Seasonal Temperature Fluctuations Induces Rapid Inactivation of

May 10, 2005 - Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of California Davis, 18830 Road 112, Tulare...
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Environ. Sci. Technol. 2005, 39, 4484-4489

Seasonal Temperature Fluctuations Induces Rapid Inactivation of Cryptosporidium parvum X U N D E L I , † E D W A R D R . A T W I L L , * ,† LISSA A. DUNBAR,† TED JONES,‡ JIMMY HOOK,† AND KENNETH W. TATE§ Veterinary Medicine Teaching and Research Center, School of Veterinary Medicine, University of CaliforniasDavis, 18830 Road 112, Tulare, California 93274, Stanford Center for DNA Sequence and Technology, Department of Biochemistry, Stanford University, Palo Alto, California 94304, and Department of Agronomy and Range Science, University of CaliforniasDavis, California 95616

This study measured the inactivation rate of bovine genotype A Cryptosporidium parvum oocysts attributable to diurnal oscillations of ambient temperature and solar radiation typical of California rangelands and dairies from spring through autumn. We first measured the relationship between air temperature and the internal temperature of bovine feces exposed to sunlight on commercial operations throughout California. Once maximum air temperature exceeded the mid 20 °C, diurnal thermal regimes of bovine fecal material exhibited peaks of over 40, 50, 60, and 70 °C. These diurnal thermal regimes were emulated using a thermocycler, with oocysts suspended in distilled water or fecal-water mix. Using oral inoculations of 105 C. parvum oocysts per neonatal Balb/c mouse (>1000fold the ID50), no infections were observed using 1 to 5-day cycles of these thermal regimes. Loss of infectivity induced by these thermal regimes was primarily due to partial or complete in vitro excystation during the first 24-h diurnal cycle and secondarily to thermal inactivation of the remaining intact or partial oocysts. These results suggest that as ambient conditions generate internal fecal temperatures g40 °C via conduction, radiation, and convection, rapid environmental inactivation occurs at a rate of g3.27 log reduction d-1 for C. parvum oocysts deposited in the feces of cattle.

Introduction Cryptosporidium parvum bovine genotype A is readily isolated from symptomatic and asymptomatic cattle (Bos taurus) and from a varying percentage of clinical cases of human cryptosporidiosis (1). Accurate estimates of the annual incidence of interspecies cycling of C. parvum between these two host species is still unknown, but it is likely that the transmission of oocysts from cattle to humans involves varying degrees of waterborne (e.g., municipal, recreational exposure), foodborne (e.g., irrigated foods), and direct routes of exposure (e.g., occupational, recreational) (2). Among these * Corresponding author phone: (559)688-1731; fax: (559)686-4231; e-mail: ratwill@vmtrc. ucdavis.edu. † School of Veterinary Medicine, University of CaliforniasDavis. ‡ Stanford University. § Department of Agronomy and Range Science, University of CaliforniasDavis. 4484

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routes of exposure, waterborne transmission remains a particular focus given the continuing concern for human illness following waterborne contamination from animal agriculture (3-10). Several steps need to occur in order for a substantial flux of waterborne, infective C. parvum oocysts to discharge from adjacent cattle operations (11). Necessary conditions likely include the deposition of a sufficiently large environmental load of oocysts in conjunction with a sufficiently intense hydrological event such that the inefficiencies of transporting infective oocysts from the terrestrial to aquatic component of the watershed can be overcome (5, 6, 12-17). For example, we and others have found that as little as a single meter of vegetated buffer can reduce the total flux of waterborne oocysts by up to 99.9%, depending on climatic, vegetation, soil, and slope conditions of the buffer strip (5, 6, 8, 16). There often exists substantial delays in time between when livestock deposit environmental loads of C. parvum and hydrological events such as rainfall, particularly under arid climatic conditions. As a consequence, the initial load of infective oocysts may be reduced by such processes as thermal or chemical inactivation and background rates of senescence (18-27). The effect of ambient temperature on the infectivity of C. parvum oocysts has traditionally been evaluated using constant thermal regimes (18-21), yet ambient temperatures experienced by C. parvum oocysts that have been excreted by a host experience diurnal oscillations when contained within a fecal matrix (7). This study measured the rate of inactivation for spring through autumn of C. parvum attributable to these diurnal swings of ambient temperature and solar radiation typical of lower elevation California animal agricultural land. Accurate rates of thermal inactivation would allow watershed and agricultural managers to better design grazing and livestock management schemes on critical watersheds, such as identifying seasonal removal dates for cattle so that sufficient time is allotted for thermal inactivation of the environmental load of C. parvum prior to the onset of fall rains (7).

