Cryptosporidium oocyst viability: Assessment and correlation with infectivity.

Abstract

Outbreaks of cryptosporidiosis have been traced to Cryptosporidium oocysts in finished drinking water. Indeed, water contaminated with oocysts may be judged perfectly safe by conventional coliform tests. Although oocysts can be specifically identified using immunofluorescence, it is not yet possible to determine their viability. The lack of a viability test means that each oocyst detected in finished water must be regarded as potentially infective even though water treatment may have killed them. The goal of this research was to develop a test for oocyst viability. In vitro excystation, oocyst morphology, vital dyes, and a monoclonal antibody were tested. In vitro excystation expressed as percent of theoretical sporozoite yield correlated best with neonatal mouse infectivity. Although not directly applicable to testing water samples, excystation provided a basis for screening other testing methods. None of the eight vital dyes tested showed any relationship between oocyst staining and viability. This was presumably due to inability of the dyes to penetrate the oocyst wall. Pretreatment strategies designed to increase oocyst wall permeability were either ineffective or damaged the oocysts in ways that rendered them nonviable. Initially, microscopic appearance appeared to be related to oocyst infectivity. However, regression analysis showed that phase contrast microscopic appearance had marginal utility for use as a viability test. Indeed, microscopic identification of internal structures of intact oocysts is not a reliable viability indicator because DAPI staining showed intact sporozoite nuclei within obviously dead oocysts that would not excyst. A monoclonal antibody (MAb OW64) was found which binds to internal sites along the oocyst suture. There was positive correlation between binding of this MAb and decreasing oocyst infectivity indicating that MAb OW64 bound preferentially to nonviable oocysts. Regression analysis showed that OW64 binding overestimated oocyst viability because many nonviable oocysts did not bind the MAb. Nevertheless, MAb OW64 is a candidate for producing an immunofluorescence based test in which oocysts that bind OW64 are nonviable whereas those that do not bind are not necessarily viable. Before such a test can be recommended, however, the nonviability of oocysts that bind OW64 must be demonstrated by neonatal mouse infectivity using oocysts sorted by a fluorescence activated cell sorter.