Impact of Sample Collection and Processing on the Infectivity of Cryptosporidium

The objective of this project was to identify potential deleterious effects of select methods of water sample collection and processing on the viability and infectivity of C. parvum oocysts. From a public health standpoint, it has become increasingly important to not only discern the presence of oocysts in untreated and treated waters, but to identify whether these oocysts are infectious and could threaten public health. However, the effects of sample collection and processing have not been previously developed or studied. Oocyst robustness after purification by various techniques (ethyl acetate, ethyl ether, cesium chloride gradients, and sucrose density gradients) as well as the effects of pretreatment with bleach and antibiotics prior to cell culture infectivity studies were evaluated. After selection of the most appropriate purification techniques, several batches of C. parvum oocysts were evaluated to determine the infectivity of both young (<30 days of age) and aged (>30 days of age) oocysts. Finally, various sampling concentration techniques such as pleated membrane filtration (Gelman and Cryptec cartridge filters), continuous flow centrifugation, and EPA Method 1623, (specifically for Cryptosporidium) including immuno-magnetic separation (IMS), were evaluated for their effects on the infectivity of C. parvum oocysts.