ArticlePDF Available

Seasonal Temperature Fluctuations Induces Rapid Inactivation of Cryptosporidium parvum

Authors:

Abstract and Figures

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 degrees C, diurnal thermal regimes of bovine fecal material exhibited peaks of over 40, 50, 60, and 70 degrees C. These diurnal thermal regimes were emulated using a thermocycler, with oocysts suspended in distilled water or fecal-water mix. Using oral inoculations of 10(5) C. parvum oocysts per neonatal Balb/c mouse (>1000-fold the ID50), no infections were observed using 1 to 5-day cycles of these thermal regimes. Loss of infectivity induced bythese 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 > or = 40 degrees C via conduction, radiation, and convection, rapid environmental inactivation occurs at a rate of > or = 3.27 log reduction d(-1) for C. parvum oocysts deposited in the feces of cattle.
Content may be subject to copyright.
Seasonal Temperature Fluctuations
Induces Rapid Inactivation of
Cryptosporidium parvum
XUNDE LI,EDWARD R. ATWILL,*,†
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 (>1000-
fold 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-1for
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
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). Tem-
perature loggers were 2.5 ×2.0 ×13.2 cm3and collected data
at 15 min intervals, with a temperature range from -35 °C
to +75 °C. Temperature loggers were placed mid-depth in
a1 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 da-
tabase 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 tem-
peratures 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
* 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.
Environ. Sci. Technol.
2005,
39,
4484-4489
4484 9ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 12, 2005 10.1021/es040481c CCC: $30.25 2005 American Chemical Society
Published on Web 05/10/2005
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
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 106and
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 (105oocysts/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 ther-
mocycler. 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 micros-
copy 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 105oocysts/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 105fresh oocysts in a 100 µL volume; negative
controls were neonatal mice inoculated with either distilled
water or 105heat-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
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
40 °C cycles 50 °C cycles 60 °C cycles 70 °C cycles
cycle
no. temp time
a
temp time temp time temp time
1 19.1 90:00 16.2 45:00 25.7 45:00 13.2 45:00
2 19.9 45:00 22.0 22:30 30.8 22:30 16.2 22:30
3 21.9 22:30 26.9 22:30 35.9 11:15 19.3 22:30
4 23.3 22:30 30.9 22:30 40.1 11:15 23.7 22:30
5 24.6 22:30 33.8 22:30 44.4 22:30 27.8 22:30
6 26.0 22:30 36.5 45:00 48.0 22:30 30.8 22:30
7 27.3 22:30 39.6 45:00 51.4 22:30 35.6 22:30
8 28.8 22:30 45.1 22:30 54.4 22:30 38.3 22:30
9 30.2 22:30 48.4 22:30 57.7 45:00 42.8 22:30
10 31.6 22:30 52.9 90:00 60.1 45:00 45.9 22:30
11 33.1 22:30 54.8 45:00 62.8 90:00 50.2 22:30
12 34.7 22:30 52.2 45:00 63.8 90:00 52.3 22:30
13 35.9 22:30 49.4 45:00 60.9 45:00 56.4 22:30
14 37.5 22:30 46.8 22:30 58.2 22:30 60.9 22:30
15 39.0 22:30 43.8 22:30 54.1 22:30 64.4 22:30
16 40.5 22:30 39.0 22:30 47.2 22:30 68.4 45:00
17 42.5 45:00 34.1 22:30 42.6 22:30 71.1 45:00
18 44.3 45:00 30.1 45:00 39.1 45:00 68.9 45:00
19 45.6 90:00 27.1 45:00 36.9 90:00 64.5 45:00
20 44.3 45:00 24.4 45:00 33.7 90:00 61.1 45:00
21 42.8 45:00 21.7 90:00 30.6 90:00 55.9 45:00
22 40.6 45:00 20.7 45:00 27.0 45:00 51.0 45:00
23 38.7 22:30 18.2 45:00 24.7 45:00 46.5 22:30
24 37.4 22:30 16.1 90:00 23.0 90:00 42.9 22:30
25 35.6 22:30 13.9 90:00 19.7 90:00 38.0 45:00
26 33.8 22:30 10.5 90:00 18.4 90:00 33.6 45:00
27 31.9 45:00 10.5 90:00 18.2 90:00 29.6 45:00
28 29.9 45:00 10.5 90:00 18.8 45:00 25.8 45:00
29 28.2 45:00 9.5 45:00 21.0 22:30 23.5 45:00
30 26.5 90:00 11.4 45:00 21.0 22:30 20.8 45:00
31 25.1 45:00 18.1 90:00
32 24.2 45:00 15.0 90:00
33 23.2 90:00 15.0 90:00
34 21.8 90:00 12.6 90:00
35 20.7 90:00 12.6 90:00
a
Time durations are expressed as minutes and seconds (min:sec)
and total time per cycle is 24 h.
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
temp
a
days
b
no. of
experiments
treated
ocysts
fresh
oocysts
inactivated
oocysts
40 °C 1 2 0/15
c
7/7
c
0/5
c
2 1 0/7 4/4 0/5
3 1 0/7 4/4 0/5
4 1 0/6 4/4 0/5
5 1 0/6 4/4 0/5
50 °C 1 3 0/18 19/19 0/18
2 2 0/17 13/13 0/14
3 2 0/14 12/12 0/11
4 1 0/7 7/7 0/8
60 °C 1 3 0/22 14/14 0/15
2 2 0/16 10/10 0/10
3 2 0/11 16/16 0/14
4 1 0/5 7/7 0/6
70 °C 1 3 0/24 16/16 0/19
2 2 0/13 10/10 0/14
3 2 0/12 10/10 0/14
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.
VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 94485
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 (107oocysts/
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 ball-
point 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 ho-
mogenizer (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 centrifu-
gation at 1500 ×g for 10 min. Fifty microliters of the final
suspension were mixed with 50 µL of fluorescent isothio-
cyanate-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 mix-
ture 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, 103,2×103,5×103,10
4, and
105fresh oocysts prepared by serial dilution from a stock
solution of 106oocysts 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 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 cycle
a
treated oocysts fresh oocysts inactivated oocysts
40 °C 0/8
b
3/3
b
0/2
b
50 °C 0/8 5/5 0/3
60 °C 0/6 4/4 0/5
70 °C 0/4 6/6 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.
