Transport of Cryptosporidium parvum Oocysts through Vegetated Buffer Strips and Estimated Filtration Efficiency

Vegetated buffer strips were evaluated for their ability toremove waterborne Cryptosporidium parvum from surface and shallowsubsurface flow during simulated rainfall rates of 15 or 40mm/h for 4 h. Log₁₀ reductions for spiked C. parvum oocystsranged from 1.0 to 3.1 per m of vegetated buffer, with buffersset at 5 to 20% slope, 85 to 99% fescue cover, soil texturesof either silty clay (19:47:34 sand-silt-clay), loam (45:37:18),or sandy loam (70:25:5), and bulk densities of between 0.6 to1.7 g/cm³. Vegetated buffers constructed with sandy loam orhigher soil bulk densities were less effective at removing waterborneC. parvum (1- to 2-log₁₀ reduction/m) compared to buffers constructedwith silty clay or loam or at lower bulk densities (2- to 3-log₁₀reduction/m). The effect of slope on filtration efficiency wasconditional on soil texture and soil bulk density. Based onthese results, a vegetated buffer strip comprised of similarsoils at a slope of $<=$ 20% and a length of $>=$ 3 m should functionto remove $>=$ 99.9% of C. parvum oocysts from agricultural runoffgenerated during events involving mild to moderate precipitation.

INTRODUCTION

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Introduction

Cryptosporidium parvum has emerged as a widespread and persistentwaterborne microbial pathogen, with specific genotypes ableto be transmitted ambidirectionally between livestock and humans(e.g., amphixenotic) (6, 42, 46, 54). Although we still do notknow the percentage of annual cases of human cryptosporidiosisthat are attributable to livestock-derived waterborne C. parvum(39), reducing the likelihood that animal agricultural operationswill contaminate surface water with infective C. parvum oocystswill help safeguard both water quality and public health (51).Several strategies exist for minimizing the likelihood thatan animal agricultural operation contaminates surface waterwith infective C. parvum oocysts. For example, one such strategyis to reduce the incidence of C. parvum infection or the intensityof fecal shedding of C. parvum oocysts by livestock populations,thereby reducing the rate of environmental loading of C. parvumper livestock unit (26, 36). These herd-health efforts remainhampered by our poor understanding of the medical ecology ofC. parvum within livestock populations (3, 4, 16, 40), how tointerrupt transmission between the biological reservoir andsusceptible animals (3, 40), and the lack of an affordable vaccinethat has been proven to be efficacious in commercial agriculturalsettings (23, 47).

A second strategy is to manage the manure produced by livestockso that the survivability and off-site transport of infectiveC. parvum are substantially reduced (3, 20, 30, 53, 55). Onestrategy being advocated for minimizing the transport potentialof C. parvum oocysts from animal manure to surface water isto place vegetated buffer strips between animal agriculturaloperations and vulnerable surface water supplies (10, 12, 15,32, 38, 51, 59, 60). Optimal design criteria for on-farm vegetatedbuffer strips currently do not exist for waterborne microbialcontaminants. Moreover, studies on the fate and transport ofzoonotic protozoal pathogens such as C. parvum in overland andsubsurface flow through such buffers are severely lacking (9,24, 37, 38, 55; E. R. Atwill, K. W. Tate, M. R. George, andN. K. McDougald, Abstr. Am. Water Resour. Assoc. Symp. RangelandManagement Water Resour., p. 446, 1998).

Our goal for this project was to assess the ability of a meter-longvegetated buffer to remove waterborne C. parvum oocysts fromoverland and shallow subsurface flow as a function of soil andvegetative characteristics, slope, and rainfall intensity. Suchdata will help provide a basis for design criteria for vegetatedbuffers that remove $>=$ 99.9% of C. parvum oocysts from agriculturalrunoff.

MATERIALS AND METHODS

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Materials and Methods

General overall study design.
The filtration efficiency of three different types of soil (sandyloam, loam, and silty clay loam) was evaluated for three slopes(5, 10, and 20%) across two two rainfall intensities (1.5 and4.0 cm/h), with three replicates per treatment, for a totalof 54 soil box trials. Mean percent vegetative cover and meanvegetative height for each box were measured the day beforethe rainfall experiment. Two to four randomized boxes were irrigatedsimultaneously by a rainfall emitter (an experimental run),and a peristaltic pump delivered an oocyst suspension to theupper end of the boxes. Once steady-state conditions were established,the water source for the peristaltic pump was switched to anoocyst suspension for 60 min. Following oocyst application,the peristaltic pump was switched back to non-oocyst water,and the rainfall and pump then continued for an additional 180min to quantify unbound and desorbed oocysts moving in overlandand subsurface flows. Overland and subsurface flows were collectedand analyzed separately.

