ASSESSMENT OF A DYE PERMEABILITY ASSAY FOR DETERMINATION OF INACTIVATION RATES OF CRYPTOSPORIDIUM-PARVUM OOCYSTS

The ability to determine inactivation rates of Cryptosporidium parvum oocysts in environmental samples is critical for assessing the public health hazard of this gastrointestinal parasite in watersheds, We comp ared a dye permeability assay, which tests the differential uptake of the fluorochromes 4'-6-diamidino-2-phenylindole (DAPI) and propidium i odide (PI) by the oocysts (G. T, Campbell, L, J, Robertson, and H. V, Smith, Appl, Environ. Microbiol. 58:3488-3493, 1992), with an in vitro excystation assay, which tests their ability to excyst and, thus, the ir metabolic potential and potential for infectivity (J, B, Rose, H, D arbin, and C, P, Gerba, Water Sci, Technol, 20:271-276, 1988), Formald ehyde-fixed (killed) oocysts and untreated oocysts were permeabilized with sodium hypochlorite and subjected to both assays, The results of the dye permeability assays were the same, while the excystation assay showed that no excystation occurred in formaldehyde-fixed oocysts, Th is confirmed that oocyst wall permeability, rather than metabolic acti vity potential, was the basis of the dye permeability viability assess ment. A previously developed protocol (L, J, Anguish and W, C, Ghiorse , Appl, Environ, Microbiol, 63:724-733, 1997) for determining viabilit y of oocysts in soil and sediment was used to examine further the use of oocyst permeability status as an indicator of oocyst viability in f ecal material stored at 4 degrees C and in water at various temperatur es, Most of the oocysts in fresh calf feces were found to be impermeab le to the fluorochromes. They were also capable of excystation, as ind icated by the in vitro excystation assay, and were infective, as indic ated by a standard mouse infectivity assay, The dye permeability assay further showed that an increase in the intermediate population of ooc ysts permeable to DAPI but not to PI occurred over time, There was als o a steady population of oocysts permeable to both dyes, Further exper iments with purified oocysts suspended in distilled water showed that the shift in oocyst populations from impermeable to partially permeabl e to fully permeable was accelerated at temperatures above 4 degrees C , This sequence of oocyst permeability changes was taken as an indicat or of the oocyst inactivation pathway, Using the dye permeability resu lts, inactivation rates of oocysts in two fecal pools stored in the da rk at 4 degrees C for 410 and 259 days were estimated to be 0.0040 and 0.0056 oocyst day(-1), respectively, The excystation assay gave simil ar inactivation rates of 0.0016 and 0.0079 oocyst day(-1), These resul ts demonstrate the utility of the dye permeability assay as an indicat or of potential viability and infectivity of oocysts, especially when combined with improved microscopic methods for detection of oocysts in soil, turbid water, and sediments (L, J, Anguish and W, C, Ghiorse, A ppl, Environ, Microbiol, 63:724-733, 1997).