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Applied and Environmental Microbiology, July 1999, p. 2820-2826, Vol. 65, No. 7
0099-2240/99/$04.00+0
Method for Detection and Enumeration of
Cryptosporidium parvum Oocysts in Feces, Manures, and
Soils
Ewa
Kuczynska and
Daniel R.
Shelton*
Environmental Chemistry Laboratory,
Agricultural Research Service, U.S. Department of Agriculture,
BARC-West, Beltsville, Maryland 20705-2350
Received 6 January 1999/Accepted 6 April 1999
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ABSTRACT |
Eight concentration and purification methods were evaluated to
determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of
recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the
NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 102, 103, 104, and 105
oocysts g of bovine feces
1. The percentages of recovery
ranged from 10.8% (25 oocysts g1) to 17.0%
(104 oocysts g1)
(r2 = 0.996). A
conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces1. Percentages of recovery were
determined for six different types of animal feces (cow, horse, pig,
sheep, deer, and chicken feces) at a single oocyst concentration
(104 oocysts g1). The percentages of recovery
were highest for bovine feces (17.0%) and lowest for chicken feces
(3.2%). Percentages of recovery were determined for bovine manure
after 3 to 7 days of storage. The percentages of recovery ranged from
1.9 to 3.5% depending on the oocyst concentration, the time of
storage, and the dispersing solution. The percentages of oocyst
recovery from soils were evaluated by using different flotation
solutions (NaCl, cold sucrose, ZnSO4), different dispersing
solutions (Triton X-100, Tween 80, Tris plus Tween 80), different
dispersion techniques (magnetic stirring, sonication,
blending), and different dispersion times (5, 15, and 30 min).
Twenty-five-gram soil samples were used to reduce the spatial
variability. The highest percentages of recovery were obtained when we
used 50 mM Tris-0.5% Tween 80 as the dispersing solution, dispersion
for 15 min by stirring, and saturated NaCl as the flotation solution.
The percentages of oocyst recovery from freshly spiked sandy loam,
silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%,
respectively. The theoretical detection limits were ca. 1 to 2 oocysts
g of soil1 depending on the soil type. The percentages of
recovery without dispersant (distilled H2O or
phosphate-buffered saline) were less than 0.1%, which indicated that
oocysts adhere to soil particles. The percentages of recovery decreased
with storage time, although the addition of dispersant
(Tris-Tween 80) before storage appeared to partially prevent
adhesion. These data indicate that the NaCl flotation method is
suitable for routine detection and enumeration of oocysts from feces,
manures, soils, or soil-manure mixtures.
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INTRODUCTION |
Cryptosporidium parvum,
the causal agent of cryptosporidiosis, is a widespread protozoan
parasite that infects numerous mammalian species. C. parvum is an important human pathogen, as evidenced by several
outbreaks of cryptosporidiosis in the past decade; the most severe of
these outbreaks occurred in Milwaukee, Wis., where more than 400,000 people were infected (13). C. parvum is
a particularly serious health threat to immunodeficient individuals (e.g., AIDS and cancer patients) because there are no effective treatments for the disease.
An important mode of C. parvum transmission to humans
is believed to be via contaminated drinking water or recreational
water. Studies have shown that waterborne C. parvum
oocysts (the infectious stage found outside the body) may remain viable
for several months (8). Although wildlife and sewage
outflows have been implicated in watershed contamination (3, 11,
16, 19), farm animals are also believed to be major contributors.
Sheep, horses, and pigs are susceptible to infection by C. parvum and shed oocysts (21, 29, 30); however, dairy
and beef calves are generally considered to present the greatest risk
because of their numbers, distribution, incidence of infection, and
high levels of oocyst excretion.
Neonatal calves are particularly susceptible to infection (scours) and
can excrete up to 30 billion oocysts or more over a 1- to 2-week
period. Based on a survey of 7,369 calves from 1,103 dairy farms
located in 28 states, Garber et al. (10) found that more
than 50% of 2-week-old calves and 22.4% of all calves (ages, 1 to 17 weeks) tested positive for C. parvum. These authors
concluded that virtually all herds with more than 100 cows are infected with C. parvum. Limited data suggests that adult cows
may also shed oocysts. Scott et al. (18) found up to 18,000 oocysts per g of feces from apparently healthy adult cows. Based on an
average content of 900 oocysts per g of feces and a total excretion of ca. 40 kg of feces per cow per day, a single adult bovine could potentially excrete more than 36 million oocysts per day.
