Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts

Several in vitro surrogates have been developed as convenient, user-friendl y alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts, Such viability assays have been used incre asingly to determine oocyst inactivation following treatment with chemical, physical, or environmental stresses. Defining the relationship between in vitro viability assays and oocyst infectivity in susceptible hosts is criti cal for determining the significance of existing oocyst inactivation data f or these in vitro assays and their suitability in future studies. In this s tudy, four viability assays were compared with mouse infectivity assays, us ing neonatal CD-1 mice. Studies were conducted in the United States and Uni ted Kingdom using fresh (<1 month) or environmentally aged (3 months at 4 d egrees C) oocysts, which were partially inactivated by ozonation before via bility and/or infectivity analyses. High levels of variability were noted w ithin and between the viability and infectivity assays in the U.S. and Unit ed Kingdom studies despite rigorous control over oocyst conditions and disi nfection experiments. Based on the viability analysis of oocyst subsamples from each ozonation experiment, SYTO-59 assays demonstrated minimal change in oocyst viability, whereas 4',6'-diamidino-2-phenylindole-propidium iodid e assays, in vitro excystation, and SYTO-9 assays showed a marginal reducti on in oocyst viability. In contrast, the neonatal mouse infectivity assay d emonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in v itro viability assays cannot be used to reliably predict oocyst inactivatio n following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable a lternative viability surrogates are identified for disinfection studies.