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Applied and Environmental Microbiology, September 2007, p. 5968-5970, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00347-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Use of Cell Culture To Assess Cryptosporidium parvum Survival Rates in Natural Groundwaters and Surface Waters{triangledown}

Rebecca L. Ives,1 Amy M. Kamarainen,1,{dagger} David E. John,2 and Joan B. Rose1*

Department of Fisheries and Wildlife, Michigan State University, East Lansing, Michigan,1 College of Marine Science, University of South Florida, St. Petersburg, Florida2

Received 12 February 2007/ Accepted 23 July 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 REFERENCES
 
Bench-scale survival studies with Cryptosporidium parvum were conducted with representative aquifer and reservoir waters of Florida. C. parvum inactivation rates ranged from 0.0088 log10/day at 5°C to –0.20 log10/day at 30°C. Temperature, surface water or groundwater type, and the interaction of these factors had statistically significant effects on the survival of C. parvum.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
REFERENCES
 
Studies have reported that low levels of viable Cryptosporidium oocysts in water present a health risk (3, 6). In surface waters, oocyst concentrations ranged from 0.0003 to 5,800 liter–1 (1) and prevalence was 60.2% of 347 samples in a 5-year study (4). Here we use cell culture to determine the survival rates of Cryptosporidium parvum in surface waters and groundwaters of Florida, which will potentially be used for aquifer storage and recovery.

Two reservoir and two aquifer waters (Bill Evers Reservoir and Avon Park well, Bradenton, FL, and Clear Lake Reservoir and Lake Lytal Park well, West Palm Beach, FL) were sampled in the summer and used for the seeded experiments. Both wells tap the Upper Floridan aquifer. Wells were purged of at least three well volumes prior to sampling. Table 1 shows the physiochemical measurements of the source water. Twenty-milliliter samples were distributed to 50-ml centrifuge tubes for each water type and temperature combination and were equilibrated to 5, 22, and 30°C prior to seeding with C. parvum (prepared as previously described [2]; Iowa isolate; Sterling Parasitology Laboratory, University of Arizona, Tucson, AZ). In the seeded waters, oocyst concentrations were 107 oocysts ml–1 by hemocytometer count and the initial infectious concentrations were between 103 and 104 infective oocysts ml–1 by the cell culture method (described below). Experiments were performed concurrently with oocysts from the same lot number to minimize differences attributed to oocyst age after shedding. C. parvum oocysts were used within 60 days of shedding from a calf. Tubes were covered to exclude light and were maintained at 5, 22, or 30°C in a refrigerator or in water baths. One-milliliter samples were analyzed in replicate at various dilutions for oocyst infectivity at 0, 1, 3, 6, 10, 20, and 25 days for each water sample at each temperature. Oocysts were assayed for infectivity on human ileocecal adenocarcinoma (HCT-8) cells (CCL-224; ATCC, Manassas VA) by using an indirect antibody focus detection method as previously described (7, 10). An EPA most probable number (MPN) program was used (http://www.epa.gov/nerlcwww/other.htm), and values were converted to percent infectivity by the following equation: (MPN ml–1/hemacytometer oocyst count ml–1 stock) x 100. Series data points for each experimental trial were converted to log10 N/N0 ratios, where N indicates the percent infectivity at time t and N0 indicates the initial percent infectivity, and fit to equation 1 to obtain inactivation rates per day:

Formula 1(1)


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  		TABLE 1. Physiochemical measurements of raw water from the sample sites

 
In data sets where the total decline was less than 1 log (90%), m (an exponential coefficient to model nonlinear kinetics) was set to 1. Since 2-log reduction periods were occasionally outside experimental durations, and kinetics could be accurately modeled with first-order models in these cases, only the first-order inactivation rate constants (k, with m at 1) were evaluated by analyses of variance using Minitab, version rel. 12. The results of two-way analysis of variance demonstrated that temperature, water type (surface water or groundwater), and the interaction of these two factors had significant effects on inactivation rate variability (P values were <0.01, <0.004, and <0.05, respectively).

Table 2 shows first-order inactivation rates and predicted number of days to achieve a 2-log reduction. Inactivation rates ranged from +0.0088 log10/day at 5°C to –0.20 log10/day at 30°C. At 5°C over 25 days, the mean across all water types showed a slight (insignificant) positive trend, possibly due to variability in the assay and oocyst maturation (2). Higher temperatures led to a decrease in the predicted number of days to achieve a 2-log reduction (Table 2). The inactivation rate at 22°C for Avon Park was found to be lower than the rate for Lake Lytal (which was similar to that observed in surface water at 22°C). This result may be due to differences in well water composition, but this possibility was not evaluated. At 5°C, native biological activity is expected to be low, as is the oocyst inactivation rate. Thus, no variability in oocyst survival is seen. At 30°C, oocyst inactivation rates are higher and temperature seems to dominate the inactivation effects. At 22°C, native biological activity or the native microbial population may be a contributing factor of oocyst inactivation. Thus, differences between waters may be seen, particularly at this temperature. At 30°C, days for a 2-log reduction of infectious oocysts were 17 and 18 days in aquifer waters and 10 and 11 days in reservoir waters. At 22°C in surface water, 30 and 45 days were estimated for a 2-log reduction. In a previous study that used cell culture methods to assess the effects of temperature on oocyst infectivity in river water, the infectivity of the oocysts in unfiltered river water at 21 to 23°C decreased 2.6 log units by 12 weeks (approximately –0.031 log10/day) (8), which was slightly lower than the rate of inactivation (–0.045 to 0.066 log10/day) predicted for surface waters here. Differences may be due to water composition or method differences, as the previous study did not use an MPN method and assessed infectivity at weekly intervals. In a previous study of longer experimental duration, 66.7% of cryptosporidia in reagent-grade water at 4°C appeared nonviable by dye exclusion after 176 days (9), an approximately 0.48-log reduction (–0.0027 log10/day). This result is similar to that of the present study, which predicts C. parvum inactivation rates of –0.0017 log10/day to –0.0037 log10/day in surface water at 5°C, and is lower than the –0.010 log10/day inactivation rate previously reported at 5°C using excystation and dye exclusion (5).