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
treatment
a
days
b
% of intact
oocysts
c
% of partial
oocysts
% of empty
oocysts
40 °C cycle 0
d
96.2 (0.4 2.3 (0.5 1.5 (0.2
1 2.5 (0.4 62.6 (4.3 34.9 (4.7
2 0.9 (0 59.2 (6.0 40.4 (5.8
3 0 53.0 (5.4 47.0 (5.3
4 0 50.2 (6.2 49.8 (6.2
5 0 49.2 (5.1 50.8 (4.9
heat inactivated oocysts
e
93.3 (0.3 4.7 (0.5 2.1 (0.3
50 °C cycle 0
d
98.6 (0.4 1.2 (0.3 0.2 (0.3
1 3.9 (1.1 41.8 (3.8 54.3 (2.9
2 0.9 (0 49.8 (5.9 49.3 (6.0
3 0.3 (0.4 43.8 (1.6 55.9 (2.0
4 0 47.5 (6.0 52.3 (5.8
heat inactivated oocysts
e
94.6 (1.4 4.6 (1.2 0.7 (0.2
60 °C cycle 0
d
95.9 (0.3 3.3 (0.4 0.8 (0.1
1 4.9 (1.0 38.1 (1.6 57.0 (2.0
2 3.5 (0.6 57.1 (6.1 38.5 (6.9
3 2.4 (0.6 49.6 (4.6 48.1 (4.2
4 0 43.4 (3.9 56.9 (4.3
heat inactivated oocysts
e
91.9 (0.7 4.0 (1.3 4.1 (0.8
70 °C cycle 0
d
94.3 (0.9 3.3 (0.6 2.4 (0.7
1 7.3 (0.7 47.4 (5.9 45.3 (6.5
2 3.2 (0.7 48.5 (1.4 48.3 (1.8
3 1.5 (0.5 48.6 (2.5 49.9 (2.7
heat inactivated oocysts
e
85.7 (0.6 9.6 (1.9 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
4to70°C, followed by 2 h duration at 70 °C).
4486 9ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 12, 2005
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 xinfected neonatal mice out of Nmice
inoculated with yinfective oocysts can be calculated from
the binomial distribution
whereby pis 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 temper-
ature treatment is calculated as δ)-log (y/105). If no
infections are observed among the neonatal mice, the solution
to yreduces to
Finally, to generate predictions of first-order decay kinetics
of oocyst infectivity attributable to diurnal thermal fluctua-
tions
whereby C0is the initial number of infective oocysts at t)
0,δis the daily log reduction of C. parvum infectivity
estimated above, tis the number of days at a specific thermal
regime, and Ctis the amount of oocysts remaining infective
after tdays 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, expos-
ing 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 105oocysts (>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 C0being the initial number of infective oocysts in a
fecal load at t)0 and tbeing 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-1for 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)att)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-1d-1, with 6550 oocysts cow-1d-1being
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 concentra-
tions of oocysts compared to adults, with C. parvum infection
highly dynamic and in part a function of age, herd manage-
ment, and season (45). Assuming a value of 106oocysts 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
ofa40°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 °Cfor5s(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
P(infection |yinfective oocysts) )1
1+e-(R+βln(y)) (1)
P(X)x))
(
N
x
)
(p)x(1 -p)N-x(2)
1
1+e-(R+βln(y)) <1-(0.05)1/N(3)
Ct)C010-δ(t)(4)
VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 94487
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 105heat-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 heat-
treated 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 temper-
ature 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 106oocysts 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
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-1accrues due to radiation,
heat conduction, and convection. Therefore, this cow-calf
herd generates a daily oocyst load of 1×108oocysts d-1
(100 cow-calf pair ×1 006 550 oocysts cow-calf pair-1d-1),
from which 2% (2 ×106oocysts) are directly deposited in a
fecal matrix within the stream. Of the remaining 98% of the
oocyst load, only 3.7 ×106oocysts (108oocysts ×0.98 ×
0.25 ×0.15) are entrainable in overland flow that are
deposited d-1about 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 ×105oocysts 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-1of
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 ×106oocysts d-1that were
deposited within the fecal matrix into the stream, presumably
with their infectivity intact.
These calculations involve a variety of simplifying as-
sumptions 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 manage-
ment 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 Pro-
gram, Division of Agriculture and Natural Resources, Uni-
versity 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.
Literature Cited
(1) McLauchlin, J.; Amar, C.; Pedraza-Dı´az, S.; Nichols, G. L.
Molecular epidemiological analysis of Cryptosporidium spp. in
the United Kingdom: results of genotyping Cryptosporidium
spp. in 1,705 fecal samples from humans and 105 fecal samples
from livestock animals. J. Clin. Microbiol. 2000,38, 3984-3990.
(2) Rosen, B. H.; Croft, R.; Atwill, E. R.; Wade, S.; Stehman, S.
Waterborne pathogens in agricultural watersheds. Technical Note
2. Watershed Science Institute, University of Vermont, Natural
Resource Conservation Service, USDA: Burlington, Vermont,
2000, pp 1-64.
4488 9ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 12, 2005
(3) MacKenzie, W. R.; Schell, W. L.; Blair, K. A.; Addiss, D. G.;
Peterson, D. E.; Hoxie, N. J.; Kazmierczak, J. J.; Davis, J. P. Massive
outbreak of waterborne Cryptosporidium infection in Milwau-
kee, Wisconsin: recurrence of illness and risk of secondary
transmission. Clin.Infect.Dis.1995,21,57-62.
(4) Dorner, S. M.; Huck, P. M.; Slawson, R. M. Estimating potential
environmental loadings of Cryptosporidium spp. and
Campylobacter spp. from livestock in the Grand River Watershed,
Ontario, Canada. Environ. Sci. Technol. 2004,38, 3370-3380.
(5) Trask, J. R.; Kalita, P. K.; Kuhlenschmidt, M. S.; Smith, R. D.;
Funk, T. L. Overland and near-surface transport of Crypto-
sporidium parvum from vegetated and nonvegetated surfaces.
