Effect of intensity of fecal pat sampling on estimates of Escherichia coli O157 prevalence.

Abstract

To evaluate site-to-site variation within fecal pats from cattle with regard to detection of Escherichia coli O157 and determine the effect on the accuracy of prevalence estimates of assay of multiple samples collected from the same fecal pat.
120 freshly voided fecal pats collected from 2 beef feedlots. Procedures-5 samples were systematically collected from each fecal pat and analyzed for E coli O157 via selective preenrichment techniques, immunomagnetic separation, and biochemical tests. Presumptive isolates were definitively identified via agglutination assays and polymerase chain reaction techniques. Best estimators of prevalence were calculated from the distribution of E coli O157-positive samples per pat.
Of the 120 fecal pats, 96, 13, 4, 2, 3, and 2 fecal pats had 0, 1, 2, 3, 4, and 5 E coli O157-positive samples, respectively. The greatest estimate of E coli O157 prevalence (20%) was achieved when all 5 samples were assessed; this estimate represented a 2.4- fold increase in prevalence, compared with that provided via analysis of 1 sample/pat (8.2%). Compared with assessment of 5 sites/pat, the relative sensitivity of detecting an E coli O157-positive fecal pat via analysis of 1 site/pat was 40.1%.
Results suggest that estimates of E coli O157 prevalence derived from sampling of 1 location/pat are likely underestimates of the true prevalence of this pathogen in fecal pats (and by extension, cattle). Additional research is warranted to confirm these results in situations of high and low prevalence and across different feedlots.

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AJVR, Vol 66, No. 12, December 20052023
E
bacterium may lead to bloody diarrhea, hemorrhagic
colitis, hemolytic uremic syndrome, and death.1,2
Although a wide variety of foods have been implicated
in E coli O157–induced disease,3-6cattle have been
scherichia coli O157 has emerged as an important
food-borne pathogen. Exposure of humans to this
identified as the major animal reservoir of the
pathogen.7,8As such, a better understanding of the
ecology and epidemiology of the microorganism in cat-
tle and their environment is needed to appropriately
assess and quantify the microbial risk posed by and
potential interventions for this pathogen.9-11
Ostensibly, it appears that the prevalence of E coli
O157 in cattle has increased over time. In part, this is
likely a result of refinement of sampling procedures,
changes in sample quantity and type used in analyses,
and the use of more sensitive microbiological meth-
ods.12-14Ultimately, studies7,8,15,16in which sensitive meth-
ods have been used have revealed the ubiquitous nature
of this organism among cattle populations. Rice et al2
determined that, compared with analysis of fecal mate-
rial, the sensitivity of detection of this pathogen in arti-
ficially inoculated cattle was even greater via collection
and analysis of rectoanal mucosal swabs. This greater
sensitivity may have been detected because lymphoid
tissue just proximal to the rectoanal junction has been
proposed as the site of colonization of E coli O157 in
cattle.17As a consequence of this limited and distal site
of colonization within the intestinal tract, E coli O157
may not be randomly distributed in fecal material.17
Moreover, Pearce et al18found evidence of variable dis-
tribution of E coli O157 in voided feces of animals kept
in pastures. Therefore, it seems possible that misclassi-
fication of the shedding status of cattle may occur at
times because samples for microbiological purposes are
collected from a relatively small proportion of fecal pats
(in those studies involving fecal pat sampling). As a
consequence, there would be an underestimate of E coli
O157 prevalence and a bias toward null in controlled
studies that are performed to evaluate strategies to
reduce carriage of E coli O157. The objective of the
study reported here was to evaluate site-to-site variation
within fecal pats from cattle with regard to detection of
E coli O157 and determine the effect on the accuracy of
prevalence estimates of collection and assay of multiple
samples from the same fecal pat.
Materials and Methods
Sample collection—Freshly voided pen-floor fecal sam-
ples were obtained from cattle maintained at 2 feedlots in the
Texas Panhandle. Each feedlot was visited on 2 occasions
(during the months of July and August), and during each
visit, 30 freshly voided fecal pats were chosen for sample col-
lection. Five samples (approx 100 g each) were systematical-
ly obtained from each pat while maintaining across-pat con-
sistency in sample collection. Briefly, the 5 samples from
individual fecal pats were collected in north to south lines
starting on the west side of the pat and progressing east (sites
1 through 5, respectively; Figure 1). Individual samples were
placed in sterile prelabeled specimen cups. Separate single-
Received March 14, 2005.
Accepted May 13, 2005.