Materials and Methods Acquiring Ambient and Fecal Matrix Temperatures. At 11 different commercial diary and cow-calf operations from throughout California, air and the internal temperature of bovine fecal pats that were exposed to sunlight were collected for 12 months using the Optic StowAway Temp Logger system (Onset Computer Corporation, Bourne, MA 02532). Temperature loggers were 2.5 × 2.0 × 13.2 cm3 and collected data at 15 min intervals, with a temperature range from -35 °C to +75 °C. Temperature loggers were placed mid-depth in a ∼1 Kg fecal pat and recorded data for approximately 2 months, at which time the logger was removed, data downloaded into a computer, and the logger replaced into a fresh fecal pat. Simulating Fecal Matrix Temperatures. From our database of air and fecal matrix temperatures from lower to middle elevation (30-760 m) regions in California, we selected four typical diurnal profiles of fecal matrix temperatures with maximum midday values of ∼45 °C, ∼56 °C, ∼68 °C, and ∼71 °C (Figure 1). These four thermal profiles correspond to maximum daytime air temperatures between the mid 20’s °C to the upper 30’s °C for fecal pats exposed to direct solar radiation. Using a custom-made UNIX fitting algorithm, we constructed a time-by-temperature 24-h step function that emulated the four diurnal thermal profiles (Table 1). These step functions were then programmed into 10.1021/es040481c CCC: $30.25

 2005 American Chemical Society Published on Web 05/10/2005

TABLE 2. Reduction in Infectivity of C. parvum Oocysts for Neonatal Balb/c Mice Due to Exposing Oocysts Suspended in Water to One or More 24-h Diurnal Temperature Cycles tempa

daysb

no. of experiments

treated ocysts

fresh oocysts

inactivated oocysts

40 °C

1 2 3 4 5 1 2 3 4 1 2 3 4 1 2 3

2 1 1 1 1 3 2 2 1 3 2 2 1 3 2 2

0/15c 0/7 0/7 0/6 0/6 0/18 0/17 0/14 0/7 0/22 0/16 0/11 0/5 0/24 0/13 0/12

7/7c 4/4 4/4 4/4 4/4 19/19 13/13 12/12 7/7 14/14 10/10 16/16 7/7 16/16 10/10 10/10

0/5c 0/5 0/5 0/5 0/5 0/18 0/14 0/11 0/8 0/15 0/10 0/14 0/6 0/19 0/14 0/14

50 °C

FIGURE 1. Representative 24-h thermal profiles measured within bovine fecal pats located on grazed rangeland from throughout California during spring through autumn, 2000.

TABLE 1. Diurnal Temperature Step Functions Modeled after 24-h Ambient Temperatures Measured Inside Bovine Fecal Pats Located on Grazed Rangeland in California, Spring through Autumn, 2000 cycle no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

40 °C cycles temp timea

50 °C cycles temp time

60 °C cycles temp time

70 °C cycles temp time

19.1 19.9 21.9 23.3 24.6 26.0 27.3 28.8 30.2 31.6 33.1 34.7 35.9 37.5 39.0 40.5 42.5 44.3 45.6 44.3 42.8 40.6 38.7 37.4 35.6 33.8 31.9 29.9 28.2 26.5 25.1 24.2 23.2 21.8 20.7

16.2 22.0 26.9 30.9 33.8 36.5 39.6 45.1 48.4 52.9 54.8 52.2 49.4 46.8 43.8 39.0 34.1 30.1 27.1 24.4 21.7 20.7 18.2 16.1 13.9 10.5 10.5 10.5 9.5 11.4