J.Environ.Qual.2004,33, 984-993.
(6) Atwill,E. R.; Hou, L.; Karle, B. M.; Harter, T.; Tate, K. W.; Dahlgren,
R. A. Transport of Cryptosporidium parvum oocysts through
vegetated buffer strips and estimated filtration efficiency. Appl.
Environ.Microbiol.2002,68, 5517-5527.
(7) Atwill, E. R.; Tate, K. W.; Dorey, P. E. Warner Ranch Integrated
Cryptosporidium parvum Assessment and Grazing Management
Plan; Vista Irrigation District, Vista, California: 2003; pp 1-24.
(8) Tate, K. W.; Das Gracas C. Pereira, M.; Atwill, E. R. Efficacy of
vegetated buffer strips for retaining Cryptosporidium parvum.
J.Environ.Qual.2004,33, 2243-2251.
(9) Teunis, P. F. M.; Medema, G. L.; Kruidnier, L.; Havelaar, A. H.
Assessment of the risk of infection by Cryptosporidium or Giardia
in drinking water from a surface water source. Water Res.1997,
31, 1333-1346.
(10) Howe, A. D.; Forster, S.; Morton, S.; Marshall, R.; Osborn, K. S.;
Wright, P.; Hunter, P. R. Cryptosporidium oocysts in a water
supply associated with a cryptosporidiosis outbreak. Emerg.
Infect.Dis.2002,8, 619-624.
(11) Atwill, E. R. Assessing the link between rangeland cattle and
water-borne Cryptosporidium parvum infection in humans.
Rangelands 1996,18,48-51.
(12) Mawdsley, J. L.; Brooks, A. E.; Merry, R. J.; Pain, B. F. Use of a
novel soil tilting table apparatus to demonstrate the horizontal
and vertical movement of the protozoan pathogen Crypto-
sporidium parvum in soil. Biol.Fertil.Soil.1996,23, 215-220.
(13) Tate,K. W.; Atwill, E. R.; George, M. R.; McDougald, N. K.; Larsen,
R. E. Cryptosporidium parvum transport from cattle fecal
deposits on California rangelands. J.Range Manage.2000,53,
295-299.
(14) Bradford, S. A.; Schijven, J. Release of Cryptosporidium and
Giardia from dairy calf manure: impact of solution salinity.
Environ.Sci.Technol.2002,36, 3916-3923.
(15) Kistemann, T.; Claben, T.; Koch, C.; Dangendorf, F.; Fischeder,
R.; Gebel, J.; Vacata, V.; Exner, M. Microbial load of drinking
water reservoir tributaries during extreme rainfall and runoff.
Appl.Environ.Microbiol.2002,68, 2188-2197.
(16) Davies, C. M.; Ferguson, C. M.; Kaucner, C.; Krogh, M.; Altavilla,
N.; Deere, D. A.; Ashbolt, N. J. Dispersion and transport of
Cryptosporidium oocysts from fecal pats under simulated rainfall
events. Appl.Environ.Microbiol.2004,70, 1151-1159.
(17) Schijven, J. F.; Bradford, S. A.; Young, S. Release of Crypto-
sporidium and Giardia from dairy cattle manure: physical
factors. J. Environ. Qual.2004,33, 1499-1508.
(18) Fayer, R. Effect of high temperature on infectivity of Crypto-
sporidium parvum oocysts in water. Appl.Environ.Microbiol.
1994,60, 2732-2735.
(19) Fujino, T.; Matsui, T.; Kobayashi, F.; Haruki, K.; Yoshino, Y.;
Kajima, J.; Tsuji, M. The effect of heating against Cryptosporidium
oocysts. J.Vet.Med.Sci.2002,64, 199-200.
(20) Pokorny, N. J.; Weir, S. C.; Carreno, R. A.; Trevors, J. T.; Lee, H.
Influence of temperature on Cryptosporidium parvum oocyst
infectivity in river water samples as detected by tissue culture
assay. J.Parasitol.2002,88, 641-643.
(21) Robertson, L. J.; Campbell, A. T.; Smith, H. V. Survival of
Cryptosporidium parvum oocysts under various environmental
pressures. Appl.Environ.Microbiol.1992,58, 3494-3500.
(22) Anderson, B. C. Effect of drying on the infectivity of crypto-
sporidia-laden calf feces for 3- to 7-day-old mice. Am.J.Vet.
Res.1986,47, 2272-2273.
(23) Jenkins, M. B.; Anguish, L. J.; Bowman, D. D.; Walker, M. J.;
Ghiorse, W. C. Assessment of a dye permeability assay for
determination of inactivation rates of Cryptosporidium parvum
oocysts. Appl.Environ.Microbiol.1997,63, 3844-3850.
(24) Jenkins, M. B.; Bowman, D. D.; Ghiorse, W. C. Inactivation of
Cryptosporidium parvum oocysts by ammonia. Appl.Environ.
Microbiol.1998,64, 784-788.
(25) Jenkins, M. B.; Walker, M. J.; Bowman, D. D.; Anthony, L. C.;
Ghiorse, W. C. Use of a sentinel system for field measurements
of Cryptosporidium parvum oocyst inactivation in soil and
animal waste. Appl.Environ.Microbiol.1999,65, 1998-2005.
(26) Freire-Santos, F.; Oteiza-Lopez, A. M.; Vergara-Castiblanco, C.
A.; Ares-Mazas, M. E. Effect of salinity, temperature and storage
time on mouse experimental infection by Cryptosporidium
parvum.Vet.Parasitol.1999,87,1-7.
(27) Walker, M.; Leddy, K.; Hager, E.; Hagar, E. Effects of combined
water potential and temperature stresses on Cryptosporidium
parvum oocysts. Appl.Environ.Microbiol.2001,67, 5526-5529.
(28) Xiao, L.; Escalante, L.; Yang, C.; Sulaiman, I.; Escalante, A. A.;
Montali, R. J.; Fayer, R.; Lal, A. A. Phylogenetic analysis of
Cryptosporidium parasites based on the small-subunit rRNA
gene locus. Appl.Environ.Microbiol.1999,65, 1578-1583.
