Environ. Sci. Technol. 1999, 33, 3134-3139
Sorption of Cryptosporidium parvum Oocysts in Aqueous Solution to Metal Oxide and Hydrophobic Substrates MARK J. WALKER* Department of Environmental and Resource Sciences, University of Nevada, Reno, Nevada 89557-0013 CARLO D. MONTEMAGNO Department of Agricultural and Biological Engineering, Cornell University, Ithaca, New York 14853
The parasite Cryptosporidium parvum (C. parvum) may be introduced into water supplies by a number of processes, including runoff and infiltration from areas where wastes from infected hosts are dispersed on soils. Soil particles may attenuate or, when suspended in flow with sufficient kinetic energy, act as transport sites for oocysts. Sorption may also be reversed by the frictional force of flowing water. As a first step toward understanding oocyst attentuation and transport, we examined partitioning in a two-phase, laminar flow microduct. This exposed aqueous suspensions of C. parvum oocysts to metal oxide and hydrophobic substrates (Al2O3, Fe2O3, SiO2, and octadecyltrichlorosilane ((OTS) CH3(CH2)17SiCl3, a hydrophobic monolayer)) on transparent glass slides. Sorption under laminar flow conditions was observed for unexposed oocysts and oocysts subjected to freeze-thaw cycling and desiccation. The results indicate differences in sorption between substrates and oocyst exposure to stresses. Regardless of exposure to stress, oocysts sorbed to Al2O3 but not to Fe2O3, OTS, and SiO2. Of the starting number of oocysts added to the microduct with Al2O3 substrates, 61% of the freezethawed, 43% of the desiccated oocysts, and 4% of the unexposed oocysts remained sorbed. The differences may be due to loss of integrity of the oocyst wall and dispersion of nucleic acids within the interior of the oocyst, as indicated by propidium iodide staining of DNA. This suggests that charge-based interactions with colloids may be most pronounced when the oocyst is no longer capable of initiating infection.
Introduction The parasite Cryptosporidium parvum (C. parvum) is a frequently found microbiological contaminant in surface waters in the United States (1) and has been found in filtered drinking water supplies (2). The dormant, environmentally resistant stage, the oocyst, is released in the feces of infected hosts and may initiate new infection in mammalian hosts far from where feces are dispersed in the environment. When exposed by drinking contaminated water, humans may contract cryptosporidiosis, a gastrointestinal upset with * Corresponding author. Phone: (775) 784-1938; fax (775) 7844789; e-mail mwalker equinox.unr.edu. 3134
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severity dependent upon the robustness of the immune response (3). Oocysts may enter raw water supplies from many sources. These include point source discharges from wastewater treatment facilities (4, 5) and nonpoint sources (6, 7). Domesticated animals, especially cows, have been studied to determine the prevalence on several geographical scales (8-10). The prevalence of infection in young stock has focused attention on potential animal sources of oocysts, particularly following outbreaks (11, 12). In fact, watershed management programs have been developed to reduce the risk of contaminating surface waters used for public drinking water supplies by controlling runoff and drainage from areas used to graze and house animals and disperse animal wastes (13). However, such programs are built upon the assumption that management practices designed for other types of pollutants (e.g., sorbed phosphorus or sediment) will also control oocysts. This assumption may not be well founded, especially because processes that lead to entrainment and transport are not well-understood (7). Animal wastes are commonly dispersed as a soil amendment. If wastes contain oocysts, areas used for land spreading could act as reservoirs that release them in runoff, provided that the kinetic energy of overland flow is sufficient to entrain and suspend. Conceptually, the level of kinetic energy in the flow must exceed a threshold that is determined partially by the effective diameter and mass of the particle under consideration. As a particle, the oocyst has been well-characterized. It is spherical, with a diameter of 4-7 µm, and estimated bouyant density greater than water (∼1.06 g/cm3). However, in the soil environment, the effective diameter and