APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 2011, p.

30023008
0099-2240/11/$12.00 doi:10.1128/AEM.02238-10
Copyright 2011, American Society for Microbiology. All Rights Reserved.

Vol. 77, No. 9

Survival of Escherichia coli O157:H7 on Cattle Hides

U.S. Department of Agriculture Agricultural Research Service, Roman L. Hruska U.S. Meat Animal Research Center,
Clay Center, Nebraska 68933-0166
Received 20 September 2010/Accepted 6 March 2011

The objective of this study was to determine the time period that Escherichia coli O157:H7 survives on the
hides of cattle. Extensive research has been conducted and is ongoing to identify and develop novel preharvest
intervention strategies to reduce the presence of E. coli O157:H7 on live cattle and subsequent transfer to
processed carcasses. If a reduction of E. coli O157:H7 levels in feces can be achieved through preharvest
intervention, it is not known how long it would take for such reductions to be seen on the hide. In the study
presented herein, three trials were conducted to follow E. coli O157:H7 hide prevalence over time. For each
trial, 36 animals were housed in individual stanchions to minimize or prevent hide contamination events.
Through prevalence determination and isolate genotyping with pulsed-field gel electrophoresis, survival of E.
coli O157:H7 on the hides of live cattle was determined to be short lived, with an approximate duration of 9 days
or less. The results of this study suggest that any preharvest interventions that are to be administered at the
end of the finishing period will achieve maximum effect in reducing E. coli O157:H7 levels on cattle hides if
given 9 days before the cattle are presented for processing. However, it should be noted that interventions
reducing pathogen shedding would also contribute to decreasing hide contamination through lowering the
contamination load of the processing plant lairage environment, regardless of the time of application.
O157:H7 in as little as 3 days (3, 37), the bacterial pathogen
may persist on the hide for longer periods of time, potentially
negating the value of the intervention if the animals are processed immediately.
E. coli O157:H7 survival in a variety of environments has
been determined. Studies have shown that E. coli O157:H7 can
persist in manure-amended soil for greater than 100 days (18,
22). Similarly, long durations of survival of up to 109 days have
been reported for E. coli O157:H7 in water environments (8,
27, 31).
The survival of E. coli O157:H7 on the hides of living cattle
is not well understood. Some studies have documented fluctuations over time in the E. coli O157:H7 population found on
cattle hides. It has been shown that the hide prevalence of E.
coli O157:H7 can decrease from 84% to 0% in 2 weeks time
for cattle housed in a feedlot pen (4). Other studies have
reported declines in hide prevalence across multiple feedlot
pens of approximately 60% in time spans of 7 and 28 days (6,
35). These studies were not designed to analyze the duration of
E. coli O157:H7 survival on hides, but the results provide
preliminary evidence that survival on cattle hides would likely
be of a shorter time frame than that for persistence in soil or
water. Information about the length of the survival is expected
to help improve the design of new intervention strategies targeted for application at the end of the feeding period to further
reduce or eliminate E. coli O157:H7 from the meat production
system. The objective of this study was to determine the length
of time of E. coli O157:H7 survival on the hides of cattle.

Escherichia coli O157:H7 remains the major food-borne

pathogen of concern for the beef industry. Extensive research
has been conducted and is ongoing to identify and develop
novel pre- and postharvest intervention strategies to reduce E.
coli O157:H7 from live cattle and processed carcasses (24, 30).
Recent work has shown that hides are the main source of beef
carcass contamination at slaughter, and as such, reductions in
the prevalence of E. coli O157:H7 on the hide are directly
correlated to lower carcass prevalence rates (7, 12, 28). While
postharvest interventions have been designed that address hide
contamination directly, the aim of preharvest intervention is to
reduce hide contamination indirectly through lowering the
prevalence and levels of E. coli O157:H7 shed in the feces of
cattle.
In order to design effective preharvest intervention strategies, it is necessary to determine the duration of survival of E.
coli O157:H7 following a hide contamination event. If a reduction of E. coli O157:H7 levels in feces can be achieved, it is not
known how long it would take for such reductions to be seen
on the hide. This information is crucial as various preharvest
interventions currently undergoing testing are to be applied to
the animal at the end of the finishing phase just prior to
shipment for processing (15, 17, 26). While short-duration
strategies may produce reductions in the fecal load of E. coli

* Corresponding author. Mailing address: U.S. Department of Agriculture Agricultural Research Service, Roman L. Hruska U.S. Meat
Animal Research Center, Clay Center, NE 68933-0166. Phone: (402)
762-4227. Fax: (402) 762-4149. E-mail: terrance.arthur@ars.usda.gov.
Present address: USDA, ARS, Animal & Natural Resource Institute, Building 201, BARC-East, 10300 Baltimore Ave., Beltsville, MD
20705-2350.
Present address: IEH Laboratories and Consulting Group, 15300
Bothell Way NE, Lake Forest Park, WA 98155.

Published ahead of print on 11 March 2011.

MATERIALS AND METHODS

Experimental design. The study was performed in three phases to establish the
duration of E. coli O157:H7 survival on the cattle hides. Phase I was conducted
to identify the approximate duration of survival of the pathogen on cattle hides.