(29) Arrowood, M. J.; Sterling, C. R. Isolation of Cryptosporidium
oocysts and sporozoites using discontinuous sucrose and
isopycnic Percoll gradients. J.Parasitol.1987,73, 314-319.
(30) Hou, L.; Li, X.; Dunbar, L.; Moeller, R.; Palermo, B.; Atwill, E. R.
Neonatal-mouse infectivity of intact Cryptosporidium parvum
oocysts isolated after optimized in vitro excystation. Appl.
Environ.Microbiol.2004,70, 642-646.
(31) Mtambo, M. M.; Wright, E.; Nash, A. S.; Blewett, D. A. Infectivity
of a Cryptosporidium species isolated from a domestic cat (Felis
domestica) in lambs and mice. Res.Vet.Sci.1996,60,61-64.
(32) Vergara-Castiblanco, C. A.; Freire-Santos, F.; Oteiza-Lopez A.
M.; Ares-Mazas, M. E. Viability and infectivity of two Crypto-
sporidium parvum bovine isolates from different geographical
location. Vet.Parasitol.2000,89, 261-267.
(33) Hardin, J.; Hilbe, J. The binomial-logit family; negative binomial
family. In Generalized Linear Models and Extensions; Stata
Press: College Station, Texas, 2001; pp 87-123, 141-158.
(34) Fayer, R.; Speer, C. A.; Dubey, J. P. The general biology of
Cryptosporidium.InCryptosporidium and Cryptosporidiosis;
Fayer, R., Ed.; CRC Press: Boca Raton, U.S.A., 1997; pp 1-42.
(35) Atwill, E. R.; Harp, J. A.; Jones, T.; Jardon, P. W.; Checel, S.;
Zylstra, M. Evaluation of periparturient dairy cows and contact
surfaces as a reservoir of Cryptosporidium parvum for calfhood
infection. Am.J.Vet.Res.1998,59, 1116-1121.
(36) Atwill, E. R.; Hoar, B.; Pereira, M. G.; Tate, K. W.; Rulofson, F.;
Nader, G. Improved quantitative estimates of low environmental
loading and sporadic periparturient shedding of Crypto-
sporidium parvum in adult beef cattle. Appl.Environ.Microbiol.
2003,69, 4604-4610.
(37) Atwill, E. R.; Pereira, M. G. C. Lack of detectable shedding of
Cryptosporidium parvum oocysts by periparturient dairy cattle.
J. Parasitol.2003,89, 1234-1236.
(38) Hoar, B. R.; Atwill, E. R.; Elmi, C.; Farver, T. B. An examination
of risk factors associated with beef cattle shedding pathogens
of potential zoonotic concern. Epidemiol.Infect.2001,127, 147-
155.
(39) Nydam, D. V.; Wade, S. E.; Schaaf, S. L.; Mohammed, H. O.
Number of Cryptosporidium parvum oocysts or Giardia spp
cysts shed by dairy calves after natural infection. Am.J.Vet.Res.
2001,62, 1612-1615.
(40) Larsen,R. E.; Buckhouse, J. C.; Moore, J. A.; Miner, J. R. Rangeland
cattle and manure placement: A link to water quality. Oregon
Acad. Sci. 1988,24,7-15.
(41) Tate, K. W.; Atwill, E. R.; McDougald, N. K.; George, M. R. Spatial
and temporal patterns of cattle feces deposition on rangeland.
J.Range Manage.2003,56, 432-438.
(42) Brush, C. F.; Ghiorse, W. C.; Anguish, L. J.; Parlange, J. Y.; Grimes,
H. G. Transport of Cryptosporidium parvum oocysts through
saturated columns. J.Environ.Qual.1999,28, 809-815.
(43) Mawdsley, J. L.; Brooks, A. E.; Merry, R. J. Movement of the
protozoan pathogen C. parvum through three contrasting soil
types. Biol.Fertil.Soil.1996,21,30-36.
(44) Harter,T.; Wagner, S.; Atwill, E. R. Colloid transport and filtration
of Cryptosporidium parvum in sandy soils and aquifer sedi-
ments. Environ. Sci. Technol. 2000,34,62-70.
(45) Atwill, E. R.; Johnson, E. M.; Pereira, M. G. C. Association of
herd composition, stocking rate, and duration of calving season
with fecal shedding of Cryptosporidium parvum oocysts in beef
herds. J.Am.Vet.Med.Assoc.1999,215, 1833-1838.
Received for review July 23, 2004. Revised manuscript re-
ceived March 7, 2005. Accepted April 12, 2005.
ES040481C
VOL. 39, NO. 12, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 94489
... The timing of pasture grazing by beef cattle was managed to create a range of total days rest between grazing and irrigation of the pasture. This allowed us to characterize the potential reduction of E. coli in tailwater attributable to such processes as the background mortality rate of E. coli, and the drying and heating of fecal pats during the summer season (Li et al. 2005). ...
... For example, the E. coli concentration was 23% lower after 9 days of rest than after 1 day of rest, but only 2% lower after each additional day of rest. This reduction was likely due to two primary processes: (1) as cattle fecal pats age, the microbial pollutants in them naturally die off (Li et al. 2005;Meays et al. 2005), and (2) as the pats dry, they develop shells that trap the bacteria inside. ...
Article
Full-text available
Microbial pollutants, some of which can cause illnesses in humans, chronically contaminate many California water bodies. Among numerous sources, runoff from irrigated pastures has been identified as an important regulatory target for improving water quality. This study examined the potential to reduce E. coli contamination from cattle in irrigated pastures. During the 14 irrigation events examined, we found that E. coli concentrations were lowest with a combination of three treatments: filtering runoff through a natural wetland, reducing runoff rates, and letting the pasture rest from grazing at least a week prior to irrigation. Integrated pasture and tailwater management are required to significantly reduce E. coli concentrations in runoff.