25.7 30.8 35.9 40.1 44.4 48.0 51.4 54.4 57.7 60.1 62.8 63.8 60.9 58.2 54.1 47.2 42.6 39.1 36.9 33.7 30.6 27.0 24.7 23.0 19.7 18.4 18.2 18.8 21.0 21.0

13.2 16.2 19.3 23.7 27.8 30.8 35.6 38.3 42.8 45.9 50.2 52.3 56.4 60.9 64.4 68.4 71.1 68.9 64.5 61.1 55.9 51.0 46.5 42.9 38.0 33.6 29.6 25.8 23.5 20.8 18.1 15.0 15.0 12.6 12.6

90:00 45:00 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 45:00 45:00 90:00 45:00 45:00 45:00 22:30 22:30 22:30 22:30 45:00 45:00 45:00 90:00 45:00 45:00 90:00 90:00 90:00

45:00 22:30 22:30 22:30 22:30 45:00 45:00 22:30 22:30 90:00 45:00 45:00 45:00 22:30 22:30 22:30 22:30 45:00 45:00 45:00 90:00 45:00 45:00 90:00 90:00 90:00 90:00 90:00 45:00 45:00

45:00 22:30 11:15 11:15 22:30 22:30 22:30 22:30 45:00 45:00 90:00 90:00 45:00 22:30 22:30 22:30 22:30 45:00 90:00 90:00 90:00 45:00 45:00 90:00 90:00 90:00 90:00 45:00 22:30 22:30

45:00 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 22:30 45:00 45:00 45:00 45:00 45:00 45:00 45:00 22:30 22:30 45:00 45:00 45:00 45:00 45:00 45:00 90:00 90:00 90:00 90:00 90:00

a Time durations are expressed as minutes and seconds (min:sec) and total time per cycle is 24 h.

a 96-well automated Thermocycler (GeneAmp PCR System 2700, Applied Biosystems, Foster City, CA 94404). Source of Wild-Type C. parvum Oocysts. Feces were collected from naturally infected calves at 9-21 days of age from a commercial dairy in Tulare, CA. Oocysts from these operations have been previously classified as bovine genotype A (28). Using an acid fast protocol on direct fecal smears, samples having more than 25 oocysts per ×400 microscopic field were washed through a series of 40, 100, 200, and 270

60 °C

70 °C

a Diurnal thermal profiles mimic temperatures in bovine fecal pats, as outlined in Figure 1. b One day is equivalent to a single 24-h cycle of diurnal temperature outlined in Figure 1. c No. of infected mice/no. of inoculated mice.

mesh sieves with Tween water (0.2% Tween 20 in deionized water [vol/vol]). The resulting suspension was centrifuged at 1500 × g for 20 min in a 250 mL centrifuge tube, the supernatant was discarded, and the pellet was resuspended in Tween water. Oocysts were purified using discontinuous sucrose gradient (29) and suspended in deionized water. The concentration of purified oocysts was determined as the arithmetic mean of 6 separate counts using a phase contrast hemacytometer. Stock solutions were prepared by the diluting of oocysts in deionized water to concentrations of 106 and 107oocysts/mL, stored at 4 °C, and used within 1 week. In Vitro Thermal Exposure for C. parvum Oocysts. For each thermal regime, 96 100-µL MicroAmp reaction tubes (Applied Biosystems, Foster City, CA 94404) were filled with oocyst stock solution (105 oocysts/tube). After completing a 24-h thermal cycle (the second or middle cycles outlined in Figure 1), a set of 15 tubes was removed from the thermocycler. The remaining tubes were subjected to a replicate 24-h thermal cycle, another set of 15 tubes was removed, and so on until oocysts were rendered noninfectious. The process of removing tubes and restarting the thermocycler was done within 1 min so that the break of continuous temperature was minimized. Oocyst suspensions from replicate temperature × duration tubes were combined, and the percentage of intact, partial, and ghost oocysts were determined using differential interference contrast microscopy at ×400 (Olympus BX 60, Olympus America, Inc., NY 11747). One hundred microliters was then used as an oral inoculumn per neonatal mouse to test for infectivity of the original 105 oocysts/tube, using the assay described below. The number of experiments conducted for each thermal regime are shown in Table 2. For positive controls, neonatal mice were given 105 fresh oocysts in a 100 µL volume; negative controls were neonatal mice inoculated with either distilled water or 105 heat-inactivated oocysts (exposed to 70 °C for 2 h) to monitor for possible detection of oocysts in intestine directly from inoculums. In addition to the experiments above, oocysts were suspended in a fecal-water mixture and exposed to a single 24-h cycle of each thermal regime (Table 3) to determine if inactivation rates were appreciably different for oocysts suspended in distilled water as apposed to a fecal matrix. Fecal-water suspension was prepared by filtering 200 g of diarrheic feces from a dairy calf with no detectable C. parvum VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. Reduction in Infectivity of C. parvum Oocysts for Neonatal Balb/c Mice Due to Exposing Oocysts in Fecal Suspensions to One 24-h Diurnal Temperature Cycle temp cyclea

