0.02-0.04 log for Cryptosporidium oocysts. Significantly higher UV 280 doses of 143.4 mJ/cm 2 led to reductions of 3.94-5.35 log for MS2 and 0.42-0.46 log for Cryptosporidium oocysts. Statistical analysis revealed that the sensitivity among the organisms to UV 280 exposure was E. coli = PhiX-174 > MS2 >> Cryptosporidium oocysts. Although experiments with WHO challenge water posed greater challenges for achieving 1 log reduction compared to filtered water, this difference only proved statistically significant for PhiX-174 and MS2 reductions. Overall, UVC-LED technology demonstrated notable efficacy in microbiological inactivation, achieving significant reductions based on WHO scheme of evaluation for POU technologies in both bacteria and viruses even in critical-quality waters. The findings emphasize the potential for extending the application of UVC-LED as a viable solution for household water treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

As an alternative to using ultraviolet (UV) lamps, which are made with mercury that is toxic to the environment and human health, UV light-emitting diodes (UV-LEDs) are expected to be effective for inactivating microorganisms in water. Although UV-LEDs have been reported to be effective against bacteria and viruses, the effectiveness of UV-LEDs against Cryptosporidium parasites has not been fully evaluated. As we report here, we have developed an in vivo quantitative inactivation assay for C. parvum oocysts using immunodeficient mice. Using the assay, we evaluated the effectiveness of treatment by UV lamp (254 nm) at approximately 1000 μJ/cm 2 (for 3 s at a distance of 95 mm) compared to inactivation by commercially available UV-LEDs (with peak wavelengths of 268, 275, 284, and 289 nm). The shed patterns of oocysts after treatment with 284- and 289-nm wavelength UV-LEDs were significantly delayed compared to that after treatment with a UV lamp. These findings provide the first suggestion that UV-LEDs are effective against these parasites, as assessed using commercially available 350-mA UV-LEDs under conditions of fixed exposure distance and time., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Prevention of coccidiosis is one of the best ways of controlling disease. Therefore, the present study was carried out to evaluate the protective effect of ultraviolet (UV)-irradiated sporulated oocysts of Eimeria species against coccidiosis in layer chickens. One hundred forty-four one-day-old layer chicks were randomly divided into 4 groups (n = 36), including non-immunized/non-challenged negative control group (NC group), non-immunized/challenged control group (NIC group), non-irradiated sporulated oocyst/challenged group (CA group), and UV-irradiated sporulated oocyst/challenged (UV group). At the age of 4 days, chickens in groups UV and CA were both orally inoculated with 1.0 × 10 4 UV-irradiated and non-irradiated sporulated oocysts of Eimeria species, respectively. Chickens in groups NIC and NC were served as positive and negative controls, respectively. Chickens in all groups were orally challenged with 7.5 × 10 4 sporulated oocysts of Eimeria species except the NC group at the age of 21 days. The results revealed that chicks receiving UV-irradiated sporulated oocysts had no signs of illness with minimal or no changes in the cecal integrity and a significantly lower oocyst shedding (OPG) than in the NIC group. Additionally, the cytokine gene expression profiles were evaluated. Expression levels of IL-2, IL-12, and IFN-γ were significantly higher in the spleen of chicks in the UV and CA groups than in the NC group post-challenge. As expected, treatment with irradiated oocysts resulted in a significant reduction in oocyst shedding and maintenance of cecal mucosal integrity. Furthermore, the body weight was higher in chickens inoculated with UV-irradiated oocysts than their non-irradiated counterparts. In conclusion, our results demonstrate that inoculation with UV-irradiated sporulated oocysts of Eimeria species can produce a substantial reduction in infection symptoms.

