Viability of sporulated oocysts of Neospora caninum after exposure to different physical and chemical treatments

The aim of the present study was to evaluate the viability of Neospora caninum sporulated oocysts after various chemical and physical treatments. Bioassays in gerbils and molecular techniques (PCR-RFLP) were used for identification of the oocysts shed by experimentally infected dogs. Sporulated oocysts were purified and divided into 11 treatment groups as follows: absolute ethanol for 1 hr; 20 C for 6 hr; 4 C for 6 hr; 60 C for 1 min; 100 C for 1 min; 10% formaldehyde for 1 hr; 10% ammonia for 1 hr; 2% iodine for 1 hr; 10% sodium hypochlorite for 1 hr; 70% ethanol for 1 hr; and one group was left untreated and kept as a positive control. All chemical treatments were performed at room temperature (37 C). A total of 33 gerbils, or 3 gerbils per treatment, were used for bioassays. After treatment, the oocysts were divided into aliquots of 1,000 oocysts and orally administered to gerbils. After 63 days, the gerbils were anesthetized and killed with 0.2 ml of T61; blood and tissue samples were collected for serological (IFAT and western blotting), molecular (real-time PCR), histopathology, and immunohistochemical tests. Treatments were considered effective only if all 5 detection techniques tested negative. High temperatures at 100 C for 1 min and 10% sodium hypochlorite for 1 hr were the only treatments that met this condition, effectively inactivating all oocysts.     

Effects of temperature and different food matrices on Cyclospora cayetanensis oocyst sporulation

Effects of temperature on the sporulation of the parasite Cyclospora cayetanensis were studied in 2 food substrates, dairy and basil. Unsporulated Cyclospora oocysts were subjected to freezing and heating conditions for time periods ranging from 15 min to 1 wk. Oocysts were then removed from the food substrates and placed in 2.5% potassium dichromate for 2 wk to allow viable unsporulated oocysts to differentiate and fully sporulate, and to determine the percentage sporulation as an indicator of viability. Sporulation occurred when oocysts resuspended in dairy substrates were stored within 24 hr at -15 C. When oocysts were placed in water or basil, sporulation occurred after incubation for up to 2 days at -20 C, and up to 4 days at 37 C. Few oocysts sporulated when incubated for 1 hr at 50 C. Sporulation was not observed in basil leaves or water at -70 C, 70 C, and 100 C. Sporulation was not affected when incubated at 4 C and 23 C for up to 1 wk, which was the duration of the experiment in both of the tested substrates.     

Treatment with beta-cyclodextrin of natural Cryptosporidium parvum infections in lambs under field conditions.

Following the unexpected activity of the excipient beta-cyclodextrin against experimental infection by Cryptosporidium parvum in suckling mice, its efficacy in the prevention and treatment of natural infections in lambs was evaluated under field conditions. Fifty-three crossbred neonatal lambs were randomly selected for the study. Treatment consisted of oral administration of an aqueous suspension of beta-cyclodextrin at a dose of 500 mg/kg of body weight. To test prophylactic efficacy, the suspension was administered at 1, 2 and 3 days of age. To evaluate therapeutic efficacy, the suspension was administered on each of the 3 days following onset of diarrhoea. Infection was monitored by daily examination of faecal samples, from birth to 30 days. The criteria studied in evaluating efficacy were: oocyst shedding, the presence of diarrhoea, and weight gain at 15 and 30 days. In the group that received prophylactic treatment with beta-cyclodextrin, there were no mortalities and, compared with control lambs, there was a decrease in the number of animals infected, a longer prepatent period and notable reduction in the patent period and the duration of diarrhoea. Therapeutic treatment also reduced the patent period and the severity of diarrhoea. beta-cyclodextrin was well tolerated by all of the treated animals.     

Effects of freeze-thaw events on the viability of Cryptosporidium parvum oocysts in soil

The effects of freeze-thaw events on the inactivation of Cryptosporidium parvum oocysts in soil were examined. Oocysts were inoculated into distilled water in microcentrifuge tubes or into chambers containing soil the water content of which was maintained at 3%, 43%, or 78% of the container capacity. The chambers and tubes were then embedded in 3 soil samples from different aspects of a hillside landscape (Experiments 1 and 2) and in 3 distinct soil types (Experiment 3) and frozen at -10 C. Containers were thawed every 3 days for a period of 24 hr in 1-9 freeze-thaw cycles over 27 days (Experiments 1 and 2) and 2-5 freeze-thaw cycles over 15 days (Experiment 3). Oocyst viability was measured using the fluorescent dyes 4'6-diaminidino-2-phenylindole and propidium iodide. Inactivation rates were greater in soils than in water and greater in dry soil than in moist and wet soils. Soil type showed no effect on inactivation. Oocysts subjected to freeze-thaw cycles had inactivation rates not significantly different from those of oocysts subjected to -10 C under static conditions. The results indicated that 99% of oocysts exposed to soils that are frozen at -10 C will become inactivated within 50 days whether or not freeze-thaw cycles occur.     