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Terrance M. Arthur,* Xiangwu Nou, Norasak Kalchayanand, Joseph M. Bosilevac,

Tommy Wheeler, and Mohammad Koohmaraie

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hide contamination could readily occur. The treated animals would be transferred to single-animal stanchions where contamination of the hide would be
minimized, if not prevented. Each stanchion had a dedicated feed and water
supply. Following sorting, but prior to placing the treated animals in the stanchions, the animals (n 39) were transferred to small holding pens (6 cattle/
pen). There were 36 individual stanchions for housing the animals during the
experiment. Three extra animals were included at this point, in the event that
some animals would not acclimate to the new environment and could not be used
for the study. After the 2-week acclimation period, the animals of the treated
group (n 36) were placed in the individual stanchions. Half (n 18) of the
animals in individual stanchions were treated with neomycin for 2 days upon
being placed in the stanchions. The day the animals were moved to the individual
stanchions was considered day 0. On day 0, hide and fecal samples were collected
from both the control and treated animals as they passed through a squeeze
chute.
(ii) Environmental samples. Prior to the arrival of the animals in either the
small holding pens or the individual stanchions, the pens and stanchions were
screened for E. coli O157:H7. Sponge samples were collected as described for
phase I. Sample processing was carried out as described below for hide samples
in phase II.
(iii) Cattle sampling. Hide and fecal samples were collected on Mondays,
Wednesdays, and Fridays for treated animals and on Mondays and Fridays for
the control animals maintained at the feedlot. Hide samples were collected for all
animals using a sterile sponge (Nasco) premoistened with BPW (Difco) and by
swabbing an area of approximately 1,000 cm2 behind the shoulder. For the
control animals, fecal samples (10 g) were collected by rectal palpation. Due to
personnel safety issues, it was not possible to obtain fecal samples by rectal
palpation from animals housed in the individual stanchions. Once the animals
were placed in the individual stanchions, fecal samples were collected by swabbing of the recto-anal junction (Spongecicle; Biotrace International Inc., Bothell,
WA).
(iv) Enumeration. E. coli O157:H7 was enumerated from hide and fecal
samples using a spiral plater (Spiral Biotech, Norwood, MA), following the
method described by Brichta-Harhay et al. (14). For hide samples, the sponge
sample was homogenized by using hand massaging prior to the addition of
enrichment medium, and 250 l of solution was removed to a microcentrifuge
tube. Each tube was vortexed and then held static for 3 min to allow the debris
to settle. Following the settling period, 50 l of sample was spiral plated onto
ntCHROMagar (CHROMagar-O157 [DRG International] supplemented with
novobiocin [5 mg/liter; Sigma] and potassium tellurite [2.5 mg/liter; Sigma])
plates. When being enumerated from fecal grab samples, the enrichment medium (90 ml TSB [Difco, Becton Dickinson] with phosphate buffer [TSB-PO4;
30 g TSB, 2.31 g KH2PO4, and 12.54 g K2HPO4 per liter of solution]) was added
to the 10-g fecal sample and the mixture was homogenized by hand massaging.
For fecal swab samples, 90 ml TSB-PO4 was added and the sample was homogenized by hand. One milliliter of each fecal sample mixture was removed to a
microcentrifuge tube and vortexed. The enumeration was then carried out as
described for hide samples.
(v) Sample processing for prevalence. Samples were processed according to
previously described methods, with slight modifications (10, 11). Hide sponge
samples were enriched with 80 ml TSB after the 250-l aliquot was removed for
enumeration. Fecal samples were enriched in the 90 ml TSB-PO4 used for
enumeration dilution. The sample bags were incubated at 25C for 2 h and then
at 42C for 6 h prior to being held at 4C overnight. Following incubation, the
samples were processed by immunomagnetic separation, in which 1-ml samples
from each enrichment were subjected to an anti-O157 immunomagnetic bead
cell concentration (Dynal). Fifty-microliter volumes of the final bead-bacterium
complexes were spread-plated onto ntCHROMagar and CT-SMAC (sorbitol
MacConkey agar [Difco] supplemented with cefixime [0.05 mg/liter] and potassium tellurite [2.5 mg/liter; Dynal]). All plates were incubated at 37C for 18 to
20 h, after which up to three suspect colonies were picked and tested by latex
agglutination (DrySpot E. coli O157; Oxoid). PCR analysis was used to confirm
that each isolate harbored genes for the O157 antigen, H7 flagella, and at least
one of the Shiga toxins (20). Isolates were maintained as frozen stocks for later
use in strain typing by pulsed-field gel electrophoresis (PFGE).
Phase III. (i) Cattle. Hide and fecal samples were collected from feedlot cattle
(n 147) for E. coli O157:H7 screening. Cattle that were positive for E. coli
O157:H7 on their hides were selected and assigned to a treated or control group
as described for phase II. In phase III, the control group consisted of 36 cattle
placed in a 50-ft by 250-ft feedlot pen.
(ii) Sampling. Hide samples were collected as described for phase II. Hide
samples were collected on days 0, 2, 4, 7, 9, and 11 for the treated group and days
0, 4, 7, 9, and 11 for the control group. Fecal samples for both the treated and