In vitro and in vivo studies were performed to assess whether Eimeria tenella (E. tenella) oocysts, exposed to low energy electron irradiation (LEEI), might be considered potential vaccine candidates against cecal coccidiosis. Sporulated oocysts were exposed to LEEI of 0.1 kGy to 10.0 kGy. Reproduction inhibition assays (RIA) were performed in MDBK cells to assess infectivity of sporozoites excysted from irradiated and non-irradiated oocysts. LEEI of 0.1 kGy or 0.5 kGy resulted in 73.2% and 86.5% inhibition of in vitro reproduction (%I RIA ), respectively. Groups of 12 one day old (D1) chicken were orally inoculated with Paracox®-8 (G1), 2.0 × 10 3 non-irradiated oocysts (G2) or 1.0 × 10 4 irradiated oocysts exposed to LEEI of 0.1 kGy (G3, G4) or 0.5 kGy (G5). Chicken of groups G1, G2, G4 and G5 were challenged 3 weeks later (D21) by a single inoculation of 7.5 × 10 4 non-attenuated oocysts of the same strain while G3 remained unchallenged. All chickens were subject to necropsy 7 days after challenge (D28) to estimate lesion scores (LS) and oocyst index (OI). A positive control (PC, non-vaccinated, challenged) and a negative control (NC, non-vaccinated, non-challenged) were kept in parallel. Chicken of group G5 had similar weight gain as the Paracox®-8 group (G1) after challenge and higher weight gains as compared to the other vaccinated groups. Feed conversion ratio (FCR) did not differ between chickens inoculated with oocysts irradiated with 0.5 kGy (G5) and negative control (NC) before challenge (1.25-1.52). After challenge FCR was 1.99 (G5) to 2.23 (G4) in the vaccinated chicken compared to 1.76 in group NC. LS and OI were significantly lower in all vaccinated groups as compared to group PC. Progeny oocysts collected from the feces of chickens following vaccination with irradiated oocysts exhibited lower in vitro infectivity/reproduction in MDBK cells with %I RIA of 89.7% and 82.4% for progeny of oocysts irradiated with 0.5 kGy and 0.1 kGy, respectively, suggesting hereditary attenuation by LEEI treatment. Seroconversion was demonstrated by ELISA before challenge (D21) in all vaccinated groups, however, chicken inoculated with irradiated oocysts displayed higher antibody levels than those inoculated with precocious oocysts (G1). In Western blot analysis chicken vaccinated with virulent (G2) or 0.1 kGy-irradiated E. tenella oocysts (G3, G4) showed more protein bands compared to G5 (0.5 kGy). We conclude that LEEI could be a promising technology for production of attenuated oocyst vaccines., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This study aimed to induce protective immunity against infection with Sarcocystis muris in experimental mice using β-irradiated sporocysts. Mice were vaccinated with 50 sporocysts of S. muris which were exposed to 1.84 μSv β-irradiation for 2, 4 and 8 h. After challenge infection, different samples were taken for evaluation. Serum and intestinal wash were assayed for IFN-γ and IgA, respectively. Mesenteric lymph nodes (MLNs) and spleen were investigated for CD4 + and CD8 + T cells using immunohistochemistry. For liver, the morphological changes in parasitic stages and the count of infiltrated CD8 + T, NK1.1 + and FasL + cells were also investigated. Real time (RT) - PCR was used for detection of liver MHC I, CD1d, IFN-γ, perforin and FasL as well as the parasite 18S ribosomal(r) RNA in liver and muscle tissues. Alterations of liver parasitic stages as well as a decrease in the infection with the parasite in both of liver and muscle tissues were dependent on radiation exposure time. An investigation for the mechanism of immunoprotection showed an increase in liver NK1.1 + & FasL + cells, serum IFN-γ and intestinal IgA, while CD4 + and CD8 + T showed a remarkable increase in MLNs and spleen. FasL expression increased in the liver dependently on radiation exposure time, while perforin, MHC I and CD1d were not. β-irradiated sporocysts with 1.84 μSv for 8 h s could induce the highest protection against infection with Sarcocystis. This could be largely relied on the increased infiltration of NK cells and associated higher expression of FasL in the liver., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Cryptosporidium is a genus of enteric protozoan parasites of medical and veterinary importance, whose oocysts have been reported to occur in different types of water worldwide, offering a great resistant to the water treatment processes. Heterogeneous solar photocatalysis using titanium dioxide (TiO2) slurry was evaluated on inactivation of Cryptosporidium parvum oocysts in water. Suspensions of TiO2 (0, 63, 100 and 200mg/L) in distilled water (DW) or simulated municipal wastewater treatment plant (MWTP) effluent spiked with C. parvum oocysts were exposed to simulated solar radiation. The use of TiO2 slurry at concentrations of 100 and 200mg/L in DW yielded a high level of oocyst inactivation after 5h of exposure (4.16±2.35% and 15.03±4.54%, respectively, vs 99.33±0.58%, initial value), representing a good improvement relative to the results obtained in the samples exposed without TiO2 (51.06±9.35%). However, in the assays carried out using simulated MWTP effluent, addition of the photocatalyst did not offer better results. Examination of the samples under bright field and epifluorescence microscopy revealed the existence of aggregates comprising TiO2 particles and parasitic forms, which size increased as the concentration of catalyst and the exposure time increased, while the intensity of fluorescence of the oocyst walls decreased. After photocatalytic disinfection process, the recovery of TiO2 slurry by sedimentation provided a substantial reduction in the parasitic load in treated water samples (57.81±1.10% and 82.10±2.64% for 200mg/L of TiO2 in DW and in simulated MWTP effluent, respectively). Although further studies are need to optimize TiO2 photocatalytic disinfection against Cryptosporidium, the results obtained in the present study show the effectiveness of solar photocatalysis using TiO2 slurry in the inactivation of C. parvum oocysts in distilled water., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The aim of the present study was to evaluate alternatives for inactivating Cryptosporidium parvum under experimental conditions. Disinfectants against this protozoan are usually based on cresols and often difficult to handle in laboratories. Four different substances (ethanol, denatured ethanol, sodium hypochlorite and peroxide) at different concentrations were tested for several exposure times (30 min, 2 h, 4 h, 12 h and 24 h). The results show an inactivation over 99% by using 10% H2O2 at an exposure time over 2 h as well as 3 and 6% NaOCl after 12 h of exposure. Furthermore, the ability of UV-C light to inactivate oocysts on smooth surfaces (e.g., laminar flow) was evaluated. To mimic laboratory conditions, oocysts were given on germ carriers. Best results (>99%) were achieved at an exposure time of 30 min (100.8 mJ/cm(2)).

Cryptosporidium spp., a significant cause of foodborne infection, have been shown to be resistant to most chemical food disinfectant agents and infective for weeks in irrigation waters and stored fresh vegetal produce. Pulsed UV light (PL) has the potential to inactivate Cryptosporidium spp. on surfaces of raw or minimally processed foods or both. The present study aimed to evaluate the efficacy of PL on viability and in vivo infectivity of Cryptosporidium parvum oocysts present on raspberries, a known source of transmission to humans of oocyst-forming apicomplexan pathogens. The skin of each of 20 raspberries was experimentally inoculated with five 10-μl spots of an oocyst suspension containing 6 × 10(7) oocysts per ml (Nouzilly isolate). Raspberries were irradiated by PL flashes (4 J/cm(2) of total fluence). This dose did not affect colorimetric or organoleptic characteristics of fruits. After immunomagnetic separation from raspberries, oocysts were bleached and administered orally to neonatal suckling mice. Seven days after infection, mice were euthanized, and the number of oocysts in the entire small intestine was individually assessed by immunofluorescence flow cytometry. Three of 12 and 12 of 12 inoculated mice that received 10 and 100 oocysts isolated from nonirradiated raspberries, respectively, were found infected. Four of 12 and 2 of 12 inoculated mice that received 10(3) and 10(4) oocysts from irradiated raspberries, respectively, were found infected. Oocyst counts were lower in animals inoculated with 10(3) and 10(4) oocysts from irradiated raspberries (92 ± 144 and 38 ± 82, respectively) than in animals infected with 100 oocysts from nonirradiated raspberries (35,785 ± 66,221, P = 0.008). PL irradiation achieved oocyst reductions of 2 and 3 log for an inoculum of 10(3) and 10(4) oocysts, respectively. The present pilot-scale evaluation suggests that PL is an effective mode of decontamination for raspberries and prompts further applicability studies in industrial contexts.

The current study investigates the use of irradiated oocysts to protect broiler chicks, raised on litter, from infection with multiple species of Eimeria. In order to determine the optimum radiation dose for each Eimeria species, 1-day-old chicks were immunized with oocysts of Eimeria maxima, Eimeria acervulina, or Eimeria tenella exposed to gamma radiation ranging from 0-500 Gy. The litter oocyst counts at 7 days postimmunization, and the effect on weight gain following a challenge infection, decreased with an optimum dose between 150-200 Gy. Based on this finding, broiler chicks were immunized with a mixture of E. maxima, E. acervulina, and E tenella that had been exposed to 150 or 200 Gy. This resulted in more than a 100-fold reduction in litter oocyst counts and significant protection from a challenge infection, as measured by improved weight gain and feed conversion ratio (FCR). Immunization of birds with oocysts receiving 200 Gy was less effective in providing protection from a challenge infection. An additional formulation of vaccines containing two different oocyst doses of the three species that had been irradiated with 150 Gy were evaluated in their ability to attenuate oocyst output and convey protection to challenge. Results were similar with both high and low numbers of irradiated oocysts. Immunized chicks shed less oocysts at 7 days postimmunization and were protected from negative effects of challenge infection as measured by FCR, changes in weight gain, lesion scores, and measurement of body composition. However, the level of protection was somewhat less than that achieved by immunization with nonirradiated oocysts. The overall conclusion is that an irradiated oocyst vaccine developed in this study can effectively protect chicks that are raised on litter from challenge infection with multiple species of Eimeria, comparable to vaccines with virulent or precocious strains.

