Inactivation Of Cryptosporidium Parvum Oocysts Research Articles

Evaluation of Cryptosporidium parvum oocyst inactivation following exposure to ultraviolet light-emitting diodes by in vitro excystation and dye staining assays.

Cryptosporidium spp. are protozoan parasites that are transmitted via fecal-oral routes and can exhibit chemical resistance. Chlorine resistance makes it very difficult to eliminate parasites present in contaminated drinking water. While the efficacy of ultraviolet light-emitting diodes (UV-LEDs) against microorganisms has been reported, the efficacy of UV-LEDs against Cryptosporidium spp. has not been fully evaluated. Here, we assessed the efficacy of UV-LEDs with peak wavelengths of 268, 275, 284, and 289nm against Cryptosporidium parvum at various exposure times, with a fixed exposure distance, using two in vitro methods. Consequently, the time required for 2 log10 inactivation through the excystation method by UV-LEDs of 268, 275, 284, and 289nm was estimated as 115.5, 104.1, 37.4, and 30.7min, respectively. The propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) staining assays estimated the inactivation time as 311.3, 275.2, 60.6, and 39.1min, respectively. Our results showed that UV-LED irradiation at longer wavelengths produced higher inactivation activity against C. parvum, which corroborates our previously reported in vivo assay results, although further study is needed to clarify the mechanism.

Inactivation of Cryptosporidium parvum oocysts and faecal indicator bacteria in cattle slurry by addition of ammonia

To determine inactivation of Cryptosporidium parvum oocysts and reduction of Escherichia coli and enterococci in cattle slurry added aqueous ammonia. Escherichia coli, enterococci and nonviable C. parvum oocysts (DAPI+PI+) were enumerated every second day for 2weeks in cattle slurry amended with 60mmoll-1 aq. ammonia and compared with untreated slurry at three temperatures. Regardless of temperature, the proportion of nonviable C. parvum oocysts increased significantly faster over time in slurry with added ammonia than raw slurry (P=0·021) corresponding to 62·0% higher inactivation (P=0·001) at day 14. Additionally, 91·8% fewer E. coli and 27·3% fewer enterococci were observed in slurry added ammonia at day 14 compared to raw slurry. The addition of aqueous ammonia to raw slurry significantly reduced the viability of C. parvum oocysts and numbers of bacterial indicators. Hence, ammonia is usable at lower pathogen concentrations in slurry before application to agricultural land. Livestock waste is a valuable source of plant nutrients and organic matter, but may contain high concentrations of pathogens like E. coli and Cryptosporidium sp. that can be spread in the environment, and cause disease outbreaks. However, die-off rates of pathogens in organic waste can increase following increasing ammonia concentrations.

Kinetic modeling of the synergistic thermal and spectral actions on the inactivation of Cryptosporidium parvum in water by sunlight

Water contamination with the enteroprotozoan parasite Cryptosporidium is a current challenge worldwide. Solar water disinfection (SODIS) has been proved as a potential alternative for its inactivation, especially at household level in low-income environments. This work presents the first comprehensive kinetic model for the inactivation of Cryptosporidium parvum oocysts by sunlight that, based on the mechanism of the process, is able to describe not only the individual thermal and spectral actions but also their synergy. Model predictions are capable of estimating the required solar exposure to achieve the desired level of disinfection under variable solar spectral irradiance and environmental temperature conditions for different locations worldwide. The thermal contribution can be successfully described by a modified Arrhenius equation while photoinactivation is based on a series-event mechanistic model. The wavelength-dependent spectral effect is modeled by means of the estimation of the C. parvum extinction coefficients and the determination of the quantum yield of the inactivation process. Model predictions show a 3.7% error with respect to experimental results carried out under a wide range of temperature (30 to 45 °C) and UV irradiance (0 to 50 W·m−2). Furthermore, the model was validated in three scenarios in which the spectral distribution radiation was modified using different plastic materials common in SODIS devices, ensuring accurate forecasting of inactivation rates for real conditions.

Evaluation of solar photocatalysis using TiO2 slurry in the inactivation of Cryptosporidium parvum oocysts in water

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.