Source and purification of C. parvum oocysts.
Naturally infected dairy calves from two local commercial dairieswere the source of wild-type C. parvum oocysts. We have determinedpreviously that these oocysts are classified as bovine genotypeA by using the genotyping scheme described by Xiao et al. (58a).By using an acid-fast procedure to detect oocysts (23), sampleswith more than 25 oocysts per x 400 microscopic field were washedthrough a series of 40, 100, 200, and 270 mesh sieves. The resultingsuspension was decanted off and centrifuged at 1,000 x g for10 min. Supernatant was discarded, and the pellet washed inTween water (0.01% Tween 80 in deionized water [vol/vol]). Adiscontinuous sucrose gradient was used to purify C. parvumoocysts from fecal suspensions (1). The concentration of purifiedoocysts was determined as the arithmetic mean of six separatecounts with a phase-contrast hemacytometer. Using tap waterfrom a local well, oocyst suspensions were diluted to 5 x 10⁴oocysts/ml for overland flow application. Oocysts were collected1 to 4 days prior to each experiment in order to minimize biasesassociated with using aged oocysts in the project (8).

Soil types and characterization.
Soils were selected to represent a wide range of soil textures(sandy loam, loam, and silty clay loam) utilized for animalhusbandry in California. The Hanford fine sandy loam, a TypicXerorthent, was selected as the sandy soil. The Argonaut loam,a Mollic Haploxeralf, was selected as the loamy soil. The Capaysilty clay, a Typic Chromoxerert, was selected as the clayeysoil. Soils were air dried, gently crushed, and passed througha 2-mm-pore-diameter sieve to remove coarse fragments. The air-dried,<2-mm-particle-diameter fraction was used for the followinganalyses (Table 1). Soil pH was measured in water (saturatedpaste suspension) following a 15-min equilibration period. Particlesize analysis was performed with a hydrometer in suspensionsdispersed in sodium hexametaphosphate-Na carbonate (18). Organiccarbon was estimated from a modified Walkley-Black analysis,assuming an organic carbon content of 58% for soil organic matter(41). Cation-exchange capacity was determined by barium acetatesaturation and calcium replacement (29). The clay-size fraction(particle diameter, <2 µm) was isolated by flocculationwith NaCl and sedimentation after organic matter removal withsodium hypochlorite (34) and free iron oxide removal with citrate-dithionite(28). X-ray diffraction analysis was performed with orientedclays mounted on glass slides by standard methods (58).

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TABLE 1. Selected soil characterization data for the three topsoil horizons used in this study

Soil box design and vegetation.
Twenty soil boxes were constructed of 0.75-in.-thick plywoodand sealed against leaks and moisture. The dimensions were 15cm wide by 100 cm long by 20 cm deep, allowing us to determinethe filtration efficiency of a meter of vegetated buffer whileallowing for reasonable root development. The box was designedto capture overland flow after it had traversed the 100-cm vegetatedbuffer, and subsurface flow was captured through a slot at thebottom of the seepage face. The junction between the soil surfaceand the inner side of the box wall was sealed with a strip ofwaterproof sealant to minimize seepage of surface flow alongthe vertical side of the box. The slope of each box was adjustedindividually to achieve the desired treatment slopes of 5, 10,and 20%. Perennial fescue seed was used to vegetate the soilboxes, with approximately 50 to 70 g of seed used per box, whichresulted in $>=$ 85% vegetative cover. On the day prior to a rainfallexperiment, we measured the height of the fescue for each boxat points 25, 50, and 75 cm down the length of the soil boxand determined a mean height from these values. Following arainfall simulation, four soil cores of 94.3 cm³ were extractedfrom each box. The combined samples from each box were driedin a convection oven at 37°C for 3 days. The samples wereweighed, and bulk density was calculated as: bulk density (g/cm³)= [net dry weight (g) of all 4 soil cores/(4 x 94.3 cm³)].

Rainfall emitter and storm intensity calculations.
The rainfall emitter was a drip-type box simulator (11) constructedof 0.5-in.-thick plywood and sealed with fiberglass. The dimensionswere 81 cm wide, 112 cm long, and 38 cm deep. The bottom ofthe rainfall emitter was fitted with a uniform grid of 22- or25-gauge needles at a density of 1 needle per 3.5 cm², or 750total needles. The distance between the emitter and surfaceof each soil box was approximately 1.8 m. This design allowedus to apply rainfall to up to four boxes simultaneously.

