The intestinal Apicomplexan parasite Cryptosporidium (Crypto) is a leading cause of diarrhea and death in malnourished young children and untreated HIV/AIDS patients in the resource-constrained world and a major cause of waterborne outbreaks in developed countries Despite the global burden of Crypto, treatment options for cryptosporidiosis are severely limited. There are several constraints to drug development for cryptosporidiosis. The host parasite interactions that mediate pathogenesis and the pathways and molecules that can be targeted for drug development are poorly understood. The lack of a primary human intestinal model that recapitulates human intestinal structure and function and supports continuous infection of wild type and transgenic Crypto is a major limitation to understanding pathogenesis and pre-clinical screening for anti- cryptosporidial interventions. We recently developed a bioengineered 3D primary human intestinal model in vitro using silk protein scaffolds seeded with human IEC lines and myofibroblasts. This model supported continuous C. parvum infection for at least 15 days and C. parvum from infected scaffolds could re-infect new scaffolds for up to three cycles. However, since IEC lines may not recapitulate normal human IEC structure and function, we incorporated human ileal enteroids (instead of IEC lines) into the system. Our preliminary data indicate that enteroids incorporated into this model which supports robust C. parvum infection. The overall goal of this project is to investigate host-parasite interactions of human Crypto infection and develop drug screening for cryptosporidiosis using 3D human ileal enteroid-based models under flow, peristaltic and low oxygen conditions. Our central hypothesis is that this model is a breakthrough technological advance that will expedite investigation of human Crypto infection and facilitate pre-clinical testing of interventions for cryptosporidiosis. The Specific Aims are to: 1) elucidate host-parasite interactions in Crypto infection in bioengineered primary 3D human enteroid-based tissue model under peristaltic, flow and low oxygen conditions in a bioreactor system and 2) evaluate an integrated 3D primary human enteroid-based “mini-gut” model under the same conditions for selected host-parasite interactions and as a pre-clinical model of cryptosporidiosis for screening of drugs and probiotics. Success in establishing 3D, enteroid-based human intestinal tissue models and achieving continuous propagation of infection will be transformative for Crypto research. The model can be used to study host- parasite-microbe interactions and test targeted interventions for cryptosporidiosis.