From the Department of Animal and Food Sciences, Texas Tech
University, Lubbock, TX 79409 (Echeverry, Brashears); the Feedlot
Research Group, Division of Agriculture, College of Agriculture,
Nursing and Natural Sciences, West Texas A&M University,
Canyon, TX 79016 (Loneragan); and the USDA:Animal Plant
Health Inspection Service:Veterinary Services:Centers for
Epidemiology and Animal Health, Mail Stop 2E7, 2150 Centre
Ave, Fort Collins, CO 80526 (Wagner).
Presented orally at the 84th Annual Meeting of the Conference of
Research Workers in Animal Diseases, Chicago, November 2003
and at the 91st Annual Meeting of the International Association for
Food Protection, Phoenix, August 2004.
Address correspondence to Dr. Loneragan.
Effect of intensity of fecal pat sampling
on estimates of Escherichia coli O157
prevalence
Alejandro Echeverry, MS; Guy H. Loneragan, BVSc, PhD; Bruce A. Wagner, PhD; Mindy M. Brashears, PhD
Objective—To evaluate site-to-site variation within
fecal pats from cattle with regard to detection of
Escherichia coli O157 and determine the effect on the
accuracy of prevalence estimates of assay of multiple
samples collected from the same fecal pat.
Sample Population—120 freshly voided fecal pats
collected from 2 beef feedlots.
Procedures—5 samples were systematically collected
from each fecal pat and analyzed for E coli O157 via
selective preenrichment techniques, immunomagnetic
separation, and biochemical tests. Presumptive iso-
lates were definitively identified via agglutination
assays and polymerase chain reaction techniques. Best
estimators of prevalence were calculated from the dis-
tribution of E coli O157–positive samples per pat.
Results—Of the 120 fecal pats, 96, 13, 4, 2, 3, and 2
fecal pats had 0, 1, 2, 3, 4, and 5 E coli O157–positive
samples, respectively. The greatest estimate of E coli
O157 prevalence (20%) was achieved when all 5 sam-
ples were assessed; this estimate represented a 2.4-
fold increase in prevalence, compared with that pro-
vided via analysis of 1 sample/pat (8.2%). Compared
with assessment of 5 sites/pat, the relative sensitivi-
ty of detecting an E coli O157–positive fecal pat via
analysis of 1 site/pat was 40.1%.
Conclusions and Clinical Relevance—Results sug-
gest that estimates of E coli O157 prevalence derived
from sampling of 1 location/pat are likely underesti-
mates of the true prevalence of this pathogen in fecal
pats (and by extension, cattle). Additional research is
warranted to confirm these results in situations of
high and low prevalence and across different feedlots.
(Am J Vet Res 2005;66:2023–2027)

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use plastic spoons were used for each sampling site. Samples
were stored on wet ice and transported to the laboratory on
the day of sample collection. Microbiological processes began
within 2 hours of sample collection.
Microbiological methods—Immediately after arrival at
the laboratory, 10 g of feces from each sample was transferred
for selective enrichment of gram-negative organisms into
90 mL of a broth containing vancomycin (8 mg/mL), cefixime
(50 ng/mL), and cefsulodin (10 mg/mL) and incubated at
37oC for 6 hours as described.19
20 µL of paramagnetic beads coated with anti–E coli O157
antibodyawas added to 1-mL aliquots of each sample and
incubated for 30 minutes at 22oC. Beads were held against the
side of the vial by a magnet and washed 3 times; 50 mL of the
bead-bacteria mixture was spread onto sorbitol MacConkey
agar plates supplemented with cefixime (0.05 µL/L) and
potassium tellurite (50 µL/L) and incubated at 37oC for 18 to
24 hours. As many as 3 typical non–sorbitol-fermenting
colonies/plate were streaked for isolation on sorbitol
MacConkey agar plates and incubated for approximately 18 to
24 hours at 37oC. Non–sorbitol-fermenting colonies (clear or
whitish) were selected and inoculated on MacConkey agar
and an E coli O157:H7–selective agar containing 4-methyl-
umbelliferyl-β β-glucuronide (MUG). After incubation, MUG-
negative colonies were transferred from the plates into tryptic
soy broth, triple sugar iron agar, and MacConkey broth and
incubated overnight in aerobic conditions at 37oC.
Presumptive E coli O157 colonies (those that were MUG neg-
ative, sorbitol negative, lactose positive, indole positive, and
triple sugar iron positive) were recovered from the backed
tryptic soy broth and confirmed by use of a commercial latex
agglutination test.bSerotype confirmation (including genes
encoding production of shiga toxins) was made by use of a
commercially available polymerase chain reaction system.c
After enrichment,
Statistical analysis—Data were analyzed by use of a
commercially available software package.dDescriptive statis-
tics were generated and reported in graphic or tabular format.