We selected rainfall intensities of 1.5 and 4.0 ml/cm²/h torepresent a 2- to 3-year or a 20- to 30-year return interval,4-h-duration storm, respectively. We selected these storms tobe representative of those in various agricultural regions throughoutthe United States (25). We developed our rainfall intensityestimates as recommended by Haan et al. (21). From a range of2- to 3-year and 20- to 30-year, 24-h rainfall amounts for agriculturalareas across the United States (25), we estimated the mean rainfallintensity for each 4-h-design storm based on the Soil ConservationService type II rainfall pattern.

Rainfall simulation experiment and collection of overland and subsurface flow.
One day before and on the morning of the experiment, each soilbox was saturated with rainfall for 2 to 6 h while the rainfallemitter was calibrated for the determined storm intensity. Onthe day of the rainfall experiment, a four-channel peristalticpump (Masterflex L/S; Cole-Palmer Instrument Company, VernonHills, Ill.) was positioned at the upper end of the boxes todeliver 10 ml of water or oocysts per min to the upper end ofeach of the boxes in order to simulate overland flow from upslopeareas. Simultaneously, the rainfall emitter was raining at thepredetermined intensity from an elevation of 1.8 m above thebox. Once steady-state conditions were established, the pumpwas switched to an oocyst suspension (5 x 10⁴ oocysts per ml)for 60 min, initiating the start of the experiment. Each boxreceived 10 ml/min from the pump, or 5 x 10⁵ oocysts/min. After60 min, the pump was switched back to water (no oocysts), andboth the simulated rain and peristaltic pump were continuedfor 180 min in order to quantify unbound or desorbed oocystsmoving in overland or subsurface flow. All 4 h of water wascollected, and the volume was recorded as 5- to 30-min compositewater samples from both overland and subsurface flow ports andanalyzed separately, as shown in Table 2. Approximately 3 x10⁷ oocysts were applied to each box.

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TABLE 2. Number of composite water samples collected per soil box during a 4-h experiment

Quantifying the concentration of C. parvum oocysts in overland and subsurface flow.
Quantitative immunofluorescent microscopy, with detailed informationon the percent recovery of spiked oocysts in the source water(13), was used to enumerate C. parvum oocysts in water samples(24). One hundred microliters of a mixture containing 10% Tween80 and 10% sodium dodecyl sulfate was added to 50-ml aliquotsof overland flow and mixed for 5 min on a hand wrist shaker.Larger volumes were tested as needed. The suspension was centrifugedat 1,000 x g for 15 min, and the supernatant was removed until1 ml of residual pellet and suspension remained. One hundredmicroliters of 100% formalin was added, and the suspension wastransferred to a 1.5-ml microcentrifuge tube, centrifuged at11,600 x g for 5 min, and supernatant was removed until 1 mlof residual pellet and supernatant remained. Ten microlitersof suspension was enumerated for C. parvum oocysts by fluorescencemicroscopy with commercially prepared well slides (MeridianDiagnostics, Inc., Cincinnati, Ohio) and a fluorescein isothiocyanate-labeledanti-Cryptosporidium immunoglobulin M antibody (Waterborne,Inc., New Orleans, La.). For subsurface samples, we used a similarprocedure, except that 100- to 250-ml aliquots were concentrateddown to 100 to 500 µl. Positive and negative controlswere included with each run.

In order to determine the percent recovery for the immunofluorescentmicroscopy procedure, we first conducted a sham soil box experimentwith no oocysts added in order to collect clean water samplesfrom each soil type into 50-ml centrifuge tubes. Water sampleswere centrifuged at 1,000 x g for 10 min, and the supernatantwas removed, leaving a small sediment pellet of 10 to 50 mgin each tube. Next, a second sham soil box experiment was conductedwith all of the same equipment and the usual oocyst suspension(5 x 10⁴ oocysts per ml), except that no soil was in the box.Specifically, the soil portion of the box was bypassed to depositthe oocyst suspension (5 x 10⁴ oocysts per ml) via synthetictubing from the oocyst delivery point near the top of the boxesto the collecting flange at the lower end of 12 boxes. The applicationrate was 10 ml/min for 5 min. The source of the oocysts andthe concentration of the oocyst stock solution were determinedas described above. The soil box effluent (oocyst suspension)was then collected into the 50-ml tubes prepared in the firstsham experiment described above, which contained the differentsoil sediments so as to more accurately measure the percentrecovery of detection of oocysts in soil box effluent. The immunofluorescentmicroscopy procedure was then performed as described above.

