Intact Cryptosporidium parvum oocysts isolated after in vitro excystation are infectious to neonatal mice

In vitro excystation is often used as a measure of viability of encysted protozoan parasites. Parasites that do not excyst in vitro are assumed to be non-viable and non-infectious, whereas those that do excyst are assumed viable. To test the validity of these assumptions, Cryptosporidium parvum oocysts were excysted in vitro using two different excystation protocols, and the non-excysted intact oocysts were isolated using flow cytometry. Non-excysted sorted oocysts readily infected neonatal CD-1 mice. Increasing the duration of the excystation assays from 1 h to 3 h resulted in a higher percent of excysted oocysts, but the remaining non-excysted parasites were still capable of infecting neonatal CD-1 mice. Our results suggest that in vitro excystation is not an accurate measure of the viability or infectious potential of C. parvum oocysts.     

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.     

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.     

Ozone inactivation of Cryptosporidium parvum in demand-free phosphate buffer determined by in vitro excystation and animal infectivity.

Inactivation of Cryptosporidium parvum oocysts by ozone was performed in ozone demand-free 0.05 M phosphate buffer (pH 6.9) in bench-scale batch reactors at 7 and 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times ranging from 5 to 15 min. The viability of the control and treated oocysts was determined by using in vitro excystation and infection in neonatal CD-1 mice. It was found that excystation consistently underestimated inactivation when compared with animal infectivity (P < or = 0.05). As inactivations increased, the difference between excystation and infectivity also increased. The inactivation kinetics of C. parvum by ozone deviated from the simple first-order Chick-Watson model and was better described by a nonlinear Hom model. The use of the Hom model for predicting inactivation resulted in a family of unique concentration and time values for each inactivation level rather than the simple CT product of the Chick-Watson model.     

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.     

Comparison of animal infectivity and nucleic acid staining for assessment of Cryptosporidium parvum viability in water

Cryptosporidium parvum oocysts were stained with the fluorogenic dyes SYTO-9 and SYTO-59 and sorted by flow cytometry in order to determine whether the fluorescent staining intensity correlated with the ability of oocysts to infect neonatal CD-1 mice. Oocysts that did not fluoresce or that displayed weak fluorescent intensity when stained with SYTO-9 or SYTO-59 readily established infections in mice, whereas those oocysts that fluoresced brightly did not. Although fluorescent staining profiles varied among different batches of oocysts, a relative cutoff in fluorescent staining intensity that correlated with animal infectivity was observed for all batches.     

Dose response of Cryptosporidium parvum in outbred neonatal CD-1 mice.

Cryptosporidium parvum infectivity in a neonatal CD-1 mouse model was used to determine the dose needed to infect 50% of the population. The 50% infective dose was estimated to be 79 oocysts. It was observed that a mean oral inoculum of 23 oocysts produced infection in 2 of 25 neonatal mice 7 days postinoculation. All animals became infected when the mean oral dose exceeded 310 oocysts per animal. The dose response of C. parvum was modeled with a logit dose-response model suitable for use in water disinfection studies.     

Effect of batch-process solar disinfection on survival of Cryptosporidium parvum oocysts in drinking water

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.     

Viability and infectivity of Cryptosporidium parvum oocysts are retained upon intestinal passage through a refractory avian host.

Six Cryptosporidium-free Peking ducks (Anas platyrhynchos) were each orally inoculated with 2.0 x 10(6) Cryptosporidium parvum oocysts infectious to neonatal BALB/c mice. Histological examination of the stomachs jejunums, ilea, ceca, cloacae, larynges, tracheae, and lungs of the ducks euthanized on day 7 postinoculation (p.i.) revealed no life-cycle stages of C. parvum. However, inoculum-derived oocysts extracted from duck feces established severe infection in eight neonatal BALB/c mice (inoculum dose, 2.5 x 10(5) per mouse). On the basis of acid-fast stained direct wet smears, 73% of the oocysts in duck feces were intact (27% were oocyst shells), and their morphological features conformed to those of viable and infectious oocysts of the original inoculum. The fluorescence scores of the inoculated oocysts, obtained by use of the MERIFLUOR test, were identical to those obtained for the feces-recovered oocysts (the majority were 3+ to 4+). The dynamics of oocyst shedding showed that the birds released a significantly higher number of intact oocysts than the oocyst shells (P < 0.01). The number of intact oocysts shed (87%) during the first 2 days p.i. was significantly higher than the number shed during the remaining 5 days p.i. (P < 0.01) and significantly decreased from day 1 to day 2 p.i. (P < 0.01). The number of oocyst shells shed during 7 days p.i. did not vary significantly (P > 0.05). The retention of infectivity of C. parvum oocysts after intestinal passage through an aquatic bird has serious epidemiological and epizootiological implications. Waterfowl may serve as mechanical vectors for the waterborne oocysts and may enhance contamination of surface waters with C. parvum. As the concentration of Cryptosporidium oocysts in source waters is attributable to watershed management practices, the watershed protection program should consider waterfowl as a potential factor enhancing contamination of the source water with C. parvum.     

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.     

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.     

Batch solar disinfection inactivates oocysts of Cryptosporidium parvum and cysts of Giardia muris in drinking water

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.     

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 degrees 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.     

