UV Disinfection sensitivity index of spores or protozoa: A model to predict the required fluence of spores or protozoa.

During UV disinfection, the required UV dose in terms of fluence depends upon the species of bacteria spore and protozoa. To rank their UV disinfection sensitivity, spore sensitivity index (SPSI) and protozoan sensitivity index (PSI) are defined. For spores, shoulder effect exists, therefore, SPSI is defined as the ratio between the ki of any spores for the linear portion of the dose response curve to the kir of Bacillus subtilis as the reference spore. After statistical analysis, the fluence of any spore can be predicted by SPSI through equation, H = (0.8358 ± 0.126)*LogI*SPSI + H0. PSI is defined as the ratio between the inactivation rate constants of a protozoa in reference to that of Cryptosporidium parvum. The equation predicting the fluence of any protozoa in reference to Cryptosporidium parvum is: H = 107.45*(3.86 ± 2.68)*LogI/PSI. Two regression equations suggest that protozoa require significantly higher UV dose than bacteria spores.

Urban water reuse: microbial pathogens control by direct filtration and ultraviolet disinfection.

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.

Virus, protozoa and organic compounds decay in depurated oysters.

AIMS: (1) Evaluate the dynamic of the depuration process of Crassostrea gigas oysters using different ultraviolet doses with different amounts of contaminants (virus, protozoa and organic contaminants) and (2) investigate the morphological changes in the oysters' tissues produced by the depuration procedures. METHODS: The oysters were allocated in sites with different degrees of contamination and analyzed after 14 days. Some animals were used as positive controls by artificial bioaccumulation with HAdV2 and MNV1 and subjected to depuration assays using UV lamps (18 or 36 W) for 168 h. The following pollutants were researched in the naturally contaminated oysters, oysters after 14 days in sites and oysters during the depuration processes: virus (HAdV, HAV, HuNoV GI/GII and JCPyV), by (RT) qPCR; protozoa (Cryptosporidium and Giardia species), by immunomagnetic separation and immunofluorescence; and organic compounds (AHs, PAHs, LABs, PCBs and organochlorine pesticides-OCs), by chromatography. Changes in the oysters' tissues produced by the depuration processes were also evaluated using histochemical analysis by light microscopy. In the artificially bioaccumulated oysters, only HAdV2 and MNV1 were investigated by (RT) qPCR before the depuration procedures and after 96 and 168 h of these procedures. RESULTS: At 14 days post-allocation, HAdV was found in all the sites (6.2 × 105 to 4.4 × 107 GC g(-1)), and Giardia species in only one site. Levels of PCBs and OCs in the oyster's tissues were below the detection limit for all samples. AHs (3.5 to 4.4 µg g(-1)), PAHs (11 to 191 ng g(-1)) and LABs (57 to 751 ng g(-1)) were detected in the samples from 3 sites. During the depuration assays, we found HAdV, Giardia and Cryptosporidium species until 168 h, independent of UV treatment. AHs, PAHs and LABs were found also after 168 h of depuration (36 W and without UV lamp). The depuration procedures did not produce changes in the oysters' tissues. In the artificially contaminated and depurated oysters, we detected HAdV until 168 h and MNV1 until 96 h of depuration. CONCLUSION: The applied depuration treatments were unable to eliminate the protozoa or to degrade the HAdV genomes but were able to degrade the MNV1 genomes. Similarly, the UV water treatment was not efficient for aliphatic hydrocarbons, PAHs and LABs, as their concentrations were equivalent or higher to the concentrations of the control samples and samples from depuration tanks without UV treatment.

Direct and indirect QMRA of infectious Cryptosporidium oocysts in reclaimed water.

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 ß-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.

Recombinant thioredoxin peroxidase from Cryptosporidium parvum has more powerful antioxidant activity than that from Cryptosporidium muris.

Cryptosporidium parvum can survive exposure to harsh environmental conditions, various disinfectants, and high doses of γ-irradiation. In an animal study, more than 25kGy of γ-irradiation was necessary to eliminate C. parvum infectivity from mice. In contrast, Cryptosporidium muris (murine Cryptosporidium), which lives in stomach epithelium, lost its infectivity in mice with 1kGy of γ-irradiation. Recently, it was found that thioredoxin peroxidase was highly expressed in C. parvum oocysts irradiated with high doses of γ-irradiation. Therefore we hypothesize that antioxidant activity of the thioredoxin peroxidase is involved in the radioresistance of C. parvum. To verify this, thioredoxin peroxidases of C. parvum (CpTPx) and C. muris (CmTPx) were expressed in Escherichia coli cells, and their antioxidant activities were compared. Both CpTPx and CmTPx belong to the 2-Cys family of peroxiredoxins. Hydrogen peroxide consumption was approximately 2- to 12-fold greater in recombinant CpTPx (rCpTPx) than in recombinant CmTPx (rCmTPx) in the presence of 0.2mM dithioerythritol or glutathione (GSH), respectively. The peroxidase activity of rCpTPx was highly enhanced by GSH, but that of rCmTPx was not. The minimum dose of rCpTPx required to protect supercoiled plasmid DNA from damage by metal-catalyzed oxidation was only 12% of that required with rCmTPx. The results showed that rCpTPx has more powerful antioxidant activity than rCmTPx. Further investigations on the role of CpTPx in the radioresistance of C. parvum are warranted.

Comparison of resistance to γ-irradiation between Cryptosporidium parvum and Cryptosporidium muris using in vivo infection.