specific gravity may be significantly different if oocysts sorb to soil particles or aggregates. This may be important if oocysts sorb to colloidal particles either while on the soil surface or while entrained in overland flows. If oocysts sorb to colloids, the effective diameter and net bouyant density of the resulting microaggregate could be expected to increase, leading to changes in settling velocity and entrainment energy needed for transport. The degree of change would be dependent upon the type and number of sorbed colloids. Sorption to soil particles at the soil-surface water interface could also attentuate oocysts, preventing entrainment and transport from sites of deposition under some conditions. As a microbiological particle, the oocyst may change when subjected to stresses in the soil matrix, with the consequence that surface properties and sorption characteristics also change. Oocysts are highly resistant to managed and natural stresses lethal to other microbes (14). Some natural stresses are lethal to oocysts, including slow and rapid freezing (15, 16), freeze-thaw cycling (17), and desiccation (18). Such stresses do not necessarily destroy oocysts. They may retain their original geometry and be detected in environmental samples, including soil (19) and water (2), but be incapable of initiating infection. With respect to sorption, studies of oocyst surface properties showed that adhesion to solid-phase substrates due to hydrophobic attraction increased as oocysts aged in water of low ionic strength (20). Static assays to assess sorption are difficult to interpret because the effects of shear stresses applied by flowing water are not considered, especially with dynamic soil/water systems that cannot be observed with a microscope. Such information is important for assessing the likelihood that oocysts, whether capable of initiating infection or not, will be entrained and transported by overland flow from areas 10.1021/es990331f CCC: $18.00
1999 American Chemical Society Published on Web 08/03/1999
FIGURE 2. Experimental apparatus used to carry out sorption trials. FIGURE 1. Design of laminar flow transparent microduct. where they have been dispersed (for example, at animal waste-spreading sites). The experimental work discussed in this paper examined oocyst sorption and response to shear stresses applied by laminar flow. The work tested the hypothesis that oocyst sorption is influenced by the chemical characteristics of substrates. The work also tested the hypothesis that prior exposure to environmental stresses lethal to oocysts affects sorption.
Experimental Description The experiments discussed below were designed to provide preliminary information about the potential for oocysts to remain sorbed to mineral or hydrophobic surfaces on soil particles in the presence of laminar flows. We applied transparent mono- and multimolecular substrates to glass slides. The slides were mounted on a milled Lucite block to create a microduct, rectangular in cross section. Oocysts injected into the microduct in suspension settled from solution to the slide surfaces. We then initiated laminar flow in the microduct and gradually increased the rate to a maximum of 8.3 × 10-3 mL/s. The maximum flow exerted an estimated 348 µN of force on oocysts sorbed to the surfaces. Following application of this force, we estimated the number of oocysts remaining, based on 35 observations of the substrate surface. We then estimated the percentage of the starting number that remained sorbed, relative to the number added to the microduct at the beginning of each experiment. In addition to varying the substrates, we also subjected oocysts to the lethal stresses of desiccation and freeze-thaw cycling and carried out experiments with the same substrates and experimental conditions. Overall, we conducted 3 replicate trials for each of the 12 conditions under study (4 substrates × 3 oocyst states). Experimental Apparatus. Laminar Flow Transparent Microduct (LFTM) System. The LFTM is a machined, polished Lucite block (Immunetics, Inc., Cambridge, MA, Model CAF-10) which allowed substrates to be mounted by continuous vacuum. The resulting microduct was rectangular in cross section with dimensions of 3.175 × 0.0.559 × 0.016 cm (length, width, height, respectively) (Figure 1). Slide surfaces of different chemical composition (described below) can be observed under carefully controlled flow conditions, and the shearing force exerted by laminar flow can be estimated using a series approximation of the Navier-Stokes equation applied to a three-dimensional system (21). We observed substrates with a Leica IRDBM inverted microscope (Leica, Inc., Deerfield, IL) in phase-contrast mode at 400× magnification (40× NPLAN long working distance objective (n.a. 0.55)). The Lucite block was mounted on a motorized