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Phases II and III utilized more frequent sampling times to define the time span
of survival more precisely.
Individual stanchions. To prevent hide contamination events during the study,
stanchions were used to house individual animals. The stanchions consisted of
tubular iron railings mounted in a concrete floor. The interior dimensions of the
stalls were 7 ft in length and 3 ft in width. Each stanchion had a mechanical head
restraint and individual food and water stations. In addition, the floor in each
stanchion was covered by rubber matting to prevent foot irritation. Animal-toanimal contact was prevented. The space was adequate for the cattle to lie down.
Individual grooming could take place, but not at the hide sample sites.
Feedlot cattle (300 to 450 kg) were used in this study. Before test cattle could
be transferred to the stanchions, they were held in small holding pens (5 to 8
cattle per pen) for 1 to 2 weeks to allow them to acclimate to human interaction
in order to prevent animal or personnel injury by taking cattle straight from the
feedlot to the individual stanchions. Based on previous observations of keeping
animals in small groups, it was presumed that hide contamination would decline
due to the cattle threshold number and animal density of these pens being below
that needed to maintain colonization. In order to maximize the number of
animals that harbored E. coli O157:H7 on their hides following this holding
period, cattle were prescreened at the feedlot to identify animals that harbored
E. coli O157:H7 on their hides.
Phase I. (i) Animals. One hundred twenty-five cattle from 5 feedlot pens were
screened for E. coli O157:H7 on their hides. E. coli O157:H7 was detected on 123
animals. One week after screening, 8 cattle from each pen were transferred to
smaller holding pens adjacent to the facility hosting the single-animal stanchions.
Another 24 cattle from three unscreened pens were also transferred to this
facility. A facility veterinarian and cattle-feeding personnel evaluated the behaviors of individual animals in small holding pens upon closer interactions with
humans. Individual animals showing aggressive behaviors or abnormal feeding
patterns were released from the small holding pens. After a week of acclimation
in the holding pens, 36 animals were placed in thoroughly cleaned individual
stanchions. Each animal was restrained by a head restraint at the time of sampling. Floors and railings of individual stanchions were sampled before hosting
animals, and no E. coli O157:H7 isolates were detected in those samples. Each
animal received standard feed and water rations for the duration of the experiment. Animal excretion was cleaned twice a day to minimize recontamination of
hides. To aid in minimizing hide recontamination, half of the animals received
two doses of neomycin (trade name Biosol) administered by the facility veterinarian on day 0 and day 1 via drinking water.
(ii) Sampling. Hide and fecal samples were collected from individual animals
on days 0, 4, 7, 11, 14, 18, 25, and 32 for detection of E. coli O157:H7. Hide
samples were collected from the accessible side of the cattle in the holding
stanchions while the animals were being restrained using a mechanical device
equipped on individual stanchions. A sterile WhirlPak sponge (Nasco, Fort
Atkinson, WI) premoistened in buffered peptone water (BPW; Difco, Becton
Dickinson Microbiology Systems, Sparks, MD) was used to sweep the shoulder
and back area in a Z pattern in one streak, followed by turning the sponge and
retracing the Z pattern in the opposite direction. The Z pattern covered approximately 1,000 cm2, and a different angle for the Z pattern was applied for each
sampling to avoid resampling the same area. Fecal samples were collected from
unperturbed fresh fecal pats in the early morning before cleaning. Floors and
railings for each stanchion were sampled using a sterile WhirlPak sponge (Nasco)
premoistened in BPW (Difco). Areas of approximately 3,000 cm2 were sampled.
(iii) Detection and enumeration. For each sponge sample (floors, hides, railings), the sponge was thoroughly massaged in 100 ml tryptic soy broth (TSB;
Difco, Becton Dickinson). For each fecal sample, 10 g fecal material was homogenized by use of hand massaging in 100 ml TSB. One milliliter of the sponge
or fecal sample was removed for direct detection of E. coli O157:H7 by immunomagnetic separation (IMS), while the rest of the sample was enriched as
described previously (10, 11). E. coli O157:H7 isolates in the preenrichment
samples were enumerated by being plated on CT-SMAC (sorbitol MacConkey
agar [Becton Dickinson] supplemented with cefixime [0.05 mg/liter] and potassium tellurite [2.5 mg/liter; Invitrogen] and ntCHROMagar [CHROMagarO157; DRG International, Mountainside, NJ] supplemented with novobiocin [20
mg/liter; Sigma, St. Louis, MO] and potassium tellurite [0.8 mg/liter; Sigma])
plates following IMS using a PickPen device. For those that produced negative
results by direct detection of preenrichment samples, 1 ml enriched samples was
similarly processed the second day for detection of E. coli O157:H7.
Phase II. (i) Animals. One hundred sixty-two crossbred cattle were screened
for E. coli O157:H7 on the hides and in feces. Animals (n 78) were selected for
further study if E. coli O157:H7 was detected on their hides. The animals were
sorted into two groups, referred to in this report as control and treated. The
control group (n 39) remained in a single feedlot pen (100 by 250 ft) where

SURVIVAL OF E. COLI O157:H7 ON CATTLE HIDES

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ARTHUR ET AL.

