Comparison of Cryptosporidium parvum Viability and Infectivity Assays following Ozone Treatment of Oocysts

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4°C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4′,6′-diamidino-2-phenylindole–propidium iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a “gold standard” until suitable alternative viability surrogates are identified for disinfection studies.     

Inter-laboratory comparison of the CD-1 neonatal mouse logistic dose-response model for Cryptosporidium parvum oocysts

cryptosporidium parvum oocyst viability can be determined by vital dyes, in vitro excystation, and cell culture; however, neonatal mouse infectivity assays are the reference method. Unfortunately, there have been few efforts to standardize methods for infectivity assays thus casting a veil of uncertainty over the significance and comparability of results. In order to address this issue, two laboratories proficient in measuring oocyst infectivity conducted independent dose titration studies with neonatal CD-1 mice using standardized protocols and a well-characterized isolate of Cryptosporidium parvum. The resulting independent logistic dose-response models derived by regression analysis were compared with each other and with a published model. The comparisons showed these dose-response functions to be reproducible under standardized conditions. It is important to standardize mouse strain, age of mice at inoculation and necropsy, oocyst isolate, and age of oocysts. However, other factors, including methods used to detect infectivity and to count oocyst doses, appear less critical. Adopting a standardized assay for oocyst infectivity will provide both a basis for comparing data from various oocyst disinfection studies and a suitable platform for evaluating new or existing in vitro viability surrogates such as excystation, vital dyes or cell culture.     

Efficacy of two peroxygen-based disinfectants for inactivation of Cryptosporidium parvum oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4',6'-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.     

Efficacy of Two Peroxygen-Based Disinfectants for Inactivation of Cryptosporidium parvum Oocysts

Two commercial peroxygen-based disinfectants containing hydrogen peroxide plus either peracetic acid (Ox-Virin) or silver nitrate (Ox-Agua) were tested for their ability to inactivate Cryptosporidium parvum oocysts. Oocysts were obtained from naturally infected goat kids and exposed to concentrations of 2, 5, and 10% Ox-Virin or 1, 3, and 5% Ox-Agua for 30, 60, and 120 min. In vitro excystation, vital dyes (4′,6′-diamidino-2-phenylindole and propidium iodide), and infectivity in neonatal BALB/c mice were used to assess the viability and infectivity of control and disinfectant-treated oocysts. Both disinfectants had a deleterious effect on the survival of C. parvum oocysts, since disinfection significantly reduced and in some cases eliminated their viability and infectivity. When in vitro assays were compared with an infectivity assay as indicators of oocyst inactivation, the excystation assay showed 98.6% inactivation after treatment with 10% Ox-Virin for 60 min, while the vital-dye assay showed 95.2% inactivation and the infectivity assay revealed 100% inactivation. Treatment with 3% Ox-Agua for 30 min completely eliminated oocyst infectivity for mice, although we were able to observe only 74.7% inactivation as measured by excystation assays and 24.3% with vital dyes (which proved to be the least reliable method for predicting C. parvum oocyst viability). These findings indicate the potential efficacy of both disinfectants for C. parvum oocysts in agricultural settings where soil, housing, or tools might be contaminated and support the argument that in comparison to the animal infectivity assay, vital-dye and excystation methods overestimate the viability of oocysts following chemical disinfection.     

Assessment of a dye permeability assay for determination of inactivation rates of Cryptosporidium parvum oocysts.

The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds. We compared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) by the oocysts (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, their metabolic potential and potential for infectivity (J.B. Rose, H. Darbin, and C.P. Gerba, Water Sci. Technol. 20:271-276, 1988). Formaldehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays. The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts. This confirmed that oocyst wall permeability, rather than metabolic activity potential, was the basis of the dye permeability viability assessment. A previously developed protocol (L. J. Anguish and W. C. Ghiorse, Appl. Environ. Microbiol. 63:724-733, 1997) for determining viability of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in fecal material stored at 4 degrees C and in water at various temperatures. Most of the oocysts in fresh calf feces were found to be impermeable to the fluorochromes. They were also capable of excystation, as indicated by the in vitro excystation assay, and were infective, as indicated by a standard mouse infectivity assay. The dye permeability assay further showed that an increase in the intermediate population of oocysts permeable to DAPI but not to PI occurred over time. There was also a steady population of oocysts permeable to both dyes. Further experiments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeable to fully permeable was accelerated at temperatures above 4 degrees C. This sequence of oocyst permeability changes was taken as an indicator of the oocyst inactivation pathway. Using the dye permeability results, inactivation rates of oocysts in two fecal pools stored in the dark at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day-1, respectively. The excystation assay gave similar inactivation rates of 0.0046 and 0.0079 oocyst day-1. These results demonstrate the utility of the dye permeability assay as an indicator of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments.     

Blind trials evaluating in vitro infectivity of Cryptosporidium oocysts using cell culture immunofluorescence

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

Chlorine dioxide inactivation of Cryptosporidium parvum oocysts and bacterial spore indicators.

