Decrease in Cryptosporidium parvum Oocyst Infectivity In Vitro by Using the Membrane Filter Dissolution Method for Recovering Oocysts from Water Samples

Abstract

Exposure of Cryptosporidium parvum oocysts to solutions used for cellulose acetate membrane (CAM) dissolution filtration reduced their infectivity in HCT-8 cells. Ethanol (95% [vol/vol] and 70% [vol/vol]) aloneand short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.Successful detection of Cryptosporidium parvum oocysts in water samples depends on efficient filtration methods to recover oocysts. Method 1623 from the U.S. Environmental Protection Agency involves filtering a 10-liter water sample using the Gelman Envirochek membrane capsule filter that traps oocysts. The filter is then shaken to recover the oocysts in an elution buffer (15,25). Other filtration methods, such as the use of polycarbonate membranes or polypropylene cartridge filters, have been described (13,(17)(18)(19). The purpose of these methods is to recover oocysts from raw or finished water samples. Suitable filtration methods should not render oocysts noninfective through chemical or mechanical treatments so infective oocysts in samples can be determined.An alternative filtration method involves capture of oocysts on a cellulose acetate membrane (CAM) that is dissolved in acetone following filtration and is subsequently centrifuged, rinsed in ethanol, and eluted in a buffer for final recovery (1). This method has an average rate of recovery up to 70.5%, making it more reliable than other methods (1, 2). When modified into a Millipore Glass Microanalysis system, the method resulted in higher oocyst recoveries, particularly when 1 liter of the elution buffer per 25 liters of low-turbidity water was used (10).The viability of C. parvum oocysts can be determined by vital dye staining, exposing oocysts to excystation solutions, and testing their infectivity by infecting mice. In recent years various cell culture methods have been developed whereby C. parvum oocysts or sporozoites are applied to cells grown in vitro (5,6,12,(20)(21)(22)24). As an alternative to the other assays, we used the HCT-8 cell line to study the effects of the various components of the CAM dissolution procedure on C. parvum infectivity.Oocysts of C. parvum (GCH1 isolate) were obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infections Diseases, National Institutes of Health, through McKessonHBOC BioServices, Rockville, Md. For all experiments, oocysts were between 2 and 7 months old. The oocysts were stored in 2.5% (wt/vol) potassium dichromate at 4°C throughout the experimentation period.Oocyst suspensions were centrifuged for 3 min at 11,750 ϫ g in sterile 1.5-ml Eppendorf tubes, and pellets were resuspended in phosphate-buffered saline (PBS), pH 7.2. Approximately 10 6 control and experimental oocysts (determined by hemocytometer counts of stock solutions) were aliquoted into tubes.To simulate the CAM dissolution method, a procedure similar to that of Aldom and Chagla (1) was used. A 47-mmdiameter CAM with an...

Detection of Cryptosporidium Parvum and Giardia Lamblia Carried by Synanthropic Flies by Combined Fluorescent in Situ Hybridization and a Monoclonal Antibody

“…24 Processing of C. parvum oocysts by the CAM-filter dissolution method does not alter their infectivity as tested in vivo by mouse bioassay, 25 although in vitro testing indicates that oocyst infectivity decreases. 26 Surface elution ensures recovery of particles derived from the exoskeleton of the fly, and homogenization releases the particles within the digestive tract. 15 The resulting pellets were stored in 200 L of 95% ethanol at 4°C 18 until analyzed by FISH and FITCconjugated MAb.…”

How to detectToxoplasma gondiioocysts in environmental samples?

“…CAM dissolution in 100% acetone is an alternative to the elution procedure [71]. However, it seems not to be appropriate for virulence studies because viability of protozoa is reduced after dissolution [72].…”

Efficacy of Common Laboratory Disinfectants on the Infectivity of Cryptosporidium parvum Oocysts in Cell Culture

“…Previous studies have shown that vital dye staining and excystation rates may overestimate the number of infectious oocysts present (2,4,8,9). Infectivity assays using animals are expensive, and it is difficult to obtain quantitative results.…”

“…Infectivity assays using animals are expensive, and it is difficult to obtain quantitative results. However, in vitro infectivity assays are useful for quantitatively measuring antimicrobial effects of disinfectants against C. parvum oocysts (4,9).…”