treated oocysts

fresh oocysts

inactivated oocysts

40 °C 50 °C 60 °C 70 °C

0/8b

3/3b

0/8 0/6 0/4

5/5 4/4 6/6

0/2b 0/3 0/5 0/5

a Diurnal thermal profiles mimic temperatures in bovine fecal pats, as outlined in Figure 1. b No. of infected mice/no. of inoculated mice.

oocysts through 4 layers of cotton gauze. The resulting fecal suspension had a specific gravity of 1.023 g/mL, pH 7.2, and near 100% moisture. One part stock solution (107 oocysts/ mL) was added to 9 parts fecal suspension, with 100 µL aliquots of the oocyst suspension exposed to the 4 thermal regimes as above. In Vivo Infectivity Assay for C. parvum Oocyst. We used a minor modification of Hou et al. (30), which was based in part on methods developed by Freire-Santos et al. (26), Mtambo et al. (31), and Vergara-Castiblanco et al. (32). Female Balb/c mice with neonatal pups were purchased from Harlan Company (San Diego, CA 92121), housed in cages fitted with air filters, and given food and water ad libitum. Intragastric inoculations of oocysts were delivered in 100 µL of deionized water to 4-day-old neonatal mice, using a 24-gauge ballpoint feeding needle. One hour prior to infection, the neonatal mice were removed from the dam to ensure that their stomachs were empty; following inoculation, the dam was returned to the pups. Litters of mice were randomly assigned to treatment groups, with positive and negative controls as described above. The average litter size was 6. To determine infection status, the entire intestine was collected and suspended in 5 mL of deionized water in a 50 mL tube and homogenized with KIKA-WERKE tissue homogenizer (IKA WERKE GMBH & CO. KG, Staufen, D-79219,

Germany). The homogenates were pelletted by centrifugation at 1500 × g for 10 min, supernatant discarded, pellet resuspended in deionized water, and filtered through a 20 µm Nylon Net filter (Millipore Co., Bedford, MA 01730) that had been fixed on a Swinnex holder (Millipore Co., Bedford, MA 01730). Filtrates were concentrated to 1 mL by centrifugation at 1500 × g for 10 min. Fifty microliters of the final suspension were mixed with 50 µL of fluorescent isothiocyanate-labeled-anti-Cryptosporidium immunoglobulin M antibodies (Meridian, Cincinnati, OH 45244) and 2 µL of 0.5% Evans blue in PBS and incubated at room temperature for 45 min in a dark box. Three duplicate wet mount slides were prepared from each sample, using 20 µL of reaction mixture per slide. Slides were examined with epifluorescent microscopy (Olympus America, Inc., New York, NY 11747). A mouse was considered infected if one or more oocysts were detected in any of the 3 slides. Tissue homogenates were shown to be twice as sensitive in detecting C. parvum infections in neonatal mice compared to histopathology (30). Dose-Dependent Infectivity of C. parvum Oocysts in Neonatal Mouse. Using the same neonatal mouse model as described above, nine litters of neonatal mice were inoculated with either 50, 100, 200, 500, 103, 2 × 103 , 5 × 103, 104, and 105 fresh oocysts prepared by serial dilution from a stock solution of 106 oocysts per milliliter. Infectivity of each dosage was expressed as the percentage of infected mice, calculated as no. of infected mice/no. of inoculated mice. Quantitative Analyses. We used logistic regression to model the dose-response curve for determining infectivity of C. parvum oocysts for our neonatal mouse bioassay (30). The logit (ln(p/1-p)), whereby p ) the probability of a neonatal mouse to become infected, functioned as the outcome variable, ln(dose) was the covariate (ln(y)), and standard errors were estimated using a robust estimator to adjust for potential lack of independence within litters of mice (33). The model can be interpreted more easily as