Physicochemical treatment efficiency for unrestricted urban water reuse was evaluated at a conventional activated-sludge wastewater treatment plant (WWTP). Pilot plant set-up consisted of an alum coagulation step, granular media upflow flocculation and direct downflow dual-media filtration followed by ultraviolet disinfection (dose of 95 mJ cm⁻²). Optimum aluminum sulfate dosage of 10 mg L⁻¹ and coagulation pH 7.0 were preset based on bench scale tests. Under WWTP stable operation, water quality met United States Environmental Protection Agency (USEPA) suggested guidelines for unrestricted urban reuse regarding turbidity (mean value 1.3 NTU) and suspended solids (mean value 2.1 mg L⁻¹). When WWTP overall plant performance dropped from 90 to 80% (although BOD value stayed below 6 mg O₂ L⁻¹, suggesting unrestricted reuse), solids breakthrough in filtrate was observed. Microorganism removal rates were: total coliforms 60.0%, Escherichia coli 63.0%, Giardia spp. 81.0%, and helminth eggs 62.5%; thus organisms still remained in filtrate. Ultraviolet (UV) disinfection efficiency was 4.1- and 3.8-log for total coliforms and E. coli, respectively. Considering low UV efficiency obtained for helminths and the survival of protozoa and helminths in the environment, effluent quality presents risk to public health if destined for unrestricted urban reuse.

Water scarcity leads to an increased use of reclaimed water, which in turn calls for an improvement in water reclamation procedures to ensure adequate quality of the final effluent. The presence of infectious Cryptosporidium oocysts (IOO) in reclaimed water is a health hazard for users of this resource. Here, we gathered information on Cryptosporidium (concentrations, infectivity and genotype) in order to perform quantitative microbial risk assessment (QMRA). Moreover, data concerning the spores of sulphite-reducing clostridia (SRC) were used to undertake QMRA at a screening level. Our results show that the probability of infection (PI) by Cryptosporidium depends on the tertiary treatment type. The mean PI using the exponential dose-response model was 3.69 × 10(-6) in tertiary effluents (TE) treated with UV light, whereas it was 3 log(10) units higher, 1.89 × 10(-3), in TE not treated with this disinfection method. With the β-Poisson model, the mean PI was 1.56 × 10(-4) in UV-treated TE and 2 log(10) units higher, 4.37 × 10(-2), in TE not treated with UV. The use of SRC to perform QMRA of Cryptosporidium showed higher PI than when using directly IOO data. This observation suggests the former technique is a conservative method of QMRA.

Water samples of 0, 5, and 100 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight in 2.5l static borosilicate solar reactors fitted with two different compound parabolic concentrators (CPCs), CPC1 and CPC1.89, with concentration factors of the solar radiation of 1 and 1.89, respectively. The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. Thus, the initial global oocyst viability of the C. parvum isolate used was 95.3 ± 1.6%. Using the solar reactors fitted with CPC1, the global viability of oocysts after 12h of exposure was zero in the most turbid water samples (100 NTU) and almost zero in the other water samples (0.3 ± 0.0% for 0 NTU and 0.5 ± 0.2% for 5 NTU). Employing the solar reactors fitted with CPC1.89, after 10h exposure, the global oocyst viability was zero in the non-turbid water samples (0 NTU), and it was almost zero in the 5 NTU water samples after 8h of exposure (0.5 ± 0.5%). In the most turbid water samples (100 NTU), the global viability was 1.9 ± 0.6% after 10 and 12h of exposure. In conclusion, the use of these 2.5l static solar reactors fitted with CPCs significantly improved the efficacy of the SODIS technique as these systems shorten the exposure times to solar radiation, and also minimize the negative effects of turbidity. This technology therefore represents a good alternative method for improving the microbiological quality of household drinking water in developing countries., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Cryptosporidium parvum is a globally distributed zoonotic parasite and an important opportunistic pathogen in immunocompromised patients. Little is known on the metabolic dynamics of the parasite, and study is hampered by the lack of molecular and genetic tools. Here we report the development of the first Agilent microarray for C. parvum (CpArray15K) that covers all predicted ORFs in the parasite genome. Global transcriptome analysis using CpArray15K coupled with real-time qRT-PCR uncovered a number of unique metabolic features in oocysts, the infectious and environmental stage of the parasite., Results: Oocyst stage parasites were found to be highly active in protein synthesis, based on the high transcript levels of genes associated with ribosome biogenesis, transcription and translation. The proteasome and ubiquitin associated components were also highly active, implying that oocysts might employ protein degradation pathways to recycle amino acids in order to overcome the inability to synthesize amino acids de novo. Energy metabolism in oocysts was featured by the highest level of expression of lactate dehydrogenase (LDH) gene. We also studied parasite responses to UV-irradiation, and observed complex and dynamic regulations of gene expression. Notable changes included increased transcript levels of genes involved in DNA repair and intracellular trafficking. Among the stress-related genes, TCP-1 family members and some thioredoxin-associated genes appear to play more important roles in the recovery of UV-induced damages in the oocysts. Our observations also suggest that UV irradiation of oocysts results in increased activities in cytoskeletal rearrangement and intracellular membrane trafficking., Conclusions: CpArray15K is the first microarray chip developed for C. parvum, which provides the Cryptosporidium research community a needed tool to study the parasite transcriptome and functional genomics. CpArray15K has been successfully used in profiling the gene expressions in the parasite oocysts as well as their responses to UV-irradiation. These observations shed light on how the parasite oocysts might adapt and respond to the hostile external environment and associated stress such as UV irradiation.

Solar water disinfection (SODIS) is a type of treatment that can significantly improve the microbiological quality of drinking water at household level and therefore prevent waterborne diseases in developing countries. Cryptosporidium parvum is an obligate protozoan parasite responsible for the diarrhoeal disease cryptosporidiosis in humans and animals. Recently, this parasite has been selected by the WHO as a reference pathogen for protozoan parasites in the evaluation of household water treatment options. In this study, the field efficacy of different static solar reactors [1.5 l transparent plastic polyethylene terephthalate (PET) bottles as well as 2.5 l borosilicate glass and 25 l methacrylate reactors fitted with compound parabolic concentrators (CPC)] for solar disinfection of turbid waters experimentally contaminated with C. parvum oocysts was compared. Potential oocyst viability was determined by inclusion/exclusion of the fluorogenic vital dye propidium iodide. The results demonstrate that static solar reactors fitted with CPCs are an excellent alternative to the conventional SODIS method with PET bottles. These reactors improved the efficacy of the SODIS method by enabling larger volumes of water to be treated and, in some cases, the C. parvum oocysts were rendered totally unviable, minimising the negative effects of turbidity., (Copyright © 2012 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

Solar radiation reduces Cryptosporidium infectivity. Biofilms grown from stream microbial assemblages inoculated with oocysts were exposed to solar radiation. The infectivity of oocysts attached at the biofilm surface and oocysts suspended in water was about half that of oocysts attached at the base of a 32-μm biofilm.