Effect of dung burial by the dung beetle Bubas bison on numbers and viability of Cryptosporidium oocysts in cattle dung

Cryptosporidium oocysts were inoculated into fresh dung (∼1.2 × 10⁴ oocysts per gram wet weight) and fed to dung beetles to assess the effect of dung burial by the dung beetle Bubas bison on the distribution of the oocysts in small cores of soil in the laboratory. The experiment consisted of five replicates of each of two treatments; controls (dung but no dung beetles) and the experimental treatment (inoculated dung and seven pairs of dung beetles). After 5 days, when approximately 90% of the dung was buried, the surface and buried dung was recovered and subsampled. The oocysts in the subsamples were recovered and enumerated using qPCR. Oocyst viability was 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). Results revealed that overall 13.7% of oocysts remained on the surface and 86.3% of oocysts were buried. The viability of oocysts in buried dung was only 10% compared to oocysts the surface dung (58%). Therefore, widespread dung burial by B. bison during the winter months could substantially reduce the numbers of Cryptosporidium oocysts available to be washed into waterways following winter rains.     

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.     

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.     

Relevance of Cryptosporidium parvum hsp70 mRNA amplification as a tool to discriminate between viable and dead oocysts

Two mRNA extraction methods were compared in this study to clarify the discrepancies found between authors regarding the presence of mRNA in inactivated Cryptosporidium parvum oocysts. Cryptosporidium parvum heat shock protein 70 (hsp70) mRNA extraction was performed by using oligo(dT)20-labeled magnetic beads or by incubating oocyst lysates with DNase I. Significant differences in mRNA recovery rates between these 2 techniques were observed when working on inactivated oocysts. We consistently detected hsp70 mRNA in oocysts heated at 60 C for 30 min and oocysts incubated in 10% formalin for 2 hr when using DNase I in the mRNA extraction procedure. In contrast, no mRNA was detected in such oocysts when magnetic beads were used for the mRNA extraction. The selective capture of long poly-A tail mRNA, when using oligo(dT)20-labeled magnetic beads, is proposed in this paper for explaining the discrepancies observed between the two mRNA extraction methods compared in this study. DNA decay in inactivated and aging oocysts makes quantitative polymerase chain reaction a potential alternative technique for assessing C. parvum oocyst viability status in environmental samples.     

The effect of gamma-irradiation on the viability of Cryptosporidium parvum

Cryptosporidium parvum is 1 of the major causative organisms in waterborne diarrheal illness. Not only does C. parvum spread ubiquitously in our environment, it is also highly resistant to harsh environmental conditions and disinfectants. Therefore, a control measure for this protozoon is urgently required. This study investigated the effect of gamma-irradiation, in the range of 1,000-50,000 Gy, on the viability of C. parvum oocysts. Oocyst viability was determined by a combined indirect immunofluorescence and nucleic acid staining and animal infectivity study. The proportion of viable oocysts estimated by nucleic acid staining ranged from 94.2 to 89.4% in the 0- to 10,000-Gy groups, whereas it was reduced significantly to 58.6 or 45.7% in the 25,000- or 50,000-Gy group, respectively, at 24 hr postirradiation. In an animal infectivity study, oocysts irradiated with less than 10,000 Gy induced infections in mice wherein there were low numbers of oocysts per gram of feces amounting to 8-10.8% of the values in control mice, whereas with 50,000 Gy-irradiated oocysts, no oocysts were produced in the mice. This study suggests that at least 50,000 Gy of gamma-irradiation is necessary for the complete elimination of oocyst infectivity in mice.     