Statistical analyses.
By using a set of composite samples as shown in Table 2, wemeasured the total number of C. parvum oocysts in overland andsubsurface flow during 4 h of rainfall for each soil box. Ourcovariates (independent variables) for each treatment combinationwere soil type (silty clay loam, loam, and sandy loam), meangrass height (centimeters), mean soil bulk density (grams percubic centimeter), percent slope (5, 10, and 20%), rainfallintensity (1.5 ml or 4.0 ml/h/cm²), and total volume of overlandand subsurface flow during the 4-h rainstorm. Percent coverwas not analyzed given the narrow range of values (85 to 99%).Mean 4-h total subsurface flow was converted to the mean infiltrationrate (10 mm/h = 1 ml/cm²/h) for each soil box. Given our 4-htime frame and approximate steady-state conditions, we ignoredthe negligible influence of evaporation, transpiration, interception,and changes in soil water storage in our calculation of infiltrationrate. The outcome (dependent) variable was the total numberof C. parvum oocysts (nonfiltered or desorbed) discharging fromthe soil box in either overland or subsurface effluent duringthe 4-h rain event, adjusted for percent recovery of the assay,as determined with the following equation:

where n is the total number of composite samplesper 4-h rain event per flow path (overland or subsurface) (Table2), the number of observed oocysts per unit volume is the totalnumber of C. parvum oocysts estimated via immunofluorescentmicroscopy for each composite sample, the total volume per timeperiod is the volume of water collected per sampling time period(5 to 30 min) for each flow path. The percent recovery for theimmunofluorescent microscopy procedure for each soil type wasestimated as described below.

A linear mixed-effects regression model (48) was used to determinethe association between each of our covariates (or fixed effects)and the total number of oocysts discharging off the vegetatedbuffer. Values for the total number of oocysts were transformedvia the natural logarithm, and the experiment (15 randomizedsets of two to four soil boxes per experiment) was modeled asa random effect. We used a forward-stepping algorithm to buildthe model, with significance for inclusion in the model setat a P value of $<=$ 0.10, with a restricted-maximum-likelihood ratiotest.

Percent recovery for the immunofluorescent microscopy procedurefor each soil type was estimated by fitting a negative binomialregression model (22) to the observed number of oocysts, withsoil type modeled as a categorical variable and the number ofspiked oocysts (n = 500) functioning as the offset (or exposure)variable. Negative binomial regression tends to provide a better-fittingmodel than Poisson regression due to typical overdispersionof oocyst count data (3, 26, 56).

RESULTS

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Results

All soil boxes had from 85 to 99% vegetative cover comprisedof perennial fescue, with vegetative heights ranging from 5to 55 in. Soil properties for the silty clay loam, loam, andsandy loam are listed in Table 1. The observed infiltrationrates (10 mm/h = 1 ml/cm²/h) for each soil type ranged from0.001 to 1.8 ml/cm²/h for silty clay loam, 0.001 to 1.9 ml/cm²/hfor loam, and 0.01 to 1.2 ml/cm²/h for sandy loam. Mean bulkdensity for each soil series ranged from 0.52 to 1.36 g/cm³for silty clay loam, 0.88 to 1.57 g/cm³ for loam, and 1.24 to1.84 g/cm³ for sandy loam. Differences in bulk density withineach soil series were due to the inherent inability to replicatesoil compaction within the range realized for these soils underfield conditions.

Compared to the initial application of approximately 3 x 10⁷oocysts, the total number of oocysts that were collected inthe effluent (nonfiltered or desorbed oocysts) ranged from 3.5x 10³ to 1.9 x 10⁷ oocysts (0.01 to 65% of initial oocyst load).The majority of oocysts in the effluent were transported inoverland flow (i.e., median percentages of oocysts that weretransported in overland compared to subsurface flow were 99.8and 0.2%, respectively). The ranges of flow rates were 1,600to 6,700 ml/h for overland flow and 0.4 to 2,800 ml/h for subsurfaceflow. The median percentages of total effluent that were collectedfrom overland or subsurface flow were 96 and 4%, respectively.