Cryptosporidium parvum, which is resistant to chlorine concentrations typically used in water treatment, is recognized as a significant waterborne pathogen. Recent studies have demonstrated that chlorine dioxide is a more efficient disinfectant than free chlorine against Cryptosporidium oocysts. It is not known, however, if oocysts from different suppliers are equally sensitive to chlorine dioxide. This study used both a most-probable-number-cell culture infectivity assay and in vitro excystation to evaluate chlorine dioxide inactivation kinetics in laboratory water at pH 8 and 21 degrees C. The two viability methods produced significantly different results (P < 0.05). Products of disinfectant concentration and contact time (Ct values) of 1,000 mg. min/liter were needed to inactivate approximately 0.5 log(10) and 2.0 log(10) units (99% inactivation) of C. parvum as measured by in vitro excystation and cell infectivity, respectively, suggesting that excystation is not an adequate viability assay. Purified oocysts originating from three different suppliers were evaluated and showed marked differences with respect to their resistance to inactivation when using chlorine dioxide. Ct values of 75, 550, and 1,000 mg. min/liter were required to achieve approximately 2.0 log(10) units of inactivation with oocysts from different sources. Finally, the study compared the relationship between easily measured indicators, including Bacillus subtilis (aerobic) spores and Clostridium sporogenes (anaerobic) spores, and C. parvum oocysts. The bacterial spores were found to be more sensitive to chlorine dioxide than C. parvum oocysts and therefore could not be used as direct indicators of C. parvum inactivation for this disinfectant. In conclusion, it is suggested that future studies address issues such as oocyst purification protocols and the genetic diversity of C. parvum, since these factors might affect oocyst disinfection sensitivity.     

An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture.

Current assay methods to detect Cryptosporidium oocysts in water are generally not able to evaluate viability or infectivity. A method was developed for low-level detection of infective oocysts by using HCT-8 cells in culture as hosts to C. parvum reproductive stages. The infective foci were detected by labeling intracellular developmental stages of the parasite in an indirect-antibody assay with a primary antibody specific for reproductive stages and a secondary fluorescein isothiocyanate-conjugated antibody. The complete assay was named the focus detection method (FDM). The infectious foci (indicating that at least one of the four sporozoites released from a viable oocyst had infected a cell) were enumerated by epifluorescence microscopy and confirmed under Nomarski differential interference contrast microscopy. Time series experiments demonstrated that the autoreinfective life cycle in host HCT-8 cells began after 12 h of incubation. Through dilution studies, levels as low as one infectious oocyst were detected. The cell culture FDM compared well to other viability assays. Vital stains and excystation demonstrated that oocyst populations less than 1% viable (by vital dyes) and having a low sporozoite yield following excystation could not infect host cells. Until now, the water industry has relied on an oocyst detection method (under an information collection regulation) that is unable to determine viability. The quantifiable results of the cell culture method described demonstrate two important applications: (i) an infectivity assay that may be used in conjunction with current U.S. Environmental Protection Agency-mandated detection methodologies, and (ii) a method to evaluate oocyst infectivity in survival and disinfection studies.     

Detection of infectious Cryptosporidium oocysts by cell culture immunofluorescence assay: applicability to environmental samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Detection of Infectious Cryptosporidium Oocysts by Cell Culture Immunofluorescence Assay: Applicability to Environmental Samples

In the past few years many waterborne outbreaks related to Cryptosporidium have been described. Current methods for detection of Cryptosporidium in water for the most part rely on viability assays which are not informative concerning the infectivity of oocysts. However, for estimation of the risk of infection with Cryptosporidium this information is required. For environmental samples the oocyst counts are often low, and the oocysts have been exposed to unfavorable conditions. Therefore, determination of the infectivity of environmental oocysts requires an assay with a high level of sensitivity. We evaluated the applicability of in vitro cell culture immunofluorescence assays with HCT-8 and Caco-2 cells for determination of oocyst infectivity in naturally contaminated water samples. Cell culture assays were compared with other viability and infectivity assays. Experiments with Cryptosporidium oocysts from different sources revealed that there was considerable variability in infectivity, which was illustrated by variable 50% infective doses, which ranged from 40 to 614 oocysts, and the results indicated that not only relatively large numbers of fresh oocysts but also aged oocysts produced infection in cell cultures. Fifteen Dutch surface water samples were tested, and the cell culture immunofluorescence assays were not capable of determining the infectivity for the low numbers of naturally occurring Cryptosporidium oocysts present in the samples. A comparison with other viability assays, such as the vital dye exclusion assay, demonstrated that surrogate methods overestimate the number of infectious oocysts and therefore the risk of infection with Cryptosporidium. For accurate risk assessment, further improvement of the method for detection of Cryptosporidium in water is needed.     

Pilot-scale evaluation of UV reactors' efficacy against in vitro infectivity of Cryptosporidium parvum oocysts

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.     

Comparison of assays for Cryptosporidium parvum oocysts viability after chemical disinfection

Abstract In vitro excystation, vital dyes (4', 6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI)), and infeictivity in neonatal CD-1 mice were used to assess the viability of Cryptosporidium parvum oocysts after chemical disinfection. In vitro excystation and DAPI/PI staining provided similar estimates of viability in bench-scale experiments, but both of these methods significantly overestimated the viability when compared with infectivity (Pr ≤ 0.01). Infectivity was the most reliable measure of the viability of C. parvum oocysts following chemical disinfection.     

