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Applied and Environmental Microbiology, December 2001, p. 5526-5529, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5526-5529.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Effects of Combined Water Potential and Temperature
Stresses on Cryptosporidium parvum Oocysts
Mark
Walker,*
Katherine
Leddy, and
Elaine
Hagar
Natural Resources Department, University of
Nevada, Reno, Nevada 89557-0013
Received 29 May 2001/Accepted 22 September 2001
 |
ABSTRACT |
Hosts infected with the parasite Cryptosporidium
parvum may excrete oocysts on soils in watersheds that supply
public drinking water. Environmental stresses decrease the numbers of
oocysts after deposition on soils. However, the rates and effects of
combined stresses have not been well characterized, especially for the purposes of estimating decrease in numbers. We subjected oocysts to
combined stresses of water potential (
4, 12, and 33 bars), above-freezing temperatures (4 and 30°C), and a subfreezing
temperature (14°C) for 1, 14, and 29 days and one to six
freeze-thaw cycles (14 to 10°C) to estimate coefficients to
characterize population degradation using multiplicative error and
exponential decay models. The experiments were carried out in NaCl
solutions with water potentials of 4, 12, and 33 bars, in
combination with temperature stresses at levels that could be expected
in natural soils. Increased water potential increased the rate of
population degradation for all temperature conditions investigated.
Enhanced degradation leads to estimated rates of population degradation
that are greater than those that have been reported and used in
previous studies conducted to assess risk of water supply contamination
from sources of C. parvum.
| |
INTRODUCTION |
Large proportions of the oocysts of
Cryptosporidium parvum shed on soils by infected hosts are
probably destroyed by environmental stresses. Important stresses
include temperature extremes, freeze-thaw cycling, and extreme water
potential (especially desiccation) (20). It is unclear how
predictably and at what rate degradation occurs when stresses are
combined. Degradation rates are important to understand for the purpose
of assessing the likelihood of contamination in the context of public
drinking water supply protection. Models developed for the purpose of
evaluating the risk of water contamination rely on estimates of
coefficients that characterize decay rates before microbial
contaminants are entrained and transported to surface waters
(13). Risk assessments conducted to evaluate the
contamination potential of sources of Cryptosporidium have relied on degradation rates from in situ studies (19).
First-order decay approaches model Cryptosporidium
degradation adequately, especially for conditions related to prolonged
exposure to fixed temperatures (9). Present estimates of
degradation coefficients are based on investigations of single stresses
or uncontrolled combinations of several stresses (9, 16).
We tested the hypothesis that the interaction between temperature and
water potential stresses enhances oocyst degradation, leading to rates
of population decay that are higher than those previously reported and
used for risk assessment. We evaluated this hypothesis using
exponential decay models, with temperature and water potential stresses
represented in a multiplicative error format (8) that
related estimates of degradation coefficients to levels of stresses applied.
| |
MATERIALS AND METHODS |
Experimental design.
We tested the effects of water
potential, temperature, and freeze-thaw cycling on rates of oocyst
degradation with a full-factorial approach. Our design included three
replicate trials at each combination of three lengths of exposure (1, 15, and 29 days), three temperatures (14, 4, and 30°C), and three
levels of water potential (4, 12, and 33 bars). We also examined
the effects of freeze-thaw cycling at the three levels of water
potential. We carried out 54 trials for above-freezing conditions
(three replicates, two temperature levels, three durations of exposure,
and three levels of water potential), 27 trials for below-freezing
conditions (three replicates, three durations of exposure, and three
levels of water potential), and 54 trials for freeze-thaw cycling
(three replicates, three levels of water potential, and six freeze-thaw cycles).
Source of oocysts.
Wild-type oocysts were extracted from
feces obtained per rectum from naturally infected Holstein
dairy calves in Fallon, Nevada. The purification procedure used
successive differential sucrose gradients in a flowthrough centrifuge,
as previously described for obtaining coccidial oocysts from poultry
litter (18). Oocyst solutions were diluted with deionized,
distilled water and stored at 4°C with 100 U of penicillin G sodium,
100 µg of streptomycin sulfate, and 0.25 µg of amphotericin B per
ml of oocyst suspension.
