Effects of low temperatures on viability of Cryptosporidium parvum oocysts.

Microcentrifuge tubes containing 8 x 10(6) purified oocysts of Cryptosporidium parvum suspended in 400 microliters of deionized water were stored at 5 degrees C for 168 h or frozen at -10, -15, -20, and -70 degrees C for 1 h to 168 h and then thawed at room temperature (21 degrees C). Fifty microliters containing 10(6) oocysts was administered to each of five to seven neonatal BALB/c mice by gastric intubation. Segments of ileum, cecum, and colon were taken for histology from each mouse 72 or 96 h later. Freeze-thawed oocysts were considered viable and infectious only when developmental-stage C. parvum organisms were found microscopically in the tissue sections. Developmental-stage parasites were not found in tissues from any mice that received oocysts frozen at -70 degrees C for 1, 8, or 24 h. All mice that received oocysts frozen at -20 degrees C for 1, 3, and 5 h had developmental-stage C. parvum; one of 6 mice that received oocysts frozen at -20 degrees C for 8 h had a few developmental-stage parasites; mice that received oocysts frozen at -20 degrees C for 24 and 168 h had no parasites. All mice that received oocysts frozen at -15 degrees C for 8 and 24 h had developmental-stage parasites; mice that received oocysts frozen at -15 degrees C for 168 h had no parasites. All mice that received oocysts frozen at -10 degrees C for 8, 24, and 168 h and those that received oocysts stored at 5 degrees C for 168 h had developmental-stage parasites. These findings demonstrate for the first time that oocysts of C. parvum in water can retain viability and infectivity after freezing and that oocysts survive longer at higher freezing temperatures.     

Effect of various environmental factors on the viability of Cryptosporidium parvum oocysts

Aims: To evaluate individual and combined effects of temperature (4, 18 and 25°C), pH (7 and 10), ammonia (5 and 50 mg l^?1) and exposure time (1, 2, 4 and 6 days) on the viability of Cryptosporidium parvum oocysts in water. Methods and Results: The viability of oocysts was evaluated using the fluorogenic vital dyes assay (4′,6‐diamidino‐2‐phenylindole and propidium iodide). All the factors analysed (temperature, pH, ammonia and exposure time) and their interaction were statistically significant (P < 0·005). Exposure of oocysts to pH 10 for 6 days at 25°C reduced oocyst viability from ～80% to 51%. Similarly, the exposure of C. parvum oocysts to 5 mg NH₃ l^?1 and 50 mg NH₃ l^?1 for 4 days reduced their viability from between ～80% to 41·5% and 14·8%, respectively. Conclusions: The interaction between pH, temperature and exposure time may have adverse effects on the survival of C. parvum oocysts in water. Low concentrations of ammonia, as commonly found in alga‐based wastewater systems, over a long period of time can produce high C. parvum oocyst inactivation rates. Significance and Impact of the Study: This study provides relevant data on the inactivation of C. parvum oocysts in alga‐based wastewater‐treatment systems in the northwest of Spain.     

Effect of high-rate algal ponds on viability of Cryptosporidium parvum oocysts.

The physicochemical conditions of high-rate algal ponds were responsible for a more than 97% reduction in the infectivity of Cryptosporidium parvum oocysts in neonatal mice. The use of semipermeable bags of cellulose showed that pH, ammonia, and/or light seems to be a major factor for the inactivation of oocysts in wastewater, supporting the importance of alga-based systems for safer reuse of treated wastewater.     

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.     

Rotifers ingest oocysts of Cryptosporidium parvum

Six genera of rotifers including Philodina, Monostyla, Epiphanes, Euchlanis, Brachionus, and Asplanchna were exposed to oocysts of Cryptosporidium parvum cleaned of fecal debris. Unstained oocysts and those stained with fluorescein-conjugated monoclonal antibody were added to suspensions of viable rotifers and were examined by phase-contrast, differential interference contrast, and fluorescence microscopy. Rotifers of all six genera were observed ingesting oocysts. A maximum of 25 oocysts was observed in the stomachs of Eauchlanis and Brachionus. Euchlanis and Epiphanes were observed excreting boluses containing up to eight oocysts. It was not determined whether rotifers digested or otherwise rendered oocysts nonviable.     

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.     

Gaseous disinfection of Cryptosporidium parvum oocysts.