TABLE 4. Percentages of Intact, Partially Excysted, and Empty (Ghosts) Oocysts Induced by Exposing C. parvum Oocysts to One or More 24-h Diurnal Temperature Cycles treatmenta 40 °C cycle

heat inactivated oocystse 50 °C cycle

heat inactivated oocystse 60 °C cycle

heat inactivated oocystse 70 °C cycle

daysb 0d 1 2 3 4 5 0d 1 2 3 4 0d 1 2 3 4 0d 1 2 3

heat inactivated oocystse

% of intact oocystsc

% of partial oocysts

% of empty oocysts

96.2 ( 0.4 2.5 ( 0.4 0.9 ( 0 0 0 0 93.3 ( 0.3 98.6 ( 0.4 3.9 ( 1.1 0.9 ( 0 0.3 ( 0.4 0 94.6 ( 1.4 95.9 ( 0.3 4.9 ( 1.0 3.5 ( 0.6 2.4 ( 0.6 0 91.9 ( 0.7 94.3 ( 0.9 7.3 ( 0.7 3.2 ( 0.7 1.5 ( 0.5 85.7 ( 0.6

2.3 ( 0.5 62.6 ( 4.3 59.2 ( 6.0 53.0 ( 5.4 50.2 ( 6.2 49.2 ( 5.1 4.7 ( 0.5 1.2 ( 0.3 41.8 ( 3.8 49.8 ( 5.9 43.8 ( 1.6 47.5 ( 6.0 4.6 ( 1.2 3.3 ( 0.4 38.1 ( 1.6 57.1 ( 6.1 49.6 ( 4.6 43.4 ( 3.9 4.0 ( 1.3 3.3 ( 0.6 47.4 ( 5.9 48.5 ( 1.4 48.6 ( 2.5 9.6 ( 1.9

1.5 ( 0.2 34.9 ( 4.7 40.4 ( 5.8 47.0 ( 5.3 49.8 ( 6.2 50.8 ( 4.9 2.1 ( 0.3 0.2 ( 0.3 54.3 ( 2.9 49.3 ( 6.0 55.9 ( 2.0 52.3 ( 5.8 0.7 ( 0.2 0.8 ( 0.1 57.0 ( 2.0 38.5 ( 6.9 48.1 ( 4.2 56.9 ( 4.3 4.1 ( 0.8 2.4 ( 0.7 45.3 ( 6.5 48.3 ( 1.8 49.9 ( 2.7 4.6 ( 1.4

a Diurnal thermal profiles mimic temperatures in bovine fecal pats, as outlined in Figure 1. b One day is equivalent to a single 24-h cycle of diurnal temperature outlined in Figure 1. c No. of intact, partially excysted, and empty oocysts, divided by no. of all oocyst forms, then multiplied by 100. Results are expressed as the arithmetic mean ((SD). d Fresh oocyst without any thermal treatment. e Acute exposure to 70 °C (5-10 min rise from 4 to 70 °C, followed by 2 h duration at 70 °C).

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P (infection | y infective oocysts) )

1 1 + e-(R+βln(y))

(1)

To estimate the log reduction of infectivity of C. parvum attributable to the various thermal treatments, one can use an estimator based on the dose-response curve and the binomial distribution. Briefly, the proportion of neonatal mice becoming infected (p) from a given dose of infective oocysts (y) can be predicted by eq 1 above. Next, the probability (P) of observing x infected neonatal mice out of N mice inoculated with y infective oocysts can be calculated from the binomial distribution

P(X)x) )

()

N (p)x (1 - p)N-x x

(2)

whereby p is calculated from eq 1. If we set P(X)x) > 0.05 (i.e., we exclude scenarios with probability of occurrence less than 0.05), we can then solve eq 2 by identifying values of y (dose of oocysts remaining infective after thermal exposure) that fulfill our constraints x, N, P > 0.05. Once y is solved, then the daily log reduction of infectivity of C. parvum attributable to a single (t)1) 24-h diurnal temperature treatment is calculated as δ ) -log (y/105). If no infections are observed among the neonatal mice, the solution to y reduces to