control animals were collected by swabbing the recto-anal junction (swab catalog
no. 10812-022; VWR International, Buffalo Grove, IL) as described by Rice et al.
(29). Immediately following sample collection, the swabs were placed into 4 ml
TSB-PO4 and held on ice during transportation to the lab. Fecal swabs were
collected from the control and treated groups on days 0, 4, 7, 9, and 11.
(iii) Laboratory procedures. Enumeration and prevalence determination of E.
coli O157:H7 was conducted for all samples as described for phase II.
(iv) Repeated hide sampling. Multiple hide samples were collected from individual animals to determine if repeated sampling of the same sample site would
affect the E. coli O157:H7 population. In the first trial, 10 animals were sampled
seven times each. In trials two and three, 12 animals were sampled eight times
each. The prevalence rates for each replication were averaged over three trials,
and the standard error was determined using Prism 5.0 GraphPad software. Hide
samples were collected and processed for enumeration and prevalence as described for phase II.
(v) PFGE. E. coli O157 isolate fingerprints generated and analyzed in this
study were based on PFGE separation of XbaI-digested genomic DNA as described by PulseNet (http://www.cdc.gov/pulsenet/protocols.htm). Agarose certified for pulsed-field gels (SeaKem gold agarose) was obtained from Cambrex Bio
Science Rockland Inc. (Rockland, ME); Tris-borate-EDTA running buffer and
proteinase K were purchased from Sigma (St. Louis, MO). XbaI was purchased
from New England BioLabs (Beverly, MA); Salmonella enterica serotype Braenderup strain H9812 was used as a control and for standardization of gels (21).
Banding patterns were analyzed and comparisons were made using BioNumerics
software (Applied Maths, Sint-Martens-Latem, Belgium), employing the Dice
similarity coefficient in conjunction with the unweighted pair group method using
arithmetic averages for clustering.

RESULTS AND DISCUSSION

The objective of this work was to determine the length of
time that E. coli O157:H7 can persist on the hides of live cattle.
In the first phase of the experiment, 36 cattle were selected for
transfer from multiple feedlot pens to individual stanchions in
which hide contamination events would be minimized if not
prevented. All stanchions were sampled prior to entry of cattle.
E. coli O157:H7 was not detected on the floors or railings of
any stanchion prior to animal entry. Upon entry into the stanchions (day 0), each animal was sampled. Hide prevalence of
E. coli O157:H7 started at 47%, was 53% on day 4, and then
decreased to 0% by day 11 (Fig. 1). From day 11 to the last
sample day (day 32), hide prevalence rose to a peak of 11% on
day 18 and then declined again. Fecal prevalence closely followed hide prevalence (Fig. 1).
The rapid decline and absence of E. coli O157:H7 on cattle
hides by day 11 indicated that the pathogen does not persist on
cattle hides for long periods of time. The mechanism of the

FIG. 2. Effects of repeated sampling on hide prevalence of E. coli

O157:H7. Multiple hide samples were collected from individual animals. The average E. coli O157:H7 prevalence (%) is presented from
three trials with at least 10 animals per trial. Error bars represent the
standard errors of the means.

reoccurrence of E. coli O157:H7 after day 11 was not known.

Possible scenarios are as follows: (i) recontamination by the
individual animal, (ii) heterogeneous distribution of hide contamination that was missed on previous samplings, or (iii)
recontamination from another source occurring at later time
periods. E. coli O157:H7 isolates were not kept for PFGE
analysis; hence, it was not possible to determine the source of
hide contamination.
In order to reduce the likelihood of missing sites of contamination due to heterogeneous distribution, the sampling
scheme was changed for phases II and III. Instead of sampling
in alternating Z patterns, samples were collected from a 1,000cm2 rectangular area behind the shoulder. To ensure that any
reductions in E. coli O157:H7 prevalence seen in phases II and
III were not due to multiple samplings of the same area of the
hide, three replicates of repeated sampling were performed.
On three separate occasions, 10 to 12 cattle not included in the
survival experiments were randomly chosen from a feedlot. As
the cattle were restrained in a squeeze chute, multiple samples
were collected from the same sample site. The results of this
sampling show that repeated sampling of the same sample site
does not lead to a decrease in E. coli O157:H7 prevalence (Fig. 2).
In phase II, a control group of cattle was added and comprised 39 animals that were kept in a single feedlot pen. The
control group was added in order to show changes in hide
prevalence when contamination events were not minimized.
Also in phase II, all animals were prescreened for E. coli
O157:H7 hide contamination and all E. coli O157:H7 isolates
were kept for PFGE analysis. Prescreening was conducted in
an attempt to increase the initial O157:H7 prevalence at day 0.
Based on the enumeration data from the screening period and
the sorting assignments, 17 animals in the treated group and 9
animals in the control group were shedding E. coli O157:H7 at
high levels (200 CFU/g feces). As the cattle were sorted, 1
week after prescreening, they were again tested for O157:H7
on their hides and in their feces. Animals destined for the
individual stanchions (treated) had hide and fecal prevalences
of 100% and 67%, respectively, with 6 animals shedding high
levels of E. coli O157:H7, while those animals that were to stay
in the feedlot had hide and fecal prevalences of 87% and 85%,
respectively, with 11 animals shedding high levels of E. coli
O157:H7 (data not shown).
The treated animals were moved to small holding pens (5 to

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FIG. 1. Hide and fecal prevalence of E. coli O157:H7 in phase I

sampling. Hide (circle) and fecal (triangle) samples were collected
from 36 cattle in individual stanchions over a 32-day period. Samples
were collected on days 0, 4, 7, 11, 14, 18, 25, and 32. The data points
represent the percentage of total samples in which E. coli O157:H7 was
detected.