These data suggest that contaminated manures from dairy or beef
cattle operations can be major sources of C. parvum
oocysts unless manure management or treatment strategies are used to
minimize oocyst viability or transport to water. In addition to direct fecal deposition, possible modes of transport to potable or
recreational water include surface transport from land-applied manures
or leaching through the soil to groundwater (e.g., karst groundwater).
Land application of manures is recommended in order to recycle
nutrients (e.g., nitrogen and phosphorus) for crop growth. The
Environmental Protection Agency has proposed manure management
recommendations to minimize nutrient transport to surface water
(7). It is important to determine if the proposed
recommendations also minimize transport of C. parvum
oocysts to surface water.
Evaluations of the efficacy manure management strategies depend on
accurate determinations of oocyst numbers in feces, manures, and soils.
Methods for detecting Cryptosporidium oocysts in fecal samples have been described previously. Fecal smears are commonly used
for clinical purposes to detect oocysts in stool samples. Although
quick and relatively quantitative, smears have limited sensitivity and
are applicable only to watery or diluted samples (samples with low
percentages of solids). Several concentration and purification methods
in which a variety of flotation solutions are used for extraction and
recovery of oocysts from fecal samples have been described (2, 5,
12, 17, 20, 22, 32). In general, these methods have not been
rigorously evaluated with respect to extraction efficiencies and/or
detection limits.
Few studies have addressed the transport of oocysts over or through
soils, in large measure because of difficulties associated with
detection and enumeration of oocysts in soil samples or soil-manure mixtures. Mawdsley et al. (14) have described a method for
extraction and enumeration of oocysts in soil in which sucrose
flotation is used. These authors reported extraction efficiencies of up to 61.6% for 1-g soil samples processed shortly after spiking; however, the extraction efficiencies declined to 4% after 24 h. Walker et al. (23) obtained comparable results by using a
procedure adapted from the method of Mawdsley et al.; in this study the percentage of recovery was 43% ± 5.7% (average ± 95%
confidence interval) for freshly spiked samples. This method is
suitable for laboratory experiments in which oocysts are likely to be
relatively homogeneously distributed throughout the soil. However, for
field scale experiments, in which the oocyst distribution is likely to
be more heterogeneous, larger sample sizes are preferable in order to
reduce spatial variability.
We describe here an evaluation of concentration and purification
methods that were used in conjunction with immunofluorescence antibody
staining for detection and enumeration of C. parvum
oocysts in feces, manures, and soils. Our goal was to identify a
relatively fast, inexpensive method that could be used for routine
detection and quantitation of low levels of oocysts in feces, manures,
soils, or soil-manure mixtures.
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MATERIALS AND METHODS |
Sample preparation.
Purified C. parvum
oocysts were obtained from infected calves as previously described
(9). Oocysts (ca. 107 oocysts ml1)
were stored in sterile phosphate-buffered saline (PBS) (pH 7.2) at
4°C until they were used. Oocyst suspensions used to spike fecal,
manure, or soil samples were prepared immediately prior to use; precise
numbers were determined with a Neubauer hemocytometer. Purified oocysts
were used in all experiments unless indicated otherwise.
One-gram aliquots of fresh calf feces and feces from lactating cows at
the Beltsville Agricultural Research Center dairy farm were spiked with
0.5-ml portions of oocyst suspensions (serial dilutions of a known
stock suspension) to give final concentrations of 2 × 103, 104, and 105 oocysts g of calf
feces1 and 25, 50, 200, 103, 104,
and 105 oocysts g of cow feces1,
respectively. One-gram aliquots of fresh feces from adult swine, sheep,
chicken, horse, and deer samples were spiked with 0.5-ml portions of an
oocyst suspension to give a concentration of 104 oocysts g
of feces1. Samples of swine, sheep, and chicken feces
were obtained from research animals at the Beltsville Agricultural
Research Center, Beltsville, Md.; horse feces were obtained from a
stable (Glen Dale Farms, Greenbelt, Md.); and deer feces from feral
animals were collected in the field at the Patuxent Wildlife Refuge,
U.S. Fisheries and Wildlife Service, Bowie, Md. Prior to spiking, the feces were examined (by the NaCl flotation method) to ensure that no
detectable oocysts were present. Samples were processed immediately after spiking. We prepared six samples of each type of feces at each concentration.