Inactivation mechanisms of cryptosporidium parvum oocysts by solar ultraviolet irradiation

Direct inactivation ofCryptosporidium parvumoocysts by UVB component of sunlight is correlated with UV fluence.

Enhanced Inactivation of Cryptosporidium parvum Oocysts during Solar Photolysis of Free Available Chlorine

Solar irradiation of aqueous solutions containing free available chlorine (FAC) dramatically enhances inactivation of Cryptosporidium parvum oocysts compared to FAC or sunlight alone. In pH 8, 10 mM phosphate buffer at 25 °C, exposure to FAC alone yields no oocyst inactivation at CTFAC ≤ 832 (mg min) L–1, while exposure to simulated sunlight alone for 60 min yields 2 log oocyst inactivation. Similar enhancement effects are observed in natural water under natural sunlight. Experiments undertaken in the presence of the reactive oxygen species (ROS) scavenger tert-butanol or in the absence of oxygen indicate that these enhancements are due to in situ ROS and ozone production via FAC photolysis.

The hydrodynamics of an ozone contactor with horizontal meandering flow to enhance disinfection efficiency

The axial dispersion of an ozone contactor is known to influence Cryptosporidium parvum oocyst inactivation and bromate formation. While low dispersion is beneficial, an extent of dispersion is not specifically addressed for the design of a new ozone contactor. C. parvum oocyst inactivation, residual ozone, CT (i.e. Concentration × Time), and bromate formation were numerically simulated with diverse dispersion number (d), ranging from 0.01 to 5, using axial dispersion reactor model and kinetic parameters obtained from sand-filtered raw water under three pH and three temperature values. C. parvum oocyst inactivation was affected sensitively by the axial dispersion number especially at the region lower than 0.5. On the contrary, residual ozone, CT, and bromate formation were slightly influenced by dispersion number. The axial dispersion number of the full-scale ozone reactor with meandering horizontal flow was obtained with a tracer test. The equation for predicting the dispersion number of an ozone contactor with horizontal meandering flow was developed in the manuscript. The experimental axial dispersion number was slightly higher than the theoretically derived one because of flow disturbance in the channel of the ozone contactor.

Effect of Daily Temperature Fluctuation during the Cool Season on the Infectivity of Cryptosporidium parvum

The present work calculated the rate of inactivation of Cryptosporidium parvum oocysts attributable to daily oscillations of low ambient temperatures. The relationship between air temperature and the internal temperature of bovine feces on commercial operations was measured, and three representative 24-h thermal regimens in the approximately 15 degrees C, approximately 25 degrees C, and approximately 35 degrees C ranges were chosen and emulated using a thermocycler. C. parvum oocysts suspended in deionized water were exposed to the temperature cycles, and their infectivity in mice was tested. Oral inoculation of 10(3) treated oocysts per neonatal BALB/c mouse (approximately 14 times the 50% infective dose) resulted in time- and temperature-dependent reductions in the proportion of infected mice. Oocysts were completely noninfectious after 14 24-h cycles with the 30 degrees C regimen and after 70 24-h cycles with the 20 degrees C regimen. In contrast, oocysts remained infectious after 90 24-h cycles with the 10 degrees C regimens. The estimated numbers of days needed for a 1-log(10) reduction in C. parvum oocyst infectivity were 4.9, 28.7, and 71.5 days for the 30, 20, and 10 degrees C thermal regimens, respectively. The loss of infectivity of oocysts induced by these thermal regimens was due in part to partial or complete in vitro excystation.

Environmental Inactivation of Cryptosporidium parvum Oocysts in Waste Stabilization Ponds

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).

Photocatalytic inactivation of Cryptosporidium parvum with TiO 2 and low-pressure ultraviolet irradiation

This study investigated the efficacy of low-pressure ultraviolet (UV) irradiation and the synergistic effect of UV/titanium dioxide (TiO 2) photocatalysis on Cryptosporidium parvum oocyst inactivation. At UV doses of 2.7, 8.0, and 40 mJ/cm 2, oocyst inactivation was 1.3, 2.6, and 3.3 log 10, respectively. Reactive oxygen species (ROS) generated by longwave UV radiation (>315 nm) and TiO 2 achieved less than 0.28-log inactivation. However, the synergistic effect of germicidal (254 nm) UV and TiO 2 resulted in 2-log and 3-log oocyst inactivation with 4.0 and 11.0 mJ/cm 2, respectively. Therefore, using TiO 2 in combination with UV reduced the dose requirement for 3-log inactivation by 56%. An approximate 1-log decrease in inactivation of oocysts was observed with nanopure water in comparison to buffered water, whereas changes in pH from 6 to 8 had little effect on the photocatalytic inactivation of oocysts in either matrix ( P>0.1).