APPL. ENVIRON. MICROBIOL.

VOL. 77, 2011

6 animals per pen) for a 2-week acclimation period. During

this time, neither the animals in the holding pens nor the
animals in the feedlot pen were sampled. Following the 2-week
acclimation, 36 treated animals were moved into individual
stanchions. At that time, each animal was sampled, as were the
control animals in the feedlot pen. The time of this sampling
was considered to be day 0. It was found that following the
2-week period, the hide prevalence for animals in the feedlot
was the same as that for the prior sampling, but the hide
prevalence for animals in the small holding pens had dropped
to 53%. As the animals were maintained in the individual
stanchions, the hide prevalence steadily dropped from 53% at
day 0 to undetectable levels by day 9 (Fig. 3). The E. coli
O157:H7 hide prevalence for the feedlot animals maintained a
level above 66% through day 7 but dropped to 39% by day 11.
The results from phase II supported the phase I results,
suggesting that in the absence of recontamination, E. coli
O157:H7 is not maintained on the hides of cattle for a lengthy
period of time. In this phase of the experiment, when recontamination was prevented, E. coli O157 levels dropped below
detectable limits after 9 days. In two of the three cases where
animals had detectable levels of E. coli O157:H7 at the day 7
sample period, hide sample results from the previous two sampling periods (days 2 and 4) were negative for E. coli O157:H7.
Either the area of hide contamination was localized and missed
in the previous samples or the hide was recontaminated. Because neither of these animals was shedding E. coli O157:H7 at
the day 7 time point, recontamination from the individual
animals was not likely. However, recontamination could have
occurred from other sources. At the time of sampling, it was
noted that there were a large number of flies congregating on
all of the cattle in the barn. Several varieties of flies have been
shown to harbor E. coli O157:H7 and even transfer the bacteria to new locations, including transmission to cattle (1, 23, 33).
PFGE analysis supported the hypothesis of recontamination
from another source. The PFGE patterns for E. coli O157:H7
isolates recovered from the day 7 hide samples from these two
animals were similar to each other, but they did not match any
PFGE patterns that had been seen on these animals previously
(Fig. 4).
For the third animal that harbored E. coli O157:H7 on its
hide at day 7, the hide samples for that animal contained E. coli

3005

FIG. 4. PFGE profiles of E. coli O157:H7 strains collected in phase

II. E. coli O157:H7 isolates collected from animals 6 and 8 that were
housed in individual stanchions. Two isolates from each animals day 0
hide samples were analyzed. On day 7, isolates were analyzed for
animal 8, while only one isolate was recovered from animal 6s day 7
hide sample.

O157:H7 on all sampling occasions up to and including day 7.

This animal was found to be shedding O157:H7 at every sampling period of this experiment. On several of the sampling
days (days 0, 4, 7, and 9), this animal was shedding E. coli
O157:H7 at high levels (range, 4.4 102 to 8.0 104 CFU/g
feces). PFGE analysis showed that the isolates recovered from
the hide were of the same genotype as those being shed in the
feces of that animal (data not shown). It is likely in this case
that recontamination of the hide occurred at some point.
These results imply that the duration of survival of E. coli
O157:H7 on hides is less than 9 days, although it may actually
be 7 days or less, but an additional trial was deemed necessary
for confirmation.
Phase III was conducted in a manner similar to that of phase
II, with the exception that no animals were treated with neomycin. Following the trend observed in phase II, hide prevalence for animals housed in the individual stanchions steadily
declined from day 0 (82%) to day 7 (8%) (Fig. 5). On day 9, the
prevalence increased to 14% and then dropped to 3% on day
11. The increase to five E. coli O157:H7-positive animals seen
on day 9 included three animals that had not yielded an O157:
H7-positive hide sample for at least the three previous sample
times. The other two animals, while positive for O157:H7 on
sample time points previous to day 9, had isolates with novel
PFGE patterns at day 9 (Fig. 6A and B). The one animal with
a positive result on day 11 had not had a positive hide result
since day 0, and the PFGE patterns for isolates collected on
days 0 and 11 were divergent (Fig. 6C).
The data presented here demonstrate that survival of E. coli

FIG. 5. Hide and fecal prevalence of E. coli O157:H7 in phase III

sampling. Hide (circle) and fecal (triangle) samples were collected
from 36 cattle in individual stanchions (solid symbols) and 36 cattle in
a feedlot pen (open symbols) over an 11-day period. Samples were
collected on days 0, 2, 4, 7, 9, and 11. The data points represent
percentage of total samples in which E. coli O157:H7 was detected.