Prevalence among sampling sites 1 through 5 was evaluated
by use of logistic regression techniques. The outcome vari-
able was a binomial response variable for each sample site. In
addition, a contrast was constructed to evaluate the periph-
eral samples (sites 1 and 5) versus the other samples (sites 2,
3, and 4).
It was also of interest to determine whether estimates of
prevalence differed when 1 sample/pat had been randomly col-
lected, 2 samples/pat had been randomly collected, and so
forth through to collection of 5 samples/pat. The probabilities
of fecal pats yielding positive test results were generated from
a hypergeometric distribution on the basis of observed data
and the various sampling scenarios (1, 2, 3, 4, or 5 random
samples/pat). In other words, the probability of 1 or more sam-
ple sites being positive for E coli O157 within a pat (fecal pat
prevalence) was calculated as 1 minus the probability of 0 sites
being positive for E coli O157, given the distribution of posi-
tive sample sites and the number of samples per pat collected.
The probabilities were used to calculate the expected number
of E coli O157–positive fecal samples and, ultimately, expected
prevalence estimates. Standard error values were calculated for
point estimates by use of binomial distribution. On the basis of
prevalence estimates, expected number of E coli O157–positive
fecal pats per visit was calculated for each sampling scenario.
The expected number of E coli O157–positive samples was
modeled as a binomial response variable (the denominator
being the number of samples collected/visit), and independent
variables evaluated were linear and quadratic terms represent-
ing number of samples per pat; feedlot and visit nested with
feedlot were modeled as a random variable by use of a random-
intercepts, random-slopes approach. The fixed component of
the model (effectively averaged over the random effects of
feedlot visit) may be written as follows:
y = exp(µ + β1xi+ β2xi
2)/(1 + exp[µ + β1xi+ β2xi
2])
where y is the expected prevalence, exp is the exponential,
: is the intercept, x is the number of fecal pats sampled (i = 1
through 5), β1is the slope for the linear effect of x, and β2is
the slope for the quadratic effect of x (as indicated by xi
The data were modeled by use of a freely available macro
program in which an events-trials binomial variable served as
the dependent variable.eUnexplained variation was parti-
tioned to the various levels of hierarchy (feedlot and visit
within feedlot). Sensitivity of detecting E coli O157 in a fecal
pat was calculated for sampling scenarios of 1, 2, 3, or 4
sites/pat, relative to sampling all 5 sites/pat. For all models, a
value of P ≤ 0.05 was considered significant.
2).
Results
Six hundred samples were collected from 120 fecal
pats (120 samples from each of the 5 sites within the
pats). Overall, E coli O157 was isolated from 49 (8.2%)
samples. Escherichia coli O157 was isolated from at
least 1 sample from 24 of the 120 (20%) pats. The bac-
terium was detected in at least 1 sample from 5 (8.3%)
and 19 (31.7%) fecal pats collected from feedlots A and
B, respectively (Table 1).
There was no evidence that prevalence varied with
sample site within fecal pats (P = 0.60); E coli O157
was isolated from 14 (11.7 ± 2.9%), 9 (7.5 ± 2.4%), 8
(6.7 ± 2.3%), 8 (6.7 ± 2.3%), and 10 (8.3 ± 2.5%) sam-
ples collected at sites 1 through 5, respectively.
Similarly, the combined estimate of prevalence
obtained from sample sites 1 and 5 did not vary signif-
icantly (P = 0.20) from the estimate obtained from
sample sites 2, 3, and 4.
The distribution of the number of E coli
O157–positive samples per fecal pat for each feedlot by
2024 AJVR, Vol 66, No. 12, December 2005
Figure 1—Photograph to illustrate the sites on fecal pats from
which samples were collected. The arrow indicates north; num-
bers identify sample sites (sites 1 and 5 were the west and east
margins of the pat). For each of 120 pats, samples were collect-
ed in order (1 though 5) by use of the same sampling protocol.

Page 3

visit was determined (Table 1). Escherichia coli O157
was not detected in any of the samples collected from
96 of 120 (80%) fecal pats. Overall, E coli O157 was
detected in 13 (10.8%), 4 (3.3%), 2 (1.7%), 3 (2.5%),
and 2 (1.7%) of the 120 fecal pats in 1, 2, 3, 4, or 5
samples, respectively.