Simultaneous simulation of pathogen inactivation and bromate formation in full‐scale ozone contactors by computer software

Ozone contactor model (OCM) software was developed as an innovative tool for full‐scale ozone bubble‐diffuser contactor design and optimization in support of current and future regulations regarding pathogen and bromate control in drinking water. The software is based on the reactive transport model that has been experimentally verified with lab‐, pilot‐, and full‐scale ozone contactors in previous studies. The OCM is designed to be user‐friendly with graphical user interfaces and operates on personal computers so that users can perform complex simulation tasks under various design and operating scenarios without extensive training. The details of software structure and usage guidelines are provided in this article. Demonstrative application of the OCM for simulating the performance of a full‐scale ozone contactor with respect to Cryptosporidium parvum oocyst inactivation and bromate formation was performed using experimental results obtained from the Linnwood Water Plant Ozone Facility (LWPOF) at the Milwaukee (Wis.) Water Works. The model was further applied to simulate the performance of this ozone contactor at different operating temperatures. The simulation results suggest that meeting inactivation requirements for C. parvum oocysts is more challenging at lower temperatures, while controlling bromate formation is more challenging at higher temperatures. Additional simulations suggest that ozone contactors be designed with the lowest possible dispersion in order to achieve target inactivation efficiency with minimum formation of bromate.

Inactivation of Cryptosporidium parvum oocysts in water using ultrasonic treatment

Ultrasound in a liquid phase cause mass and heat transfer across the liquid through cavitational processes which act as nanoreactors to generate unstable mechanical equilibrium. The effect of 1 MHz ultrasound on the inactivation of Cryptosporidium parvum was investigated. Continuous irradiation of ultrasound (20 min) increased temperature due to cavitational phenomena. Ultrasound irradiation of liquid containing C. parvum showed significant quantitative changes in pH, temperature and inactivation of C. parvum (102.7 oocysts killed/s) with a minimum energy consumption (0.05 oocysts/s).

Uncertainty in prediction of disinfection performance

Predicting the disinfection performance of a full-scale reactor in drinking water treatment is associated with considerable uncertainty. In view of quantitative risk analysis, this study assesses the uncertainty involved in predicting inactivation of Cryptosporidium parvum oocysts for an ozone reactor treating lake water. A micromodel is suggested which quantifies inactivation by stochastic sampling from density distributions of ozone exposure and lethal ozone dose. The ozone exposure distribution is computed with a tank in series model that is derived from tracer data and measurements of flow, ozone concentration and ozone decay. The distribution of lethal ozone doses is computed with a delayed Chick–Watson model which was calibrated by Sivaganesan and Marinas [2005. Development of a Ct equation taking into consideration the effect of Lot variability on the inactivation of Cryptosporidium parvum oocysts with ozone. Water Res. 39(11), 2429–2437] utilizing a large number of inactivation studies. Parameter uncertainty is propagated with Monte Carlo simulation and the probability of attaining given inactivation levels is assessed. Regional sensitivity analysis based on variance decomposition ranks the influence of parameters in determining the variance of the model result. The lethal dose model turns out to be responsible for over 90% of the output variance. The entire analysis is re-run for three exemplary scenarios to assess the robustness of the results in view of changing inputs, differing operational parameters or revised assumptions about the appropriate model. We argue that the suggested micromodel is a versatile approach for characterization of disinfection reactors. The scheme developed for uncertainty assessment is optimal for model diagnostics and effectively supports the management of uncertainty.