A linear mixed-effects model was fitted to the data and usedto evaluate the association between biotic and abiotic factorsand the natural logarithm of the total number of nonfilteredand desorbed oocysts in the effluent of overland or subsurfaceflow. Examples of the raw data for the breakthrough curve forC. parvum are shown in Fig. 1 and 2. Prior to statistical modeling,values for total enumerated oocysts were adjusted for the percentrecovery of our assay (Table 3). Factors significantly associatedwith total oocysts in the effluent of overland flow (proportionalto the inverse of filtration efficiency) were soil type andpercent slope of the vegetated buffer, but this associationwas strongly influenced by the bulk density (Table 4 and Fig.3, 4, and 5). For example, at a 5% slope, for every increaseof bulk density of 0.1 g/cm³, the total numbers of oocysts inthe effluent of overland flow (oocysts avoiding filtration ordetaching during transport across the buffer) were increasedby approximately 12% [e^{(1.14 x 0.1)} - 1 = 0.12], 49% {e^{[(1.14+ 2.83) x 0.1]} - 1 = 0.49}, and 21% {e^{[(1.14 + 0.77) x 0.1]}- 1 = 0.2112} for silty clay loam, loam, and sandy loam, respectively.Alternatively, at a 5% slope and for every increase of 0.1 g/cm³in bulk density, the log₁₀ reductions were reduced by approximately2.5, 8.0, and 5.0% for silty clay loam, loam, and sandy loam,respectively. In other words, increasing the soil's bulk densitysubstantially reduced the ability of the buffer to remove waterborneoocysts in overland flow (Fig. 3 to 5). In general, buffersconstructed of sandy loam compared to the other two soils wereleast effective at removing oocysts in overland flow. For buffersconstructed of silty clay loam and loam, positioning the soilbox at a higher slope was in general more effective at removingoocysts than buffers at a 5% slope, but this trend did not holdat higher bulk densities nor for the sandy loam (Fig. 3 to 5).Given that soil type, slope, and the interactions with bulkdensity were in the regression model, infiltration rate, percentvegetative cover, vegetative height, rate of total overlandflow, and precipitation rate were not associated with filtrationefficiency.

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FIG. 1. Raw data for discharge of C. parvum oocysts from overland flow. The slope was 5%, and the 4-h precipitation rate was 1.5 ml/cm²/h.

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FIG. 2. Raw data for discharge of C. parvum oocysts from subsurface flow. The slope was 5%, and the 4-h precipitation rate was 1.5 ml/cm²/h.

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TABLE 3. Recovery for the oocyst delivery and collection system in combination with the direct immunofluorescent assay

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TABLE 4. Final linear mixed-effects regression models^a for abiotic factors associated with vegetated buffer strip filtration of waterborne C. parvum oocysts in overland flow

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FIG. 3. Modeled filtration efficiency of a 1-m vegetated buffer as a function of bulk density and percent slope with silty clay loam. The initial load was 3 x 10⁷ oocysts.

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FIG. 4. Modeled filtration efficiency of a 1-m vegetated buffer as a function of bulk density and percent slope with loam. The initial load was 3 x 10⁷ oocysts.

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FIG. 5. Modeled filtration efficiency of a 1-m vegetated buffer as a function of bulk density and percent slope with sandy loam. The initial load was 3 x 10⁷ oocysts.

Factors significantly associated with total oocysts in the effluentof subsurface flow were soil type and infiltration rate, butthis association was strongly influenced by the rainfall intensity(Table 5 and Fig. 6). For every increase of 0.1 ml/cm²/h (1mm/h) in the infiltration rate, the total number of oocystsin the effluent of subsurface flow was increased by 86% [e^{(6.21x 0.1)} - 1 = 0.86] and 29% {e^{[(6.21-3.67) x 0.1]} - 1 = 0.29}for lower (-1.5 ml/cm²/h) and higher (-4.0 ml/cm²/h) rainfallintensities, respectively. Buffers constructed of sandy loamresulted in 81% more oocysts in subsurface effluent [e^(0.60)- 1 = 0.81] relative to buffers constructed of silty clay loam.All other biotic and abiotic factors were not significant.

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TABLE 5. Final linear mixed-effects regression models^a for abiotic factors associated with vegetated buffer strip filtration of waterborne C. parvum in subsurface flow

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FIG. 6. Modeled number of total oocysts in subsurface flow effluent as a function of infiltration rate, as predicted by the linear mixed-effects regression model. The initial load was 3 x 10⁷ oocysts, stratified by soil type and precipitation rate (1.5 or 4.0 ml/cm²/h).

Significant random effects were present from experiment to experimentin our study, whereby two to four soil boxes were positionedunderneath the rainfall simulator per experiment. Approximately67 and 45% of the total variation for log total oocysts in theeffluent of overland and subsurface flows, respectively, wereproduced by interexperiment variation. Such variation is notuncommon for these types of experiments; for example, enumerationof C. parvum oocysts via microscopy from replicate samples caninduce substantial intersample variation in oocyst counts (3,26, 56). Both the coefficients and their associated standarderrors for our regression model were adjusted for these randomeffects (48).

