Impact of purification procedures on the viability and infectivity of Cryptosporidium parvum oocysts

The development of new technologies for Cryptosporidium oocyst detection as well as inactivation and removal is at the forefront of research objectives for the drinking and wastewater industries. One of the major issues associated with testing new technology is determining oocyst viability and infectivity before and after treatment. Because oocysts must be isolated from feces, preparation and pretreatment procedures may affect oocyst infectivity and potentially confound results obtained during survival and disinfection studies. The principal objective of this study was to evaluate the effects of preparation and pretreatment on C. parvum oocyst (Ames, Iowa isolate) infectivity in an in vitro cell culture assay. In vitro excystation, sporozoite yield, and vital dye exclusion using DAPI and PI were used to test viability. A matrix of purification procedures using two defatting agents (ethyl acetate and ethyl ether), cesium chloride (CsCl) and Sheather's solution was evaluated. Effects of immuno-magnetic separation (IMS) and bleach treatment were also assessed. Oocysts purified using CsCl alone showed the most consistent infection from experiment to experiment, compared to preparations that were purified using a defatting agent. Defatting agents, IMS and bleach treatment had no detrimental effects on oocyst infectivity, however, considerable variability between oocyst lots was observed. This study determined that both purification processes and age affect oocyst infectivity.