To determine percentages of recovery from bovine manure, 100-g aliquots
of a manure slurry (containing feces, urine, and water)
that was
collected fresh from the Beltsville Agricultural Research
Center dairy
barn were each spiked with 1 ml of an oocyst suspension
to give a final
concentration of 10
3 or 10
4 oocysts g of
manure
1. After thorough mixing with a magnetic stirrer
the manure was
stored at 4°C.
To determine percentages of recovery from soils, 25-g aliquots of
air-dried soil were each spiked with 0.5 ml of a purified
oocyst
suspension. Experiments were conducted with the following
three soil
types: sandy loam, silty clay loam, and clay loam.
A textural analysis
was conducted by using the hydrometer method
(
27). The sandy
loam soil contained 66.9% sand, 16.6% silt,
and 16.5% clay, and its
organic matter content was 1.1%; the silty
clay loam soil contained
18.4% sand, 53.2% silt, and 28.4% clay,
and its organic matter
content was 3.1%; and the clay loam soil
contained 25.7% sand, 42.5%
silt, and 31.8% clay, and its organic
matter content was 2.8%. Except
as noted below, experiments were
conducted with the sandy loam soil.
All experimental treatments
were replicated six times unless indicated
otherwise.
Extraction procedures used for feces.
Twenty-milliliter
portions of PBS were added to samples after they were thoroughly mixed
with oocysts. Fecal suspensions were filtered through a stainless steel
mesh sieve (pore size, 45 µm) and rinsed with ca. 30 ml of PBS. The
suspensions were centrifuged at 500 × g for 10 min in
50-ml polypropylene centrifuge tubes. Each resulting supernatant was
decanted, the sediment was resuspended in PBS, and the process was
repeated once. Most of the sediments were resuspended in 5 ml of PBS to
form a slurry; the exceptions were (i) after discontinuous sucrose
gradient centrifugation, when the sediment was resuspended in 5 ml of
2.5% aqueous potassium dichromate
(K2Cr2O7), and (ii) after flotation
with ZnSO4 · 7H2O, when the sediment was
resuspended in 5 ml of distilled water.
The following four flotation solutions were examined: MgSO
4
(575 g liter
1; specific gravity, 1.27),
ZnSO
4 · 7H
2O (703 g
liter
1; specific gravity, 1.3), cold sucrose (700 g
liter
1; specific gravity, 1.18), and NaCl (360 g
liter
1; specific gravity, 1.21). Sediment slurries were
emulsified with
45-ml portions of the flotation solutions and
centrifuged at 500
×
g for 10 min. The upper 5 ml of
each supernatant was transferred
to a 50-ml tube. The other methods
examined included cesium chloride
gradient centrifugation,
discontinuous Sheather's gradient centrifugation,
discontinuous
Percoll gradient centrifugation, and formalin-ethyl
acetate
sedimentation.
For cesium chloride gradient centrifugation (
12), solutions
were prepared from stock solutions of CsCl (specific gravity,
1.8) and
Tris buffer (50 mM Tris, 10 mM EDTA; pH 7.2) by using
the following
proportions of CsCl and Tris buffer: 1:1 (density,
1.4 g
ml
1), 1:7 (density, 1.1 g ml
1), and
1:15 (density, 1.05 g ml
1). Three milliliters of
each CsCl solution was layered into a
15-ml tube. The sediment was
centrifuged at 1,500 ×
g for 10 min
in Tris buffer,
the supernatant was removed, and the sediment
was resuspended in 1 ml
of Tris buffer. The contents of each tube
were overlaid with 1 ml of
suspension, and the tubes were centrifuged
at 16,000 ×
g for 60 min at 4°C. Following centrifugation, the
band
between the 1.1- and 1.05-g ml
1 densities in each tube
was aspirated with a glass pipette and
transferred to a 50-ml
tube.
For discontinuous Sheather's gradient centrifugation (
2), a
sucrose solution (500 g of sucrose and 6.5 g of phenol in 320
ml
of water) was diluted 1:2 and 1:4 with sterile PBS. Ten milliliters
of
the 1:2 dilution was transferred to a 50-ml centrifuge tube
and
overlaid with 10 ml of the 1:4 dilution. The contents of each
tube were
overlaid with 5 ml of a PBS suspension in
K
2Cr
2O
7, and
the tubes were
centrifuged at 1,500 ×
g for 30 min at 4°C.