... In general, as elevation increases: a) temperature decreases as a result of the air expansion under lower pressure; b) relative humidity decreases as a consequence of lower air temperature and pressure; and c) UV radiation increases due to decreasing density of molecules and particles in the atmosphere. Temperature is a key factor for the survival of intact oocysts on the ground (Fayer, 1994;Jenkins et al., 1999;Olson et al., 1999;Nasser et al., 2007) and in feces (Jenkins et al., 1999;Olson et al., 1999;Kato et al., 2002;Li et al., 2005Li et al., , 2010. Exposure to ambient temperatures in excess of 30°C to 40°C has been shown to inactivate or reduce oocyst infectivity (Anderson, 1985;Fayer, 1994;Li et al., 2005Li et al., , 2010. ...
... Temperature is a key factor for the survival of intact oocysts on the ground (Fayer, 1994;Jenkins et al., 1999;Olson et al., 1999;Nasser et al., 2007) and in feces (Jenkins et al., 1999;Olson et al., 1999;Kato et al., 2002;Li et al., 2005Li et al., , 2010. Exposure to ambient temperatures in excess of 30°C to 40°C has been shown to inactivate or reduce oocyst infectivity (Anderson, 1985;Fayer, 1994;Li et al., 2005Li et al., , 2010. Moreover, oocysts have been shown to be susceptible to freeze/thaw cycles (Jenkins et al., 1999) and freezing (Kato et al., 2002). ...
Article
Full-text available
Wildlife are increasingly recognized as important biological reservoirs of zoonotic species of Cryptosporidium that might contaminate water and cause human exposure to this protozoal parasite. The habitat range of the yellow-bellied marmot (Marmota flaviventris) overlaps extensively with the watershed boundaries of municipal water supplies for California communities along the foothills of the Sierra Nevada. We conducted a cross-sectional epidemiological study to estimate the fecal shedding of Cryptosporidium oocysts by yellow-bellied marmots and to quantify the environmental loading rate and determine risk factors for Cryptosporidium fecal shedding in this montane wildlife species. The observed proportion of Cryptosporidium positive fecal samples was 14.7% (33/224, positive number relative to total number samples) and the environmental loading rate was estimated to be 10,693 oocysts animal-1 day-1. Fecal shedding was associated with the elevation and vegetation status of their habitat. Based on a portion of the 18s rRNA gene sequence of 2 isolates, the Cryptosporidium found in Marmota flaviventris were 99.88%–100% match to multiple isolates of C. parvum in the GenBank.
... Cryptosporidium parvum oocysts are resistant to many environmental stressors and most of the traditional drinking water treatment technologies and wastewater treatment processes (Atwill et al., 2003;Fayer et al., 2000). Although their resistance to many environmental stressors can ensure their potentially infectious viability for months (Fayer, 2004;Fayer et al., 1997;Jenkins et al., 2002;Kato et al., 2004;King & Monis, 2007;Robertson et al., 1992), dessication, heat, and ultraviolet (UV) light have been reported to decrease the infectivity of C. parvum oocysts (Brookes et al., 2004;Jenkins et al., 2002;Li et al., 2005;Olson et al., 1999;Robertson et al., 1992). The release of Cryptosporidium in the environment through direct excretion and application of manure or wastewater to farm fields is a potential source of contamination of the soil-subsurface environment as well as surface water and groundwater resources Harter et al., 2000;Mawdsley, Brooks, Merry, & Pain, 1996;Santamaria et al., 2012;Zopp et al., 2016). ...
Article
Full-text available
A series of laboratory experiments was performed to investigate the transport and retention of viable C. parvum oocysts in soil columns homogeneously packed with loamy sand soils (Lewiston and Greenson series) and sandy loam soils (Sparta and Gilford series), and under hydrologic conditions involving the presence of an anionic surfactant—Aerosol 22 in artificial rainfall. To characterize the effect of surfactant on the mobility of C. parvum oocysts in the soils used in this study, these results were compared with previous laboratory‐scale column experiments of a previous study using soils contaminated with oocysts, under conditions identical to those described here except for the absence of the surfactant in the percolating water. Quantitative polymerase chain reaction was used for the detection and quantification of C. parvum oocysts in soil leachates to assess their breakthrough and in soil matrices to characterize their spatial distribution. Alterations in the rate and extent of transport of C. parvum oocysts were discerned per the physicochemical parameters analyzed—soil types, soil chemistry, and surfactant—and resulted in either the enhancement or hinderance of C. parvum oocysts adsorption to surfaces, and their movement in soils. In the case of loamy sand soils, the transport of C. parvum oocysts through the soil matrices increased with the application of surfactant for Sparta series and remained at a similar level for Gilford series. Regarding sandy loam soils, the movement of C. parvum oocysts through the soil matrices increased for Greenson series and decreased for Lewiston series with the application of surfactant. Mechanisms governing transport of C. parvum oocysts in soils are influenced by soil types and soil water chemistry. Surfactant affected the transport and retention behaviors of C. parvum oocysts in soils. qPCR was used for the detection and enumeration of C. parvum oocysts in natural soil and water samples.
... In this investigation, the water temperatures in WWTPs were 6°C-9°C in winter and 17°C-23°C in summer, and 0°C-4°C was measured in the unfrozen surface water in winter. Previous studies have reported that both heating (40°C-70°C) and freezing (-20°C-70°C) treatments induced rapid inactivation of C. parvum and C. muris (Li et al., 2005;Neumayerová and Koudela, 2008). Li et al. (2004) reported that C. parvum oocysts could survive and remain infectious for 8-24 weeks in cool water (0°C-20°C). ...
Article
This study investigated the occurrence, species, infectivity and removal efficiency of Cryptosporidium spp. across typical wastewater treatment train. Samples from different process units were collected seasonally and synchronously from four wastewater treatment plants (WWTPs) in Northeastern China. Live Cryptosporidium oocysts were identified in most samples from both influent (97.50%) and effluent (90.00%) wastewaters of the four WWTPs, at an average density of 26.34 and 4.15 oocysts/L, respectively. The overall removal efficiency was 84.25%, and oocysts were mainly removed (62.01%) by the modified secondary sedimentation process. Ten Cryptosporidium species were identified in the effluent samples. C. andersoni, C. bovis, and C. ryanae were the three most prevalent species. Oocyst viability assays indicated no reduction of excystation rate during the primary and secondary wastewater treatments (varied in the range of 63.08%–68.50%), but the excystation rate declined to 52.21% in the effluent after disinfection. Notably, the Cryptosporidium oocysts showed higher infection intensity in the cold season (winter and spring) than that in summer and autumn. The influences of environmental temperature on virulence factors of Cryptosporidium were further examined. It was observed that more extracellular secretory proteins were bound on the oocyst surface and several virulence genes were expressed relatively strongly at low temperatures, both of which could facilitate oocyst adhesion, invasion, and host immune evasion. This research is of considerable interest since it serves as an important step towards more accurate panoramic recognition of Cryptosporidium risk reduction in WWTPs, and especially highlights the potential health risk associated with Cryptosporidium in cold regions/seasons.
