Morphological and autofluorescence assessment of oocysts differentiate live from dead coccidian parasites

To assess and mitigate foodborne risk, regulatory agencies and produce growers require the means not merely to detect but moreover determine the viability of foodborne eukaryotic pathogens such as Cyclospora cayetanensis. Viability assessment would also aid those employing live attenuated vaccines against coccidiosis, a major problem in poultry production. Therefore, we sought to identify morphological changes differentiating viable from non-viable coccidian oocysts, employing Eimeria acervulina as a tractable model, enabling empirical validation by means of in vivo challenge infections in the natural chicken host. High resolution microscopic examinations identified granular structures that autofluoresce under UV exposure in dead oocysts, greatly intensifying overall autofluorescence in dead oocysts. We harnessed this intensification as a basis to sort live from dead oocysts using a Fluorescence-Activated Cell Sorting (FACS) cell sorter, validating their distinction by documenting infectivity in chickens using the former, and minimal shedding with the latter. Our rapid, sensitive, and robust assay holds promise for application to other species of coccidia, including those important to livestock and public health.