We conducted three analyses to verify that the
Cryptosporidium species in the stock was most likely
C. parvum. We examined samples of stock at a magnification
of ×1,000 by using differential interference contrast microscopy to
verify that oocyst dimensions, geometry, and morphology corresponded
with that expected of C. parvum oocysts (approximately
spherical and 4 to 7 µm in diameter [17]). We
successfully applied fluorescein isothiocyanate-labeled, single-stage,
anti-Cryptosporidium immunoglobulin M (Waterborne, Inc., New
Orleans, La.). Finally, using PCR, we successfully amplified C. parvum DNA target sequences from stock solutions with forward and
reverse primers provided by the National Institutes of Health AIDS Reagent Program (catalog no. 1558, forward primer,
5'-CCGAGTTTGATCCAAAAAGTTACGAAG-3'; and reverse primer,
5'-GCTCCTCATATGCCTTATTGAGTA-3'), using a previously described procedure (11).
We used stock diluted with distilled, deionized water containing
approximately 50,000 oocysts/ml (estimated from 20 counts
conducted on
separate 100-µl aliquots of stock). All experiments
were carried out
with stock solutions from the same batch, which
was less than 2 months
old before completion of all experiments.
Immediately prior to carrying
out trials, we characterized stock
with respect to numbers present and
proportions potentially effective,
based on the criteria described
below. Proportions considered
to be potentially infective are denoted
v0.
Water potential solutions.
One of the impediments to
carrying out studies of the degradation of C. parvum oocysts
in soils is poor recovery. Recovery efficiencies decrease significantly
with time (for example, less than 2% is recovered 7 days after
addition to soils [10, 12]). It is unclear whether
oocysts lost during soil sample processing represent a fraction that
has been destroyed or whether they remain capable of infecting a new
host in liquid or solid portions discarded during the extraction
process. To avoid this uncertainty, we used osmotic potential as a
surrogate for total water potential in soils. Stress on microbes due to
water potential in soils is the result of the additive effects of
matric and osmotic potentials (14). Previous work with
fungi indicates that osmotic potential adequately simulates total water
potential due to the combined effects of matric and osmotic potentials
(1). Use of solutions avoids problems of uneven
distribution of oocysts in soil, heterogeneity in soil moisture
distribution, and inefficiencies in soil extraction methods. By the use
of solutions, intact oocysts could be recovered by microfiltration.
We conducted preliminary trials with NaCl and sucrose solutions. We
evaluated differences at three temperatures with respect
to the
relationship between estimates of the degradation coefficient
(for
k, the coefficient in the exponential model representing
population change, see equation 1) and temperature and water potential.
Preliminary results indicated that NaCl and sucrose solutions
yielded
equivalent results. For example, the results of trials
conducted at 4, 25, and 30°C (
n = 41 trials total) at water
potentials
of
6,
13,
20, and
30 bars produced an estimate of
k =
0.002
· bars
0.003 · temperature (regression significance,
P < 0.001;
for coefficient significance, bars,
P = 0.005; and
temperature,
P < 0.001). These estimates are within
the 95% confidence intervals
for estimates derived from similar
conditions in experiments carried
out with NaCl (Table
1). Accordingly, we narrowed the scope of
our experiments to NaCl.
We prepared 0.08, 0.27, and 0.77 molal solutions of distilled,
deionized water with NaCl, which produced total water potential
conditions of
4,
12, and
33 bars, as determined by thermocouple
psychrometry (
15). These levels simulate soil matric
potentials
that lie between field capacity (
0.1 bars), the wilting
point
(
15 bars), and extremely dry conditions (less than
15 bars)
(
2). The solutions of 0.08, 0.27, and 0.77 M NaCl had
estimated
freezing points of
0.3,
0.8, and
2.9°C, respectively,
which
were well above the minimum temperature (
14°C) used for
experiments.
Isolation of oocysts from suspensions.