1 1 + e-(R+βln(y))

< 1 - (0.05)1/N

(3)

Finally, to generate predictions of first-order decay kinetics of oocyst infectivity attributable to diurnal thermal fluctuations

Ct ) C010-δ(t)

(4)

whereby C0 is the initial number of infective oocysts at t ) 0, δ is the daily log reduction of C. parvum infectivity estimated above, t is the number of days at a specific thermal regime, and Ct is the amount of oocysts remaining infective after t days of thermal treatment.

Results Oral inoculation of 50 and 100 fresh C. parvum oocysts resulted in 12.5% (1/8) and 87.5% (7/8) infected neonatal Balb/c mice. Inoculation of 200, 500, 1000, 2000, 5000, 10 000, or 100 000 oocysts per neonatal mouse resulted in 100% infections. The coefficients for the logistic regression model depicting the dose-response curve are R ) -24.244 (95% CI: -25.03, -23.46) and β ) 5.695 (95% CI: 5.50, 5.89), resulting in an estimated ID50 for neonatal Balb/c mice of 70.6 oocysts. Based on the assessment procedures outlined above, exposing C. parvum oocyst to as few as one and up to five 24-h cycles of the 40 °C, 50 °C, 60 °C, or 70 °C thermal regimes (Figure 1) resulted in 100% loss of infectivity when mice were given 105 oocysts (>1000 the ID50) (Table 2). Oocysts suspended in a fecal-water mixture and exposed to a single 24-h cycle of each thermal regime shown in Figure 1 likewise exhibited 100% loss of infectivity when mice were given 105 oocysts (Table 3), resulting in no measurable difference in the inactivation rate for oocysts suspended in either distilled water or a fecal suspension. Based on eqs 1-3 and the sample sizes outlined in Table 2, the estimated log reduction of infectivity (δ) attributable to a single 24-h cycle of the 40 °C, 50 °C, 60 °C, or 70 °C thermal regimes was g3.27, g3.28, g3.30, and g3.31, respectively. Therefore, the decay of C. parvum infectivity as a function of daily temperature fluctuations inside a fecal matrix are predicted to range from C010-3.27(t) to C010-3.31(t), with C0 being the initial number of infective oocysts in a

fecal load at t ) 0 and t being the number of days occurring at a specific fecal thermal regime (40 °C, 50 °C, 60 °C, or 70 °C). Premature partial or complete excystation (outside of a host) was the primary mechanism of thermal inactivation. Exposure to a single 24-h cycle of 40 °C, 50 °C, 60 °C, or 70 °C thermal regimes resulted in >90% excystation of oocysts prior to in vivo inoculation, with almost all remaining intact oocysts excysting at least partially during the subsequent 24-h cycle (Table 4).