Following
centrifugation, the upper yellow potassium dichromate layer
was
discarded, while the pellet and next two layers (yellow turbid
and
white clear layers) were transferred to 50-ml
tubes.
For discontinuous Percoll gradient centrifugation (
17),
Percoll gradients were prepared in 15-ml centrifuge tubes, and each
gradient consisted of the following four 2-ml layers: 100% Percoll
(density, 1.13 g ml
1), 75% Percoll in distilled
water (density, 1.09 g ml
1), 33% Percoll (density,
1.05 g ml
1), and 10% Percoll (density, 1.01 g
ml
1). The contents of each tube were overlaid with 1 ml
of suspension,
and the tubes were centrifuged at 650 ×
g for 15 min at 4°C. The
layer between the 1.09- and 1.05-g
ml
1 densities in each tube was aspirated with a glass
pipette and
transferred to a 50-ml centrifuge
tube.
For formalin-ethyl acetate sedimentation (
31), sediment
slurries were mixed with 9 ml of neutral buffered 10% formalin and
then with 4 ml of ethyl acetate. Samples were shaken in an inverted
position for 30 s and then centrifuged at 500 ×
g
for 2 min. The
upper three layers were transferred to 50-ml tubes,
enough PBS
was added to bring the volume to 50 ml, and the tubes were
centrifuged
twice at 500 ×
g for 10 min. The sediment
in each tube was resuspended
in MgSO
4 and centrifuged at
500 ×
g for 10 min, and the upper
5 ml was transferred
to a 50-ml
tube.
Following each procedure, the volume of the oocyst suspension was
brought to 50 ml with distilled water, and the preparation
was
centrifuged at 500 ×
g for 10 min. The pellet was
washed once
more with 50 ml of water and then with 15 ml of water, and
then
it was centrifuged in a 1-ml Eppendorf tube at ca.
1,500 ×
g for
3 min. The final pellet was resuspended
in 100 µl of distilled
water.
Extraction procedures used for manure.
One-gram aliquots of
manure were processed on days 3 and 6 (104 oocysts
g1) or days 4 and 7 (103 oocysts
g1). Samples were diluted with 50 ml of 50 mM Tris and
0.5% (vol/vol) Tween 80 or 50 ml of PBS and dispersed for 15 min with
a magnetic stirrer. Manure solutions were filtered through a stainless
steel mesh sieve (pore size, 45 µm) and washed with ca. 50 ml of
Tris-Tween 80 or PBS. After centrifugation (500 × g
for 10 min) in 100-ml tubes, the supernatants were decanted, and the
sediments were transferred to 50-ml tubes and processed by using the
NaCl flotation method as described above. Percentages of recovery and
standard deviations were calculated based on six replicates.
Extraction procedures used for soils.
The flow diagram in
Fig. 1 shows the generic extraction
procedure used for soils. The flotation solutions evaluated included ZnSO4 · 7H2O, cold sucrose, and NaCl.
All other experiments were conducted by using the NaCl flotation
method. The dispersing solutions evaluated included distilled water,
PBS (pH 7.2), 1% (wt/vol) Tween 80, 1% (wt/vol) Triton X-100, and 50 mM Tris and 0.5% (vol/vol) Tween 80. Dispersion times of 5, 15, and 30 min were evaluated in conjunction with magnetic stirring and Tris-Tween
80. Samples were processed as previously described.
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FIG. 1.
Flow diagram showing the NaCl flotation method for
extracting oocysts from soils. S.G., specific gravity; IFA,
immunofluorescence antibody.
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The dispersion methods evaluated included magnetic stirring for 15 min,
sonication for 15 min with a model 2210 Ultrasonic
Cleaner (Branson,
Danbury, Conn.) at 47 kHz, and blending for
2 min with a Waring blender
at low speed (19,000 rpm). Twenty-five-gram
soil samples were obtained
from the upper 2-cm portions of sandy
loam or loam soil cores
previously used for leaching experiments
(unpublished data). Three
samples per core were obtained approximately
48 h after leaching,
and the percentages of recovery were determined
for each dispersion
method (
n = 2). Since the percentages of oocyst
recovery from the sandy loam and loam soils were almost identical,
the
data are presented below as the average levels of recovery
for both
cores. The samples were processed as described above
by using
Tris-Tween 80 dispersing solution and magnetic stirring
for 15
min.