Visit-level and overall estimators of prevalence
given various sampling scenarios were assessed
(Table 2). If 1 sample/pat was randomly selected, the
overall best estimate of prevalence was 8.2% (95%
confidence interval [CI], 3.3 to 13.1). However, if 2
samples were collected at random per pat, the overall
estimate of prevalence increased to 12.3% (95% CI,
6.4 to 18.2). This represented an absolute increase in
the estimate of prevalence of 4.1% and a relative
increase of 50%. The relative sensitivity of detecting
E coli O157 when 1 sample was randomly selected,
compared with detection of the pathogen by use of 2
samples, was 66.7% (95% CI, 58.6 to 75.4). When 5
samples/pat were included in the sampling scenario,
the estimate of prevalence was 20% (95% CI, 12.8 to
27.2), which is a 2.4-fold increase in prevalence, com-
pared with that determined when just 1 sample/pat
was collected. The relative sensitivity of correctly clas-
sifying a fecal pat as E coli O157–positive increased
with the increasing number of sample sites per pat.
Relative to sampling at 5 sites/pat, the fecal pat–level
sensitivity of detection was 40.1% (95% CI, 31.3 to
48.9), 61.5% (95% CI, 52.8 to 70.2), 76.5% (95% CI,
68.9 to 84.1), and 86.5% (95% CI, 80.4 to 92.6) for
sampling scenarios that included 1, 2, 3, or 4 sites/pat,
respectively. The relative sensitivities of including 1
sample/pat, compared with 5 samples/pat, for each of
the visits were as follows: 27% (feedlot A, visit 1),
19.4% (feedlot A, visit 2), 44.6% (feedlot B, visit 1),
and 46.5% (feedlot B, visit 2).
The estimate of prevalence was quadratically asso-
ciated (P = 0.05) with the number of samples per pat
included in the analysis. The fixed component of the
model, which is effectively the mean of the random-
intercepts, random-slopes model, was as follows:
y = exp(–3.24 + 0.57xi– 0.05xi
(1 + exp[–3.24 + 0.57xi– 0.05xi
2)/
2])
Feedlot accounted for 69.4% of random variation
in the model, whereas visit within feedlot accounted
for 24.4% of random variation. In a separate model in
which the mean prevalence for each visit was forced
into the model, feedlot and visit within feedlot
accounted for 0.0% and 45.0% of random variation,
respectively.
Discussion
On the basis of results of the present study, it
seems probable that animal- or pat-level estimates of
E coli O157 prevalence derived via simple fecal pat
sampling substantially underestimate the true preva-
lence. Overall, E coli O157 was isolated from 24 of the
120 (20%) fecal pats. If 1 sample/pat had been ran-
domly collected, the expected prevalence would have
been considerably less (ie, 8.2%). With each addition-
al sample collected per pat, the expected prevalence
estimate increased. However, the absolute and relative
increases in estimates diminished with increasing sam-
ple number. This is reflected in the negative quadratic
parameter estimate (ie, –0.05). Ideally, an optimum
number of samples per pat that maximized prevalence
would have been discovered; however, on the basis of
the data described herein, the optimum number of
samples was 5.7, which was outside the inferential lim-
its of the data. We chose to collect samples from 5 sites
AJVR, Vol 66, No. 12, December 20052025
Table 1—Distribution of number of Escherichia coli O157–positive samples per fecal pat (5 sites/pat)
collected during each of 2 visits to 2 beef feedlots.
Feedlot AFeedlot B
No. of E coli O157–positive
samples/patVisit 1Visit 2Visit 3Visit 4Overall
0
1
2
3
4
5
27
2
1
0
0
0
3 30 0
28
2
0
0
0
0
3 30 0
17
5
3
2
3
0
3 30 0
24
4
0
0
0
2
3 30 0
96
13
4
2
3
2
T To ot ta al l p pa at ts s s sa am mp pl le ed d1 12 20 0
Table 2—Estimates of E coli O157 prevalence (%) and 95% confidence intervals calculated from data
collected from 1, 2, 3, 4, or 5 randomly sampled sites per fecal pat for each of 2 visits to 2 beef
feedlots.
Feedlot AFeedlot B
No. of sites
assessed/patVisit 1Visit 2Visit 3Visit 4 Overall
1
2
3
4
5
2.7 (0.0–8.4)
5.0 (0.0–12.8)
7.0 (0.0–16.1)
8.7 (0.0–18.7)
10.0 (0.0–20.7)
1.3 (0.0–5.4)
2.7 (0.0–8.4)
4.0 (0.0–11.0)
5.3 (0.0–13.4)
6.7 (0.0–15.6)
19.3 (5.2–33.5)
29.7 (13.3–46.0)
35.7 (18.5–52.8)
40.0 (22.5–57.5)
43.3 (25.6–61.1)
9.3 (0.0–19.7)
12.0 (0.4–23.6)
14.7 (2.0–27.3)
17.3 (3.8–30.9)
20.0 (5.7–34.3)
8.2 (3.3–13.1)
12.3 (6.4–18.2)
15.3 (8.9–21.8)
17.3 (11.0–24.7)
20.0 (12.8–27.2)
Values in parentheses represent the 95% confidence interval.