... PCR amplification of Cryptosporidium oocyst DNA from aged scat samples (i.e., not collected per rectum) were hampered in some older samples in this study due to either low concentrations of oocysts, aged and possibly damaged oocysts, and/or the many inhibitors present in feces, as experienced by our laboratory and reported by others [43][44][45]. For example, feces exposed to moderate to high ambient temperature, in part due to exposure to solar radiation, can lead to excystation of oocysts [46,47] and subsequent loss of DNA in the fecal matrix, thereby impeding PCR. In addition, during the course of this study the decision was made to cease attempts at PCR amplification for scat samples with less than 20 to 25 oocysts given the low success rate for these types of samples. ...
Article
Full-text available
Between October 2013 and May 2016, 506 scat samples were collected from 22 species of wildlife located in a protected watershed of a major municipal water supply in the Pacific Northwest, USA. Overall prevalence of Cryptosporidium in the wildlife scat was 13.8% (70/506), with 15 species of wildlife found positive for Cryptosporidium. Prevalence of Cryptosporidium varied among species of wildlife, with higher prevalences observed in cougars (50.0%), mountain beavers (40.0%), and bobcats (33.3%), but none of these species are riparian-dependent. Genotyping of Cryptosporidium by sequencing PCR amplicons from the 18S rRNA gene were successful for seven species of wildlife, including bobcat, unknown predator, black-tailed deer, deer mouse, snowshoe hare, mountain beaver, and western spotted skunk. BLAST and phylogenetic analyses indicated that multiple species and genotypes of Cryptosporidium were present, with some isolates possibly co-circulating within and between wildlife populations in this protected watershed. Evidence of oocyst exchange between infected prey and their predators was also found. During the study period, several zoonotic Cryptosporidium species and genotypes that are uncommon in humans were detected in bobcat (99.58% identical to Cryptosporidium felis), unknown predator (100% identical to Cryptosporidium canis), snowshoe hare (100% identical to Cryptosporidium sp. skunk genotype), and mountain beaver (100% identical to Cryptosporidium ubiquitum). Novel sequences were also found in mountain beaver. To our knowledge, this is the first published report of a unique genotype or species of Cryptosporidium in mountain beaver (Aplodontia rufa).
... This observed high prevalence is likely maintained by populations of deer mice coinfecting each other, given that these hosts shed in their feces a median of 5.3 3 10 4 oocysts m À2 day À1 in this region of California. Assuming that the oocyst dose needed for 50% infection of deer mice is similar to the value of 71 C. parvum oocysts for BALB/c mice (35), interanimal transmission could become endemic owing to daily foraging and constant contact of susceptible mice with this environmental load of oocysts. Under steady state conditions (stable population and seasonally stable rates of disease), assuming a prevalence of 20 to 40% during the produce growing season and a mean shedding duration of oocysts of around 7 days, the estimated incidence density in these rodent populations would be 0.036 to 0.095 (number of incident cases of Cryptosporidium infection per day). ...
Article
Full-text available
Deer mice ( Peromyscus maniculatus ) are abundant and widely distributed rodents in North America that occupy diverse habitats, including agricultural landscapes. Giardia and Cryptosporidium are common parasites in wildlife including deer mice, which may play a role in on-farm contamination of produce. An important step in assessing the risk of produce contamination by Cryptosporidium and Giardia shed by deer mice is to determine the prevalence, levels, and genotypes of (oo)cysts in mouse feces. A total of 63 (30.3%) and 53 (25.5%) of 208 deer mice trapped on 12 farms on the California Central Coast were positive for Cryptosporidium and Giardia, respectively. Of these mice, 41 (19.7%) contained both parasites. The odds of Cryptosporidium shedding were 2.5 to 5 times higher for mice trapped in autumn than for mice trapped in summer or spring. Female mice had a higher prevalence and two- to threefold higher levels of Cryptosporidium and Giardia compared with male mice. Female adults and female juveniles had the highest rates of contamination of the environment with Cryptosporidium and Giardia, respectively. We estimated that 20 infected deer mice inhabiting 1 ha of a typical leafy green produce farm in the study region could shed approximately 5.3 × 10(8) Cryptosporidium and 10.5 × 10(8) Giardia, respectively, per day into the environment. The small-subunit rRNA gene loci from a subset of protozoan isolates were sequenced and compared with existing sequences in GenBank. Multiple genotypes of Cryptosporidium and Giardia were found, and BLAST analyses suggest that Giardia and the majority of Cryptosporidium genotypes in deer mice circulate within various rodent populations, but some Cryptosporidium isolates possess zoonotic potential.
... Tarazona et al. (1998) reported that inoculation of 10 4 or more C. parvum oocysts results in 100% infection in BALB/c mice. We determined previously that the 50% infective dose (ID 50 ) for C. parvum in neonatal BALB/c mice was 70.6 oocysts (Li et al., 2005) and mice inoculated with 1000 oocysts resulted in 100% infection . Previously we have shown that inoculation up to 10 4 Sbey03c oocysts failed to infect neonatal BALB/c mice and the current results confirm this, indicating that Cryptosporidium sp. ...