The percentages of recovery as a function of storage time were
evaluated by using oocysts obtained from diluted calf diarrhea
samples;
the final concentration used was 10
4 oocysts per 25 g
of soil. Five milliliters of distilled water
or dispersing solution (50 mM Tris and 0.5% [vol/vol] Tween 80)
was added to 25 g of
air-dried soil (moisture content, ca. 20%),
and then 0.5 ml of the
oocyst suspension was added immediately.
The soil samples were stored
at 4°C until they were processed.
The samples were processed after
1 h and 1, 3, 7, 10, 14, and
21 days as described above by using
the Tris-Tween 80 dispersing
solution and magnetic stirring for 15
min.
Oocyst detection and enumeration.
After each Eppendorf tube
was thoroughly vortexed, three 10-µl aliquots (of the 100-µl
sample) were pipetted into slide wells (diameter, 5 mm), dried with a
slide warmer, and stained by the direct immunofluorescence antibody
method by using a commercial kit (Merifluor; Meridian Diagnostic, Inc.,
Cincinnati, Ohio). Samples were examined with an epifluorescence
microscope (Olympus) by using a magnification of ×200. The number of
oocysts per gram of feces or manure or per 25 g of soil was
determined by multiplying the average number of oocysts counted in
three wells by 10. Percentages of recovery and standard deviations were
calculated based on six replicates unless indicated otherwise.
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RESULTS |
Recovery from feces and manure.
The percentages of oocyst
recovery from calf feces varied from <1 to 18.7% for the eight
concentration and purification methods evaluated (Table
1). The NaCl and sucrose flotation
methods gave significantly higher percentages of recovery (P < 0.05) at the lowest oocyst concentration used (2 × 103 oocysts g1). The NaCl flotation method
gave significantly higher percentages of recovery (P < 0.05) at oocyst concentrations of 104 and
105 oocysts g1 than most of the other methods
gave; the only exception was CsCl gradient centrifugation. In general,
the percentages of recovery were highest for the lowest oocyst
concentration (2 × 103 oocysts g1).
Subjectively, CsCl gradient centrifugation and discontinuous Percoll
gradient centrifugation resulted in the smallest amounts of fecal
debris in final oocyst preparations, formalin-ethyl acetate sedimentation, MgSO4 flotation, ZnSO4
flotation, and sucrose flotation resulted in the most fecal debris, and
Sheather's discontinuous gradient centrifugation and NaCl flotation
resulted in intermediate amounts of fecal debris. The flotation methods
were more cost and time efficient than the gradient centrifugation
methods because of the inexpensive materials and fewer, less complex
procedures. However, except for NaCl flotation, they generally resulted
in larger amounts of fecal debris. The NaCl flotation method was chosen
for further evaluation because of the higher percentages of recovery,
intermediate amounts of fecal debris, relatively short processing times
(<3 h), and low expense associated with it.
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TABLE 1.
Percentages recovery from calf feces at three oocyst
concentrations obtained with the following eight concentration and
purification methods: cesium chloride gradient centrifugation,
Sheather's discontinuous sucrose gradient centrifugation,
discontinuous Percoll gradient centrifugation, formalin-ethyl
acetate sedimentation, MgSO4 flotation, ZnSO4
flotation, sucrose flotation, and NaCl flotation
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Percentages of recovery from adult bovine feces were determined by
using oocyst concentrations ranging from 25 to 10
5 oocysts
g of feces
1 (Table
2). The
percentages of recovery from adult feces were
generally comparable to
the percentages of recovery from calf
feces and were comparable at
different oocyst concentrations.
The coefficients of variation were
consistent at oocyst concentrations
of >10
3 oocysts
g
1 but increased as the oocyst concentrations decreased.
Linear
regression plots (log number of oocysts added versus log number
of oocysts recovered) gave an
r2 value
of 0.996. A conservative estimate of the detection limit
in bovine
feces is ca. 30 oocysts g
1 (reciprocal of fraction
recovered [ca. 0.12] × fraction of suspension
counted [0.3]). The
percentages of recovery from different animal
feces varied from 3.2%
for chicken feces to 17% for bovine feces
(Table
3).
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TABLE 2.
