Cultivating the endogenous life cycle of <i>Eimeria tenella</i> in chicken intestinal organoids

Coccidiosis, caused by <i>Eimeria</i> spp., leads to substantial economic losses in the poultry industry globally. These protozoan parasites invade the intestinal epithelium of birds, impairing nutrient absorption, causing diarrhoea and potentially leading to mortality. The complex endogenous life cycle of <i>Eimeria</i> spp., particularly the gametogony phase, presents significant challenges for <i>in vitro</i> cultivation. This study aimed to develop mature chicken intestinal organoids as an <i>in vitro</i> model capable of supporting the complete endogenous life cycle of <i>Eimeria tenella</i>. Two commercially available culture media, 3dGRO L-WRN conditioned medium (L-WRN) and IntestiCult™ Organoid Growth Medium (OGM), were evaluated for their ability to support chicken intestinal organoid development. The results demonstrated that basolateral-out organoids embedded in Matrigel and cultured in the L-WRN medium expanded more rapidly. In contrast, those apical-out organoids in the OGM developed more microvilli structures on enterocytes. Apical-out organoids, initially cultured in L-WRN medium and subsequently matured in OGM, were selected as the optimal host for the <i>Eimeria</i> infection model. Sporozoites of <i>E. tenella</i> successfully invaded the organoids and progressed through both the schizogony and gametogony phases. Moreover, the parasites produced a new generation of oocysts in this study. The presence of schizonts, gametocytes, and sporulated oocysts confirmed that the model can support the full endogenous life cycle of the parasite <i>in vitro</i>. This organoid-based infection model serves as a promising platform for studying host-pathogen interactions and developing novel interventions to control avian coccidiosis.