There was reasonable agreement between the observed number oftotal nonfiltered and desorbed oocysts in overland flow andthe values predicted by the linear mixed-effects model (Fig.7). Therefore, coefficients from the linear mixed-effects modelswere used to calculate the mean filtration efficiency (log₁₀reduction) associated with the use of vegetated buffers. Thesepredictions were calculated as a function of soil type, slopeof buffer, and interactions with bulk density, given that thesefactors were significant in the regression model (Fig. 3 to5). Hence, the mean log₁₀ reduction of waterborne C. parvumoocysts (bovine genotype A) associated with a 100-cm-long vegetatedbuffer under these experimental conditions ranged from 1.0 to3.1 (Table 6). Oocyst counts in subsurface flow were excludedfrom these calculations, because in almost all cases, totaloocyst counts in subsurface flow did not exceed 10,000 oocysts(0.03% of total), resulting in a negligible effect on the calculatedlog₁₀ reduction of waterborne C. parvum oocysts.

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FIG. 7. Observed number of total C. parvum oocysts in overland flow effluent compared to predictions generated by the linear mixed-effects regression model.

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TABLE 6. Predictions of log₁₀ reduction of waterborne C. parvum oocysts in overland flow attributable to 1 meter of vegetated buffer strip filtration

DISCUSSION

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Discussion

Vegetated buffer strips are being advocated by agencies suchas the U.S. Department of Agriculture as a practical methodto reduce waterborne transport of C. parvum and other zoonoticmicrobial pathogens from animal agricultural operations to nearbysurface water supplies (51). The assumption is that by placinga vegetated buffer of adequate width between livestock populationsand adjacent surface water supplies, microorganisms that areshed in the feces or urine of livestock will be removed (strainedor adsorbed) as they are transported via advective and dispersiveprocesses through the vegetated buffer (10, 12, 15, 32, 59,60). In testing this assumption, we determined that a 100-cm-longvegetated buffer with 85 to 99% perennial fescue cover and underconditions involving a 5 to 20% slope, silty clay loam, loam,or sandy loam and precipitation rates of 1.5 or 4.0 ml/cm²/h,produced a 1.0- to 3.1-log₁₀ mean reduction in waterborne C.parvum oocysts (bovine genotype A) (Table 6).

To put these estimated values for the filtration efficiencyof a vegetated buffer into a public health perspective, a varietyof domestic and wild mammalian species, including humans, havebeen shown to shed in excess of 10⁶ C. parvum oocysts per individualper day during the course of infection (5, 31, 57). In comparison,the 50% infective dose (ID₅₀) for livestock-derived C. parvumfor healthy human adult volunteers ranges from approximately10 to 1,000 (42). This suggests that a length of several metersof vegetated buffer may be needed to generate adequate waterquality protection from relatively few infected individualsthat are located in close proximity to surface water supplies.Appropriate modifications would also need to be made to bufferlength for such factors as higher animal densities or higherrates of endemic cryptosporidiosis for specific mammalian populations(2, 4, 5, 40). Alternatively, the risk of waterborne transmissionof C. parvum from entire livestock production systems (feedlot,cow to calf, dairy, etc.) at large distances from source watersupplies, such as $>=$ 30 m, should be quite minimal if the filtrationefficiency of the intervening buffer has been properly maintained(e.g., adequate soil porosity and vegetative cover) and if thepreferential flow paths and large macropores have been keptto a minimum (e.g., few rills, gullies, and complexes of smallmammal burrows).

We calculated previously that at a 95% confidence level, themean number of C. parvum oocysts being shed in the feces ofCalifornia adult beef cows was around 1.7 x 10⁵ oocysts peranimal per day (26). With these estimates, a California herdcomprised of 500 adult beef cows (no calves or yearlings) wouldon average produce an estimated maximum 8.5 x 10⁷ oocysts/day.Given our range of a 1.0- to 3.1-log₁₀ reduction produced bya meter of vegetated buffer, 3 to 7 m of equivalent buffer wouldbe needed to reduce the translocation potential of freshly depositedC. parvum oocysts to below 1 oocyst for events involving lowto moderate precipitation ( $<=$ 4 cm/h). Such predictions are stillcrude approximations, due to the absence from our calculationsof additional parameters that markedly influence microbial waterquality risk from entire livestock operations (20, 26, 27, 55).Most notably, obtaining valid and precise methods for characterizingmicrobial risk from oocysts in aged fecal material remains problematicdue to the observation that the translocation potential of C.parvum oocysts in aged fecal material (2 to 7 days old duringsummer) appears to be 3 log₁₀ less than that of fresh fecalmaterial, indicating that the majority of bovine-derived oocystsare trapped in aged fecal material (unpublished observation).In addition, exposure of oocysts to a variety of common environmentalstressors can result in rapid loss of viability or infectivity(50). Furthermore, much of the fecal material produced by cattleis deposited in hydrologically remote areas, even when cattleuse surface water supplies (e.g., streams) as their source ofdrinking water (17, 33). Although these additional processescollectively function to decrease the risk to water qualityfor a given C. parvum loading rate and buffer width, this reductionin risk to water quality is offset somewhat by the relativelylarge amount of aged compared to fresh fecal material locatedon the soil surface of livestock production systems (27).