Impact of zooplankton grazing on the excystation, viability, and infectivity of the protozoan pathogens Cryptosporidium parvum and Giardia lamblia

Very little is known about the ability of the zooplankton grazer Daphnia pulicaria to reduce populations of Giardia lamblia cysts and Cryptosporidium parvum oocysts in surface waters. The potential for D. pulicaria to act as a biological filter of C. parvum and G. lamblia was tested under three grazing pressures (one, two, or four D. pulicaria grazers per 66 ml). (Oo)cysts (1 x 10(4) per 66 ml) were added to each grazing bottle along with the algal food Selenastrum capricornutum (6.6 x 10(4) cells per 66 ml) to stimulate normal grazing. Bottles were rotated (2 rpm) to prevent settling of (oo)cysts and algae for 24 h (a light:dark cycle of 16 h:8 h) at 20 degrees C. The impact of D. pulicaria grazing on (oo)cysts was assessed by (i) (oo)cyst clearance rates, (ii) (oo)cyst viability, (iii) (oo)cyst excystation, and (iv) oocyst infectivity in cell culture. Two D. pulicaria grazers significantly decreased the total number of C. parvum oocysts by 52% and G. lamblia cysts by 44%. Furthermore, two D. pulicaria grazers significantly decreased C. parvum excystation and infectivity by 5% and 87%, respectively. Two D. pulicaria grazers significantly decreased the viability of G. lamblia cysts by 52%, but analysis of G. lamblia excystation was confounded by observed mechanical disruption of the cysts after grazing. No mechanical disruption of the C. parvum oocysts was observed, presumably due to their smaller size. The data provide strong evidence that zooplankton grazers have the potential to substantially decrease the population of infectious C. parvum and G. lamblia in freshwater ecosystems.     

Infectivity of Cryptosporidium parvum oocysts following cryopreservation.

This study was undertaken to investigate the cryopreservation of Cryptosporidium parvum oocysts. Oocysts purified from mouse feces were suspended in distilled water, 10% glycerin, and 2.5% potassium dichromate. They were stored at -20 C and -80 C for 2, 7, and 30 days, respectively. In addition to the purified oocysts, the feces of C. parvum-infected mice were preserved under the same conditions described above. Purified and fecal oocysts were thawed at 4 C, and their viability was assessed by a nucleic acid stain, excystation test, tissue culture infectivity test, and infectivity to immunosuppressed adult mice. Oocysts purified from fecal material prior to cryopreservation lost most of their viability and all of their infectivity for tissue culture and mice. However, when oocysts were cryopreserved in feces, between 11.7 and 34.0% were judged to be viable and retained their infectivity for mice when stored at -20 C (but not -80 C) for 2, 7, and 30 days. Clearly, fecal material provides a cryoprotective environment for C. parvum oocysts stored at -20 C for at least 30 days.     

Long-term survival of Cryptosporidium parvum oocysts in seawater and in experimentally infected mussels (Mytilus galloprovincialis).

Transmission of infectious oocysts of Cryptosporidium parvum via surface- and drinking-water supplies has been reported and many surface waters flow into the sea, potentially causing runoff of animal-infected faeces. Eating raw mussels is a common practice in many countries, increasing the public's risk of acquiring enteric pathogens. The aims of the present study were to estimate how long C. parvum oocysts remain infectious in artificial seawater, to determine if the oocysts are retained in mussel tissues (Mytilus galloprovincialis), and how long they maintain their infectivity. Oocysts were incubated in artificial seawater at 6-8 degrees C under moderate oxygenation and the infectivity of oocysts was tested five times, over a 12 month period after incubation in seawater, in BALB/c mice. Each pup was inoculated per os with 10(5) oocysts and killed 5 days p.i. Oocysts remained infectious for 1 year. Forty mussels held in an aquarium containing artificial seawater filtered out more than 4 x 10(8) oocysts in a 24 h period. Oocysts were detected in the gill washing up to 3 days p.i., in the haemolymph up to 7 days p.i., and in the intestinal tract up to 14 days p.i. Oocysts collected from the gut of mussels 7 and 14 days p.i. were observed to have infected mice. These results suggest that C. parvum oocysts can survive in seawater for at least 1 year and can be filtered out by benthic mussels, retaining their infectivity up to 14 days, so seawater and molluscs are a potential source of C. parvum infection for humans.     

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.     

Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.

A viability assay for oocysts of Cryptosporidium parvum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples.     

Viability of Cryptosporidium parvum oocysts: correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.

A viability assay for oocysts of Cryptosporidium parvum based on the inclusion or exclusion of two fluorogenic vital dyes, 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide, was developed by using several different isolates of oocysts. Correlation of this assay with viability measured by in vitro excystation was highly statistically significant, with a calculated correlation coefficient of 0.997. In this research, two similar excystation protocols were utilized, and no significant difference between excystation protocols was detected. Percent excystation of oocyst suspensions could be increased or reduced by inclusion of a preincubation treatment in either excystation protocol, and this alteration was also demonstrated in the viability assay. Oocysts which excluded both dyes would not excyst in vitro unless a further trigger was provided and were more resistant to acid or alkali treatment. The results of this research provide a reproducible, user-friendly assay which is applicable to individual oocysts and also provides a useful adjunct for identification of oocysts in water and environmental samples.