HCT-8 cells were seeded at a density of 2 × 104 cells per well in 24-well flat-bottom plates. Monolayers were allowed to grow at 37 °C in a 5% CO2 atmosphere and to reach 90% confluence before the infectivity assay. Excysted or not, oocysts (104 per well) were inoculated onto monolayers. During this study, the excystation procedure was adapted from those described elsewhere such as (i) 1.5% sodium taurocholate (NaTC; Sigma, Steinheim, Germany) solution in RPMI 1640 medium at 37 °C for 1 h [21]; (ii) 2.63% aqueous sodium hypochlorite at room temperature for 10 min [18]; and (iii) the combination of 2.63% aqueous sodium hypochlorite at room temperature for 10 min, then centrifugation at 15,000× g for 10 min, and 1.5% NaTC solution in RPMI 1640 medium at 37 °C for 1 h. After the excystation, the parasitic suspension (oocysts and sporozoites) was centrifugated at 15,000× g for 10 min and resuspended in a final volume of 1 mL in co-culture medium (RPMI 1640 medium supplemented with 45 mg/mL of glucose (Sigma), 40 μg/mL of para-aminobenzoic acid (Sigma), 40 μg/mL of 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid “HEPES” (Sigma), 0.35 mg/mL of ascorbic acid (Sigma), 1 UI/mL of insulin (Sigma), 100 UI/mL of penicillin (Panpharma, Luitré-Dompierre, France), and 100 pg/mL streptomycin (Panpharma) before inoculation onto the monolayer. Parasitic suspensions were deposited in flat-bottom 24-well plates with and without cells in a co-culture medium and incubated at 37 °C in a 5% CO2 atmosphere. Infection was stopped at 0, 16, 24, 48, and 72 h post-inoculation. Oocysts inoculated into wells without HCT-8 cells were used as “background” controls for each time point. Experiments were done at least in triplicate. Control assays with unexcysted and heat-inactivated (95 °C for 15 min) oocysts were performed in conjunction with each series of tests.

The limit of CC–qPCR detection was determined using a dilution series of C. parvum parasites at concentrations of 104 to 100 (one) oocysts. Unexcysted oocysts were directly inoculated onto monolayers; the assay was stopped at 48 h post-inoculation. Experiments were performed in triplicate and repeated thrice.

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