We isolated oocysts
by vacuum-filtering an oocyst suspension through a 0.2-µm-pore-size
diameter filter at a rate of approximately 1 ml per min. The entire
volume of each replicate, including approximately 1 ml of distilled,
deionized water used to rinse tubes after initial filtration, was filtered.
We tested recovery by comparing results of 10 replicate trials with
direct counts of 20-µl aliquots of stock solution. Results
from the
two methods were not significantly different (using the
criterion of
= 0.05) for estimates of concentrations and the
percentage of
the population that was potentially infective. Thus,
all oocysts were
recovered without bias in estimates of
v0.
Determination of potential infectivity and quantification.
Oocyst degradation was defined by several indicators of disruption of
the oocyst wall and nuclei of sporozoites, including appearance at
×1,000 magnification (obvious breaches in the oocyst wall), or the
inclusion of propidium iodide (PI) within the oocyst (e.g., references
4 and 9).
We applied PI directly to filters, as previously described
(
9) (10 µl of PI solution (1 g of PI:10 ml of 0.1 M
phosphate-buffered
saline, pH 7.2, with an incubation period of 90 min
at 37°C).
This assay leads to conservative results with respect to
degradation
coefficients, because it overestimates the number of
potentially
infective oocysts present relative to other methods for
demonstrating
infectivity, e.g., the use of experimental animals
(
3). Following
application of PI, we applied fluorescently
labeled anti-
Cryptosporidium immunoglobulin M according to
the manufacturer's instructions
(Waterborne, Inc.) and mounted filters
on thick agar-coated slides
beneath a coverslip with 20 µl of
fluorescence preservative gel
mounting medium (gel and mount; Fisher
Scientific, Pittsburgh,
Pa.). We examined a minimum of 100 locations
per
slide.
Oocyst suspensions and exposures to temperature regimes.
Oocysts were suspended in NaCl solutions by mixing 100 µl of oocyst
stock (containing 5 × 104 oocysts per ml)
with 1 ml of NaCl solution in 1.5-ml sealable polypropylene microfuge
tubes. Triplicate suspensions were placed in aluminum heating blocks at
30°C or in plastic microcentrifuge tube racks in a refrigerator
compartment at 4°C for 1, 14, and 29 days. For below-freezing
temperatures (14°C), oocysts were suspended in NaCl solutions and
placed in a freezer compartment in a plastic microcentrifuge tube rack
for 1, 14, and 29 days. The tubes were in the central area of a
frost-free freezer compartment, away from compartment walls and
insulated from temperature fluctuations by stored materials. At the end
of each time period, triplicates of each water potential condition were
removed and thawed for 1 h at room temperature (22 ± 2°C).
For freeze-thaw cycling trials, oocysts were suspended in NaCl
solutions and placed in the freezer compartment at
14°C. Freeze
cycles lasted for 12 h, prior to the entire batch being thawed
in
a refrigerated water bath at 10°C for 2 h. We thawed tubes
in a
10°C water bath to ensure that observed changes in oocyst
characteristics represented freeze-thaw cycling rather than prolonged
exposure to relatively high temperatures (e.g., room temperature).
Triplicate sets were subjected to one to six cycles of freezing
and
thawing.
Data analysis.
We represented population degradation with
exponential decay models in a multiplicative error format
(8). The dependent variable was the number of oocysts
potentially capable of infecting a new host
(n3) following exposure to stresses,
relative to the starting number (n1)
of oocysts capable of infecting a new host. Independent variables
included fixed temperatures, exposure times, and levels of water potential.
Data analysis considered the following general model for outcomes of
each experiment:
k = 
X,
for
X = (
X1,X2...Xn), in
which
k = ln(
n3/
n1)/
t
(the estimated degradation coefficient,
day
1),
t 
time of exposure (days) or number of freeze-thaw
cycles,
n1 v0 ·
n0,
n0 number of
oocysts initially added to suspension,
v0 proportion of initial
population of oocysts considered potentially
infective (0.92 and 0.90, for above- and below-freezing trials
and freeze-thaw cycling trials,
respectively),
n2 number of
oocysts recovered from suspension following exposure to stresses,
v2 proportion of recovered
population of oocysts considered
potentially infective,
n3 v2 ·
n2,
vector of coefficients
estimated using linear regression
(
1,
2...
n) associated with
levels of applied stresses,
including duration of exposure and
X vector of values
representing levels of applied
stresses.
