Cryptosporidium spp. and Giardia spp. are intestinal parasites that can be transmitted through water contaminated by feces. Over a 16 month period, surface waters were examined in Kansas in order to measure local geographic distribution and seasonal fluctuation of oocyst and cyst populations. Cryptosporidium and/or Giardia were found in 86 percent of the locations surveyed. Between 1946 and 1980, giardiasis resulted in more cases of illness than any other known agent of waterborne disease (Lippy and Waltrip, 1984). While only two outbreaks were attributed to Giardia lamblia between 1965 and 1969, 56 outbreaks of waterborne giardiasis were reported between 1980 and 1985, demonstrating a substantial increase in the frequency, and perhaps recognition of the importance, of this flagellate. Cryptosporidium parvum, another intestinal pathogen, has recently been linked to human illness through waterborne transmission (Rose, 1988; Sterling, 1990; Smith and Rose, 1990). The disease is normally self-limiting in immunocompetent individuals, but may become life-threatening in immunocompromised individuals (Fayer et al., 1990; Reese et al., 1982; Ungar, 1990). Giardia lamblia and C. parvum exist in the environment in the form of cysts and oocysts, respectively. Despite the impact of these microorganisms on human health, little has been known about the epidemiology and prevalence of either parasite, especially C. parvum in distribution (Lippy and Waltrip, 1984). Recent outbreaks of cryptosporidiosis in New Mexico (Anonymous, 1986), Texas (D'Antonio et al., 1985), British Columbia (IsaacRenton et al., 1987), Sheffield and Great Yarmouth, England (Brown et al., 1989; Rush et al., 1987), and Georgia (Hayes et al., 1989) have suggested that this parasite may be an important cause of undiagnosed enteric disease. Recently, concentration techniques have been developed whereby both I To whom reprint requests should be addressed. This content downloaded from 207.46.13.105 on Wed, 25 May 2016 06:49:25 UTC All use subject to http://about.jstor.org/terms 102 TRANSACTIONS OF THE KANSAS ACADEMY OF SCIENCE G. lamblia and C. parvum can be detected in surface waters (Rose et al., 1985, 1986, 1988, 1989; Sterling et al., 1987; Stetzenbach et al., 1988). Surveys using these techniques have shown both G. lamblia and C. parvum to be potential sources of human disease in Arizona (Madore et al., 1987; Stetzenbach et al., 1988), Washington, and California (Ongerth and Stibbs, 1987). Since few studies have examined surface waters in the midwest United States for the presence of these pathogens, a study was conducted to determine whether Giardia and Cryptosporidium were present in significant numbers in surface waters in Kansas. MATERIALS AND METHODS Sample collection. One hundred gallon samples of water were pumped through polypropylene wound cartridge filters (Micro Wynd II, AMF/CUNO Division, Meriden, CT) at a rate of 3.3 gal/min (Musial et al., 1987; Rose et al., 1985). Filters were processed within 48 hours of collection and stored at 4?C. Samples were taken from three sites at Tuttle Creek State Park and River Pond Area, two sites at Milford Lake, the Big Blue River, and two sites on the Konza Prairie (Fig. 1). Filter elution. Filtered particles and microorganisms were removed by backflushing with 2.7 liters of 0.1% Tween 80 (Musial et al., 1987; Rose, 1988; Rose et al., 1986). Filters were teased apart and placed into eluate on a shaker for 10 min at 370C. Filter fibers were removed and the eluate concentrated by centrifugation at 1200 x g for 10 min. Cyst/oocyst extraction. Samples were washed in phosphate buffered saline (PBS), 1% sodium dodecyl sulfate (SDS), distilled water, and then 10% formalin. Samples were resuspended in 10 ml 1% SDS/1% Tween 80 and split in half. One half was layered onto 15 ml of Sheather's solution (500 g sucrose, 320 ml water, 9.7 ml liquid phenol) and the other half onto 15 ml Percoll-sucrose solution (5 ml 0.25 M sucrose with 5 ml Percoll). Each half was then centrifuged at 1200 x g and 800 x g, respectively, and the supernatants removed for assay. Cyst/oocyst quantification. Supernatants from the gradients were reconcentrated to a volume of 2.5 ml and air dried as five, 10 Aul aliquots onto microscope slides using egg albumin as an adhesive. Slides, including positive controls, were processed for indirect immunofluoresence viewing using the Hydrofluor@ combo detection assay (Hydrofluor Combo, Meridan Diagnostics, Inc.) except that 3 percent bovine serum albumin in PBS was found to be a superior blocking agent over Tween 20. Parasites on slides were then counted and original cyst/oocyst concentrations extrapolated. Efficiency studies. One hundred gallons of chlorinated tap water were seeded with known numbers of Giardia cysts or Cryptosporidium oocysts (2.5 x 105 and 1.0 x 106 cysts/oocysts, respectively). Samples were processed identically to river water samples. This content downloaded from 207.46.13.105 on Wed, 25 May 2016 06:49:25 UTC All use subject to http://about.jstor.org/terms VOLUME 94, NUMBERS 3-4 103
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