Page 4

within each pat because the volume of fecal material in
pats of feedlot cattle made collection of more than five
100-g samples/pat impractical in many instances. It
would, however, have been possible to increase the
number of samples at the expense of sample volume. If
we had done so, it may have been possible to identify
the optimum number of samples per pat that maxi-
mizes prevalence and falls within the inferential limits
of the data.
Feedlot and visit within feedlot accounted for
69.4% and 24.4% of the random variation observed in
the model, respectively, which indicated that feedlot-
level factors may explain some of the apparent varia-
tion in 1-sample sensitivity (relative to 5 samples)
detected between the 2 feedlots. Feedlot-level factors
that may have contributed to the results are uncertain,
and unfortunately, information beyond feedlot location
was not collected. Despite this, when mean visit preva-
lence was forced into the model, the proportion of vari-
ation attributable to feedlot was reduced to 0%.
Therefore, it is possible that differences in prevalence,
and not feedlot per se, may account for most of the
variation in 1-sample sensitivity. If so, feedlot-level fac-
tors that affect prevalence would be of interest. In the
present study, all visits were conducted during July and
August, months in which prevalence of E coli O157 is
typically greatest. Factors contributing to the observed
variation are uncertain but are clearly important in the
epidemiology of E coli O157 in feedlots; without
doubt, more research needs to be performed to better
understand these factors. Despite the unexplained
model variation in our study, estimates of prevalence
increased with the number of samples per pat for all 4
visits. This suggests that sampling strategies that
include multiple samples per pat should result in
improvements in prevalence accuracy in most situa-
tions. However, it should be noted that the present
study was limited in the number of fecal pats from
which samples were collected and only 2 feedlots were
included. More research is needed to validate these
results across a range of E coli O157 prevalence and
across feedlots.
In cattle, the likely site for long-term colonization
with E coli O157 is in lymphoid tissue just proximal to
the rectoanal junction.17One consequence of this distal
colonization site within the intestinal tract is that dis-
tribution of E coli O157 in fecal material may not be
uniform; the distance between the colonization site
and the anus would presumably be insufficient to allow
thorough homogenous distribution of the pathogen
within the fecal material before defecation. Moreover,
Naylor et al17 determined that there was a greater con-
centration of E coli O157 on the fecal pat surface than
within the fecal pat. In addition, variation in the con-
centration of E coli O157 in feces of animals kept on
pasture has been identified.18,20If indeed there is
nonuniform distribution of E coli O157 within fecal
material of feedlot cattle, it may have accounted for the
improved pat-level (and ultimately animal-level) esti-
mates of prevalence when the number of samples per
pat assessed was increased. Although we propose that
the data of the present study support the findings of
other investigators that there is a nonuniform distribu-
tion of E coli O157 within fecal material, the distribu-
tion of E coli O157 was not directly evaluated in our
study and can only be inferred. Unfortunately, the
semiliquid consistency of feces from cattle fed high-
concentrate diets (which are typically used in modern
commercial feedlots) made comparison of surface and
internal pat material impractical. We did not detect sig-
nificant variation in E coli O157 prevalence estimates
between sampling sites within pats nor did we find evi-
dence that the margins of pats had greater prevalence
than other samples. More research is needed to direct-
ly evaluate variation in concentration of E coli O157
within feces of feedlot cattle.
Sampling 5 sites within a pat offered substantial
improvements in prevalence accuracy, compared with
estimates based on data from only 1 site. However,
increased sampling intensity also substantially
increased costs associated with supplies and isolation
of the organism. In future studies, the number of sam-
ples per pat to be collected should depend on the
desire for increased accuracy, number of pats from
which samples are to be collected, and financial limita-
tions of the study.
a.
b.
c.
d.
e.
Dynabeads anti–E coliO157, Dynal Biotech Inc, Brown Deer, Wis.
Escherichia coli O157:H7 latex test, REMEL Inc, Lenexa, Kan.
BAX detection system, DuPont Qualicon, Wilmington, Del.
SAS for Windows, version 8.2, SAS Institute Inc, Cary, NC.
GLIMMIX macro for the SAS System, release 8. Available at:
www.ftp.sas.com/techsup/download/stat/glmm800.html.
Accessed Mar 16, 2003.
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