stage (CellRobotics, Inc., Albuquerque, NM), for precise and reproducible positioning. Flow was introduced into the microduct from a 5-mL reservoir after initial inoculation. In-line flowmeters (Gilmont Instruments, Inc., Barrington, IL, Catalog No. GF 9760, Catalog No. GF 9060) mounted in series were able to gauge flows from 0.0002 to 1.1 mL/min. Images from the microscope, captured by video camera, were transmitted to a Sony Trinitron Super Fine Pitch high-resolution monitor with an affixed grid that presented a measurement scale in gradations equivalent to 0.0010 cm, as determined by a Graticules Ltd. stage micrometer (1 mm in 0.01-mm divisions (Tonbridge, Kent, England)). A vibration-isolation breadboard (Newport Corp., Irvine, CA) served as a platform for the microscope and flow system, preventing accidental disturbance by external vibrations. A syringe pump (Harvard Model 944, South Natick, MA) equipped with a 5-cm3 glass syringe (Japan), placed on foam pads approximately 1 m below the stage, regulated the flows (Figure 2). Reagents and Materials. Oocysts. Oocysts were isolated from the feces of naturally infected Holstein calves, using a technique developed by Vetterling (22), applied by Dr. D. Bowman of the College of Veterinary Medicine at Cornell University. Oocysts were separated from dilute feces using a series of discontinuous sucrose gradients in a flow-through centrifuge. A final flotation with a discontinuous Percoll gradient provided high concentrations of purified oocysts (108/mL), which were stored in distilled water with 100 U/mL penicillin G. sodium, 100 µg/mL streptomycin sulfate, and 0.25 µg of amphotericin B/mL of oocyst suspension. A single stock of purified oocysts was used for all experiments, and all experiments were carried out within 2 months of purification. Substrates Applied to Glass Slides. The substrates included transparent multimolecular layers of Al2O3 (100 Å thickness), Fe2O3 (500 Å thickness), SiO2 (NoChromixcleaned glass slides), and octadecyltrichlorosilane ((OTS) CH3(CH2)17SiCl3; a hydrophobic self-assembling monomolecular carbon polymer). The silicon dioxide and hydrophobic monolayer substrates were prepared by immersing glass slides for at least 24 h in a solution of 36 N H2SO4 and NoChromix, followed by rinsing and 30 min of sonication in the same stock of distilled, deionized water used for experiments (described below) and 2-3 min of immersion in boiling water (also from stock reserved for flow cell experiments) followed by light wiping with a paper tissue to remove any residue. The hydrophobic OTS substrate was applied by immersing cleaned slides in a 1 mM OTS/anhydrous hexadecane solution in a nitrogen atmosphere using a procedure modified from Kleinfeld et al. (23) by H. Craighead and S. Turner (Department of Applied Engineering Physics, Cornell University). Slides were rinsed and sonicated in a chloroform VOL. 33, NO. 18, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. DAPI/PI Status of Oocyst Stocks Subjected to Stresses oocyst treatment
D-P-
unexposed frozen desiccated
95.2 1.5 0
Percent D+PP+ 1.8 0.3 0
2.5 93.83 70.8
empty
% potentially infective
0.5 4.2 29.3
97.0 1.8 0
bath to remove excess hexadecane and cured by baking on a hot plate at 115 °C for 10 min. The Cornell Nanofabrication Facility prepared Al2O3 coatings by initial ultrasonication of slides in a solution of surfactant and deionized water, followed by deionized water rinse, nitrogen drying, 3 min of oxygen plasma cleaning, and 8 min of exposure to an argon ion beam. Aluminum oxide was deposited at a rate of 11 Å/minute using flow rates of 5 cm3/min aluminum and 19 cm3/min oxygen under 2.5 mTorr pressure for 9 min to form a layer 100 Å thick. Slides coated with iron oxide (Fe2O3) were prepared by Towne Technologies, Inc. (Somerville, NJ) using a proprietary process to form a 500 Å thick layer, after initial cleaning of 1.5-mmthick glass with a surfactant. Procedures. Application of Simulated Stresses