Recovery of oocysts from adult bovine feces at different
initial concentrations when the NaCl flotation method was used
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The percentages of recovery from a bovine manure slurry were determined
as a function of the storage and dispersing solution
(Table
4). The percentages of recovery were
substantially lower
than the percentages of recovery for fresh bovine
feces. The percentages
of recovery when PBS was used as the dispersing
solution were
consistent (2.3 to 2.5%) regardless of the oocyst
concentration
or storage time. The percentages of recovery when
Tris-Tween 80
was used as the dispersing solution were initially higher
(3.2
to 3.5%) but decreased after an additional 3 days of storage (to
1.9 to 2.1%). The coefficients of variation were consistently
higher
at lower oocyst concentrations but were generally consistent
at a given
concentration regardless of the storage time.
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TABLE 4.
Recovery of oocysts from a bovine manure slurry as a
function of concentration, storage, and dispersion solution when
the NaCl flotation method was used
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Recovery from soils.
Initial experiments were conducted with
NaCl, ZnSO4, and cold sucrose flotation solutions
(n = 6) by using a Tris-Tween 80 dispersing solution
and magnetic stirring for 15 min. The percentages of recovery were as
follows: ZnSO4, 0.8% ± 0.4%; cold sucrose, 2.8% ± 0.9%; and NaCl, 16.6% ± 2.3%. The percentages of recovery with NaCl
were significantly higher (P < 0.05) than the
percentages of recovery with ZnSO4 or cold sucrose.
PBS, H
2O, and three detergent dispersing solutions were
evaluated in conjunction with magnetic stirring for 15 min. The order
for percentage of oocyst recovery was as follows: PBS and
H
2O
< 1% Tween 80 <1% Triton X-100 < 50 mM
Tris-0.5% Tween 80 (Table
5). The
percentages of recovery with Tris-Tween 80 were significantly
higher
(
P < 0.05) than the percentages of recovery with the
other
dispersants. The time of dispersion was evaluated by using 50
mM
Tris-0.5% Tween 80. The percentages of recovery increased ca.
threefold from 5 min (5.7% ± 0.5%) to 15 min (18.1% ± 1.9%) but
did not increase after 30 min (18.0% ± 3.2%). The percentages
of
recovery after 15 or 30 min were significantly higher (
P <
0.05) than the percentages of recovery after 5 min.
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TABLE 5.
Percentages of recovery of purified oocysts from sandy
loam soil as a function of the dispersing solution
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Different methods of dispersion were evaluated by using soil leaching
cores ca. 48 h after experimental treatments were used
(unpublished data). The numbers of oocysts detected were as follows:
blender, 14.0 ± 0.5 oocysts/10 µl; sonication, 21.5 ± 0.7 oocysts/10
µl; and magnetic stirrer, 25.0 ± 1.4 oocysts/10
µl. Because of
limited replication (
n = 2), we could
not conclude that the percentages
of oocyst recovery with magnetic
stirring were significantly different
than the percentages of oocyst
recovery with
sonication.
Percentages of oocyst recovery were determined for freshly spiked sandy
loam, silty clay loam, and clay loam soils (Fig.
2).
The percentages of recovery decreased
as the clay content increased.
A linear regression analysis of
percentage of recovery versus
clay content gave an
r2 value of 0.993. Percentages of
recovery were evaluated as a function
of storage with and without the
addition of a dispersing solution
(Tris-Tween 80). With or without the
dispersing solution, the
percentages of recovery decreased ca.
threefold after 1 h (Fig.
3). The
percentages of recovery continued to slowly decrease for
10 days,
although the percentages of recovery were generally higher
when the
dispersing solution was added.
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FIG. 2.
Mean percentages of oocyst (purified) recovery from
freshly spiked sandy loam, silty clay loam, and clay loam soils. The
error bars indicate standard deviations (n = 6).
The initial oocyst concentration was 104 oocysts per
25 g of soil.
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FIG. 3.
Plot of percentages of oocyst (from diluted calf
diarrhea) recovery from sandy loam soil versus storage time with and
without dispersing solution (disp. sol.) (Tris-Tween 80). The error
bars indicate standard deviations (n = 6). The initial
oocyst concentration was 104 oocysts per 25 g of soil.
The first sample was obtained 1 h after spiking.
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DISCUSSION |
The goal of these experiments was to identify a method which can
be used for routine detection and enumeration of low levels of
C. parvum oocysts in feces, manures, soils, and
soil-manure mixtures. Primary consideration was given to percentages of
recovery, the amounts of debris in the final oocyst preparations,
processing time, and expense.