The analyses applied regression through the origin to represent the
assumption that the absence of temperature and water potential
stresses
would not affect degradation rates. The measure of multiple
correlation
often cited for linear regression (
r2)
is inappropriate for regression through the origin and is not
presented
to support the results of data analysis. Analysis of
results tested
experimental hypotheses by applying the significance
of linear
regression relationships (using
F, with a rejection
criterion of
P = 0.05) and significance of estimated
coefficients
(using
t, with a rejection criterion of
P
= 0.05). Individual
and combined stresses were assumed significant
if the regression
relationship and coefficient estimates were
significant at
P 
0.05. All analyses were carried out
with Minitab Release 12 (Minitab,
Inc., State College, Pa.).
| |
RESULTS |
Suspensions at temperatures of >0°C.
Water potential and
temperature effects interact to increase the magnitude of the
degradation coefficient relative to values expected from previous work
(19). The regression model (Table 1) representing the
relationship between the degradation coefficient at 4° and 30°C and
bar water potential was statistically significant at P < 0.001. Coefficient estimates for bars water potential and temperature were statistically significant at P = 0.003 and
P < 0.001, respectively. These results indicate that,
with increasing water potential stress, estimates of the degradation
coefficient also increase (Fig. 1). As
reported by other researchers (9), estimates of
degradation coefficients increased in direct proportion to temperature
increases. However, the results indicate that water potential stresses
enhance the degradation rate, even under conditions in which
temperature stresses are relatively minor.
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|
FIG. 1.
Estimates of degradation coefficients (k,
day1) as a function of temperature (in degrees Celsius)
and water potential (1 bar [...], 5 bars
[-.-], and 10 bars [ ]), based on coefficient
estimates reported for above-freezing conditions in Table 1.
|
|
Suspensions at 14°C.
A linear model of population decay
(assuming the form of equation 1) considered the proportion of oocysts
remaining as a variable dependent upon water potential at 14°C. The
fitted model (Table 1) was significant at P < 0.001, with the coefficient for bars water potential significant at P
= 0.001. The results indicate that water potential enhances the
rates of reduction of oocyst numbers (Fig.
2).
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|
FIG. 2.
Estimates of population degradation dynamics for
exposure to subfreezing temperatures at water potential conditions of
0.5 bar (solid gray line) and 1.0 bar (solid black line) and
freeze-thaw cycling conditions at water potential conditions of 0.5
bar (dotted gray line) and 1.0 bar (dotted black line). Graph depicts
proportion of potentially infective oocysts remaining relative to the
number added for each trial
(n3/n1) versus duration
of exposure (days or number of freeze-thaw cycles). Note that estimates
of degradation for freeze-thaw cycling are extrapolated beyond
experimental conditions, which considered a maximum of six freeze-thaw
cycles.
|
|
Suspensions subjected to freeze-thaw cycling.
The fitted model
was significant at P < 0.001, with the coefficient for
bars water potential significant at P < 0.001 (Table 1). The model suggests that water potential enhances the effects of
freeze-thaw cycling (Fig. 2).
| |
DISCUSSION |
Relative effects of stresses.
Water potential stresses enhance
degradation due to above- and subfreezing temperature effects and
freeze-thaw cycling. The values that we estimated are larger (on an
absolute scale) than values previously used for risk assessment
modeling related to public water supply protection (19)
(Fig. 3). Among the stresses applied,
freeze-thaw cycling is extreme and the effects of water potential are
roughly four times those noted for freezing alone. The difference
between effects of freeze-thaw cycling and simple freezing may be
caused by mechanical damage to the oocyst wall (6). We
expected such damage to increase as oocysts were subjected to more
cycling events.