The Effects of pH,Buffers, Bile and Bile Acids on Excystation of Sporozoites of Various Eimeria Species*

SYNOPSIS. Oocysts of Eimeria bovis were found to undergo excystation when subjected at 39 C to a pretreatment consisting of exposure for 24 hr to CO₂ and air (50–50), and a treatment for 7 hr with a mixture of bile and trypsin. At pH's of 6.0 thru 10.0 with tris-maleate buffer, excystation occurred over the entire range of pH tested, with the highest levels at pH 7.5-8.5. No adverse or inhibitive effect on excystation or the viability of the sporozoites was observed. Disintegration of sporozoites occurred within the sporocysts of intact oocysts at each of the pH levels studied when boric acid-borax, ammediol, and glycine-sodium hydroxide buffers were used in the treatment medium. Phosphate buffer inhibited excystation when used in the excysting medium. Excystation occurred at levels above 90% in all dilutions of taurocholic, glycocholic, glycotaurocholic, and cholic acids included in the study (0.5-10.0%) except for the 10% and 5% dilutions of cholic acid and the 10% dilution of glycotaurocholic acid. In the latter 3 dilutions, sporozoites within the sporocysts of intact oocysts disintegrated. Excystation levels above 90% were observed in the 50% and 10% dilutions of fresh bovine bile, and in the 5% dilution of lyophilized bovine bile. Lower levels of excystation occurred in greater dilutions of both kinds of bile. No excystation occurred when any of the bile acids, fresh bovine bile or lyophilized bile were used without trypsin, except for fresh bile that contained a heavy suspension of bacteria and fungi. In a medium containing trypsin and heat-treated bile, heat-treated bile acids, or no bile, 2.5–8% of the oocysts excysted. The findings indicate that satisfactory excystation can be obtained with a treatment medium containing tris-maleate at pH 7.5–8.5, 0.25% trypsin, and 1% of one of the bile acids.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.     

Cultivation of Eimeria tenella in Japanese quail embryos (Coturnix coturnix japonica).

Complete development of Eimeria tenella in Japanese quail embryos was observed. Sporozoites were inoculated into the allantoic cavity of 7-day-old Japanese quail embryos (Coturnix coturnix japonica), after which the infected embryos were incubated at 41 C. In the chorioallantoic membrane mature first generation schizonts, mature second generation schizonts, and gametes were detected at 48 hr postinoculation of sporozoites (PI), 84 hr PI, and 126 hr PI, respectively. Mature gametes and zygotes were found at 132 hr PI, and oocysts were detected at 138 hr PI. Mortality of embryos increased with increment of inoculum size of sporozoites. LD50 was 1.7 x 10(2) sporozoites. Oocyst production was also dependent on inoculum size. Oocysts harvested from embryos sporulated. The oocysts were inoculated into 13-day-old chickens, and oocysts, capable of sporulating normally, were recovered from ceca 7 days after inoculation.     

Survival of Toxoplasma gondii oocysts in Eastern oysters (Crassostrea virginica)

Toxoplasma gondii has recently been recognized to be widely prevalent in the marine environment. It has previously been determined that Eastern oysters (Crassostrea virginica) can remove sporulated T. gondii oocysts from seawater and that oocysts retain their infectivity for mice. This study examined the long-term survival of T. gondii oocysts in oysters and examined how efficient oysters were at removing oocysts from seawater. Oysters in 76-L aquaria (15 oysters per aquarium) were exposed to 1 x 10(6) oocysts for 24 hr and examined at intervals up to 85 days postexposure (PE). Ninety percent (9 of 10) of these oysters were positive on day 1 PE using mouse bioassay. Tissue cysts were observed in 1 of 2 mice fed tissue from oysters exposed 21 days previously. Toxoplasma gondii antibodies were found in 2 of 3 mice fed oysters that had been exposed 85 days previously. In another study, groups of 10 oysters in 76-L aquaria were exposed to 1 x 10(5), 5 x 10(4), or 1 x 10(4) sporulated T. gondii oocysts for 24 hr and then processed for bioassay in mice. All oysters exposed to 1 x 10(5) oocysts were infected, and 60% of oysters exposed to 5 x 10(4) oocysts were positive when fed to mice. The studies with exposure to 1 x 10(4) oocysts were repeated twice, and 10 and 25% of oysters were positive when fed to mice. These studies indicate that T. gondii can survive for several months in oysters and that oysters can readily remove T. gondii oocysts from seawater. Infected filter feeders may serve as a source of T. gondii for marine mammals and possibly humans.     