Discussion We have determined that once climatic conditions generate internal fecal temperatures g40 °C, rapid environmental inactivation occurs at rates of g3.27 to g3.31 log reduction d-1 for C. parvum oocysts deposited in the feces of beef and dairy cattle. These ambient conditions for rapid oocyst inactivation began to occur as maximum daytime air temperature reached the mid 20 to upper 30 °C for rangeland locations and dairy farms throughout California (data not shown). This suggests that as air temperature reaches the mid 20 to upper 30 °C, oocysts lodged within bovine fecal matrices and exposed to solar radiation are being inactivated at a daily rate (t) of at least 10-3.27(t), such that e0.00054 of the previous day’s oocyst load remains infective following either of the four 24-h fluctuating diurnal temperature regimes shown in Figure 1. Calculations for the number of days needed to generate 100% theoretical inactivation are conditional on the initial load of oocysts (C0) at t ) 0 and need to be adjusted for the ongoing rate of daily oocyst loading that typically occurs by infected livestock. For example, we recently calculated the daily environmental loading rate of C. parvum for adult beef cows on California range to be 3900 to 9200 oocysts cow-1 d-1, with 6550 oocysts cow-1 d-1 being a central value (36). After 1 day of a 40 °C diurnal temperature fluctuation shown in Figure 1, on average 3.5 oocysts remain infective (6550 × 10-3.27(1) ) 3.5 infective oocysts) from a cow’s daily load of oocysts. After the second day, the remaining 3.5 oocysts are theoretically inactivated (6550 × 10-3.27(2) , 1 infective oocyst), indicating that infective oocysts deposited by an adult beef cow on these western rangeland systems do not accumulate beyond 1 day during spring through fall when air temperatures exceed the mid 20 to upper 30 °C. Infected calves typically shed higher concentrations of oocysts compared to adults, with C. parvum infection highly dynamic and in part a function of age, herd management, and season (45). Assuming a value of 106 oocysts calf-1 d-1, about 540 oocysts remain infective (106 × 10-3.27(1) ) 537 infective oocyst) from a calf’s daily load of oocysts after 1 day of a 40 °C diurnal temperature fluctuation shown in Figure 1. After the second day, the remaining 540 oocysts are theoretically inactivated (106 × 10-3.27(2) < 1 infective oocyst). Field validation would need to be conducted in order to accept these predictions as accurate. This rapid loss of infectivity attributed to elevated thermal conditions is consistent with previous work on the survival of C. parvum oocysts in the environment. For example, using neonatal mice to determine infectivity, C. parvum oocysts in distilled water became noninfective if heated to72.4 °C for g1 min or heated to 64.2 °C for g2 min (18). Likewise, C. parvum oocysts became noninfective if heated at 55 °C for 15 s, 60 °C for 15 s, and 70 °C for 5 s (19). In contrast to this previous work which used short incubations of elevated temperature delivered as an abrupt heat-wave function (very rapid rise to a constant peak temperature), naturally occurring diurnal swings of temperature for bovine fecal material exposed to solar radiation during spring through autumn experience a much slower rise and a considerably longer duration of temperature above 40 °C, often in excess of 6-8 h (Figure 1 and Table 1). Despite this slower rate of increase VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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in temperature for these diurnal thermal regimes, which might have allowed infective oocysts to stabilize to these higher temperatures, none of the neonatal mice inoculated with 105 heat-treated oocysts exhibited active C. parvum infections based on our in vivo assay. Apparently, heat tolerance does not occur in any significant manner for C. parvum under these experimental conditions. The primary cause of inactivation of oocyst infectivity under these experimental conditions was >95% premature partial or complete excystation outside of the host, whereby the released sporozoites have a very limited ability to survive in the environment while passively or actively questing for a host. This phenomena of in vitro excystation being stimulated by warm aqueous solutions in the absence of reducing conditions and pancreatic enzymes or bile salts has been observed previously (34). We constructed our heattreated 70 °C controls to somewhat mimic previous work on thermal inactivation of C. parvum oocysts (18, 19), in that oocyst suspensions were taken from 4 to 70 °C in 5 to 10 min. Under these acute thermal conditions we observed only minor amounts of excystation (5-10%), which would have lead us to the faulty conclusion that the primary cause of inactivation attributable to diurnal fluctuations of temperature was direct thermal inactivation of oocysts rather then premature excystation and subsequent inactivation of the released sporozoites. Our primary motivation for estimating rates of inactivation for C. parvum across California agricultural locations was to further the development of a risk assessment framework for waterborne infective C. parvum attributable to animal agricultural systems, like herds of beef cattle grazing on open range (11). The primary processes governing the risk of contamination of infective C. parvum for adjacent bodies of water include the rate of environmental loading from the animal agricultural commodity of concern (4, 35-39), proportion of the daily fecal load deposited directly in the waterway (40), the spatial distribution of the remaining fecal material on the landscape and in proximity to waterways (41), the efficiency of oocyst escapement from the fecal matrix (14, 16, 17), overland and subsurface transport for oocysts deposited on the terrestrial component of the watershed (5, 6, 8, 12, 13, 16, 42-44), and the rate of environmental inactivation of oocyst infectivity (21-25, 27). Using this framework and a variety simplifying assumptions, crude estimates for the risk of contamination of waterborne infective C. parvum attributable to these extensive livestock production systems can be roughly calculated by extending the example presented above regarding decay kinetics of oocyst infectivity for beef cattle and their calves. Consider a herd of 100 cow-calf pairs grazing a mountain pasture in July in the Sierra Nevada Range, with occasional afternoon thunderstorms leading to some combination of Hortonian- and Dunne-type overland flow conditions that hydrologically connect the surface of the grazed meadow to an adjacent perennial stream. As in the example above, we will assume adult beef cows and young calves on California rangeland shed approximately 6550 and 106 oocysts animal-1 d-1, respectively (36). Using similar values as those observed for cow-calf herds in eastern Oregon (40), we will presume that 98 and 2% of the daily fecal load is deposited on the meadow and in the stream, respectively. Furthermore, about 25% of the daily fecal load in the meadow is deposited in the variable source area (41) where a fence has been positioned 3 m upslope and parallel to our perennial steam, with the remaining fecal load being hydrologically remote and therefore ignored. Our mountain meadow with loamy soils might generate ∼2.0 log reduction per meter of meadow buffer during overland flow conditions (6), and the mean interstorm interval is set at 8 days in July. Last, about 15% of oocysts are capable of escapement from the fecal matrix 4488