Sequential helminth egg inactivation using a solar driven advanced oxidation process (AOP) followed by chlorine was achieved. The photo-assisted Fenton process was tested alone under different H(2)O(2) and/or Fe(II) concentrations to assess its ability to inactivate Ascaris suum eggs. The effect of free chlorine alone was also tested. The lowest egg inactivation results were found using Fe(II) or H(2)O(2) separately (5 and 140 mmol L(-1), respectively) in dark conditions, which showed about 28% inactivation of helminth eggs. By combining Fe(II) and H(2)O(2) at the same concentrations described earlier, 55% of helminth egg inactivation was achieved. By increasing the reagent's concentration two-fold, 83% egg inactivation was achieved after 120 min of reaction time. Process efficiency was enhanced by solar excitation. Using solar disinfection only, the A. suum eggs inactivation reached was the lowest observed (58% egg inactivation after 120 min (120 kJ L(-1))), compared with tests using the photo-Fenton process. The use of the photo-Fenton reaction enhanced the process up to over 99% of egg inactivation after 120 kJ L(-1) when the highest Fe(II) and H(2)O(2) concentration was tested. Practically no effect on the helminth eggs was observed with free chlorine alone after 550 mg min L(-1) was used. Egg inactivation in the range of 25-30% was obtained for sequential processes (AOP then chlorine) using about 150 mg min L(-1).

Water samples of 0, 5, and 30 nephelometric turbidity units (NTU) spiked with Cryptosporidium parvum oocysts were exposed to natural sunlight using a 25-L static solar reactor fitted with a compound parabolic collector (CPC). The global oocyst viability was calculated by the evaluation of the inclusion/exclusion of the fluorogenic vital dye propidium iodide and the spontaneous excystation. After an exposure time of 8 hours, the global oocyst viabilities were 21.8 ± 3.1%, 31.3 ± 12.9%, and 45.0 ± 10.0% for turbidity levels of 0, 5, and 30 NTU, respectively, and these values were significantly lower (P < 0.05) that the initial global viability of the isolate (92.1 ± 0.9%). The 25-L static solar reactor that was evaluated can be an alternative system to the conventional solar water disinfection process for improving the microbiological quality of drinking water on a household level, and moreover, it enables treatment of larger volumes of water (> 10 times).

In the genus Cryptosporidium, there are more than 14 species with different sizes and habitats, as well as different hosts. Among these, C. parvum and C. hominis are known to be human pathogens. As C. parvum can survive exposure to harsh environmental conditions, including various disinfectants or high doses of radiation, it is considered to be an important environmental pathogen that may be a threat to human health. However, the resistance of other Cryptosporidium species to various environmental conditions is unknown. In this study, resistance against γ-irradiation was compared between C. parvum and C. muris using in vivo infection in mice. The capability of C. muris to infect mice could be eliminated with 1,000 Gy of γ-irradiation, while C. parvum remained infective in mice after up to 1,000 Gy of γ-irradiation, although the peak number of oocysts per gram of feces decreased to 16% that of non-irradiated oocysts. The difference in radioresistance between these 2 Cryptosporidium species should be investigated by further studies.

Cryptosporidium parvum is known as one of the most highly resistant parasites to gamma irradiation. To morphologically have an insight on the radioresistance of this parasite, ultrastructural changes in C. parvum sporozoites were observed after gamma irradiation using various doses (1, 5, 10, and 25 kGy) following a range of post-irradiation incubation times (10 kGy for 6, 12, 24, 48, 72, and 96 hr). The ultrastructures of C. parvum oocysts changed remarkably after a 10-kGy irradiation. Nuclear membrane changes and degranulation of dense granules were observed with high doses over 10 kGy, and morphological changes in micronemes and rhoptries were observed with very high doses over 25 kGy. Oocyst walls were not affected by irradiation, whereas the internal structures of sporozoites degenerated completely 96 hr post-irradiation using a dose of 10 kGy. From this study, morphological evidence of radioresistance of C. parvum has been supplemented.

Cryptosporidium parvum is a waterborne protozoan parasite that is found intracellularly in host animals, including humans, and causes severe diarrhea, which can lead to the death of an immunocompromised individual. Previously, we found that this organism is highly radioresistant as it can productively infect mice after exposure to a 10-kGy dose of γ-radiation. To understand how C. parvum avoids radiation damage, we characterized its protein expression patterns 6, 24, and 48 h after a 10-kGy dose of γ-radiation using two-dimensional PAGE. The gels showed 10 silver-stained spots that increased or decreased in size following γ-irradiation. Five proteins contained in these spots were identified using MALDI-TOF MS peptide fingerprinting, and two of these showed an increase in expression after γ-irradiation. These proteins were identified by LC-MS/MS as proteasome subunit alpha type 4 (NTN hydrolase fold) and thioredoxin peroxidase-like protein. The roles of these two upregulated proteins as related to the radioresistance of C. parvum remain to be evaluated., (Copyright © 2010 Elsevier Inc. All rights reserved.)

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.

Cryptosporidium parvum is a well-known waterborne intracellular protozoan that causes severe diarrheal illness in immunocompromised individuals. This organism is highly resistant to harsh environmental conditions and various disinfectants, and it exhibits one of the highest known resistances to gamma irradiation. We investigated rejoining of gamma-ray-induced DNA damage in C. parvum by neutral comet assay. Oocysts were gamma irradiated at various doses (1, 5, 10, and 25kGy) and were incubated for various periods (6-96h) after exposure to 10kGy. The comet tail moment showed that the number of DNA double-strand breaks increased concomitantly with the gamma irradiation dose. When investigating rejoining after irradiation at 10kGy, double-strand breaks peaked at 6h postirradiation, and rejoining was highest at 72h postirradiation. The observed rejoining pattern suggests that repair process occurs slowly even when complex DNA double-strand breaks in C. parvum were induced by high dose irradiation, 10kGy.

