Decrease in Cryptosporidium parvum oocyst infectivity in vitro by using the membrane filter dissolution method for recovering oocysts from water samples.

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]) alone and short exposure times to acetone decreased infectivity. These findings contrast with similar experiments using excystation assays and infectivity in mice.

Phenotypic and genotypic characterization of Cryptosporidium species and isolates.

Recent outbreaks of cryptosporidiosis from contaminated water supplies have led to a need for the detection of Cryptosporidium oocysts from various hosts and contaminating sources. The presence of nonpathogenic species or strains of Cryptosporidium is important for diagnostic purposes as there is a potential for false- positive detection of pathogenic parasites. The present review focuses on phenotypic differences and recent advances in genotypic analyses of the genus Cryptosporidium with an emphasis on detecting various isolates and identifying differences in Cryptosporidium parvum and other species in this genus. The information currently available demonstrates important patterns in DNA sequences of Cryptosporidium, and our understanding of macro- and microevolutionary patterns has increased in recent years. However, current knowledge of Cryptosporidium genetic diversity is far from complete, and the large amount of both phenotypic and genotypic data has led to problems in our understanding of the systematics of this genus.

Improving the rate of infectivity of Cryptosporidium parvum oocysts in cell culture using centrifugation.

Centrifugation was evaluated as a method to improve infectivity assays of Cryptosporidium parvum in cell culture using the focus detection method, an immunofluorescence-based method for detecting infectious C. parvum oocysts in vitro. Human ileocecal adenocarcinoma (HCT-8) cells were grown for 48 hr on 13-mm cover slips in 24-well microtiter plates and infected with bleach-treated C. parvum oocysts. Plates were centrifuged at 228 g for 10 min and incubated at 37 C for 5, 12, 18, 24, and 48 hr. Foci of infection were stained by immunofluorescence and enumerated using epifluorescent microscopy. Results were compared to noncentrifuged controls. Foci in centrifuged samples could be enumerated after 18 hr. According to most probable number (MPN) analysis, the number of infectious oocysts estimated at 48 hr (13,326 infectious oocysts) was reached by 18 hr in centrifuged samples. After 48 hr, there was no significant difference (P < 0.05) between centrifuged and noncentrifuged samples enumerated by number of foci or the MPN of infectious oocysts. Centrifugation may expedite detection during C. parvum infectivity assays. Furthermore, multiwell plate formats are more cost effective than traditional chamber slides.

DNA fingerprinting of Cryptosporidium parvum isolates using amplified fragment length polymorphism (AFLP).

The genetic variability of 10 Cryptosporidium parvum isolates of human and animal origin was investigated using amplified fragment length polymorphism (AFLP). Analysis of fluorescent dye-labeled amplified products was carried out using an ABI PRISMS 377 DNA sequencer and ABI PRISMS GeneScan software. One-hundred and twelve primer combinations were evaluated using a single C. parvum isolate. The patterns generated were highly reproducible. For subsequent study, a subset of 9 primer pairs that yielded 30-90 DNA fragments after the polymerase chain reaction, within the size range of 50-500 bp, was used to screen the 10 C. parvum isolates, including 7 bovine, 1 equine, and 2 of human origin. The animal isolates produced identical fingerprint patterns with every primer combination tested. Of the 2 human isolates tested, 1 of the isolates, passaged in calves, generated the same AFLP DNA banding patterns as the animal isolates, whereas the other isolate, obtained directly from human feces, produced unique patterns. Polymorphism, detected by comparison of the fingerprint patterns of the latter human isolate with the common pattern shared by all other isolates, ranged from 17 to 35% for the 9 primer pairs. The results show that AFLP is a useful method for differentiating C. parvum isolates into 2 distinct genotypes.