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|
FIG. 3.
Estimates of population degradation dynamics for
above-freezing conditions, based on temperature-dependent degradation
coefficients (19) and temperature- and water
potential-dependent degradation coefficients (reported for
above-freezing temperatures [Table 1]). Estimates show differences
between temperature-based estimates of the decay coefficient
k at 4 (solid gray line) and 20°C (dotted gray line)
and temperature and water potential estimates at 4 (solid black line)
and 20°C (dotted black line) over the course of 30 days with water
potential stresses of 0.5 bars. Graph depicts proportion of
potentially infective oocysts remaining relative to the number added
for each trial (n3/n1)
versus duration of exposure (days).
|
|
The results suggest that water potential conditions encountered under
field conditions are likely to lead to more rapid degradation
of oocyst
populations than might be expected from studies of degradation
in calf
feces, distilled water with antibiotics, and reverse osmosis
water at
low temperatures (
9) (Fig.
3). For example, at 4°C
and
1 bar water potential, the estimated degradation coefficient
is
0.046 day
1, which is approximately an order
of magnitude higher than previous
estimates (
9) that have
been used to model the fate and transport
of
Cryptosporidium
in public water supply watersheds (
19).
Approaches to risk assessment modeling have relied on estimates of
degradation coefficients that are conservative with respect
to public
health protection, although use of estimates derived
from previous
studies may be excessively conservative. Because
water potential in the
soil environment is likely to vary seasonally,
with changes in soil
moisture content, using a static degradation
coefficient to
characterize changes in oocyst populations may
not represent the actual
processes accurately. Water potential
stresses in soils are
predominantly exerted in combination as
matric potential due to soil
moisture content and osmotic potentials
due to chemistry of soil
solution (
14). As an alternative, results
of these
experiments suggest that conservative approaches could
be based on
minimum degradation rates expected for water potential
and temperature
conditions at the soil surface, which may yield
results that are
conservative and are based on understanding of
local conditions of
expected soil temperature and water
potential.
The multiplicative error model is used commonly to model the
exponential growth and death phases of microbial population life
cycles. This model assumes that stresses lead to changes in numbers
that are best described by a single coefficient,
k, which
assumes
negative values for the death phase (
8) and
represents a constant
rate of reduction per time step. If the
exponential models fitted
above are accurate descriptions of population
degradation, then
those oocysts not destroyed by environmental stresses
represent
a subpopulation of concern. The results of these experiments
and
model fitting imply that small proportions of oocyst populations
survive stresses. Because numbers of oocysts excreted by infected
hosts
may be extremely large (for example, ~10
10 over
the course of illness in dairy calves [
7], while the
estimated infectious dose is ~10
2
[
5]), small proportions of populations may represent
very
large numbers with respect to the potential to
infect.
The implications of having small proportions of oocysts remaining
infective following exposure to combined stresses is unclear.
Prolonged
exposure to natural stresses may render oocysts vulnerable
to water
disinfection processes. If so, the effects may enhance
raw water
disinfection processes. However, oocysts that survive
stresses may be a
resistant subpopulation that is more difficult
to remove by
disinfection processes commonly used for water treatment.
In either
case, experiments to evaluate performance of existing
and new
disinfection processes could consider using prestressed
populations to
evaluate treatment efficiency. Such experimental
populations may best
represent the treatment challenge faced by
systems that potentially are
influenced by nonpoint sources of
oocysts.
| |
ACKNOWLEDGMENTS |
This research was supported in part by Hatch and USDA-NRI grants
(Projects NEV0533F and NEV052HL, respectively).
We thank the reviewers for thoughtful and helpful comments.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: University of
Nevada, Natural Resources Department (MS370-FA 132), 1664 Virginia
Ave., Reno, NV 89557-0013. Phone: (775) 784-1938. Fax: (775) 784-4789. E-mail: mwalker{at}equinox.unr.edu.
| |
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Applied and Environmental Microbiology, December 2001, p. 5526-5529, Vol. 67, No. 12
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.12.5526-5529.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Top
Abstract
Introduction