Cryptosporidium parvum is an enteric coccidian parasite that is recognised as a frequent cause of water-borne disease in humans. We report for the first time on use of the in vitro HCT-8 cell culture-quantitative PCR (qPCR) assay and the in vivo SCID-mouse bioassay for evaluating critical factors that reduce or eliminate infectivity of C. parvum after irradiating oocysts in saline solution under varying operational conditions with pulsed UV light. Infections post UV treatments were detected by immunofluorescence (IF) microscopy and by quantitative PCR in cell culture, and by IF staining of faeces and by hematoxylin and eosin staining of intestinal villi in mice. There was a good agreement between using cell culture-qPCR and the mouse assay for determining reduction or elimination of C. parvum infectivity as a consequence of varying UV operating conditions. Reduction in infectivity depended on the intensity of lamp discharge energy applied, amount of pulsing and population size of oocysts (P < or = 0.05). Conventional radiometer was unable to measure fluence or UV dose in saline samples due to the ultra-short non-continuous nature of the high-energy light pulses. Incorporation of humic acid at a concentration above that found in surface water (i.e., < or =10 ppm) did not significantly affect PUV disinfection capability irrespective of parameters tested (P < or = 0.05). These observations show that use of this HCT-8 cell culture assay is equivalent to using the 'gold standard' mouse-based infectivity assay for determining disinfection performances of PUV for treating C. parvum in saline solution., (Copyright 2010 Elsevier B.V. All rights reserved.)

Aim: To assess the efficiency of a medium-pressure UV reactor under full-scale water treatment plant (WTP) conditions on the infectivity of Cryptosporidium parvum oocysts in an Naval Medical Research Institute (NMRI) suckling mice infectivity model., Methods and Results: Six/seven-day-old mice were administered orally 2-10x10(4)Cryptosporidium parvum oocysts. Compared with nonirradiated oocysts, 40 mJ cm(-2) UV irradiation of ingested oocysts resulted 7 days later in a 3.4-4.0 log10 reduction in the counts of small intestine oocysts, using a fluorescent flow cytometry assay., Conclusion: Present data extend to industrial conditions previous observations of the efficiency of UV irradiation against Cryptosporidium parvum oocyst in vivo development., Significance and Impact of the Study: Present results suggest that in WTP conditions, a medium-pressure UV reactor is efficient in reducing the infectivity of Cryptosporidium parvum oocysts, one of the most resistant micro-organisms present in environmental waters.

Control of waterborne gastrointestinal parasites represents a major concern to water industries worldwide. In developed countries, pathogens in drinking water supplies are normally removed by sand filtration followed by chemical disinfection. Cryptosporidium spp. are generally resistant to common disinfection techniques and alternative control strategies are being sought. In the current study, the photocatalytic inactivation of C. parvum oocysts was shown to occur in buffer solution (78.4% after 180 min) and surface water (73.7% after 180 min). Viability was assessed by dye exclusion, excystation, direct examination of oocysts and a novel gene expression assay based on lactate dehydrogenase 1 (LDH1) expression levels. Collectively, this confirmed the inactivation of oocysts and scanning electron microscopy (SEM) confirmed cleavage at the suture line of oocyst cell walls, revealing large numbers of empty (ghost) cells after exposure to photocatalytic treatment.

Toxoplasma gondii, a ubiquitous parasitic protozoan, is emerging as an aquatic biological pollutant. Infections can result from drinking water contaminated with environmentally resistant oocysts. However, recommendations regarding water treatment for oocyst inactivation have not been established. In this study, the physical method of radiofrequency (RF) power was evaluated for its ability to inactivate T. gondii oocysts in water. Oocysts were exposed to various RF energy levels to induce 50, 55, 60, 70 and 80 degrees C temperatures maintained for 1 min. Post-treatment oocyst viability was determined by mouse bioassay with serology, immunohistochemistry and in vitro parasite isolation to confirm T. gondii infections in mice. None of the mice inoculated with oocysts treated with RF-induced temperatures of > or =60 degrees C in an initial experiment became infected; however, there was incomplete oocyst activation in subsequent experiments conducted under similar conditions. These results indicate that T. gondii oocysts may not always be inactivated when exposed to a minimum of 60 degrees C for 1 min. The impact of factors such as water heating time, cooling time and the volume of water treated must be considered when evaluating the efficacy of RF power for oocyst inactivation.

Objective: To investigate the efficacy of the solar water disinfection (SODIS) method for inactivating Cryptosporidium parvum oocysts in turbid waters using 1.5 l polyethylene terephthalate (PET) bottles under natural sunlight., Methods: All experiments were performed at the Plataforma Solar de Almería, located in the Tabernas Desert (Southern Spain) in July and October 2007. Turbid water samples [5, 100 and 300 nephelometric turbidity units (NTU)] were prepared by addition of red soil to distilled water, and then spiked with purified C. parvum oocysts. PET bottles containing the contaminated turbid waters were exposed to full sunlight for 4, 8 and 12 h. The samples were then concentrated by filtration and the oocyst viability was determined by inclusion/exclusion of the fluorogenic vital dye propidium iodide. Results After an exposure time of 12 h (cumulative global dose of 28.28 MJ/m(2); cumulative UV dose of 1037.06 kJ/m(2)) the oocyst viabilities were 11.54%, 25.96%, 41.50% and 52.80% for turbidity levels of 0, 5, 100 and 300 NTU, respectively, being significantly lower than the viability of the initial isolate (P < 0.01)., Conclusions: SODIS method significantly reduced the potential viability of C. parvum oocysts on increasing the percentage of oocysts that took up the dye PI (indicator of cell wall integrity), although longer exposure periods appear to be required than those established for the bacterial pathogens usually tested in SODIS assays. SODIS.

Species belonging to the genera Cryptosporidium are recognized as waterborne pathogens. Solar water disinfection (SODIS) is a simple method that involves the use of solar radiation to destroy pathogenic microorganisms that cause waterborne diseases. A notable increase in water temperature and the existence of a large number of empty or partially excysted (i.e. unviable) oocysts have been observed in previous SODIS studies with water experimentally contaminated with Cryptosporidium parvum oocysts under field conditions. The aim of the present study was to evaluate the effect of the temperatures that can be reached during exposure of water samples to natural sunlight (37-50 degrees C), on the excystation of C. parvum in the absence of other stimuli. In samples exposed to 40-48 degrees C, a gradual increase in the percentage of excystation was observed as the time of exposure increased and a maximum of 53.81% of excystation was obtained on exposure of the water to a temperature of 46 degrees C for 12 h (versus 8.80% initial isolate). Under such conditions, the oocyst infectivity evaluated in a neonatal murine model decreased statistically with respect to the initial isolate (19.38% versus 100%). The results demonstrate the important effect of the temperature on the excystation of C. parvum and therefore on its viability and infectivity.