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during rainfall (17), and the daily thermal regime for bovine fecal material in the meadow resembles the 40 °C profile in Figure 1, so g3.27 log reduction d-1 accrues due to radiation, heat conduction, and convection. Therefore, this cow-calf herd generates a daily oocyst load of ∼1 × 108 oocysts d-1 (100 cow-calf pair × 1 006 550 oocysts cow-calf pair-1 d-1), from which 2% (2 × 106 oocysts) are directly deposited in a fecal matrix within the stream. Of the remaining 98% of the oocyst load, only ∼3.7 × 106 oocysts (∼108 oocysts × 0.98 × 0.25 × 0.15) are entrainable in overland flow that are deposited d-1 about 3 m from the stream’s edge. During the 8-day interstorm interval, the accumulating oocyst load from the 100 pairs of cow-calves is subjected to 3.27 log reduction d-1, resulting in 9.26 × 105 oocysts remaining infective as the thunderstorm commences at 6 a.m. on the 9th day. As infective oocysts are released from the fecal matrix and enter overland flow, they are subjected to 2.0 log reduction m-1 of meadow buffer, resulting in ∼93 infective oocysts reaching the stream’s edge from 3 m away, or a ∼7 log reduction for the overall terrestrial load of oocysts that accumulated between storm events. This relatively small amount of infective oocysts (n)93) from the 8.25-day terrestrial load is in very sharp contrast to the 2 × 106 oocysts d-1 that were deposited within the fecal matrix into the stream, presumably with their infectivity intact. These calculations involve a variety of simplifying assumptions and ignore both uncertainty and variability in numerous parameters, yet the comparison between these two loads underscores the need to minimize in-stream fecal deposition from infected cattle (or from any infected host for that matter) and to motivate continuing research and rancher compliance for how best to design grazing management tools that result in 100% of the fecal load being deposited on the terrestrial component of the watershed. There is growing evidence that multiple processes with log reduction capacities exist on our grazed and agricultural landscapes, ranging from thermal inactivation, oocyst attachment, and subsurface straining to chemical inactivation and oocyst predation. To maximize the benefits from maintaining animal agriculture within the United States (e.g., rural employment, food safety, and security) while simultaneously minimizing environmental and public health costs associated with these extensive grazing systems, these processes that attenuate pathogen loads should be fully leveraged in the design and implementation of modern grazing practices.

Acknowledgments This work was conducted under the auspices of the Bernice Barbour Communicable Disease Laboratory, with financial support from the Bernice Barbour Foundation, Hackensack, N.J., as a grant to the Center of Equine Health, University of California, Davis. Additional financial support was provided by the Sustainable Agriculture Research & Education Program, Division of Agriculture and Natural Resources, University of California, and the California Beef Council. We are grateful to the dedicated livestock and natural resource farm advisors of the University of California Cooperative Extension for collecting temperature profiles from bovine fecal pats from throughout California.

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Received for review July 23, 2004. Revised manuscript received March 7, 2005. Accepted April 12, 2005. ES040481C

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