The conceptual model motivating these experiments on bufferstrip filtration is that C. parvum oocysts (or any gastrointestinalmicrobe) are deposited in fecal material, whereby a precipitationevent of sufficient intensity erodes the upper layer of thefecal pat and releases oocysts onto the wetted soil surface.When rainfall intensity exceeds the infiltration capacity ofthe site, these waterborne oocysts are entrained into the prevailingsurface flow (e.g., Hortonian overland-sheet flow, preferentialflow in rills, and exfiltration in saturated variable sourceareas), wherein they are transported both downslope and infiltrateinto the subsurface, the distance and comparative rates beingthe result of a variety of hydrogeological and other local factorsgoverning overland flow over an infiltrating surface (55). Giventhat vegetative buffer strips are intended to intercept andremove these waterborne contaminants before they reach a specifieddownslope site, processes that enhance infiltration of contaminatedsurface runoff into the subsurface should improve buffer stripfiltration. Therefore, for a given soil texture, we hypothesizethat the ability of a vegetated buffer strip to remove waterbornezoonotic microorganisms from agricultural runoff is in parta function of the bulk density or porosity and pore size distributionof the underlying soil. For a specific soil, as bulk densityis decreased, total porosity is increased. In general, thiswill function to increase effective porosity, leading to anincrease in intrinsic permeability, increasing hydraulic conductivity,and thereby causing the infiltration rate (i) of the vegetatedbuffer to increase in some monotonic fashion. Under naturalconditions, variations of bulk density in surface soils arecaused by variation in plant and root density, microbial activity,and density and activity of soil macroorganisms (e.g., insectsand worms), as well as by the amount of cultivation. In thesoil box experiments, variations in bulk density are due tononhomogeneous packing of the artificially filled boxes. Likemacrobiotic activities in natural soils, artificial packingresults in reduced bulk density due primarily to an increasein the macropore and mesopore space, not due to an increasein the micropore space. On the other hand, bulk density differencesbetween different soil textures (i.e., sand, silt, and clay)can be largely attributed to an increase in micropore space,which does not increase the intrinsic permeability or infiltrationcapacity of the soil.

Under constant rainfall intensity (r) where r > i and oncefield saturated steady-state conditions exist (as in our experiment),buffers with lower bulk densities due to increased macro- andmesopore space should exhibit increased saturated hydraulicconductivity, leading to an increase in the proportion of totalflow infiltrating into the subsurface. This enhanced partitioningof total flow into the subsurface should improve the abilityof a buffer to remove via straining and adsorption waterbornezoonotic microorganisms that are being transported via advectiveand dispersive processes. Derivations of this general hypothesishave been presented previously for a variety of surface-subsurfaceinterfacial systems, including, for example, the removal ofsilt particles from sheet flow on rangeland (45).

For our project, this hypothesis was generally correct for eachof our three soils. As the bulk density was decreased, likelydue to the increased density of micro-, macro-, and mesopores,greater amounts of the oocysts were removed from the surfaceeffluent exiting the buffer (Fig. 3 to 5). At the same time,despite the fact that an increase in the infiltration rate wasassociated with increased numbers of oocysts in the subsurfaceeffluent (Fig. 6), but this increase in oocysts in the subsurfaceoutflow was insignificant compared to the decrease in unfilteredoocyst in the surface outflow. These observations suggest thatmanagement practices that decrease the bulk density and increasethe infiltration capacity of agricultural soils could benefitwater quality with respect to microbial contamination. Alternatively,land use practices that compact the soil or reduce its porositymay have a negative impact on microbial water quality by reducingthe ability of vegetated buffers, grazed pastures, paddocks,and grazed rangeland to remove microbial contaminants from overlandor shallow subsurface flow. For example, Blackburn (7) and Giffordand Hawkins (19) summarized a body of work illustrating thatimproperly managed livestock production practices can lead tosoil compaction that reduces soil porosity and infiltrationcapacity. Reduced infiltration capacity was associated withincreased surface runoff, soil erosion, and transport of nonpointsource pollutants. Soil bulk density is a common measure ofsoil compaction, and it has been long established that soilbulk density is negatively correlated with soil porosity andinfiltration capacity, while being positively correlated withsurface runoff (35, 43, 44, 49).