Modeling Cryptosporidium parvum oocyst inactivation and bromate in a flow-through ozone contactor treating natural water

A reactive transport model was developed to simultaneously predict Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of natural water. A mechanistic model previously established to predict bromate formation in organic-free synthetic waters was coupled with an empirical ozone decay model and a one-dimensional axial dispersion reactor (ADR) model to represent the performance of a lab-scale flow-through ozone bubble-diffuser contactor. Dissolved ozone concentration, bromate concentration (in flow-through experiments only), hydroxyl radical exposure and C. parvum oocyst survival were measured in batch and flow-through experiments performed with filtered Ohio River water. The model successfully represented ozone concentration and C. parvum oocyst survival ratio in the flow-through reactor using parameters independently determined from batch and semi-batch experiments. Discrepancies between model prediction and experimental data for hydroxyl radical concentration and bromate formation were attributed to unaccounted for reactions, particularly those involving natural organic matter, hydrogen peroxide and carbonate radicals. Model simulations including some of these reactions resulted in closer agreement between predictions and experimental observations for bromate formation.

Modeling Cryptosporidium parvum Oocyst Inactivation and Bromate Formation in a Full-Scale Ozone Contactor

The inactivation of Cryptosporidium parvum oocysts and the formation of bromate were assessed simultaneously by performing experiments with a full-scale ozone bubble-diffuser contactor used for drinking water disinfection. Fluorescence-dyed polystyrene microspheres were used as surrogates for C. parvum oocysts. Semi-batch ozonation experiments were performed to determine the fluorescence-intensity decay of individual microspheres, which was measured by flow cytometry. The results obtained with the microspheres were correlated to the inactivation kinetics of C. parvum oocysts by choosing an appropriate threshold fluorescence intensity below which microspheres were considered to be equivalent to nonviable oocysts. A mathematical model was then used to predict the inactivation efficiency and bromate formation. The contactor hydrodynamics were characterized by running tracer tests, and the kinetic parameters for ozone decomposition and bromate formation were obtained by performing batch experiments. Model predictions were in good agreement with full-scale experimental results. Additional model simulations revealed that ozone contactors should be designed with the lowest possible backmixing so that the target inactivation efficiency can be achieved with the lowest possible formation of bromate.

Synergistic inactivation of Cryptosporidium parvum using ozone followed by monochloramine in two natural waters

The effect of sequential exposure to ozone followed by monochloramine on inactivation of Cryptosporidium parvum oocysts suspended in untreated natural surface water from two different sources was studied in bench-scale batch reactors. Animal infectivity using neonatal CD-1 mice was used to measure oocyst inactivation. A statistically significant synergistic effect on oocyst inactivation was measured in both natural water samples studied. The magnitude of the effect measured in the natural water with lower turbidity, colour, and organic carbon concentration was comparable to that previously reported for oocysts suspended in buffered de-ionized water but was reduced considerably in the natural water with higher turbidity, colour and organic carbon concentration. Synergy increased with initial pH and with the degree of ozone pre-treatment but was independent of temperature. For water treatment plants with adequate disinfectant contact times, ozone followed by monochloramine may be a practical means of achieving additional C. parvum inactivation, however, the influence of water quality characteristics should be considered.

Development of a Ct equation taking into consideration the effect of lot variability on the inactivation of Cryptosporidium parvum oocysts with ozone

Cryptosporidium parvum oocysts are prevalent in surface water and ground water under the influence of surface water, and are difficult to inactivate using free chlorine, the most common disinfectant currently used for treating drinking water. In contrast, it has been shown that ozone is a more effective disinfectant than chlorine. US EPA is currently evaluating a treatment rule, which addresses the control of C. parvum oocysts in drinking water. The use of Ct (average disinfectant concentration multiplied by characteristic contact time) values is being considered as one of the options for demonstrating adequate control of this microbial contaminant. The purpose of this study is to incorporate the variability in inactivation kinetics among different lots of oocysts and to develop a statistical model for Ct based on first-order delayed Chick–Watson inactivation kinetics. A Bayesian approach is used to estimate the delayed Chick–Watson kinetic parameters. A log-linear regression analysis is then used to represent the effect of temperature on the resulting kinetic parameters. The overall model developed in this study provides an approach for water utilities and regulatory agencies to decide on the level of safety needed when developing treatment requirements for the inactivation of C. parvum oocysts with ozone as part of broader risk assessment considerations.