Carbon Dioxide as the Initial Stimulus for Excystation of Eimeria tenella Oocysts*

SYNOPSIS. The stimulus necessary to initiate in vitro excystation of the chicken coccidium Eimeria tenella was provided by exposure of intact sporulated oocysts to an atmosphere of carbon dioxide. This stimulus produced a thinning and indentation at the micropylar region and oocysts became permeable to trypsin and bile. Sporozoites became active and began to escape from sporocysts into the oocyst cavity and then to the outside thru the altered micropyle after incubation in the enzyme-bile mixture. Activation of sporozoites when CO₂-pretreated oocysts were incubated in trypsin and bile, was used as the criterion to determine the number of oocysts responding to the initial stimulus. Thus, activation of sporozoites within intact oocysts was an indirect measurement of the number of oocysts stimulated during CO₂-pretreatment. Approximately 90% of the oocysts contained active sporozoites after 18 hr of pretreatment with carbon dioxide and 8 hr incubation in trypsin and bile at 38 or 41 C, respectively. Pretreatment of oocysts with air, N₂, O₂, or He resulted in 8% or less activation during incubation in trypsin and bile. Approximately 83% of the oocysts responded to the stimulus during 8 hr CO₂-pretreatment at 41 C, whereas at 38 C, 16 hr of pretreatment were required for a similar response. The stimulus did not elicit a response from oocysts held at 23 C during the pretreatment gasphase. No significant difference occurred in number of oocysts containing active sporozoites after sufficient CO₂-pretreatment for maximum stimulation of oocysts and incubation in trypsin and bile at 38 or 41 C.     

Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil

Toxoplasma gondii oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples.     

Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation.

Centrifugation was evaluated as a method to improve infectivity assays of Cryptosporidium parvum in cell culture using the focus detection method, an immunofluorescence-based method for detecting infectious C. parvum oocysts in vitro. Human ileocecal adenocarcinoma (HCT-8) cells were grown for 48 hr on 13-mm cover slips in 24-well microtiter plates and infected with bleach-treated C. parvum oocysts. Plates were centrifuged at 228 g for 10 min and incubated at 37 C for 5, 12, 18, 24, and 48 hr. Foci of infection were stained by immunofluorescence and enumerated using epifluorescent microscopy. Results were compared to noncentrifuged controls. Foci in centrifuged samples could be enumerated after 18 hr. According to most probable number (MPN) analysis, the number of infectious oocysts estimated at 48 hr (13,326 infectious oocysts) was reached by 18 hr in centrifuged samples. After 48 hr, there was no significant difference (P < 0.05) between centrifuged and noncentrifuged samples enumerated by number of foci or the MPN of infectious oocysts. Centrifugation may expedite detection during C. parvum infectivity assays. Furthermore, multiwell plate formats are more cost effective than traditional chamber slides.     

Cyclodextrin-facilitated bioconversion of 17 beta-estradiol by a phenoloxidase from Mucuna pruriens cell cultures.

After complexation with beta-cyclodextrin, the phenolic steroid 17 beta-estradiol could be ortho-hydroxylated into a catechol, mainly 4-hydroxyestradiol, by a phenoloxidase from in vitro grown cells of Mucuna pruriens. By complexation with beta-cyclodextrin the solubility of the steroid increased from almost insoluble to 660 microM. The bioconversion efficiency after 72 hr increased in the following order: freely suspended cells (0%), immobilized cells (1%), cell homogenate (6%), phenoloxidase preparation (40%). Mushroom tyrosinase converted 17 beta-estradiol, as a complex with beta-cyclodextrin, solely into 2-hydroxyestradiol, with a maximal yield of 30% after 6-8 hr. Uncomplexed 17 beta-estradiol was not converted at all in any of these systems.     

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.     

High-yield amplification of Cryptosporidium parvum in interferon gamma receptor knockout mice

To date, large-scale production of Cryptosporidium parvum oocysts has only been achieved by amplification in neonatal calves and sheep. Many laboratories currently depend on supplies from external sources and store oocysts for prolonged periods which results in progressive loss of viability. Six to 8-week-old interferon gamma receptor knockout (IFN gamma R-KO) mice on a C57BL/6 background were inoculated by gavage (2000 oocysts/animal). Fecal pellets were collected daily from 7 days post-infection (p.i.) up to 2 weeks p.i. Intestinal oocyst yield was assessed at days 11, 12 and 14 p.i. by homogenization of intestinal tissues. Ether extraction and one or more NaCl flotations were used to purify oocysts. Total recoveries averaged 2.6 x 10(6) oocysts/mouse from fecal material and 3.8 x 10(7) oocysts/mouse from intestinal tissues. Overall, 2.3 x 10(9) purified oocysts were obtained from 60 mice. Recovered oocysts were capable of sporulation and were shown to be infectious both in vitro and in vivo. Oocyst amplification was achieved in only 11-14 days with minimal expense. The simplicity of this method presents a practical alternative for the routine passage, maintenance and storage of C. parvum in biomedical laboratories.