The E. tenella INRAE PAPt36 strain (Et-INRAE) was used for all experiments except for studying the effect of the microbiota on parasite invasion. E. tenella strain was isolated from a poultry farming in France in 1974. Initially referenced as E. tenella strain PAPt38 is now renamed as E. tenella-INRAE strain. The strain was maintained by regular passages on chicken in the PFIE facility since then (Laurent et al., 2001). For parasite invasion studies, Et-INRAE was transfected with nano luciferase and mcherry genes under the E. tenella actin promoter [Et-INV; (Yan et al., 2009; Swale et al., 2019)]. The mcherry gene allows sorting by flow cytometry of sporulated oocysts after inoculation of transfected sporozoites to chickens and for parasite amplification. The nano luciferase activity (NanoLuc® Luciferase, Promega) facilitates the detection of low load of parasites and accurate parasite quantification in tissues. Purification of sporozoites was performed as described by (Shirley, 1995). Briefly, 0.5 mm sterilized glass beads (Carl Roth, Karlsruhe, Germany) were added to sporulated oocysts. The oocyst wall was broken by vortexing for 17 s. Released sporocysts were washed with PBS and incubated in standard excystation medium (trypsin 0.25% and biliary salts 0.5% in PBS; pH 7.4) at 41°C for 1 h. Sporozoites were then washed in PBS and ready for cloacal inoculation.

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