Cryptosporidium parvum is a well-known waterborne and opportunistic intracellular protozoan parasite that causes diarrheal illness. In this study, we quantitatively investigated reduction of the infectivity of C. parvum after gamma irradiation and repair of the infectivity during incubation time after irradiation. C. parvum oocysts were subjected to gamma irradiation at various doses (1, 5, 10, and 25 kGy), and the in vitro infectivity was measured by real-time PCR every day up to 7 days after irradiation. The in vitro infectivity of C. parvum on human ileocecal adenocarcinoma cells (HCT-8) was effectively reduced (> 2 log(10)) by irradiation at 10 kGy or more. However, in the experiment to find out repair of the infectivity, recovery was not noted until day 7 post-incubation.

The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P < 0.01). Inactivation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day(-1).

Apicomplexan parasites differ from their host by possessing at least two distinct types (long and short) of replication protein A large subunits (RPA1). Different roles for the long and short types of RPA1 proteins have been implied in early biochemical studies, but certain details remained to be elucidated. In the present study, we have found that the Cryptosporidium parvum short-type RPA1 (CpRPA1A) was highly expressed at S-phase in parasites during the early stage of merogony (a cell multiplication process unique to this group of parasites), but otherwise present in the cytosol at a much lower level in other cell-cycle stages. This observation indicates that CpRPA1A is probably responsible for the general DNA replication of the parasite. On the other hand, the long-type CpRPA1B protein was present in a much lower level in the early life cycle stages, but elevated at later stages involved in sexual development, indicating that CpRPA1B may play a role in DNA recombination. Additionally, CpRPA1B could be up-regulated by UV exposure, indicating that this long-type RPA1 is probably involved in DNA repair. Collectively, our data implies that the two RPA1 proteins in C. parvum are performing different roles during DNA replication, repair and recombination in this parasite.

Clinical toxoplasmosis in humans has been epidemiologically linked to the consumption of drinking water contaminated by Toxoplasma gondii oocysts. We evaluated killing of T. gondii oocysts after ultraviolet (UV) or ozone treatments by bioassay in mice and/or cell culture. A 4-log inactivation of the oocyst/sporozoite infectivity was obtained for UV fluences >20 mJ cm(-2). In contrast, oocysts were not inactivated by ozone with an exposure (Ct) up to 9.4 mg min l (-1) in water at 20 degrees C. In conclusion, UV treatment can be an effective disinfection method to inactivate T. gondii oocysts in drinking water, but ozone did not show promise in this research.

Aims: To determine the effect of solar radiation on Cryptosporidium parvum in tap and environmental waters., Methods and Results: Outdoor tank experiments and a cell culture infectivity assay were used to measure solar inactivation of C. parvum oocysts in different waters. Experiments conducted on days with different levels of solar insolation identified rapid inactivation of oocysts in tap water (up to 90% inactivation within the first hour). Increased dissolved organic carbon content in environmental waters decreased solar inactivation. The role of solar ultraviolet (UV) in inactivation was confirmed by long-pass filter experiments, where UV-B was identified as the most germicidal wavelength. Reductions in oocyst infectivity following solar radiation were not related to a loss of excystation capacity., Conclusions: Solar UV can rapidly inactivate C. parvum in environmental waters., Significance and Impact of the Study: This is the first study to assess natural sunlight inactivation of C. parvum oocysts in surface waters and drinking water using an infectivity measure and determines the wavelengths of light responsible for the inactivation. The findings presented here provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in aquatic environments and identify solar radiation as a critical process affecting the oocyst survival in the environment.

Cryptosporidium parvum is an organism that threatens public health in the water industry. It is critical to develop improved detection methods as well as disinfection methods for protecting against cryptosporidiosis, which is caused by C. parvum. In this study, we investigated the ability of pulsed-light irradiation at 200-900 nm to inactivate C. parvum. Absolute quantitative real-time PCR was performed with cDNA made from total RNA extracted from C. parvum oocysts or HCT-8 cells infected with C. parvum oocysts in vitro. C. parvum oocysts in 100-mL quartz flasks were positioned 20, 30, and 40 cm from the light source, and the duration of irradiation was either 5 or 60 s. The reductions in oocyst viability (4.9 log10) and infectivity (6 log10) were maximal when the C. parvum oocysts were irradiated 20 cm from the pulsed-light source for 60 s, for which the UV dose was 278 mJ/cm2. The minimum dose of pulsed-UV light required for effective reduction in C. parvum infectivity (2 log10) was 15 mJ/cm2, which was achieved by 5 s of irradiation at 30 cm from the light source. This study confirmed that short-duration pulsed-UV light is an effective disinfection measure for C. parvum.

Schistosoma mansoni occurs in tropical regions where levels of ultraviolet B (UVB; 290-320 nm) light are elevated. However, the effects of UVB on parasite transmission are unknown. This study examines effects of UVB on the miracidia and sporocysts of S. mansoni, focusing specifically on intramolluscan development, infectivity, and the ability to photoreactivate (repair DNA damage using visible light). Histology revealed that miracidia irradiated with 861 J x m(-2) underwent abnormal development after penetrating Biomphalaria glabrata snails. Total number of sporocysts in snail tissues decreased as a function of time postinfection (PI), among both nonirradiated and irradiated parasites; however, this decrease was greater in the latter. Moreover, whereas the proportion alive of nonirradiated sporocysts increased PI, that of irradiated sporocysts, i.e., derived from irradiated miracidia, decreased. Irradiation of miracidia with UVB resulted in decreased prevalence of patent infection (defined by presence of daughter sporocysts) in a dose-dependent manner, and no infections occurred at a dose of 861 J x m(-2). Like many aquatic organisms, including the snail host, parasites were able to photoreactivate if exposed to visible light following UVB irradiation, even subsequent to penetrating snails. These photoreactivation results suggest cyclobutane-pyrimidine dimers in DNA as the primary mechanism of UVB damage, and implicate photoreactivation, rather than nucleotide excision, as the main repair process in S. mansoni.

An experimental protocol was developed to assess the efficacy of two UV reactors (medium-pressure UVaster), and a low-pressure reactor) on the infectivity of Cryptosporidium parvum oocysts under conditions mimicking small- or medium-size water distribution units. The protocol included purification of large amounts of viable oocysts from experimentally infected calf feces, pilot spiking, sample concentration and purification after UV radiation, oocyst quantification and in vitro evaluation of oocyst infectivity on HCT-8 cells. Water samples were collected at intervals upstream and downstream from the UV reactor after spiking. Oocysts were concentrated by centrifugation, purified by immunomagnetic capture and quantified using laser-scanning cytometry. An enhanced in vitro infectivity test on HCT-8 cells was developed, where oocysts were pretreated in order to obtain maximized in vitro infectivity, and infectious foci were enumerated after immunofluorescence staining after 3 days of culture. This method was superior to viability measured by excystation for assessing oocyst infectivity. The infectivity rate of untreated oocysts ranged between 9% and 30% in replicate experiments. The method allowed us to determine inactivation rates >4.92 (log) with UVaster and >4.82 with the LP reactor after exposition of oocysts to an effective dose of 400 J m(-2) at flow rates of 15 and 42 m(3) h(-1), respectively.

