A most-probable-number assay for enumeration of infectious Cryptosporidiumparvum oocysts

Cryptosporidium is globally established as a contaminant of drinking and re creational waters. A previously described cell culture infectivity assay ca pable of detecting infectious oocysts was adapted to quantify viable oocyst s through sporozoite invasion and clustering of foci, Eight experiments wer e performed by using oocysts less than 4 months of age to inoculate host HC T-8 cell monolayers, Oocysts were diluted in a standard 5- or 10-fold multi ple dilution format, levels of infection and clustering were determined, an d the most probable number (MPN) of infectious oocysts in the stock suspens ion was calculated. The MPN was compared to the initial oocyst inoculum to determine the level of correlation. For oocysts less than 30 days of age, t he correlation coefficient (r) was 0.9726 (0.9306 to 0.9893; n = 20), A two -tailed P value (alpha = 0.05) indicated that P was less than 0.0001. This strong correlation suggests that the MPN can be used to effectively enumera te infectious oocysts in a cell culture system. Age affected the degree of oocyst infectivity. Oocyst infectivity was tested by the focus detection me thod (FDM)-MPN assay and in BALB/c mice before and after treatment with pul sed white light(PureBrite), The FDM-MPN assay and animal infectivity assays both demonstrated more than a 4 log,, inactivation. Municipal water system s and a host of other water testing organizations Could utilize the FDM-MPN assay for routine survival and disinfection studies.