With respect to our set of three soils (silty clay loam, loam,and sandy loam), buffers constructed of sandy loam were leasteffective at removing oocysts from both overland and subsurfaceflow. Relative to the silty clay loam and loam, sandy loam hadsubstantially greater amounts of sand (70%) than the other twosoils ( $<=$ 45%) and was dominated by macropores (pore diameters>0.08 mm) compared to the other two soils that were dominatedby mesopores (0.03 to 0.08 mm) or micropores (<0.03 mm).Moreover, the silty clay loam and loam featured significantmacropore space due to artificial packing, shrinking and swelling,and macrobiotic activity. At the same time, the sandy loam exhibitedthe highest bulk density. The sandy loam was the soil that wasmost closely packed to resemble natural conditions and had probablythe fewest preferential and macropore flow paths due to shrinkingand swelling, macrobiotic activity, or packing nonuniformity.We found previously by using 10-cm columns with saturated, sandysediments that increasing the effective grain diameter from180 to 1,400 µm was associated with a 2-log₁₀ increasein the number of C. parvum oocysts eluting from the column (24).An additional soil difference was that sandy loam had a higherpH, 6.7, compared to pHs of 6.0 and 5.5 for silty clay loamand loam, respectively. Drozd and Schwartzbrod (14) estimatethe zeta potential on the surface of fresh C. parvum oocystsin river water to be -23.7, -21.4, and -19.5 for the pH levelsobserved for these three soils (silty clay loam, loam, and sandyloam), respectively. Hence, the higher pH associated with thesandy loam will generate a more negatively charged soil surfacein combination with a more negatively charged oocyst wall, creatingless favorable conditions for oocyst attachment to the soilsurface compared to those in the other soils. Similarly, elevatedpH has been shown to inhibit attachment of negatively chargedbacteria onto mineral surfaces under saturated conditions (52).

In general, oocyst removal efficiencies were greater for boxeswith higher slopes (10 and 20%) than for boxes with a 5% slope,with this trend particularly strong at lower bulk densities.This effect of slope on oocyst removal could not be explainedby a different infiltration rate (and hence, overland flow rate),due to the observation that the infiltration rate under steady-stateconditions did not significantly change with slope within theconditions of this experiment. If we assume relatively uniformsoil moisture and given that the overland flow rates were similaracross the three slopes used in these experiments, removal efficiencyshould increase monotonically (albeit not necessarily linearly)with increasing slope due to a decreased thickness of the Hortoniansheet flow and a subsequent increased likelihood of oocyst filtration.The decreased thickness of the Hortonian sheet flow resultswhen the overland flow rate (liters/time = Q) is held constant,but sheet-flow velocity (Q/area) is increased due to the higherslope. Interestingly, we did not observe a monotonic increasein filtration efficiency as slope was increased. To explainthis nonmonotonic behavior, we speculate that the soil moisturenear the soil surface was relatively uniform only for boxesset at a 5% slope. At slopes of 10% and particularly 20%, thesoil moisture near the soil surface was lower for upslope sectionscompared to downslope sections within the soil box, due to theboundary conditions imposed by the soil box on the subsurfacedrainage flow. Thus, infiltration rates were nonuniform alongthe soil box surface for boxes with higher slopes, resultingin the observed nonmonotonic behavior of filtration efficiencyas a function of slope. Resolving this interplay between slope,thickness of Hortonian sheet flow, and heterogeneities of theinfiltration rate on filtration efficiency of a vegetative bufferwill require a more controlled study than was performed herein.

Based on the data presented above and within the limitationsof our experimental design, vegetated buffer strips of similarsoils with bulk densities between 0.6 to 1.7 g/cm³, $<=$ 20% slope,and widths of at least 3 m should generally function to remove99.9% of C. parvum oocysts from overland flow generated duringevents involving low to moderate precipitation ( $<=$ 4 cm/h). Theseminimal design criteria for a 3-log₁₀ reduction are recommendedunder the assumption that vegetated buffers will be activelymaintained such that overland and subsurface preferential flowpaths are kept to a minimum. Communities with high levels ofwater quality risk intolerance, substantial amounts of recreationalcontact with surface water, or watersheds with high rates ofC. parvum loading may want to strengthen these criteria in orderto further minimize overland flow of C. parvum.

ACKNOWLEDGMENTS

This project was funded by a grant from the International LifeSciences Institute North America, Technical Committee on FoodMicrobiology.

FOOTNOTES

* Corresponding author. Mailing address: Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of California-Davis, Tulare, California 93274. Phone: (559) 668-1731. Fax: (559) 686-4231. E-mail: ratwill{at}vmtrc.ucdavis.edu.

REFERENCES

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