Decreased virulence in egressed parasites. Free parasites were collected after egress and counted using blood cell counting plates. (A) Egressed parasites were added to freshly prepared PCKs and incubated for 12 h; free sporozoites were suspended in 500 μL DMEM, and 10 μL suspensions were analyzed by flow cytometry after incubation. Data are means ± SEM of five replicates, representing three independent experiments. **p < 0.01. (B) Six birds were infected with egressed parasites (2 × 10⁴) via the cloacal route. Oocyst output per bird was quantified using a MacMaster chamber at 6–9 days after infection. Bars indicate means ± SEM. **p < 0.01.