The oocysts of C. parvum (KKU isolate) maintained in specific pathogen-free C57BL female mice were purified according to the method of previous papers [14,15]. The purified oocysts were surface sterilized by placing them in 10% sodium hypochlorite solution for 10 min, and they were stored for less than 2 wk at 4℃ before the experiment. As we found that C. parvum sporozoites maintained their infectivity up to 13 days in this study, we irradiated sporozoites directly and used them for the viability and in vitro infectivity studies. For excystation, oocysts were incubated in acidified Hanks' balanced salt solution (pH 2.75) for 10 min at 37℃ in a shaking incubator, followed by incubation in 4 mM sodium taurocholate (Sigma, St. Louis, Missouri, USA) in phosphate-buffered saline (PBS, pH 7.4) at 37℃ for 15 min [16]. A 1.5-ml microcentrifuge tube containing 8 × 10⁷ of C. parvum sporozoites with 1 ml of Dulbecco's modified Eagle's medium (DMEM) (Sigma) was immersed in a 50-ml tube filled with distilled water, both to obtain the backscattered ray and to reduce the temperature increase due to energy absorption from the high-dose radiation. Irradiation was performed at room temperature (22℃ for 2 hr with a ⁶⁰Co IR221 High Performance Tote Irradiator (MDS Nordion, Ottawa, Canada). The irradiation doses were 1, 5, 10, and 25 kGy, with dose rates ranging from 5 × 10² to 1.25 × 10⁴ Gy/hr. The control group was kept at room temperature during irradiation. The temperatures of the room and irradiated sample tubes were measured before and immediately after irradiation.

Human ileocecal adenocarcinoma cells (HCT-8; Korean Cell Line Bank, Seoul, Korea) were incubated in DMEM (Sigma) with 10% heat-inactivated fetal bovine serum (Gibco BRL, Gaithersburg, Maryland, USA) supplemented with L-glutamine (Sigma), 15 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.5), 14 mM sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. For routine cell passages, the HCT-8 cells were incubated at 37℃ in a humidified incubator containing 6% CO₂ and 94% air. Before inoculation of excysted oocysts (sporozoites), HCT-8 cells at a confluence of approximately 70% in a 35-mm dish were carefully rinsed with 0.1 M PBS (pH 7.2). Sporozoites (8 × 10⁶) diluted with prewarmed cryptosporidia infection medium [17] were inoculated gently onto the HCT-8 cells. After 2 hr of incubation, the infected HCT-8 cells were gently washed 3 times with 0.1 M PBS and then reincubated for another 22 hr in a humidified incubator containing 6% CO₂ and 94% air. The infection was stopped at each time point in the experiment evaluating time point of maximal infection. For the study of infectivity after gamma irradiation, infection was stopped at 24 hr after inoculation of sporozoites. After removing the culture medium, the cell monolayer was washed with 0.1 M PBS and used for RNA purification.

C. parvum viability was assayed by treating with 4 cycles of freezing (liquid nitrogen for 2 min) and thawing (95℃ for 2 min) to extract total RNA, and the infectivity was assayed by lysing C. parvum-infected HCT-8 cells in Trizol® reagent (Invitrogen, Carlsbad, California, USA). Total RNA was extracted in accordance with the manufacturer's instructions as described by Lee et al. [13]. For reverse transcription (RT), the reaction mixtures included 0.5-1.0 µg of total RNA, 10 pmol of oligo (dT)₂₀ primer (Bioneer, Daejon, Korea), 1 × Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV) RT reaction buffer, 0.25 mM dNTP mixture (TaKaRa, Otsu, Shiga, Japan), 1 U of recombinant RNasin ribonuclease inhibitor (Promega, Madison, Wisconsin, USA), and 10 U of M-MLV RT enzyme (Enzynomics, Daejeon, Korea). RT reactions were carried out for 1 hr at 42℃ and were stopped by incubation at 70℃ for 10 min.

Plasmid pGEMT-CP2 was prepared as previously described [13]. Absolute qPCR reactions were performed in a 20-µl volume using cDNA as a template. Reaction mixtures included 0.1 × LightCycler® FastStart HybProbe master mix (Roche, Mannheim, Germany), each C. parvum primer set at 0.5 µM (CP2-F, 3'-AAAACAAAAACTACTCAGACTCAAG-5', CP2-R, 3'-GGGTTGTAAACTTCATTGACTTT-5'; Bioneer), and each probe set at 0.1 µM (CP2FL, TCAATATCAGAGGTTACTTCAACTTCGC-FL; CP2LC, 705-TGGGACTGGTAAAACTATGACGTCTTC-P; TIB MOLBIO, Germany). qPCR was carried out with a LightCycler® real-time PCR system (Roche). Each qPCR mixture was subjected to an initial denaturation at 95℃ for 10 min, followed by 55 cycles of denaturation at 95℃ for 5 sec, annealing at 55℃ for 15 sec, and extension at 72℃ for 8 sec, with a final cooling step at 40℃ for 30 sec. The results were analyzed by LightCycler® software (version 4.05, Roche). DNase/RNase-free water was included as a negative control.

The viability and infectivity changes in non-irradiated C. parvum oocysts according to storage duration were measured by qPCR using a CP2 gene-specific primer and probe with cDNA converted from total RNA extracted from oocysts and HCT-8 cells infected with C. parvum. The viability of C. parvum oocysts at 4 wk was not changed, but it was reduced rapidly thereafter, and was less than 3% after 3 months compared to 1-wk-old oocysts (Fig. 1A). The infectivity of C. parvum oocysts remained at up to 98% after 3 months of storage compared to 1-wk-old fresh oocysts (Fig. 1A), but it was reduced to 71% and 45% after 6 and 9 months, respectively, and to only 0.7% after 12 months (Fig. 1A). The number of parasites infected in the HCT-8 cell line was maximal (7.2 × 10⁷) at 6 to 24 hr after inoculating cells (Fig. 1B), which represented increases of 9- and 3-folds in the inoculation dose (8 × 10⁶ sporozoites) and the number of infected parasites at 2 hr after inoculation (2.4 × 10⁷), respectively. However, the number of infected parasites decreased rapidly, with no parasites being detected at 7 days after infection (Fig. 1B).

The infectivity of C. parvum sporozoites was maintained for 13 days (the last day we studied) after excystation when they were preserved at 4℃ in cell culture medium (DMEM) without FBS (Fig. 1C). Based on the above results of biological characteristics of non-irradiated C. parvum oocysts, we irradiated sporozoites directly and observed the in vitro infectivity at 24 hr post-inoculation of parasites into host cells.

The temperatures of the irradiated sample tubes were not different before and immediately after irradiation such as in a range of 21.7 to 22.0℃ The effective reduction of in vitro infectivity (> 2 log₁₀) was achieved by more than 10 kGy irradiation (Fig. 2). During 7 days after irradiation, there was no recovery of infectivity in irradiated group with more than 10 kGy (Fig. 2). In the irradiated group with less than 5 kGy, in vitro infectivity was not reduced effectively and effective reduction of infectivity (> 2 log₁₀) was not achieved during 7 days after irradiation (Fig. 2).