Using ultraviolet light for disinfection of finished water

Ultraviolet light is now recognised to be very effective for inactivation of Cryptosporidium parvum oocysts; however, its application for disinfection of finished water necessitates validation of UV reactors prior to their installation. Although reactor performance will likely be assessed using non-pathogenic microorganisms as biodosimetry surrogates, it would be prudent for the water industry to simultaneously measure Cryptosporidium oocysts inactivation in controlled bench-scale studies using the water matrix intended for disinfection. The likelihood of that occurring is dependent upon the availability of infectivity measurement procedures that are more user-friendly than the mouse infectivity assays currently used. This study describes a modified cell culture procedure that would enable reliable measurement of changes in oocysts' infectivity following their UV treatment. Also, a number of different biodosimetry surrogates were examined and one selected for comparing the UV doses delivered between bench-scale and full-scale biodosimetry studies. Impacts of UV disinfection on production of disinfection byproducts, effects of lamp ageing on effectiveness of disinfection and the costs associated with employing this technology were also examined.

Cryptosporidium parvum oocyst inactivation in field soil and its relation to soil characteristics: analyses using the geographic information systems

The need exists to understand the environmental parameters that affect inactivation of Cryptosporidium parvum oocysts in soil under field conditions. The inactivation of C. parvum oocysts placed in the natural environment was studied at a dairy farm in western New York State, USA. Seventy sampling points were arranged in a grid with points 150 m apart using the Geographic Information System. The sampling points were distributed among three distinct areas: woodland, corn field and pasture. Purified oocysts were inoculated into chambers filled with soil from each sampling point, and buried in the surface of each respective sampling point. To compare C. parvum oocyst survival with another organism known to survive environmental stresses, Ascaris suum eggs were also placed in soil contained in chambers and buried at the same sampling points as the oocysts. As controls oocysts and eggs in distilled water were also placed at each sampling point. Oocyst and egg viability, soil pH and percent gravimetric water content were measured at all sampling points at 0, 60 and 120 day sampling periods. Soil organic content was determined for each sampling point. At 120 days after placement, mean viability of C. parvum oocysts was 10% although at a few sampling points, 30% of oocysts were still potentially infective; whereas 90% of A. suum eggs were viable at all sampling points. Statistically significant differences were not observed among the three different sampling areas, and no statistically significant predictors were found by regression analysis. Results exemplified the heterogeneity of soil parameters and oocyst viability across a landscape; such results make predictive models for C. parvum inactivation problematical. The long-term survival of C. parvum oocysts in soil under field conditions, as this study demonstrated, emphasizes their potential as a risk to contaminate surface waters.

Simultaneous prediction of Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of synthetic waters.

A model was developed to simultaneously assess Cryptosporidium parvum oocyst inactivation and bromate formation during ozonation of synthetic solutions in batch and flow-through reactors. The model incorporated 65 elementary chemical reactions involved in the decomposition of ozone and the oxidation of bromine species and their corresponding rate or equilibrium constants reported in the literature. Ozonation experiments were performed with a laboratory-scale batch reactor to evaluate the model with respect to the rate of ozone decomposition and bromate formation. The model was found to provide a good representation of experimental results when the ozone decomposition initiation reaction with hydroxide ion was assumed to produce superoxide radical instead of the alternatively proposed product hydrogen peroxide. The model was further developed to simulate the performance of a flow-through bubble-diffuser reactor with an external recirculation line. Each compartment of the reactor (bubble column and recirculation line) was assumed to behave as a plug flow reactor as supported by tracer test results, and an empirical correlation was used to represent the rate of ozone gas transfer in the bubble column. Model predictions of the performance of the flow-through ozone bubble-diffuser contactor were in good agreement with experimental results obtained for bromate formation and C. parvum oocyst inactivation under all conditions investigated. Additional model simulations revealed that hydrodynamic conditions had a more pronounced effect on C. parvum oocyst inactivation than on bromate formation. In contrast, pH had a strong effect on bromate formation without affecting the inactivation efficiency of C. parvum oocysts for a given level of exposure to ozone. These findings suggested that bromate formation could be minimized while achieving target inactivation levels for C. parvum oocysts by designing ozone reactors with hydrodynamic conditions approaching that of an ideal plug flow reactor and by lowering the pH of the target water.