This Small Business Innovation Research Phase I project will investigate the technical feasibility of an in vitro excystation-based automated cartridge assay for rapid detection of viable Cryptosporidium oocysts in water. Briefly, viable oocysts will be subjected to an in vitro excystation step to release sporozoites and the antigens for detection on a patent-pending self- contained cartridge immunoassay system. The Phase I research will focus on demonstrating the feasibility of specific detection of viable oocysts with a direct detection limit (i.e., without sample preconcentration) of d100-1000 oocysts/mL within in 1-2 h, which will be improved in Phase II to achieve the detection of d100 oocysts/10 L with sample preconcentration. The proposed assay will ultimately be highly automated and provide quantitative results for viable oocysts within 4 h from sample acquisition, largely reducing the complexity and lengthiness of current standard methods and allowing for near real-time monitoring of contamination as water leaves a drinking water treatment facility. While the United States has one of the safest water supplies in the world, our water still plays an important role i the transmission of human disease. Parasitic protozoa such as Cryptosporidium, Giardia, and Cyclospora are responsible for 21% of all waterborne illnesses. Cryptosporidium, in particular, presents unique challenges to community water systems since oocysts are resistant to standard disinfection processes (e.g., chlorination, ozonation), are small enough to pass through conventional water plant filters, and have a low infectious dose. The public health significance of dead Cryptosporidium oocysts is minimal; however, when the oocysts are infective, the risk to public health can be enormous. Unfortunately, Current EPA and conventional ELISA and PCR methods cannot determine the viability or infectivity of detected oocysts. In vitro excystation is commonly used to determine the viability of Cryptosporidium. However, in vitro excystation of Cryptosporidium is almost exclusively followed by microscopic examination to determine the number of excysted oocysts, which is tedious and time consuming and requires a sample purification step. If successfully developed, the proposed assay will address an unmet need in the market for rapid detection of viable Cryptosporidium in water. While the proposed research focuses on detection of viable Cryptosporidium only, the in vitro excystation assay principle would also be applicable to detection of other viable waterborne disease-causing protozoan parasites, e.g. Giardia and Cyclospora, significantly broadening its application and commercial appeal.