In our hands, the NaCl flotation method generally gave the highest
percentages of recovery from calf feces; in addition, the processing
times with this method were relatively short (<3 h), and this method
was the least expensive method tested. Although other methods gave
cleaner preparations (CsCl and Percoll gradient centrifugation) or
resulted in less variability (MgSO4 flotation), they were
deficient in other areas considered. Commonly used methods, such as
formalin-ethyl acetate sedimentation (6, 24-26, 30, 31) and
sucrose flotation (5, 15, 20, 26), were generally inferior
because of lower percentages of recovery, greater background fecal debris levels, or greater variability. Xiao and Herd
(28) have described a method ("quantitative FA")
which requires no flotation or gradient centrifugation procedure. Using
a concentration of 103 oocysts g of calf
feces1, these authors obtained a percentage of recovery
of 14.8% and a coefficient of variation of 47.1%. Given the
simplicity of their method (one clarification step and one
centrifugation step) and the excellent percentages of recovery at
higher oocyst concentrations, it appears that this method is preferable
for relatively highly contaminated fecal samples. However, as described
by Xiao and Herd, the quantitative FA method has a detection limit of
ca. 700 oocysts g1 (reciprocal of 0.15 × 0.01). By
comparison, the detection limit of our method is ca. 30 oocysts
g1 because of the 10-fold concentration step and because
30% of the final oocyst suspension is counted.
The percentages of oocyst recovery from fresh bovine feces were
relatively consistent over a wide range of oocyst concentrations (25 to
105 oocysts g1) when the NaCl flotation
method was used; the r2 value was
0.996. Therefore, it appears to be reasonable to quantify oocyst
concentrations based on a single correction factor (percentage of
recovery) for a given feces type; separate correction factors must be
determined for different fecal types. Note that previously described
methods have been evaluated by extracting fecal samples immediately
after spiking. Consequently, the reported percentages of recovery may
overestimate the levels of recovery from stored fecal samples depending
on the extent of oocyst adhesion to fecal solids.
Since manures typically are stored for different periods of time, we
attempted to simulate a contaminated manure slurry by spiking a
preparation with an infected calf diarrhea sample, thoroughly mixing
it, and then storing it. The percentages of oocyst recovery were
substantially lower for the stored bovine manure than for fresh fecal
samples, indicating that correction factors for feces are not
applicable to manures. We suspect that the lower percentages of oocyst
recovery were primarily due to enhanced adhesion of oocysts to fecal
particles during storage, although the possibility that decomposition
occurred cannot be ruled out. Experiments were conducted with
Tris-Tween 80 as the dispersing solution in an attempt to improve the
percentages of recovery. The percentages of recovery were initially
higher (on days 3 and 4), but then they decreased. It is unclear why
this occurred. Low percentages of recovery complicate attempts to
quantify oocyst loading rates from manures (the numbers of oocysts
applied per square meter or hectare). For example, if it is assumed
that manure accumulates from a 100-animal herd for 1 week, that the
fecal excretion rate is 40 kg of feces cow1
day1, that the preparation is diluted ca. 50% with urine
and water, and that the percentage of recovery is 2.5%, a minimum of
ca. 7.5 billion oocysts would be required to obtain a positive sample, and each 0.1% change in the percentage of recovery would correspond to
ca. 300 million oocysts.
Experiments were conducted to assess the levels of oocyst recovery from
soils with several previously described flotation solutions, including
NaCl, ZnSO4, and cold sucrose. The ideal flotation solution
should have (i) a relatively high specific gravity, (ii) low viscosity,
and (iii) the ability to disperse clay and silt soil particles. In
theory, because of the great difference in the densities of oocysts and
soil particles (ca. 1.05 and 2.65 g ml1,
respectively), separation of oocysts from soil particles should be
simple. In contrast to a previous report (1), our data
indicates that oocysts adhere to soil particles, as shown by
percentages of recovery of <0.1% when distilled water or PBS was used
as the dispersant. Consequently, the levels of oocyst recovery depend on both the physical separation of oocysts from soil particles and the
dispersion of soil particles during sedimentation, as well as the
specific gravity of the flotation solution. For example, if it is
assumed that an oocyst with a spherical radius of 2.5 µm adheres to a
soil particle that is half its size (spherical radius, 1.25 µm), the
resulting oocyst-soil particle aggregate should have a composite
density of ca. 1.24 g ml1.
