Applied and Environmental Microbiology, August 2003, p . 4983-4984, Vol . 69, No . 8

Infectious Cryptosporidium parvum Oocysts in Final Reclaimed Effluent

Angela L . Gennaccaro,^* Molly R . McLaughlin, Walter Quintero-Betancourt, Debra E . Huffman, and Joan B . Rose

Department of Marine Science, University of South Florida, St . Petersburg, Florida 33701

Received 6 December 2002/ Accepted 2 June 2003

Cryptosporidium parvum, a coccidian protozoan parasite, is a potential contaminant of reclaimed water . C . parvum oocysts have been found to be persistent in the environment and resistant to chlorination . Because of this, physical removal by chemical pretreatment and filtration is the primary means of reducing the level of oocysts in environmental water (6) . A possible risk to human health exists if filtration fails to function efficiently . This risk is greater still with reclaimed water, as to date no monitoring for C . parvum oocysts has been required and little information is available on the filtration efficiency in these facilities . Recently, in the state of Florida, monitoring for protozoan parasites, including Cryptosporidium, once every 2 years for larger facilities and once every 5 years for smaller facilities has been mandated . Sampling is recommended at a single point following disinfection (2) .

In one study, C . parvum oocysts were detected in untreated wastewater (67% of the samples were positive) and in reclaimed water (25% of final effluent samples were positive) (5) . However, only the presence of the oocysts was evaluated using fluorescence microscopy . Robertson et al . evaluated wastewater samples for viable C . parvum by using vital stains; 35% of the influent samples and 46% of the effluent samples contained viable oocysts (4) . In the last few years, the focus detection method-most-probable-number (FDM-MPN) cell culture technique has been developed to test the oocyst infectivity because the previously employed methods did not accurately reflect the infectious nature of the oocysts (7) . The objective of the present study was to demonstrate the presence of infectious C . parvum oocysts in final reclaimed effluent from six reclamation facilities in the United States by using the FDM-MPN cell culture technique .

Samples were collected from influent, secondary effluent, postfiltration, and final disinfected effluent waters . Six reclamation facilities in the United States, utilizing a variety of filtration systems (shallow- or deep-bed sand and anthracite filters or fabric disk filters) and disinfection methods (chlorine gas or UV radiation), were monitored . Three facilities were monitored five times over a 1-year time period . Three additional facilities were monitored over a 5-month time period . One to 400 liters of sample, depending on the site, was filtered through Envirochek HV filters (Pall Gelman Laboratories, Ann Arbor, Mich.) . After filtration, elution, and centrifugation using the guidelines provided by EPA method 1623 (9), oocysts were concentrated into a pellet by immunomagnetic separation (Dynabeads Anti-Crypto Kit; Dynal Biotech, Inc., Lake Success, N.Y.) .

Half of the concentrated pellet was analyzed by the FDM-MPN cell culture technique (7) . (The other half of the concentrated pellet was processed by microscopic screening of the sample after staining with monoclonal antibodies [anti-Cryptosporidium; Waterborne Inc., New Orleans, La.] tagged with fluorescein isothiocyanate, using an indirect fluorescent-antibody assay [IFA] to determine total counts of oocysts.) The concentrate was first bleach treated at room temperature for 8 min with a 10.5% solution of 4% sodium hypochlorite in phosphate-buffered saline (PBS; pH 7.2) to eliminate bacterial contamination as well as to trigger excystation of the oocysts . The sample was then washed once with PBS and centrifuged, after which the pellet was suspended in 1 ml of prewarmed growth medium (RPMI 1640 [Fisher Scientific, Pittsburgh, Pa.] plus additives) and placed directly on human ileocecal adenocarcinoma HCT-8 cell monolayers in eight-well chamber slides . The slides were incubated at 37°C in a 5% CO₂ incubator . After 90 min, additional growth medium was added to each well, and the slides were incubated for another 40 to 48 h .

After incubation, the slides were removed, washed with PBS, and fixed in 100% methanol . The monolayers were rehydrated for 30 min in blocking buffer (PBS with 2% goat serum [Atlanta Biologicals, Norcross, Ga.] and 10% of a 0.002% solution of Tween 20) and stained with rat anti-C . parvum sporozoite antibody (Waterborne Inc.) followed by fluorescein isothiocyanate-labeled anti-rat immunoglobulin G (Sigma Aldrich, Inc., St . Louis, Mo.) . The slides were evaluated under an Olympus BH-2 epifluorescence microscope at 200x and 400x magnification (excitation, 340 to 380 nm; 420-nm barrier or suppression filter) for the presence of infectious foci .