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

The results of a batch-process solar disinfection (SODIS) and solar photocatalytic disinfection (SPCDIS) on drinking water contaminated with Cryptosporidium are reported. Cryptosporidium parvum oocyst suspensions were exposed to natural sunlight in Southern Spain and the oocyst viability was evaluated using two vital dyes [4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)]. SODIS exposures (strong sunlight) of 8 and 12h reduced oocyst viability from 98% (+/-1.3%) to 11.7% (+/-0.9%) and 0.3% (+/-0.33%), respectively. SODIS reactors fitted with flexible plastic inserts coated with TiO2 powder (SPCDIS) were found to be more effective than those which were not. After 8 and 16 h of overcast and cloudy solar irradiance conditions, SPCDIS reduced oocyst viability from 98.3% (+/-0.3%) to 37.7% (+/-2.6%) and 11.7% (+/-0.7%), respectively, versus to that achieved using SODIS of 81.3% (+/-1.6%) and 36.0% (+/-1.0%), respectively. These results confirm that solar disinfection of drinking water can be an effective household intervention against Cryptosporidium contamination.

Inactivation of Toxoplasma gondii oocysts occurred with exposure to pulsed and continuous UV radiation, as evidenced by mouse bioassay. Even at doses of >or=500 mJ/cm2, some oocysts retained their viability.

When filter-feeding shellfish are consumed raw, because of their ability to concentrate and store waterborne pathogens, they are being increasingly associated with human gastroenteritis and have become recognized as important pathogen vectors. In the shellfish industry, UV depuration procedures are mandatory to reduce pathogen levels prior to human consumption. However, these guidelines are based around more susceptible fecal coliforms and Salmonella spp. and do not consider Cryptosporidium spp., which have significant resistance to environmental stresses. Thus, there is an urgent need to evaluate the efficiency of standard UV depuration against the survival of Cryptosporidium recovered from shellfish. Our study found that in industrial-scale shellfish depuration treatment tanks, standard UV treatment resulted in a 13-fold inactivation of recovered, viable C. parvum oocysts from spiked (1 x 10(6) oocysts liter (-1)) Pacific oysters. Depuration at half power also significantly reduced (P < 0.05; ninefold) the number of viable oocysts recovered from oysters. While UV treatment resulted in significant reductions of recovered viable oocysts, low numbers of viable oocysts were still recovered from oysters after depuration, making their consumption when raw a public health risk. Our study highlights the need for increased periodic monitoring programs for shellfish harvesting sites, improved depuration procedures, and revised microbial quality control parameters, including Cryptosporidium assessment, to minimize the risk of cryptosporidiosis.

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 x 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 x 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 x cm-2 of UV light. Similar results were obtained with C. hominis. C. parvum oocysts exposed to 10 to 40 mJ x 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.

We used the fluorescent dye monochlorobimane (MCB) which binds glutathione (GSH) to localize between 2 and 6 distinctly labelled nuclear and cytoplasmic GSH foci in recently excreted and aged, intact Cryptosporidium parvum oocysts and sporozoites. Buthionine sulfoximine (BSO), a potent and specific inhibitor of GSH, was used to determine whether GSH is synthesized in BSO-treated C. parvum oocysts, by labelling treated oocysts with MCB. Both visual and electronic quantifications were performed. At 5 mM BSO, a significant inhibition of MCB fluorescence, reflecting reduced MCB uptake, was observed in GSH-depleted oocysts (mean +/- S.D. 35 +/- 3.7) compared with controls (3.3 +/- 1.2, P = 0). This clear reduction occurred only in viable oocysts. 1 mM BSO-treated oocysts exhibited weak or no MCB fluorescence, although they were viable (excluded propidium iodide, PI)), and intact and contained sporozoites by differential interference contrast microscopy (DIC). MCB was used in conjunction with PI to determine C. parvum oocyst viability. Oocysts labelled with MCB/PI or 4'6-diamidino-2-phenyl indole (DAPI)/PI produced comparable labelling patterns. Viable oocysts were labelled with MCB or DAPI whereas dead oocysts were labelled with PI only. The localization of GSH in viable, intact oocysts and excysted sporozoites and UV light-irradiated oocysts and sporozoites revealed no changes in MCB uptake at levels up to 40 mJ.cm(-2) irradiation. Although GSH can be detected following MCB localization in both the nucleus and cytoplasm of sporozoites, and can be specifically depleted by BSO treatment, MCB is unlikely to be useful as a surrogate for detecting UV damage in UV-treated Cryptosporidium oocysts.

Aim: To determine whether batch solar disinfection (SODIS) can be used to inactivate oocysts of Cryptosporidium parvum and cysts of Giardia muris in experimentally contaminated water., Methods and Results: Suspensions of oocysts and cysts were exposed to simulated global solar irradiation of 830 W m(-2) for different exposure times at a constant temperature of 40 degrees C. Infectivity tests were carried out using CD-1 suckling mice in the Cryptosporidium experiments and newly weaned CD-1 mice in the Giardia experiments. Exposure times of > or =10 h (total optical dose c. 30 kJ) rendered C. parvum oocysts noninfective. Giardia muris cysts were rendered completely noninfective within 4 h (total optical dose >12 kJ). Scanning electron microscopy and viability (4',6-diamidino-2-phenylindole/propidium iodide fluorogenic dyes and excystation) studies on oocysts of C. parvum suggest that inactivation is caused by damage to the oocyst wall., Conclusions: Results show that cysts of G. muris and oocysts of C. parvum are rendered completely noninfective after batch SODIS exposures of 4 and 10 h (respectively) and is also likely to be effective against waterborne cysts of Giardia lamblia., Significance and Impact of the Study: These results demonstrate that SODIS is an appropriate household water treatment technology for use as an emergency intervention in aftermath of natural or man-made disasters against not only bacterial but also protozoan pathogens.