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  		TABLE 2. First-order inactivation rates and predicted number of days to a 2-log decline of C. parvum in natural water trials

 
Averaged over all temperatures, inactivation was –0.083 and –0.044 log10/day in reservoir and aquifer waters, respectively. Surface waters are subject to greater annual temperature fluctuations than groundwaters, but seasonal changes were not evaluated in this study. Cryptosporidium entering surface waters will likely persist longer in the winter months as the in situ temperatures decrease.

The results of this study have implications for aquifer storage and recovery projects. It has been suggested that untreated surface waters (possibly containing Cryptosporidium) could be stored via aquifers for later recovery. The specific conditions, particularly temperature, need to be evaluated in order to better predict attenuation during subsurface storage. Storage time and differences in surface water and groundwater temperatures will produce both a gradient of conditions as waters mix and a range of inactivation rates. Thus, stochastic models are needed to better predict attenuation rates during aquifer storage of untreated waters.


   ACKNOWLEDGMENTS
 
Funding was provided for this study by the Southwest Florida Water Management District and the South Florida Water Management District.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Fisheries and Wildlife, 13 Natural Resources Building, Michigan State University, East Lansing, MI 48824. Phone: (517) 432-4412. Fax: (517) 432-1699. E-mail: rosejo{at}msu.edu Back

{triangledown} Published ahead of print on 3 August 2007. Back

{dagger} Present address: Center for Limnology, University of Wisconsin, Madison, WI 53706. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 REFERENCES
 

  1. Bagley, S. T., M. T. Auer, D. A. Sterm, and M. J. Babiera. 1998. Sources and fate of Giardia cysts and Cryptosporidium oocysts in surface waters. Lake Reservoir Manag. 14:379-392.
  2. Coulliette, A. D., D. E. Huffman, T. R. Slifko, and J. B. Rose. 2006. Cryptosporidium parvum: treatment effects and the rate of decline in oocyst infectivity. J. Parasitol. 92:58-62.[CrossRef][Medline]
  3. DuPont, H. L., C. L. Chapell, C. R. Sterling., P. O. Okhuysen, J. B. Rose, and W. Jakubowski. 1995. The infectivity of Cryptosporidium parvum in healthy volunteers. N. Engl. J. Med. 332:855.[Abstract/Free Full Text]
  4. LeChevalier, M. W., and W. D. Norton. 1995. Giardia and Cryptosporidium in raw and finished water. J. Am. Water Works Assoc. 87:54-68.
  5. Medema, G. J., M. Bahar, and F. M. Schets. 1997. Survival of Cryptosporidium parvum, Escherichia coli, faecal enterococci, and Clostridium perfringens in river water: influence of temperature and autochthonous microorganisms. Water Sci. Tech. 35:249-252.
  6. Okhuysen, P. C., C. L. Chappell, J. H. Crabb, C. R. Sterling, and H. L. DuPont. 1999. Virulence of three distinct Cryptosporidium parvum isolates for healthy adults. J. Infect. Dis. 180:1275-1281.[CrossRef][Medline]
  7. Pokorny, N. J., S. C. Weir, R. A. Carreno, J. T. Trevors, and H. Lee. 2002. Influence of temperature on Cryptosporidium parvum oocyst infectivity in river water samples as detected by tissue culture assay. J. Parasitol. 88:641-643.[CrossRef][Medline]
  8. Robertson, L. J., A. T. Campbell, and H. V. Smith. 1992. Survival of Cryptosporidium parvum oocysts under various environmental pressures. Appl. Environ. Microbiol. 58:3494-3500.[Abstract/Free Full Text]
  9. 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.[Abstract/Free Full Text]
  10. Slifko, T. R., D. Friedman, J. B. Rose, and W. Jakubowski. 1997. An in vitro method for detecting infectious Cryptosporidium oocysts with cell culture. Appl. Environ. Microbiol. 63:3669-3675.[Abstract]

Applied and Environmental Microbiology, September 2007, p. 5968-5970, Vol. 73, No. 18
0099-2240/07/$08.00+0     doi:10.1128/AEM.00347-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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