and Assessment of Infectivity Potential. Oocyst stocks were exposed to freeze-thaw cycling in stock water in a 2.2-mL screw-top tube. The sealed tube was held at subfreezing temperature (-3 °C) for 48 h and then cycled to a 6 °C compartment for 48 h. This cycle was repeated three times. To desiccate oocysts, 0.36 mL of stock was placed in a 15-mL polypropylene tube, with cap off at 6 °C. This was done to avoid introducing a temperature effect on oocyst degradation. Complete desiccation took place in 36 days. The change in oocyst potential infectivity was evaluated using a combination of stains with high affinity for nucleic acids (propidium iodide (PI) and 4,6-diamadino-4-phenylindole (DAPI) as described by Campbell et al. (24) and Jenkins
et al. (25)). Oocysts were classified according to dye uptake, with D- indicating a lack of DAPI staining of the nuclei and lack of propidium iodide staining in the oocyst, D+ indicating the presence of DAPI in the nuclei of sporozoites, P+ indicating the presence of propidium iodide in the oocyst, and EMPTY indicating that the oocyst was identified by a two-stage, fluorochrome-conjugated antibody labeling kit (EnSys, Inc., Research Triangle Park, NC), but oocysts contained no sporozoites or internal structure when examined using differential interference contrast microscopy. A total of 100 oocysts were classified in each of 6 replicate subaliquots of the original sample extract. Oocysts in the classes D- and D+ are considered to be capable of initiating infection (25). The average results per class from six replicate analyses for unexposed, freeze-thawed, and desiccated oocysts are reported in Table 1. The estimated percents of potentially infective were 97.0, 1.8, and 0.0 for unexposed, freeze-thawed, and desiccated oocysts, respectively. Stock Water and Solutions for Experiments. Stock solutions for experimental use in the LFTM were prepared by adding oocysts to distilled, deionized water (deionized using Barnstead D8904 and Barnstead D8922 filters) reserved for flow cell experiments. Water for suspending oocysts was from the same stock for all experiments. The stock was of neutral pH (7.0) and very low ionic strength (4.8 × 10-5 mol/L estimated from the relationship µ ) 1.35 × 10-2EC, with EC ≡ conductivity (µS) (26)) based upon 10 replicate measurements with a Hanna Instrument HI 8633 conductivity meter (Woonsocket, RI) and 10 replicate measurements with an Accumet Model 15 pH meter. Concentrations of oocysts ranged from 1.5 × 106/mL to 1.2 × 107/mL, with 0.050 mL used per trial. The lowest concentrations were used for trials with Al2O3 to avoid errors in counting caused by excessive sorption. LFTM Inoculation. With the microscope light source off (to avoid heating the substrate and stock water while at rest), 0.050 mL of oocyst stock solution was injected into the flow cell. After inoculation, flow at a rate of 3.3 × 10-5 mL/s
FIGURE 3. Percentage of oocysts added to LFTM sorbed to substrates. 3136
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FIGURE 4. Percentages of oocysts sorbed to aluminum oxide substrates. distributed oocysts over the length of the substrate for 75 min. This permitted at least two complete volume exchanges in the flow cell and allowed complete settling of oocysts to the substrate (assuming negligible resuspension by the flow field, which is reasonable for laminar flow systems), as estimated by Stoke’s law (with a mid-temperature range for kinematic viscosity of 20 °C, and the oocyst diameter of 0.0005 cm). Oocysts settle at approximately 0.0014 cm/s, which indicates that the maximum expected settling time for the flow cell (with depth ) 0.016 cm.) is approximately 11 s. Therefore, all oocysts introduced ino the LFTM had contact with substrates prior to exiting the microduct through the outlet ports. Shear Stresses Applied to Sorbed Oocysts. Experiments were carried out by gradually increasing the flow rate in the microduct to 8.3 × 10-3 mL/s. This flow rate was chosen after exploratory experiments conducted with SiO2 and OTS substrates indicated that sorption was easily reversed by very low shear stresses. The flow rate corresponded to an average velocity within the microduct of 0.93 cm/s, and a velocity near the substrate surface of approximately 0.08 cm/s, estimated as discussed below. The volumetric flow rate was transformed to an estimate of shear stress (in microNewtons (µN)) by estimating the velocity of flow at 0.0025 mm above the substrate (half the diameter of an oocyst) and transforming the velocity to an estimate of force using Stoke’s law. The velocity at any point in a duct with rectangular cross section can be determined by a series approximation of the Navier-Stokes equations representing flow in a three-dimensional system (27). The estimates are valid for laminar flow conditions, which, at fluid temperatures from 16 to 22 °C, are expected for flows of less than 5 mL/s in a duct system with the geometry of the LFTM. We estimated flow velocities at three points orthogonal to the direction of flow (y ) 0.0000, 0.1100, and 0.2200 cm from the centerline of the cell) and found them to be within approximately 10% of one another. This corresponds with the parabolic distribution of flow velocity with no slip boundaries at the surfaces of the flow cell but
also indicated that the change with lateral position was unlikely to introduce a high degree of variation in shear stresses at the observed locations on the substrates. Observations Used To Develop Estimates of Sorbed Percentage of Oocysts. Three replicate experiments were carried out for each of the 12 combinations of substrates and oocysts. After maximum flow rates and shear stresses were applied (8.3 × 10-3 mL/s; approximately 348 µN), the flow was reduced to 0.12 × 10-3 mL/s to ensure that unsorbed oocysts would remain in motion. We then estimated the average densities by observing the number of oocysts in 0.01× 0.01-cm areas at nodes of a grid formed using spatial increments of dx ) 0.40 cm and dy ) 0.11 cm, with x orthogonal to the direction of flow and y parallel to the direction of flow (Figure 1). The average density per square centimeter was then calculated and used to estimate the total number of oocysts remaining on a substrate by multiplying the estimated average density by the area between the inlet and outlet ports of the microduct (1.78 cm2). The sorbed percentage was estimated as the average density divided by the amount added to the microduct at the beginning of the experiment.
Results and Discussion Sorbed Percentages of Oocysts: Differences by Substrate and Treatment. Experimental work examined sorption to multi- and monomolecular chemical substrates (Al2O3, Fe2O3, SiO2, and CH3(CH2)17SiCl3 (OTS) (a self-assembling monolayer 18-carbon polymer). It also examined changes in sorption caused by the stresses of freeze-thaw cycling and desiccation, relative to sorption observed for unstressed oocysts. The data were analyzed using a general linear model with substrate, treatment, and substrate-treatment interactions as factors. The model, developed using Minitab, Release 12 (Minitab, Inc., State College, PA, 1997), indicated that substrate, treatment, and substrate-treatment interactions were determinants of the percent of the number of oocysts added that would remain after application of maximum shear stresses applied (significant at p ) 0.0+). The results for the VOL. 33, NO. 18, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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individual oocyst treatment/substrate combinations indicate that negligible sorption took place on the hydrophobic OTS substrate and Fe2O3 and SiO2 substrates, regardless of prior exposure of the oocyst to simulated environmental stresses. However, the percent of oocysts remaining on Al2O3 substrates was significantly different than zero and appeared to change with increasing disruption of the oocyst from simulated environmental stresses. The estimated percents of starting numbers of oocysts sorbed to substrates are reported in Table 1 and depicted with 95% confidence intervals in Figure 3. Confidence intervals about the estimated percentages indicate that oocyst sorption by a substrate follows the trend Al2O3 > (Fe2O3 ≈ OTS ≈ SiO2 ≈ 0%). Within the Al2O3 substrate, freeze-thawed ≈ desiccated > unexposed > 0% (Figure 3). The 95% confidence intervals about the mean percentages indicate no significant differences between the oocysts subjected to desiccation and freeze-thaw cycling, though sorption associated with each type of stress is significantly different than zero. In addition, a small percentage of unstressed stock oocysts (4%) was sorbed to Al2O3 substrates. However, the stock used for trials contained oocysts that would be considered incapable of initiating