Development and application of a quantitative, specific assay for Cryptosporidium parvum oocyst detection in high-turbidity environmental water samples.

Chlorine-resistant Cryptosporidium parvum oocysts in drinking water play an important role in the epidemiology of cryptosporidiosis. Current methods of detecting these organisms in water are insensitive, labor-intensive, highly subjective, and severely limited by sample turbidity. We describe here an alternative technique utilizing electrochemiluminescence (ECL) technology for detecting C. parvum oocysts in environmental water samples. This method is quantitative, reproducible, and requires only minimal sample processing. Currently, the ECL assay can detect as few as one oocyst in one milliliter of concentrated test sample with sample turbidity of up to 10,000 nephelometric turbidity units. Water and sewer samples collected during a cryptosporidiosis outbreak were tested by ECL assay. Cryptosporidium parvum oocysts were found in the source water at the time of outbreak, and a sharply decreasing level of oocysts in sewer samples was observed over a three-month period following the outbreak.

Immunoassay for viable Cryptosporidium parvum oocysts in turbid environmental water samples.

Cryptosporidium parvum oocysts in drinking water have been implicated in outbreaks of diarrheal disease. Current methods for monitoring environmental exposures to C. parvum only account for total number of oocysts without regard for the viability of the parasite. Measurement of oocyst viability, as indicated by an oocyst's ability to excyst, is useful because over time oocysts lose the ability to excyst and become noninfective. Thus, correlating the number of viable oocysts in drinking water with incidence and risk for disease should be more reliable than using the total number of oocysts. We have developed a quantitative assay capable of detecting low numbers of excystable, sporozoite-releasing C. parvum oocysts in turbid water samples. Monoclonal (CP7) and polyclonal antibodies have been developed against a sporozoite antigen released only during excystation or when the oocyst is mechanically disrupted. CP7 is specific for C. parvum and does not react with C. baileyi, C. muris, C. serpentis, Giardia spp., Eimeria spp., or E. nieschulzi. In this assay, oocysts in the test sample are first excysted and then centrifuged. The soluble sporozoite antigen is captured by CP7 attached to a magnetic bead. The captured antigen is then detected by ruthenium-labeled polyclonal antibodies via electrochemiluminescence. The CP7 viability assay can detect as few as 50 viable oocysts in a 1-ml assay sample with a turbidity as high as 200 Nephelometric turbidity units. This sensitive, turbidity-tolerant assay for oocyst viability may permit a better assessment of the disease risk associated with the presence of environmental oocysts.

Serum-free culturing of adult Schistosoma mansoni in dialysis bags for the production of excretory/secretory antigens.

Antigenic excretory/secretory (E/S) products from Schistosoma mansoni are potentially important in the development of diagnostic assays used to detect circulating antigens in schistosomiasis. The E/S products to be used as antigen(s) for this development must, by necessity, be free of exogenous proteins. The ability to extend serum-free in vitro culture of adult worms is, therefore, essential. Adult worms were perfused from mice, washed in serum-free RPMI-1640 with antibiotics, and placed in sterile dialysis bags, molecular weight cut-off 10 kDa, at a concentration of 100 worms in 1 ml of serum-free, supplemented RPMI-1640. Each bag was then placed in a flask of supplemented RPMI-1640 with 10% fetal calf serum in a humidified incubator at 37 C, 7% CO2. At days 1, 3, 5, 8, and 12, worms were collected; E/S culture medium was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Worm survival rates were 85% after 1 day in culture, dropping gradually to 65% on day 8, and then to 38% on day 12. Silver stain for total protein and immunoblot exposed to positive human infection serum showed E/S culture media from days 3 and 5 having the least complex banding pattern. The quantitative specific activity of E/S, as measured by antigen-limiting Falcon assay screening test system-enzyme-linked immunosorbent assays against human infection serum, indicates E/S antigenicity closely follows the attrition of worms and, therefore, may be directed against the release of somatic antigens by dead worms. Culturing S. mansoni in dialysis tubing is useful in deriving E/S products.