Among many waterborne diseases the giardiasis and cryptosporidiosis are of particular public health interest, because Giardia cysts and Cryptosporidium oocysts can persist for long periods in the environment, and both pathogenic protozoa have been implicated as the cause of many outbreaks of gastroenteritis in the last 25 years. In order to evaluate the efficiency of cysts and oocysts' removal by the activated sludge process, and by UV reactor in inactivating cysts and oocysts in one wastewater treatment plant (WWTP) of Campinas, three sampling points were selected for study: (1) influent, (2) treated effluent without UV disinfection and (3) treated effluent with UV disinfection. Giardia spp. cysts prevailed with higher density in the three different sample types. Cryptosporidium spp. oocysts were observed in only two samples of influent and just one sample of treated sewage with UV disinfection. In the animal infectivity assay for Giardia spp, one mouse of the UV treated group revealed trophozoites in intestinal scrapings. The results of the present study indicate that treatment by activated sludge process delivered a reduction of 98.9% of cysts and 99.7% of oocysts and UV disinfection was not completely efficient regarding the inactivation of Giardia cysts in the case of the WWTP studied.

Shellfish have been identified as a potential source of Cryptosporidium infection for humans. The inactivation of C. parvum and other pathogens in raw molluscan shellfish would provide increased food safety for normal and at-risk consumers. The present study examined the efficacy of two alternative food-processing treatments, e-beam irradiation and microwave energy, on the viability of C. parvum oocysts in Eastern Oysters (Crassostrea virginica), which were artificially infected with the Beltsville strain of C. parvum. The effects of the treatments were evaluated by oral feeding of the processed oyster tissues to neonatal mice. Significant reductions (P<0.05) in infectivity were observed for in-shell and shucked oysters treated with e-beam irradiation at doses of 1.0, 1.5, or 2 kGy vs. untreated controls. A dose of 2 kGy completely eliminated C. parvum infectivity and did not adversely affect the visual appearance of the oysters. Oyster tissue treated with microwave exposures of 1 s (43.2 degrees C), 2 s (54.0 degrees C), and 3 s (62.5 degrees C) showed a reduction in C. parvum mouse infectivity, but the effects were not significantly different (P>0.05) from controls. Microwave energy treatments at 2 and 3 s showed extensive changes in oyster meat texture and color. Thus, because of lack of efficacy and unacceptable tissue changes, microwave treatment of oysters is not considered a viable food-processing method.

Numerous studies have demonstrated the efficiency of ultraviolet (UV) radiation for the inactivation of oocysts of Cryptosporidium parvum. In these studies inactivation is measured as reduction in oocysts. A primary goal is to estimate the UV radiation required to achieve a high degree of inactivation. Different lots of Cryptosporidium parvum oocysts are used in these studies, and the inactivation rate may vary depending on the lot of oocysts used. The goal of this paper is to account for the error in estimating the amount of inactivation after exposure to UV radiation, and for the effect of lot variability in determining the required UV radiation. A Bayesian approach is used to simultaneously model the logistic dose-response model and the UV inactivation kinetic model. The oocysts lot variability is incorporated using a hierarchical Bayesian model. Posterior distributions using Markov Chain Monte Carlo method is used to obtain estimates and Bayesian credible interval for the required UV radiation to achieve a given inactivation level of Cryptosporidium parvum oocysts.

The Cryptosporidium spp. UV disinfection studies conducted to date have used Cryptosporidium parvum oocysts. However, Cryptosporidium hominis predominates in human cryptosporidiosis infections, so there is a critical need to assess the efficacy of UV disinfection of C. hominis. This study utilized cell culture-based methods to demonstrate that C. hominis oocysts displayed similar levels of infectivity and had the same sensitivity to UV light as C. parvum. Therefore, the water industry can be confident about extrapolating C. parvum UV disinfection data to C. hominis oocysts.

The results of batch-process solar disinfection (SODIS) of Cryptosporidium parvum oocysts in water are reported. Oocyst suspensions were exposed to simulated sunlight (830 W m(-2)) at 40 degrees C. Viability assays (4',6'-diamidino-2-phenylindole [DAPI]/propidium iodide and excystation) and infectivity tests (Swiss CD-1 suckling mice) were performed. SODIS exposures of 6 and 12 h reduced oocyst infectivity from 100% to 7.5% (standard deviation = 2.3) and 0% (standard deviation = 0.0), respectively.

The purpose of this study was to determine whether gamma-irradiated Cryptosporidium parvum oocysts could elicit protective immunity against cryptosporidiosis in dairy calves. Cryptosporidium parvum Iowa strain oocysts (1 x 10(6) per inoculation) were exposed to various levels of gamma irradiation (350-500 Gy) and inoculated into 1-day-old dairy calves. The calves were examined daily for clinical signs of cryptosporidiosis, and fecal samples were processed for the presence of C. parvum oocysts. At 21 days of age, the calves were challenged by oral inoculation with 1 x 10(5) C. parvum oocysts and examined daily for oocyst shedding and clinical cryptosporidiosis. Calves that were inoculated with C. parvum oocysts exposed to 350-375 Gy shed C. parvum oocysts in feces. Higher irradiation doses (450 or 500 Gy) prevented oocyst development, but the calves remained susceptible to C. parvum challenge infection. Cryptosporidium parvum oocysts exposed to 400 Gy were incapable of any measurable development but retained the capacity to elicit a protective response against C. parvum challenge. These findings indicate that it may be possible to protect calves against cryptosporidiosis by inoculation with C. parvum oocysts exposed to 400-Gy gamma irradiation.

To evaluate the effectiveness of UV irradiation in inactivating Cryptosporidium parvum oocysts, the animal infectivities and excystation abilities of oocysts that had been exposed to various UV doses were determined. Infectivity decreased exponentially as the UV dose increased, and the required dose for a 2-log(10) reduction in infectivity (99% inactivation) was approximately 1.0 mWs/cm(2) at 20 degrees C. However, C. parvum oocysts exhibited high resistance to UV irradiation, requiring an extremely high dose of 230 mWs/cm(2) for a 2-log(10) reduction in excystation, which was used to assess viability. Moreover, the excystation ability exhibited only slight decreases at UV doses below 100 mWs/cm(2). Thus, UV treatment resulted in oocysts that were able to excyst but not infect. The effects of temperature and UV intensity on the UV dose requirement were also studied. The results showed that for every 10 degrees C reduction in water temperature, the increase in the UV irradiation dose required for a 2-log(10) reduction in infectivity was only 7%, and for every 10-fold increase in intensity, the dose increase was only 8%. In addition, the potential of oocysts to recover infectivity and to repair UV-induced injury (pyrimidine dimers) in DNA by photoreactivation and dark repair was investigated. There was no recovery in infectivity following treatment by fluorescent-light irradiation or storage in darkness. In contrast, UV-induced pyrimidine dimers in the DNA were apparently repaired by both photoreactivation and dark repair, as determined by endonuclease-sensitive site assay. However, the recovery rate was different in each process. Given these results, the effects of UV irradiation on C. parvum oocysts as determined by animal infectivity can conclusively be considered irreversible.