Effects of Freeze-Thaw Cycles on the Viability of Cryptosporidium parvum Oocysts in Soil

Preliminary analysis of data from three experiments on the effects of freeze-thaw events on the viability of Cryptosporidium parvum oocysts in soil was performed. The percentages of viable Cryptosporidium parvum oocysts that had undergone several freeze-thaw cycles in soil were measured, and inactivation rates due to the number of cycles were calculated. Wild type oocysts obtained from naturally infected calves were used. Soils containing oocysts in soil-filled sentinel chambers were frozen at temperatures ranging from −10°C to −20°C. Cycles up to a 27-day period were produced by thawing the containers of soil containing the Sentinel chambers every 3 days for a period of 24 hours. The viability of oocysts was measured using a dye-permeability assay with the fluorescent dyes DAPI and PI. The results showed that the inactivation rates were greater for oocysts in soil than for oocysts in water. The inactivation rates of oocysts in low moisture content soils were greater than in high moisture content soils. When comparisons were made between oocysts undergoing inactivation with several repeated freeze-thaw events were compared to those undergoing only a single freeze-thaw event, the effect appeared to be due mainly to the amount of time frozen, not to the number of cycles that occurred. The high correlation between the time that oocysts were frozen and the rate of oocyst inactivation suggests that the time at different below freezing temperature might be used as an indicator of oocyst survival in soil under field conditions.