Article
Full-text available
Previously we reported the unique Cryptosporidium sp. "c" genotype (e.g., Sbey03c, Sbey05c, Sbld05c, Sltl05c) from three species of Spermophilus ground squirrel (Spermophilus beecheyi, Spermophilus beldingi, Spermophilus lateralis) located throughout California, USA. This follow-up work characterizes the morphology and animal infectivity of this novel genotype as the final step in proposing it as a new species of Cryptosporidium. Analysis of sequences of 18S rRNA, actin, and HSP70 genes of additional Cryptosporidium isolates from recently sampled California ground squirrels (S. beecheyi) confirms the presence of the unique Sbey-c genotype in S. beecheyi. Phylogenetic and BLAST analysis indicates that the c-genotype in Spermophilus ground squirrels is distinct from Cryptosporidium species/genotypes from other host species currently available in GenBank. We propose to name this c-genotype found in Spermophilus ground squirrels as Cryptosporidium rubeyi n. sp. The mean size of C. rubeyi n. sp. oocysts is 4.67 (4.4-5.0) μm × 4.34 (4.0-5.0) μm, with a length/width index of 1.08 (n = 220). Oocysts of C. rubeyi n. sp. are not infectious to neonatal BALB/c mice and Holstein calves. GenBank accession numbers for C. rubeyi n. sp. are DQ295012, AY462233, and KM010224 for the 18S rRNA gene, KM010227 for the actin gene, and KM010229 for the HSP70 gene.
Article
Cryptosporidium spp. and Giardia duodenalis are common protozoal parasites in livestock including beef cattle on rangeland and irrigated pasture. A statewide cross-sectional study was conducted to determine the prevalence, species or genotype, and risk factors for fecal shedding of Cryptosporidium and Giardia by cattle from California cow-calf operations. Species and genotypes of Cryptosporidium and Giardia were determined by molecular fingerprinting. Prevalence of Cryptosporidium (19.8%) and Giardia (41.7%) in fecal samples from calves were approximately twice as high as fecal samples from cows (9.2% and 23.1%, respectively). In addition to age, multivariable logistic regression showed that higher stocking density and a higher number of replacement heifers were positively associated with fecal shedding of Cryptosporidium while longer calving interval, a winter/spring calving season, and higher numbers of replacement heifers were positively associated with shedding of Giardia. The dominant species and genotypes of Cryptosporidium and Giardia in feces from these cow-calf herds were Cryptosporidium ryanae (75%) and assemblage E for Giardia duodenalis (90%), which have low impact on public health compared with other zoonotic species/genotypes of these two parasites. We identified host and potential management practices that can be used to protect cattle health and reduce the risk of surface water contamination with protozoal parasites from cow-calf operations. In addition, this work updated the scientific data regarding the predominance of low zoonotic genotypes of Cryptosporidium and Giardia shed in the feces of commercial cow-calf herds on California rangeland and irrigated pasture.
Article
Water is a major route of transmission for Cryptosporidium and oocysts commonly occur in surface and recreational waters as a consequence of fecal contamination from Wildlife or anthroponotic sources. There are many characteristics possessed by Cryptosporidium oocysts that allow them to persist in aquatic environments, including recreational waters, and to bypass water treatment processes. These types of events lead to outbreaks of cryptosporidiosis, caused by direct exposure to contaminated recreational water (such as swimming pools) or by drinking contaminated potable water. Previous chapters have discussed the epidemiology of Cryptosporidium in relation to waterborne transmission and also the sources and presence of oocysts in drinking and recreational waters. This chapter will review the processes contributing to the removal and inactivation of Cryptosporidium oocysts from surface waters and wastewaters, including natural processes that occur in surface waters and engineered processes used for the production of drinking water or for the treatment of wastewater.
Article
Full-text available
The objective of this study was to identify and model environmental and management factors associated with cattle feces deposition patterns across annual rangeland watersheds in the Sierra Nevada foothills. Daily cattle fecal load accumulation rates were calculated from seasonal fecal loads measured biannually on 40 m(2) permanent transects distributed across a 150.5 ha pasture in Madera County, Calif. during the 4 year period from 1995 through 1998. Associations between daily fecal load per season, livestock management, and environmental factors measured for each transect were determined using a linear mixed effects model. Cattle feces distribution patterns were significantly associated with location of livestock attractants, slope percentage, slope aspect, hydrologic position, and season. Transects located in livestock concentration areas experienced a significantly higher daily fecal load compared to transects outside of these concentration areas (P < 0.001). Percent slope was negatively associated with daily fecal load, but this association had a significant interaction with slope aspect (P = 0.02). Daily fecal load was significantly lower during the wet season compared to the dry season (P = 0.002). Daily fecal loading rates across hydrologic positions were dependent upon season. Our results illustrate the opportunities to reduce the risk of water quality contamination by strategic placement of cattle attractants, and provide a means to predict cattle feces deposition based upon inherent watershed characteristics and management factors.
Article
Full-text available
Cryptosporidium parvum is a fecal borne protozoan parasite that can be carried by and cause gastrointestinal illness in humans, cattle, and wildlife. The illness, cryptosporidiosis, can be fatal to persons with compromised immune systems. At question is the potential for C. parvum in cattle fecal deposits on rangeland watersheds to contaminate surface water. First, C. parvum oocysts must be released from fecal deposits during rainfall, becoming available for transport. In 1996, we examined the transport of C. parvum oocysts in overland flow from fecal deposits under natural rainfall and rangeland conditions at the San Joaquin Experimental Range in Madera County, Calif. Our null hypothesis was that C. parvum oocysts are not released from fecal pats and transported 1 m downslope as overland flow with rainfall. Paired plots were located on 10, 20, and 30% slope sites. Each plot was loaded with four, 200 g fecal pats dosed with 105 oocysts g-1. Pats were placed 1.0 m above the base of each plot. Composite runoff samples from each plot were analyzed for oocyst concentration following each of 4 storm events. Oocysts were transported during each storm. Slope was a significant factor in oocyst transport, with oocyst transport increasing with slope. Although not significant, there was an apparent flushing effect of oocysts across storms, with the majority transported in the first 2 storms. A pilot rainfall simulation experiment also revealed a flushing phenomenon from pats during individual rainfall events. C. parvum oocysts in fecal pats on rangeland can be transported from fecal deposits during rainfall events, becoming available for transport to water-bodies. Future studies need to examine surface and subsurface transport of oocysts on rangeland hillslopes for distances greater than 1 m.
