The number of Cyclospora outbreaks due to contamination of produce has continued to rise over the last decade with 2018 marking the first outbreak associated with domestic produce. This increased risk has led to numerous studies focused on surveillance of Cyclospora in raw sewage, treated wastewater, irrigation water, and on produce. Since 2014, CPS has invested $1.6M in Cyclospora research, with efforts primarily focused on surveillance. Results of this investment confirmed that Cyclospora is endemic in the U.S. and can be recovered from wastewater and irrigation water, which poses a significant risk for produce contamination. Despite this risk, few studies have investigated methods to inactivate Cyclospora due to two bottlenecks: 1) the inability to culture the organism has limited the number of oocysts that can be collected for inactivation studies; and 2) the methods for determining oocyst viability are laborious, requiring a trained parasitologist to microscopically validate the sporulation of oocysts. The objectives of this 2 year project are: 1) systematically evaluate inactivation of Cyclospora oocysts by gamma radiation, ultraviolet radiation (UV), ozonation, and chlorine dioxide gas (ClO2); 2) develop a high-throughput, automated method for determining inactivation of Cyclospora oocysts; and 3) employ the automated method to screen a library of chemical compounds for inactivation of Cyclospora. To meet these objectives, a parallel workflow will be established, where inactivation of Cyclospora by the four primary strategies will be evaluated using manual microscopy as the “gold standard” to determine inactivation, while the high-throughput, automated method is simultaneously developed. By standardizing the methods used to determine inactivation it will be possible to compare the results between the different methods. The inactivation protocol will consist of 1×104 oocysts/treatment in water with the same concentration of natural organic matter (NOM), at a starting pH of 7.0, and constant temperature of 25°C. The efficiency of an inactivation method will be reported as the number of intact oocysts after application of the test method and the % sporulation of oocysts. This will capture both physical disruption of oocysts and inactivation of intact oocysts. Both metrics are important to characterize the efficiency of inactivation and decreasing the overall risk of Cyclospora infection. In year 2, the automated system will be used to evaluate inactivation of Cyclospora oocysts from a library of chemical targets that will be identified (Objective 3), but not tested, in year 1. Using the automated system, inactivation of Cyclospora oocysts will be measured for each of the target compounds with the number of compounds tested determined by the availability of oocysts. This study will identify ≥ 2 methods for inactivation of Cyclospora oocysts to reduce or eliminate the risks associated with agricultural water inputs. In addition, the successful methods will be evaluated for the feasibility of their use on produce and in the produce processing environment, with consideration paid to cost, availability, effectiveness, and regulation. Further, by developing a system for automated determination of oocyst viability, it will be possible for other researchers to engage in further Cyclospora inactivation studies with reduced effort.