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FIG. 3. Hide and fecal prevalence of E. coli O157:H7 in phase II

sampling. Hide (circle) and fecal (triangle) samples were collected
from 36 cattle in individual stanchions (solid symbols) and 39 cattle in
a feedlot pen (open symbols) over an 11-day period. Samples were
collected on days 0, 2, 4, 7, 9, and 11. The data points represent the
percentage of total samples in which E. coli O157:H7 was detected.

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ARTHUR ET AL.

O157:H7 on the hides of cattle is short lived, with an approximate duration of less than 9 days. Similar results were produced using in vitro testing of cattle hide fragments. Small et al.
inoculated pieces of cattle hide and monitored the E. coli
O157:H7 population reduction over time (32). The authors
reported that it would take between 5 and 8 days for a reduction in 90% of the pathogens population on the surfaces of
cattle hides (32). It can be seen from the data presented herein
and previously (32) that the decrease in E. coli O157:H7 on
cattle hides occurs rapidly. It is possible that E. coli O157:H7
population reductions occurring in less than 9 days may be
sufficient to minimize or eliminate finished product contamination simply by lowering hide contamination to levels that are
managed by the in-plant interventions. However, in these studies, recontamination was much easier to prevent than would be
the case in an actual production setting, where even if fecal
shedding were stopped instantaneously, it is likely that there
would still be some hide recontamination from the production
environment. Therefore, our recommendation of a 9-day time
period between application of a short-course intervention and
harvest of the animals is based on achieving maximum reductions in the E. coli O157:H7 populations on cattle hides.
Survival time of E. coli O157:H7 in other matrices has been
shown to be considerably longer. E. coli O157:H7 has been
shown to persist in multiple environments (soil, water, and
bovine feces) for over 90 days (19, 31). At this point, the
mechanism of bacterial reduction on the hides of cattle is not

known. The simplest scenario would involve cell desiccation,

because the hide has no moisture retention capabilities. Williams et al. determined that survival of E. coli O157:H7 in
inoculated feces that were applied to metal and wood surfaces
was dependent on moisture content and temperature (36).
They showed that survival at 20C under desiccating conditions
was less than or equal to 7 days. The conditions leading to the
7-day survival in the study by Williams et al. would be similar
to those found on the hides of cattle (36). Desiccation does not
always lead to reduced cell survival, as shown previously by
Varma et al. (34), where an outbreak-related E. coli O157
strain was isolated from several samples of dust located in the
rafters of a barn that had hosted an agricultural fair 42 weeks
earlier (34). Therefore, further research is required to understand the mechanism by which E. coli O157:H7 bacteria are
reduced on the hides of cattle in less than 9 days.
It should be noted that the animals kept in individual stanchions were in a covered building. While the temperature and
humidity were not controlled and the building remained open
to the outside atmosphere, the animals were not subjected
other environmental factors, such as direct sunlight or precipitation. This may have influenced the survival times for the
bacterial pathogen on the cattle hides.
The presence of E. coli O157:H7 on the hides of cattle when
presented for processing has been shown to be a primary determinant of carcass contamination (7, 12, 13, 28). Postharvest
antimicrobial interventions directly targeting the hide have
proven effective in reducing the transfer of pathogenic bacteria
from the hide to the carcass (12, 25, 28). However, despite
effective multihurdle intervention schemes utilized at processing, end product contamination still occurs, indicating the need
for additional, effective interventions. The goal of preharvest
intervention design is to reduce the pathogen prevalence and
load in the feces of the animal. In doing so, the prevalence and
load of the target bacteria will be reduced indirectly on the
hides of animals through less-frequent contact with contaminated feces.
Some preharvest intervention methods (i.e., vaccination and
probiotics) are designed to be applied over a long-duration
time span to maintain fecal pathogen levels in a reduced state
(16, 26, 30). Other preharvest intervention types (i.e., antibiotics and sodium chlorate) are implemented at the end of the
finishing period immediately before cattle are sent to processing (3, 15, 17). It is in the latter scenario that existing hide
contamination may render short-duration preharvest interventions ineffective for prevention of carcass contamination. In
vitro studies utilizing inoculated rumen fluid showed that E.
coli O157:H7 was reduced from 106 CFU/ml to less than detectable levels in 24 h by the addition of sodium chlorate (3).
When administered to live cattle as a feed additive within 20 h
of harvest, reductions in fecal prevalence of E. coli O157:H7
were observed, while no effects were seen for recovery of E.
coli O157:H7 from hide or carcass samples (2). Based on the
data presented herein, a 20-h time period between intervention
and slaughter would not provide enough time for reductions in
fecal prevalence to be translated into lower levels of hide
prevalence.
In addition, the role of the processing plant lairage environment as a major source of E. coli O157:H7 contamination can
be a confounding factor in the interpretation of preharvest

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FIG. 6. PFGE profiles of E. coli O157:H7 strains collected in phase

III. Two animals (2 and 11) which were positive for O157:H7 on
sample time points previous to day 9 had isolates with novel PFGE
patterns at day 9. (A) Two isolates each from animal 2s hide samples
collected on days 0, 4, 7, and 9 were analyzed by PFGE. (B) E. coli
O157:H7 isolates collected from animal 11 on days 0, 7, and 9 were
analyzed by PFGE. (C) One animal (14) had a positive hide sample
result on day 11. E. coli O157:H7 isolates collected on day 11 were
divergent from those collected on the only other positive sampling
occasion, day 0.