We obtained the highest percentages of recovery with NaCl flotation. We
suspect that this was due primarily to the ability of monovalent
cations to disperse soil particles, which minimized entrapment of
oocysts or oocyst-soil particle aggregates during sedimentation.
Substantially lower percentages of recovery were obtained with
ZnSO4 flotation, despite the higher specific gravity of
ZnSO4. We suspect that this was due primarily to the
tendency of divalent cations to precipitate soil particles, which
entrapped oocysts or oocyst-soil particle aggregates during
sedimentation. We also obtained lower percentages of recovery with cold
sucrose flotation. We suspect that this was due to a combination of
lower specific gravity and higher viscosity. Oocyst detection was
difficult due to high soil particle background levels in wells
resulting from poor sedimentation of soil particles.
Different dispersing solutions, dispersion times, and dispersion
procedures were evaluated to optimize levels of oocyst recovery. Our
results obtained with dispersing solutions are consistent with the
results of Mawdsley et al. (14), who obtained their highest
levels of recovery with Tris-Tween 80. The highest levels of oocyst
recovery were obtained with magnetic stirring, which is a relatively
mild procedure. By comparison, the highest levels of bacterial recovery
from soils are typically observed with blending (4). We
suspect that the shear forces created by blending were too severe for
oocyst walls. After blending, large numbers of what appeared to be
fluorescent wall fragments were observed in oocyst preparations.
The percentage of oocyst recovery was linearly correlated
(r2 = 0.993) with clay content
(soil particle diameters, 
2 µm). These data suggest that for
mineral soils, it may be possible to predict levels of oocyst recovery
based on soil texture data. More soil types are required, however, to
verify this relationship. The detection limit of our method is ca. 1 to
2 oocysts g of soil1 depending on the soil type
(reciprocal of percentage of recovery × fraction counted). The
detection limit reported by Mawdsley et al. (14) was 529 oocysts g of clay loam soil1. Walker et al.
(23) obtained detection limits of <40 oocysts g of silt
loam soil1 by including a final concentration step.
Although our extraction procedure is somewhat more complicated than
that of Walker et al. (23), the larger sample size means
that fewer samples can be used.
Levels of oocyst recovery from soils also depend on incubation or
storage time. The percentages of recovery from sandy loam soil
decreased to <1% within 10 days. Our results are similar to those of
Mawdsley et al. (14), who observed a >99% decrease in the
level of recovery after 1 week of incubation. It is unclear to what
extent this is due to adhesion to soil particles or to decomposition.
The addition of a dispersing solution (Tris-Tween 80) to soil samples
enhanced the levels of oocyst recovery, suggesting that the initial
decreases in percentages of recovery were due primarily to adhesion to
soil particles. In addition, storage of soil samples at 4°C should
have minimized the decomposition rates. Adhesion of oocysts to soil
particles does not preclude decomposition. It does, however, complicate
attempts to estimate decomposition rates and to quantify oocyst loading rates.
It is unclear whether the NaCl flotation method is compatible with
oocyst viability testing. Gradient centrifugation methods, such as the
CsCl and discontinuous Percoll methods, are most commonly used to
purify oocysts from feces for viability testing (9). ZnSO4 and cold sucrose flotation methods have also been
shown to be compatible with viability testing, although they
selectively concentrate viable oocysts (5). It is
questionable whether there is any method which is suitable for
quantitative recovery of oocysts from a wide range of environmental
matrices and is compatible with viability testing.
In conclusion, the NaCl flotation method appears to be suitable for
routine detection and enumeration of C. parvum oocysts in a variety of environmental matrices, including feces, manures, soils, and soil-manure mixtures. Further research is needed to elucidate the mechanisms of oocyst-manure and oocyst-soil interactions in order to improve the levels of recovery and to estimate
decomposition and mortality rates in manures and soils.
| |
ACKNOWLEDGMENTS |
We thank Valerie McPhatter and Nicolle Farmer for technical
assistance and Ron Fayer, Jim Trout, and Colleen Carpenter (Immunology and Disease Resistance Laboratory, Beltsville Agricultural Research Center, Beltsville, Md.) for oocyst preparations.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Environmental
Chemistry Laboratory, Agricultural Research Service, U.S. Department of
Agriculture, Bldg. 001, BARC-West, 10300 Baltimore Ave., Beltsville, MD
20705-2350. Phone: (301) 504-6582. Fax: (301) 504-5048. E-mail: dshelton{at}asrr.arsusda.gov.
| |
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Abstract
Introduction