infection (approximately 3%, using the criteria of propidium iodide inclusion and lack of internal structure (Table 1)). This proportion of oocysts is low but may have been the result of very slow degradation of the oocyst wall while in storage in water. Estimates of the changes in oocyst population capability of the initiating infection under such conditions are very low. For example, Jenkins et al. (25) estimated that oocyst populations changed at a rate of approximately 0.20.4% per day in seived feces in cold storage. The substrates can be considered as simplified analogues for soil textural classes and hydrophobic organic matter. Sands, largely composed of primary silicate minerals such as quartz (SiO2), are the resistant products of parent rock decomposition (28). With further weathering and further decomposition, the lattice structure of the primary silicate minerals is reconfigured to contain aluminum, iron, and titanium oxides (28, 29). Silts and clays are enriched in oxides, hydroxides, and hydroxyoxides of Al, Fe, and Ti, relative to Si. This leads to an abundance of oxides of iron and aluminum in clay and silt soil textures. The Al2O3, Fe2O3, SiO2, and OTS substrates are simple analogues of textural classes and hydrophobic organic matter that yield insights about likely partitioning in natural soil systems. The differences in the sorbed percentages to Al2O3 substrates suggest that the potential for sorption increased as the internal oocyst structure was disrupted by external stress. A large percentage of the oocysts exposed to freezethaw cycling appeared to have disrupted nuclei with nucleic acids spread throughout the interior of the oocyst as indicated by a high proportion of P+ oocysts (Table 1). In addition, many oocysts had observable slight-to-large fissures in the oocyst wall, possibly due to ice expansion during freezing. A study of cryopreservation of oocysts suggested damage from ice crystal formation left the cell membrane intact while disrupting the internal structures, rendering the oocyst noninfective (30). If oocyst nucleic acids were exposed through fissures in the wall, the magnitude of the negative charge on or near the surface would increase due to the presence of negatively charged phosphate groups in DNA and RNA molecules. This would lead to enhanced sorption to Al2O3, which has a positive charge at neutral pH (29). Approximately a third (29.25%) of the desiccated oocysts were empty with no staining of nucleic acids. The absence of internal structure indicates that desiccation followed by rehydration ruptured and removed the contents of the oocyst in a large proportion (29.25%) of the oocysts exposed. However, a large proportion of oocysts (70.75%) was also 3138
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P+, suggesting that the effect of diffused and exposed nucleic acids enhanced the negative charge of the oocysts, but to a smaller degree than was noted for freeze-thawed oocysts. The unexposed oocysts had a small proportion of oocysts stained by propidium iodide (2.50%). A graph of the proportion of propidium iodide stained oocysts versus the percentage sorbed to Al2O3 substrates (Figure 4) supports this as a working hypothesis. The results suggest that lethal stresses that leave the oocyst intact but remove the capacity to initiate infection may increase the potential for charge-based interactions in the soil environment, especially if positively charged sites are present. This suggests that the presence of clay- and siltsized particles, highly enriched in oxides and hydroxyoxides, may have a significant effect on the transport or attenuation of noninfective oocysts in overland flow or infiltration. However, infective, intact oocysts may enter overland flow as biocolloids without an associated solid-phase sorption site.
Acknowledgments This work was supported in part by funds provided by the USDA Competitive Research Grants Program (Award No. 9337102-8958) and the New York City Department of Environmental Protection through the Watershed Agricultural Program for Watershed Protection and the Department of Agricultural and Biological Engineering, Cornell University. We are grateful to Dr. D. Bowman (School of Veterinary Medicine, Cornell University) for providing oocysts for experimental work and to L. Yeghiazarian (ABEN, Cornell University) for assistance in estimating shear stresses in the LFTM.