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VOL. 77, 2011

SURVIVAL OF E. COLI O157:H7 ON CATTLE HIDES

ACKNOWLEDGMENTS
This project was funded in part by The Beef Checkoff.
We thank Julie Dyer, Bruce Jasch, Kim Kucera, Frank Reno, and
Greg Smith for technical support and Marilyn Bierman for secretarial
support.
Names are necessary to report factually on available data; however,
the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the
product to the exclusion of others that may also be suitable.
REFERENCES
1. Ahmad, A., T. G. Nagaraja, and L. Zurek. 2007. Transmission of Escherichia
coli O157:H7 to cattle by house flies. Prev. Vet. Med. 80:7481.
2. Anderson, R. C., et al. 2005. Effects of experimental chlorate preparations as
feed and water supplements on Escherichia coli colonization and contamination of beef cattle and carcasses. Food Microbiol. 22:439447.
3. Anderson, R., et al. 2000. Bactericidal effect of sodium chlorate on Escherichia coli O157:H7 and Salmonella Typhimurium DT104 in rumen contents
in vitro. J. Food Prot. 63:10381042.
4. Arthur, T. M., et al. 2009. Longitudinal study of Escherichia coli O157:H7 in
a beef cattle feedlot and the role of high shedders in hide contamination.
Appl. Environ. Microbiol. 75:65156523.

5. Arthur, T. M., et al. 2007. Transportation and lairage environment effects on

prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and
carcasses of beef cattle at processing. J. Food Prot. 70:280286.
6. Arthur, T. M., et al. 2010. Evaluation of a direct fed microbial product effect
on the prevalence and load of Escherichia coli O157:H7 in feedlot cattle. J.
Food Prot. 73:366371.
7. Arthur, T. M., et al. 2004. Escherichia coli O157 prevalence and enumeration
of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various
steps in commercial beef processing plants. J. Food Prot. 67:658665.
8. Avery, L. M., A. P. Williams, K. Killham, and D. L. Jones. 2008. Survival of
Escherichia coli O157:H7 in waters from lakes, rivers, puddles and animaldrinking troughs. Sci. Total Environ. 389:378385.
9. Reference deleted.
10. Barkocy-Gallagher, G. A., et al. 2005. Methods for recovering Escherichia
coli O157:H7 from cattle fecal, hide, and carcass samples: sensitivity and
improvements. J. Food Prot. 68:22642268.
11. Barkocy-Gallagher, G. A., et al. 2002. Development of methods for the
recovery of Escherichia coli O157:H7 and Salmonella from beef carcass
sponge samples and bovine fecal and hide samples. J. Food Prot. 65:1527
1534.
12. Bosilevac, J. M., et al. 2004. Prevalence of Escherichia coli O157 and levels
of aerobic bacteria and Enterobacteriaceae are reduced when hides are
washed and treated with cetylpyridinium chloride at a commercial beef
processing plant. J. Food Prot. 67:646650.
13. Brichta-Harhay, D. M., et al. 2008. Salmonella and Escherichia coli O157:H7
contamination on hides and carcasses of cull cattle presented for slaughter in
the United States: an evaluation of prevalence and bacterial loads by immunomagnetic separation and direct plating methods. Appl. Environ. Microbiol. 74:62896297.
14. Brichta-Harhay, D. M., et al. 2007. Enumeration of Salmonella and Escherichia coli O157:H7 in ground beef, cattle carcass, hide and fecal samples
using direct plating methods. J. Appl. Microbiol. 103:16571668.
15. Callaway, T. R., et al. 2002. Sodium chlorate supplementation reduces E. coli
O157:H7 populations in cattle. J. Anim. Sci. 80:16831689.
16. Callaway, T. R., et al. 2008. Probiotics, prebiotics and competitive exclusion
for prophylaxis against bacterial disease. Anim. Health Res. Rev. 9:217225.
17. Elder, R. O., et al. 2002. Intervention to reduce fecal shedding of enterohemorrhagic Escherichia coli O157:H7 in naturally infected cattle using neomycin sulfate. J. Anim. Sci. 80(Suppl. 1):15. (Abstract.)
18. Franz, E., et al. 2008. Manure-amended soil characteristics affecting the
survival of E. coli O157:H7 in 36 Dutch soils. Environ. Microbiol. 10:313
327.
19. Gagliardi, J. V., and J. S. Karns. 2002. Persistence of Escherichia coli
O157:H7 in soil and on plant roots. Environ. Microbiol. 4:8996.
20. Hu, Y., Q. Zhang, and J. C. Meitzler. 1999. Rapid and sensitive detection of
Escherichia coli O157:H7 in bovine faeces by multiplex PCR. J. Appl. Microbiol. 87:867876.
21. Hunter, S. B., et al. 2005. Establishment of a universal size standard strain
for use with the PulseNet standardized pulsed-field gel electrophoresis protocols: converting the national databases to the new size standard. J. Clin.
Microbiol. 43:10451050.
22. Jiang, X., J. Morgan, and M. P. Doyle. 2002. Fate of Escherichia coli
O157:H7 in manure-amended soil. Appl. Environ. Microbiol. 68:26052609.
23. Keen, J. E., T. E. Wittum, J. R. Dunn, J. L. Bono, and L. M. Durso. 2006.
Shiga-toxigenic Escherichia coli O157 in agricultural fair livestock, United
States. Emerg. Infect. Dis. 12:780786.
24. Koohmaraie, M., et al. 2007. Interventions to reduce/eliminate Escherichia
coli O157:H7 in ground beef. Meat Sci. 77:9096.
25. Koohmaraie, M., et al. 2005. Post-harvest interventions to reduce/eliminate
pathogens in beef. Meat Sci. 71:7991.
26. LeJeune, J. T., and A. N. Wetzel. 2007. Pre-harvest control of Escherichia coli
O157 in cattle. J. Anim. Sci. 85:7380.
27. McGee, P., et al. 2002. Survival of Escherichia coli O157:H7 in farm water: its
role as a vector in the transmission of the organism within herds. J. Appl.
Microbiol. 93:706713.
28. Nou, X., et al. 2003. Effect of chemical dehairing on the prevalence of
Escherichia coli O157:H7 and the levels of aerobic bacteria and Enterobacteriaceae on carcasses in a commercial beef processing plant. J. Food Prot.
66:20052009.
29. Rice, D. H., H. Q. Sheng, S. A. Wynia, and C. J. Hovde. 2003. Rectoanal
mucosal swab culture is more sensitive than fecal culture and distinguishes
Escherichia coli O157:H7-colonized cattle and those transiently shedding the
same organism. J. Clin. Microbiol. 41:49244929.
30. Sargeant, J. M., M. R. Amezcua, A. Rajic, and L. Waddell. 2007. Pre-harvest
interventions to reduce the shedding of E. coli O157 in the faeces of weaned
domestic ruminants: a systematic review. Zoonoses Public Health 54:260277.
31. Scott, L., P. McGee, J. J. Sheridan, B. Earley, and N. Leonard. 2006. A
comparison of the survival in feces and water of Escherichia coli O157:H7
grown under laboratory conditions or obtained from cattle feces. J. Food
Prot. 69:611.
32. Small, A., C. A. Reid, and S. Buncic. 2003. Conditions in lairages at abattoirs
for ruminants in southwest England and in vitro survival of Escherichia coli

