Inactivation of Cryptosporidium Oocysts for Use in Immunological and Molecular Assay Applications

Cryptosporidium spp. are obligate, intracellular parasites that can cause life-threatening diarrhea among children and immunocompromised adults. Infection with the parasite is transmitted orally by thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Previous studies reported the inactivation of oocysts by disinfectants such as sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia. Effective concentrations of these chemicals to reduce oocyst viability can be toxic and not practical for downstream research uses of inactivated oocysts in assay development. The aim of this study was to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory. In addition, the utility of non-viable oocysts in immunological and molecular assays was examined. Inactivation experiments were performed on purified C. parvum oocysts subjected to heat (75°C/10 min) or treated with increasing concentrations of ethanol and methanol in time increments up to 24 h. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations >70% of these alcohols were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence, even after storage for 30 days at 4°C. Stored oocysts were also suitable for use in qPCR assays, either directly or following DNA extraction. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory. The ultimate goal of future work is to provide the researcher with non-infectious oocysts that can be readily used as reference reagents in the development of diagnostic assays.