Literature Cited (1) LeChevallier, M. W.; Norton, W. D.; Lee, R. G. Appl. Environ. Microbiol. 1991, 57 (9), 2610-2616. (2) LeChevallier, M. W.; Norton, W.; Lee, R. Appl. Environ. Microbiol. 1991. 57(9): 2617-2621. (3) Current, W. L.; Garcia, L. S. Clin. Microbiol. Rev. 1991, 4 (3), 325-358. (4) Madore, M.; et al. J. Parasitol. 1987, 73 (4), 702-705. (5) Smith, H. V.; Rose, J. B. Parasitol Today 1990, 6 (1), 8-12. (6) Atwill, E. R. Rangelands 1996, 18 (2), 48-51. (7) Walker, M.; Montemagno, C.; C. Jenkins, C. Water Resour. Res. 1998, 34 (12), 3383-3392. (8) McCluskey, B. J. Animal Health Insight 1992 (Winter), 1-5. (9) Anderson, B. C.; Hall, R. F. J. Am. Vet. Med. Assoc. 1982, 181 (5), 484-485. (10) Garber, L.; Salmon, M.; Hurd, H.; Keefe, T.; Schlater, J. J. Am. Vet. Med. Assoc. 1994, 205 (1), 86-91. (11) Hayes, E. B.; Matte, T.; O’Brien, T.; McKinley,T.; Logsdon, G.; Rose, J.; Ungar, B.; Word, D.; Pinsky, P.; Cummings, M.; Juranek, D. New Eng. J. Med. 1989, 320 (21), 1372-1376. (12) Mackenzie, W. R.; Hoxie, N.; Proctor, M.; Gradus, M.; Blair, K.; Peterson, D.; Kazmierczak, J.; Addiss, D.; Fox, K.; Rose, J.; Davis, J. New Engl. J. Med. 1994, 331 (3), 161-167. (13) Roberts, M.; Getman, D.; Kolb, B.; Beardsley, E.; Adelman, A. Investigations of Sources of Giardia and Cryptosporidium to Enhance Watershed Management for Wachusett Reservoir. In Watershed Management: Planning for the 21st Century; American Society of Civil Engineers: San Antonio, TX, 1995. (14) Guiver, K. J. Instit. Wat. Environ. Manage. 1991, 5, 99-101. (15) Robertson, L.; Campbell, A.; Smith, H. Appl. Environ. Microbiol. 1992, 58 (11), 3494-3500. (16) Fayer, R.; Nerad, T. Appl. Environ. Microbiol. 1996, 62 (4), 14311433. (17) Sanin, F. D.; Vesilind, P. A.; Martel, C. J. Water Res. 1994, 28 (11), 2393-2398. (18) Anderson, B. C. Am. J. Vet. Res. 1986, 47 (10), 2272-2273. (19) Anguish, L.; Ghiorse, W. Appl. Environ. Microbiol. 1997, 63 (2), 724-733. (20) Brush, C. F. Surface and Transport Properties of Cryptosporidium parvum Oocysts. Agricultural and Biological Engineering; Cornell University: Ithaca, NY, 1997; p 175. (21) Yih, C. S. Fluid MechanicssA Concise Introduction to the Theory; West River Press: Ann Arbor, MI, 1977. (22) Vetterling, J. J. Parasitol. 1969, 55 (2), 412-417.
(23) Kleinfeld, D.; Kahler, K. H.; Hockberger, P. E. J. Neurosci. 1988, 8 (11), 4098-4120. (24) Campbell, A. T.; Robertson, L. J.; Smith, H. V. Appl. Environ. Microbiol. 1992, 58 (11), 3488-3493. (25) Jenkins, M. B.; Anguish, L.; Walker, M.; Bowman, D.; Ghiorse, W. Appl. Environ. Microbiol. 1997, 63 (10), 3844-3850. (26) Tchobanoglous, G.; Schroeder, E. Water Quality Management. Water Quality, 2nd ed.; Reading, MA, Addison-Wesley: 1987; Vol. 1, p 768. (27) Yih, C.-S. Fluid Mechanics: A Concise Introduction to the Theory, Corrected ed.; West River Press: Ann Arbor, MI, 1977.
(28) Brady, N. C. The Nature and Property of Soils, 10th ed; Macmillan Publishing Co.: New York, 1991. (29) Stumm, W.; Morgan, J. Aquatic Chemistry, 3rd ed.; John Wiley and Sons: New York, 1996. (30) Fayer, R.; Nerad, T.; Rall, W.; Lindsay, D.; Blagburn, B. J. Parasitol. 1991, 77 (3), 357-361.
Received for review March 24, 1999. Revised manuscript received June 18, 1999. Accepted June 21, 1999. ES990331F
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