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intervention effects on hide and carcass prevalence during processing. Arthur et al. have shown that over 80% of the E. coli
O157:H7 isolates recovered from beef carcasses during processing did not come from the feedlot of origin for the associated cattle (5). It was determined that cattle acquire a large
amount of hide contamination following arrival at the processing plant as animals move through multiple common spaces of
the lairage environment. In this scenario, effects of preharvest
interventions in lowering the hide prevalence are likely negated by the time the animals begin processing. Hence, data
from hide and carcass samples collected during processing
should be interpreted with caution when evaluating the efficacy
of preharvest interventions. In order to avoid these confounding effects, processing scenarios which prevent cohabitation of
the lairage environment by treated and untreated animals must
be employed.
When considering the role of preharvest intervention in the
context of lairage environment contributions to hide contamination, it should be noted that any intervention that reduces
fecal shedding of E. coli O157:H7 would be beneficial in reducing hide contamination at harvest by reducing the contamination potential in the lairage environment. The incoming
fecal load of cattle presented for processing is a main determinant of the lairage environments potential to serve as a
contamination source for cattle hides. Therefore, an effective
preharvest intervention would serve to reduce E. coli O157:H7
hide contamination in both the production and lairage environments. Only in the production environment would the duration of E. coli O157:H7 survival on cattle hides be relevant.
In the lairage environment, the critical issue is to minimize or
prevent hide contamination, not to allow time for die-off the
pathogen.
In summary, E. coli O157:H7 persistence on cattle hides is
short lived. This fact should be taken into account when designing preharvest antimicrobial intervention schemes to maximize the effectiveness of the intervention in preventing carcass
contamination. Any preharvest interventions that are to be
administered at the end of the feeding period will achieve
maximum effect in reducing E. coli O157:H7 levels on cattle
hides if given 9 days before the cattle are presented for processing.

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35. Wells, J. E., et al. 2009. Prevalence and level of Escherichia coli O157:H7 in
feces and on hides of feedlot steers fed diets with or without wet distillers
grains with solubles. J. Food Prot. 72:16241633.
36. Williams, A. P., L. M. Avery, K. Killham, and D. L. Jones. 2005. Persistence
of Escherichia coli O157 on farm surfaces under different environmental
conditions. J. Appl. Microbiol. 98:10751083.
37. Woerner, D. R., et al. 2006. Pre-harvest processes for microbial control in
cattle. Food Prot. Trends 26:393400.

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FUNDA O
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O157, Salmonella Kedougou, and Campylobacter jejuni on lairage-related

substrates. J. Food Prot. 66:15701575.
33. Talley, J. L., et al. 2009. Association of Escherichia coli O157:H7 with filth
flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies
(Diptera: Muscidae). J. Food Prot. 72:15471552.
34. Varma, J. K., et al. 2003. An outbreak of Escherichia coli O157 infection
following exposure to a contaminated building. JAMA 290:27092712.

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