A Most-Probable-Number Assay for Enumeration of Infectious Cryptosporidium parvum Oocysts

Cryptosporidium is globally established as a contaminant of drinking and recreational waters. A previously described cell culture infectivity assay capable of detecting infectious oocysts was adapted to quantify viable oocysts through sporozoite invasion and clustering of foci. Eight experiments were performed by using oocysts less than 4 months of age to inoculate host HCT-8 cell monolayers. Oocysts were diluted in a standard 5- or 10-fold multiple dilution format, levels of infection and clustering were determined, and the most probable number (MPN) of infectious oocysts in the stock suspension was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, the correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20). A two-tailed P value (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumerate infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection method (FDM)-MPN assay and in BALB/c mice before and after treatment with pulsed white light (PureBrite). The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log₁₀ inactivation. Municipal water systems and a host of other water testing organizations could utilize the FDM-MPN assay for routine survival and disinfection studies.     

Environmental temperature controls Cryptosporidium oocyst metabolic rate and associated retention of infectivity

Cryptosporidium is a significant cause of water-borne enteric disease throughout the world and represents a challenge to the water industry and a threat to public health. In this study we report the use of a cell culture-TaqMan PCR assay to measure oocyst inactivation rates in reagent-grade and environmental waters over a range of temperatures. While oocysts incubated at 4 degrees C and 15 degrees C remained infective over the 12-week holding period, we observed a 4 log(10) reduction in infectivity for both 20 and 25 degrees C incubation treatments at 12 and 8 weeks, respectively, for all water types examined, a faster rate of inactivation for oocysts than previously reported. This temperature-dependent inactivation was further investigated using a simple and rapid ATP assay described herein. Time course experiments performed in reagent-grade water at incubation temperatures of 4, 15, 20, 25, 30, and 37 degrees C identified a close relationship between oocyst infectivity and oocyst ATP content, demonstrating that temperature inactivation at higher temperatures is a function of increased oocyst metabolic activity. While water quality did not affect oocyst inactivation, biological antagonism appears to be a key factor affecting oocyst removal from environmental waters. Both the cell culture-TaqMan PCR assay and the ATP assay provide a sensitive and quantitative method for the determination of environmental oocyst inactivation, providing an alternative to the more costly and time-consuming mouse infection assay. The findings presented here relating temperature to oocyst inactivation provide valuable information for determining the relative risks associated with Cryptosporidium oocysts in water.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Effects of ozone, chlorine dioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.

Purified Cryptosporidium parvum oocysts were exposed to ozone, chlorine dioxide, chlorine, and monochloramine. Excystation and mouse infectivity were comparatively evaluated to assess oocyst viability. Ozone and chlorine dioxide more effectively inactivated oocysts than chlorine and monochloramine did. Greater than 90% inactivation as measured by infectivity was achieved by treating oocysts with 1 ppm of ozone (1 mg/liter) for 5 min. Exposure to 1.3 ppm of chlorine dioxide yielded 90% inactivation after 1 h, while 80 ppm of chlorine and 80 ppm of monochloramine required approximately 90 min for 90% inactivation. The data indicate that C. parvum oocysts are 30 times more resistant to ozone and 14 times more resistant to chlorine dioxide than Giardia cysts exposed to these disinfectants under the same conditions. With the possible exception of ozone, the use of disinfectants alone should not be expected to inactivate C. parvum oocysts in drinking water.     

Aging of Cryptosporidium parvum oocysts in river water and their susceptibility to disinfection by chlorine and monochloramine.

Cryptosporidium parvum oocysts were aged in waters from both the St. Lawrence River and the Ottawa River. In situ survival experiments were carried out by incubating the oocysts in either dialysis cassettes or microtubes floated into an overflow tank. A significant portion of the oocysts survived in the test waters for several weeks. Oocyst survival in the St. Lawrence River was better in membrane-filtered (0.2 microm-pore diameter) water than in unfiltered water, suggesting that biological antagonism may play a role in the environmental fate of the parasite. Oocysts aged in river waters under in situ conditions and control oocysts kept refrigerated in synthetic water (100 ppm as CaCO3); pH 7.0) were subjected to the same disinfection protocol. Aged oocysts were at least as resistant as, if not more resistant than, the control oocysts to disinfection. This indicates that the oocysts surviving in the water environment may be just as difficult to inactivate by potable water disinfection as freshly shed oocysts. Therefore, water treatment should not be based on the assumption that environmental oocysts may be more easily inactivated than freshly shed oocysts. First-order kinetics die-off rates varied from one river to another (from 0.013 to 0.039 log(10).day(-1)) and from one experiment to another with water from the same river collected at different times. Calculation of the die-off rates based on either in vitro excystation or in vitro excystation in combination with total counts (overall die-off rates) showed that the assessment of oocyst viability by microscopic methods must account for the total oocyst loss observed during long-term inactivation assays of river waters.