Purified oocysts of Cryptosporidium parvum suspended in approximately 400 microliters of phosphate-buffered saline or deionized water in microcentrifuge tubes were exposed at 21 to 23 degrees C for 24 h to a saturated atmosphere of ammonia, carbon monoxide, ethylene oxide, formaldehyde, or methyl bromide gas. Controls were exposed to air. Oocysts in each tube were then rinsed and resuspended in fresh, deionized water, and 1 million oocysts exposed to each gas were orally administered to each of three to six neonatal BALB/c mice in replicate groups. Histologic sections of ileum, cecum, and colon tissues taken from each mouse 72 h after oral administration of oocysts were examined microscopically to determine if infection had been established. All 15 mice given oocysts exposed to carbon monoxide had numerous developmental stages of cryptosporidium in all three intestinal segments. Of 10 mice given oocysts exposed to formaldehyde, 6 had a few developmental stages of cryptosporidium in the ileum. No mice given oocysts exposed to ammonia, ethylene oxide, or methyl bromide were found to be infected. These findings indicate the efficacy of these low-molecular-weight gases (ammonia, ethylene oxide, and methyl bromide) as potential disinfectants for C. parvum oocysts where soil, rooms, buildings, tools, or instruments might be contaminated.     

Infectious Cryptosporidium parvum oocysts in final reclaimed effluent

Water samples collected throughout several reclamation facilities were analyzed for the presence of infectious Cryptosporidium parvum by the focus detection method-most-probable-number cell culture technique. Results revealed the presence of infectious C. parvum oocysts in 40% of the final disinfected effluent samples. Sampled effluent contained on average seven infectious oocysts per 100 liters. Thus, reclaimed water is not pathogen free but contains infectious C. parvum.     

Recovery and viability of Cryptosporidium parvum oocysts and Giardia intestinalis cysts using the membrane dissolution procedure

Previously, the cellulose acetate membrane filter dissolution method was reported to yield Cryptosporidium parvum oocyst recoveries of 70.5%, with recovered oocysts retaining their infectivity. In contrast, high spike doses (approximately 1 x 10(5) Cryptosporidium parvum oocysts and Giardia intestinalis cysts) yielded recoveries ranging from 0.4% to 83.9%, and 3.2% to 90.3%, respectively, in this study. Recoveries with low spike doses (approximately 100 (oo)cysts) continued to demonstrate high variability also. Efforts to optimize the method included increased centrifugation speeds, suspension of the final concentrate in deionized water for organism detection on well slides, and analysis of the entire concentrate. A comparison of two monoclonal antibodies was also conducted to identify potential differences between antibodies in detection of organisms. Archived source and finished water samples were spiked, yielding variable recoveries of C. parvum oocysts (11.8% to 71.4%) and G. intestinalis cysts (7.4% to 42.3%). Effects of organic solvents used in the membrane dissolution procedure on the viability of recovered (oo)cysts was determined using a fluorogenic vital dyes assay in conjunction with (oo)cyst morphology, which indicated > 99% inactivation. These data indicate that the membrane dissolution procedure yields poor and highly variable (oo)cyst recoveries, and also renders the majority of recovered organisms non-viable.     

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.     

Biofilms reduce solar disinfection of Cryptosporidium parvum oocysts

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

Effects of combined water potential and temperature stresses on Cryptosporidium parvum oocysts.

Hosts infected with the parasite Cryptosporidium parvum may excrete oocysts on soils in watersheds that supply public drinking water. Environmental stresses decrease the numbers of oocysts after deposition on soils. However, the rates and effects of combined stresses have not been well characterized, especially for the purposes of estimating decrease in numbers. We subjected oocysts to combined stresses of water potential (-4, -12, and -33 bars), above-freezing temperatures (4 and 30 degrees C), and a subfreezing temperature (-14 degrees C) for 1, 14, and 29 days and one to six freeze-thaw cycles (-14 to 10 degrees C) to estimate coefficients to characterize population degradation using multiplicative error and exponential decay models. The experiments were carried out in NaCl solutions with water potentials of -4, -12, and -33 bars, in combination with temperature stresses at levels that could be expected in natural soils. Increased water potential increased the rate of population degradation for all temperature conditions investigated. Enhanced degradation leads to estimated rates of population degradation that are greater than those that have been reported and used in previous studies conducted to assess risk of water supply contamination from sources of C. parvum.     

