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Applied and Environmental Microbiology, July 2000, p. 2972-2980, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Comparison of Cryptosporidium parvum Viability and Infectivity Assays following Ozone Treatment of Oocysts

Z. Bukhari,1,* M. M. Marshall,2 D. G. Korich,2 C. R. Fricker,3 H. V. Smith,4 J. Rosen,5 and J. L. Clancy1

Clancy Environmental Consultants, Inc., St. Albans, Vermont1; University of Arizona, Tucson, Arizona2; Thames Water Utilities, Reading,3 and Scottish Parasite Diagnostic Laboratory, Glasgow,4 United Kingdom; and Technology Planning and Management Corporation, Scituate, Massachusetts5

Received 28 December 1999/Accepted 8 March 2000

Several in vitro surrogates have been developed as convenient, user-friendly alternatives to mouse infectivity assays for determining the viability of Cryptosporidium parvum oocysts. Such viability assays have been used increasingly 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 critical for determining the significance of existing oocyst inactivation data for these in vitro assays and their suitability in future studies. In this study, four viability assays were compared with mouse infectivity assays, using neonatal CD-1 mice. Studies were conducted in the United States and United Kingdom using fresh (<1 month) or environmentally aged (3 months at 4°C) oocysts, which were partially inactivated by ozonation before viability and/or infectivity analyses. High levels of variability were noted within and between the viability and infectivity assays in the U.S. and United Kingdom studies despite rigorous control over oocyst conditions and disinfection 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 iodide assays, in vitro excystation, and SYTO-9 assays showed a marginal reduction in oocyst viability. In contrast, the neonatal mouse infectivity assay demonstrated significantly higher levels of oocyst inactivation in the U.S. and United Kingdom experiments. These comparisons illustrate that four in vitro viability assays cannot be used to reliably predict oocyst inactivation following treatment with low levels of ozone. Neonatal mouse infectivity assays should continue to be regarded as a "gold standard" until suitable alternative viability surrogates are identified for disinfection studies.

* Corresponding author. Mailing address: Clancy Environmental Consultants, Inc., P.O. Box 314, St. Albans, VT 05478. Phone: (802) 527-2460. FAX: (802) 524-3909. E-mail: zbukhari{at}together.net.

Applied and Environmental Microbiology, July 2000, p. 2972-2980, Vol. 66, No. 7
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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