Article
Various physical factors affecting the release rate of naturally occurring Cryptosporidium parvum oocysts and Giardia duodenalis cysts from dairy manure disks to sprinkled water were studied. The investigated factors included temperature (5 or 23°C), manure type (calf manure, a 50% calf and 50% cow manure mixture, and a 10% calf and 90% cow manure mixture), and water application method (mist or drip) and flow rate. Effluent concentrations of manure and (oo)cysts were always several orders of magnitude below their initial concentration in the manure, decreased gradually, and exhibited persistent concentration tailing. Release of manure and (oo)cysts were found to be related by a constant factor, the so-called release efficiency of (oo)cysts. A previously developed (oo)cyst release model that included these release efficiencies provided a satisfactory simulation of the observed release. An effect of temperature on the release of manure and (oo)cysts was not apparent. The manure and (oo)cyst release rates from cow manure decreased faster than those from calf manure, and (oo)cyst release efficiencies from cow manure were higher than those from calf manure. In comparison with mist application, dripping water resulted in higher release rates of manure and (oo)cysts and in higher (oo)cyst release efficiencies due to the increased mechanical forces associated with droplet impact. Mist application at a higher flow rate resulted in faster release, but did not affect the (oo)cyst release efficiencies. The data and modeling approach described herein provide insight and an enhanced ability to describe the influence of physical factors on (oo)cyst release. Please view the pdf by using the Full Text (PDF) link under 'View' to the left. Copyright © 2004. American Society of Agronomy, Crop Science Society of America, Soil Science Society . ASA, CSSA, SSSA
Article
Understanding microbial pathogen transport patterns in overland flow is important for developing best management practices for limiting microbial transport to water resources. Knowledge about the effectiveness of vegetative filter strips (VFS) to reduce pathogen transport from livestock confinement areas is limited. In this study, overland and near-surface transport of Cryptosporidium parvum has been investigated. Effects of land slopes, vegetation, and rainfall intensities on oocyst transport were examined using a tilting soil chamber with two compartments, one with bare ground and the other with brome (Bromus inermis Leyss.) vegetation. Three slope conditions (1.5, 3.0, and 4.5%) were used in conjunction with two rainfall intensities (25.4 and 63.5 mm/h) for 44 min using a rainfall simulator. The vegetative surface was very effective in reducing C. parvum in surface runoff. For the 25.4 mm/h rainfall, the total percent recovery of oocysts in overland flow from the VFS varied from 0.6 to 1.7%, while those from the bare ground condition varied from 4.4 to 14.5%. For the 63.5 mm/h rainfall, the recovery percentages of oocysts varied from 0.8 to 27.2% from the VFS, and 5.3 to 59% from bare-ground conditions. For all slopes and rainfall intensities, the total (combining both surface and near-surface) recovery of C. parvum oocysts was considerably less from the vegetated surface than those from the bare-ground conditions. These results indicate that the VFS can be a best management practice for controlling C. parvum in runoff from animal production facilities. Please view the pdf by using the Full Text (PDF) link under 'View' to the left. Copyright © 2004. American Society of Agronomy, Crop Science Society of America, Soil Science Society . sASA, CSSA, SSSA
Article
We present theoretical and experimental work on Cryptosporidium parvum oocysts to characterize their transport behavior in saturated, sandy sediments under strictly controlled conditions. Column experiments are implemented with three different sands (effective grain size:  180, 420, and 1400 μm) at two different saturated flow rates (0.7 and 7 m/d). The experiments show that C. parvum oocysts, like other colloids, are subject to velocity enhancement. In medium and coarse sands, the oocysts travel 10−30% faster than a conservative tracer. The classic clean-bed filtration model is found to provide an excellent tool to estimate the degree of C. parvum filtration. Experimentally determined collision efficiencies, α, range from 0.4 to 1.1. The magnitude of α is consistent with the known physical and chemical properties of the oocyst and the transport medium and compares well with, e.g., measured collision efficiencies of similarly sized E. coli bacteria. However, a significant amount of the initial deposition appears to be reversible leading to significant asymmetry and tailing in the oocyst concentration breakthrough curve. We are able to show that the observed late-time oocyst elution can qualitatively be explained by postulating that a significant fraction of the oocyst filtration is reversible and subject to time-dependent detachment.
Article
The potential for transfer of the protozoan pathogen Cryptosporidium parvum through soil to land drains and, subsequently, water courses following the application of livestock waste to land was monitored in the laboratory using simulated rainfall and intact soil cores. Following irrigation over a 21-day period, Cryptosporidium parvum oocysts applied to the surface of soil cores (initial inoculum concentration 1108 oocysts core–1) were detected, albeit in low numbers, in the leachates from clay loam and silty loam soils but not in that from a loamy sand soil. Variations in leaching patterns were recorded between replicate cores. At the end of the study soil cores were destructively sampled to establish the location of oocysts remaining within the soil. Distribution within cores was similar in all three soil types. The majority (72.8+-5.2%) of oocysts were found in the top 2 cm of soil, with numbers decreasing with increasing depth to 13.22.8%, 8.391.4%, and 5.361.4% at depths of 10, 20, and 30 cm, respectively.
Article
A novel greenhouse based soil tilting table apparatus was used to investigate the potential for movement of the protozoan pathogen Cryptosporidium parvum both through and across a low permeability soil following the application of contaminated livestock waste to land. Soil blocks supported at an angle of 7.5% by the soil table were inoculated at one end with oocyst seeded slurry and subsequently irrigated at regular intervals over a 70-day period. Movement of the pathogen in runoff was demonstrated for at least 21 days and in one case in excess of 70 days from the time of inoculation. Water was also lost following percolation down through the soil profile and significant numbers of oocysts were also lost via this route, average numbers leached decreasing from 8.360.56106 at day 1 to 2.270.73104 at day 70. At the end of the study cores were removed from the soil blocks to determine the location of oocysts remaining within the soil. Numbers decreased down through the soil profile and as the distance from the point of inoculation increased so that 70 cm from the point of inoculation no oocysts could be detected in the soil at any depth. This implies that oocysts contained in runoff stay in the aqueous phase and do not precipitate out onto the soil surface, suggesting that even if the distances travelled are increased there may still be a significant pollution threat.