Efficacy of UV irradiation in inactivating Cryptosporidium parvum oocysts

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

Influence of surface characteristics on the stability of Cryptosporidium parvum oocysts

Microelectrophoresis is a common technique for probing the surface chemistry of the Cryptosporidium parvum oocyst. Results of previous studies of the electrophoretic mobility of C. parvum oocysts in which microelectrophoresis was used are incongruent. In this work we demonstrated that capillary electrophoresis may also be used to probe the surface characteristics of C. parvum oocysts, and we related the surface chemistry of C. parvum oocysts to their stability in water. Capillary electrophoresis results indicated that oocysts which were washed in a phosphate buffer solution had neutrally charged surfaces. Inactivation of oocysts with formalin did not influence their electrophoretic mobility, while oocyst populations that were washed in distilled water consisted of cells with both neutral and negative surface charges. These results indicate that washing oocysts in low-ionic-strength distilled water can impart a negative charge to a fraction of the oocysts in the sample. Rapid coagulation experiments indicated that oocysts did not aggregate in a 0.5 M NaCl solution; oocyst stability in the salt solution may have been the result of Lewis acid-base forces, steric stabilization, or some other factor. The presence of sucrose and Percoll could not be readily identified on the surface of C. parvum oocysts by attenuated total reflectance-Fourier transform infrared spectroscopy, suggesting that these purification reagents may not be responsible for the stability of the uncharged oocysts. These findings imply that precipitate enmeshment may be the optimal mechanism of coagulation for removal of oocysts in water treatment systems. The results of this work may help elucidate the causes of variation in oocyst surface characteristics, may ultimately lead to improved removal efficiencies in full-scale water treatment systems, and may improve fate and transport predictions for oocysts in natural systems.     

Ultrastructural changes in Cryptosporidium parvum oocysts by gamma irradiation

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

Detection of viable Cryptosporidium parvum oocysts by PCR.

PCR was used to detect and specifically identify a gene fragment from Cryptosporidium parvum. An 873-bp region of a 2,359-bp DNA fragment encoding a repetitive oocyst protein of C. parvum was shown to be specifically amplified in C. parvum. An excystation protocol before DNA extraction allowed the differentiation between live and dead Cryptosporidium parvum oocysts.     

The effect of blueberry extracts on Giardia duodenalis viability and spontaneous excystation of Cryptosporidium parvum oocysts, in vitro

The protozoan parasites Giardia duodenalis and Cryptosporidium parvum are common causes of diarrhoea, worldwide. Effective drug treatment is available for G. duodenalis, but with anecdotal evidence of resistance or reduced compliance. There is no effective specific chemotherapeutic intervention for Cryptosporidium. Recently, there has been renewed interest in the antimicrobial properties of berries and their phenolic compounds but little work has been done on their antiparasitic actions. The effect of various preparations of blueberry (Vaccinium myrtillus) extract on G. duodenalis trophozoites and C. parvum oocysts were investigated. Pressed blueberry extract, a polyphenolic-rich blueberry extract, and a commercially produced blueberry drink (Bouvrage) all demonstrated antigiardial activity. The polyphenol-rich blueberry extract reduced trophozoite viability in a dose dependent manner. At 167 microgml(-1), this extract performed as well as all dilutions of pressed blueberry extract and the Bouvrage beverage (9.6+/-2.8% live trophozoites remaining after 24h incubation). The lowest dilution of blueberry extract tested (12.5% v/v) contained >167 microgml(-1) of polyphenolic compounds suggesting that polyphenols are responsible for the reduced survival of G. duodenalis trophozoites. The pressed blueberry extract, Bouvrage beverage and the polyphenolic-rich blueberry extract increased the spontaneous excystation of C. parvum oocysts at 37 degrees C, compared to controls, but only at a dilution of 50% Bouvrage beverage, equivalent to 213 microgml(-1) gallic acid equivalents in the polyphenolic-rich blueberry extract. Above this level, spontaneous excystation is decreased. We conclude that water soluble extracts of blueberries can kill G. duodenalis trophozoites and modify the morphology of G. duodenalis and C. parvum.     

A comparison of enumeration techniques for Cryptosporidium parvum oocysts

A variety of methods have been used to enumerate Cryptosporidium parvum oocysts from source or drinking waters. The reliability of these counting methods varies, in part, with suspension density, sample purity, and other factors. Frequently, the method of determination of suspension density is not reported by authors. To confound the problem, each method of counting has large inherent variation. There is a relationship between suspension density, overall number of organisms counted, and counting mechanism accuracy that should be accounted for when selecting a counting mechanism. This study selected a maximum acceptable coefficient of variation (CV) to be 10%. A method was considered unreliable if this standard was not achieved. Flow cytometry achieved this standard at 486 oocysts/ml. Counting with a Coulter counter achieved this level of reliability at about 1,230 oocysts/ml. Neither chamber slides nor fluorescent antibody-stained well slides ever demonstrated less than 10% CV. However, estimates of the minimum required concentrations were 5,100 oocysts/ml and approximately 6,500 oocysts/ml, respectively. The hemacytometer provided counts accurate to a 10% CV at a concentration of at least 60,000 organisms/ml. Of the methods tested, flow cytometry provided the least amount of variability at low suspension densities.     

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).