The number of positive wells for each sample was entered into the MPN program, downloaded from the EPA website, to determine the number of infectious oocysts per milliliter (8) . When no infectious foci were observed, the MPN program determined the detection limit for the assay . The number of infectious oocysts per 100 liters was determined from the initial volume collected and the concentrate volume analyzed after immunomagnetic separation .

C . parvum oocysts were found in all sites monitored throughout the treatment process (Table 1) . As well, and more importantly, infectious C . parvum oocysts were found in all sampling sites (Table 1) . Average concentrations of 6,910 oocysts/100 liters by IFA and 993 infectious oocysts/100 liters by MPN were found in the influent (raw wastewater) . Therefore, roughly 14% of all oocysts observed were infectious in nature . At the conclusion of treatment, average concentrations of 28 oocysts/100 liters by IFA and 7 infectious oocysts/100 liters by FDM-MPN were detected . Roughly 25% of the oocysts detected were infectious in nature . Average recovery efficiencies of 5.5% from influent samples for four trials (standard deviation, ±1.3), 15.3% from secondary effluent samples for four trials (standard deviation, ±2.9), and 15% from postfiltered and final disinfected effluent samples for 12 trials (standard deviation, ±11.2) have been observed in our laboratory using gamma-irradiated oocysts labeled with Texas Red . Thus, it is important to note that higher concentrations of infectious oocysts may be present in all samples, but especially final disinfected reclaimed effluent samples, since recovery efficiencies are not 100% .

 
During the past decade, there has been an increase in awareness of the risk of illness resulting from C . parvum because of outbreaks such as that in Milwaukee, Wisconsin, in 1993 (3) . By 1995, surveillance for cryptosporidiosis in the human population had begun in the United States . Between 1995 and 1998, the mean incidence per 100,000 ranged from 0.9 to 3 (1) . The relationship to water transmission is not known . In 1999, Florida began to require periodic sampling for Cryptosporidium and Giardia in reclaimed water systems (10) . In this paper, we report initial findings of infectious C . parvum oocysts in final reclaimed effluent as determined by the FDM-MPN method . Additional monitoring to produce a more statistically significant database and research to determine the best treatment processes are suggested as the next steps . Eventually, standards for monitoring of reclaimed water for Cryptosporidium should be considered . The FDM-MPN method will be a useful tool for future monitoring requirements to determine the presence of infectious oocysts and the associated health risk .

Present address: United States Geological Survey, Center for Coastal and Regional Marine Studies, St . Petersburg, FL 33701 .

Present address: Department of Fisheries and Wildlife, Michigan State University, East Lansing, MI 48824 .

1. Dietz, V . J., and J . M . Roberts. 2000 . National surveillance for infection with Cryptosporidium parvum, 1995-1998: what have we learned? Public Health Rep . 115:358-363.
2. Florida Department of Environmental Protection. 1999 . Reuse of reclaimed water and land application, p . 1-123 . In Florida Administrative Code . Florida Department of Environmental Protection, Tallahassee.
3. MacKenzie, W . R., N . J . Hoxie, M . E . Proctor, M . S . Gradus, K . A . Blair, D . E . Peterson, J . J . Kazmierczak, D . G . Addiss, K . R . Fox, J . B . Rose, and J . P . Davis. 1994 . A massive outbreak in Milwaukee of Cryptosporidium infection transmitted through the public water supply . N . Engl . J . Med . 331:161-167.
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7. Slifko, T . R., D . E . Huffman, and J . B . Rose. 1999 . A most-probable-number assay for enumeration of infectious Cryptosporidium parvum oocysts . Appl . Environ . Microbiol . 65:3936-3941.
8. U.S . Environmental Protection Agency. 1992 . Guidelines for water reuse . EPA/625/R-92/004 . Office of Water, U.S . Environmental Protection Agency, Washington, D.C.
9. U.S . Environmental Protection Agency. 1999 . Method 1623: Cryptosporidium and Giardia in water by filtration/IMS/FA . EPA/821/R-99/006 . Office of Water, U.S . Environmental Protection Agency, Washington, D.C.
10. York, D . W., and L . Walker-Coleman. 2000 . Pathogen standards for reclaimed water . Water Environ . Tech . 12:58-61.

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