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.

Effect of solar disinfection on viability of intestinal protozoa in drinking water.

The effect of solar disinfection on the viability of intestinal protozoa Giardia lamblia, Microsporidia sp., Cryptosporidium parvum, Cyclospora cyatenensis and Entamoeba histolytica in drinking water was studied as compared to chlorine disinfection. The protozoa were collected from stool samples, to infect to the distilled water. Chlorinated water samples were prepared at concentration of 4 ppm, and the parasites were incubated overnight at room temperature with the treated water. Sun treatment was applied for 2 exposures (6 & 24 hrs), in summer and winter. Sun treated water samples were put in tubes and exposed to sun. The 2 disinfection methods were tested in plastic and glass test tubes. Parasites viability was assessed by viability assay using trypan blue stain (0.4%), and bioassay infectivity tests in experimentally laboratory bred mice. Results proved that all parasites' viability was not affected by chlorine, following solar disinfection treatment, parasites became dark blue in colour and deformed by trypan blue stain. High parasites death was recorded for all parasites except Microsporidia sp. Bioassay infectivity test showed a statistically significant reduction in mean number of all parasites in intestinal sections compared to controls. The best results were tubes exposure to sun for 24 hrs in summer, where G. lamblia, C. parvum and C. cyatenensis were inactivated or absence in intestinal sections. No statistically significant difference was between the use of plastic and glass tubes, either in chlorine or sun treated parasites. So, solar disinfection proved a simple, cheap and effective means for improving water for human use, particularly in developing countries.

Detection of UV-induced thymine dimers in individual Cryptosporidium parvum and Cryptosporidium hominis oocysts by immunofluorescence microscopy.

To investigate the effect of UV light on Cryptosporidium parvum and Cryptosporidium hominis oocysts in vitro, we exposed intact oocysts to 4-, 10-, 20-, and 40-mJ 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.

Evaluation of activated sludge treatment and the efficiency of the disinfection of Giardia species cysts and Cryptosporidium oocysts by UV at a sludge treatment plant in Campinas, south-east Brazil.

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.

A Bayesian analysis of mouse infectivity data to evaluate the effectiveness of using ultraviolet light as a drinking water disinfectant.

Modelling disinfectant performance using Bayesian hierarchical methods can overcome problems with traditional methods and lead to improved estimates. Animal and cell-culture assays are used to estimate the degree of inactivation of a microorganism produced by a given disinfectant dose. Assay data traditionally are analyzed with logistic model or most probable number (MPN) method. These methods are limited particularly when assays show all (or no) animals or cells to be infected-estimates are reported as greater than (or less than) a measurement limit (i.e., censored data). The proposed Bayesian approach (1) properly models the propagation of uncertainty through the data analysis/modelling process, resulting in reduced model uncertainty, and (2) uses appropriate probability distribution models for the response variables, avoiding the censored data problem and more accurately describing statistical error when estimating dose-response behavior. This paper applies the Bayesian hierarchical models to logistic and MPN data from published papers for the ultraviolet (UV) inactivation of Cryptosporidium. Results are compared to those from three alternative models. The Bayesian model estimates a significantly lower UV dose for a given level of Cryptosporidium inactivation than the alternative models, due mainly to the reduced model uncertainty.

UV inactivation of Cryptosporidium hominis as measured in cell culture.

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.

Risk and control of waterborne cryptosporidiosis.

Cryptosporidium remains at the forefront of studies on waterborne disease transmission and abatement. The impact of environmental land use patterns which contribute animal and human waste, climatic precipitation leading to a strong association with outbreaks, and community infrastructure and water treatment are now recognized as contributing factors in the potential for waterborne spread of the protozoan. Advances in detection methodologies, including the ability to genotype various strains of this organism, have shown that human wastes are often the source of the contamination and cell culture techniques have allowed insight into the viability of the oocyst populations. Currently water treatment has focused on UV and ozone disinfection as most promising for the inactivation of this protozoan pathogen.

Interaction between Cryptosporidium oocysts and water treatment coagulants.

The electrokinetic properties of gamma-irradiated Cryptosporidium oocysts in the presence of coagulants (ferric chloride and alum) and coagulant aids (DADMAC based cationic polyelectrolytes) have been studied. The zeta potential of the oocysts was unaffected by the addition of ferric chloride at all pH values (3-10) studied. Addition of alum resulted in reversal of the oocysts charge, which suggests that the initial stage in the coagulation process leading to floc formation proceeds via the adsorption of hydrolysed aluminium species. The cationic polyelectrolyte Magnafloc LT35 was adsorbed onto iron flocs at doses of 0.1 mg/L even against an electrostatic barrier. The cationic polyelectrolyte only adsorbed and caused charge reversal at the oocyst surface at around 0.4 mg/L, suggesting a lower affinity for this surface. These results indicate that the oocysts, unlike inorganic colloidal materials such as metal oxides, appear to possess a lower surface density of active or charged sites. The lower density of sites, combined with the rapid precipitation of iron salts, may be responsible for the lack of specific adsorption of either hydroxylated ferric species or primary iron hydroxide particles on the oocysts. Further, this suggests that a process of sweep flocculation, where oocysts are engulfed in flocs during coagulation and floc formation, is the more likely mechanism involved. By comparison, it is likely that the specific interaction of hydrolysed aluminium species with the oocysts surface would result in a stronger link at the oocyst-floc interface and that the flocculation process may initially proceed via charge neutralisation.