Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Prevalence and molecular characterization of Escherichia

coli O157:H7 on Irish lamb carcasses, fleece and in faeces
samples
M. Lenahan1,2, S. O’Brien1, K. Kinsella1, T. Sweeney2 and J.J. Sheridan1
1 Teagasc, Ashtown Food Research Centre, Ashtown, Dublin, Ireland
2 School of Agriculture, Food Science and Veterinary Medicine, University College Dublin, Belfield, Dublin, Ireland

Keywords
carcass, Escherichia coli O157:H7, faeces,
fleece, sheep.
Correspondence
M. Lenahan, Ashtown Food Research Centre,
Ashtown, Dublin 15, Ireland.
E-mail: mary.lenahan@teagasc.ie
2007 ⁄ 0065: received 16 January 2007,
revised 17 May 2007 and accepted 21 May
2007
doi:10.1111/j.1365-2672.2007.03476.x
Abstract
Aims: To determine the prevalence, seasonal variation and virulence character-
istics of Escherichia coli O157:H7 in lambs presented for slaughter in Ireland.
Methods and Results: Over a 13-month period, pre- and postchill carcass
swabs, faeces and fleece samples from 1600 lambs were examined for the pres-
ence of E. coli O157:H7. Escherichia coli O157:H7 was isolated from 5Æ75%
(23 ⁄ 400) of fleece samples, 1Æ5% (6 ⁄ 400) of pre- and 1% (4 ⁄ 400) of postchill
carcass swabs but was not isolated in faeces (0 ⁄ 400). The present study detec-
ted no evidence of seasonal variation. Polymerase chain reaction analysis
showed that both the vt1 and vt2 genes associated with clinical illness were car-
ried by five of the E. coli O157:H7 isolates, while 24 of the remaining isolates
carried the vt2 gene only. Phage typing detected four different subtypes: PT 32
(48Æ48%), PT 8 (12Æ12%), PT 31 (12Æ12%) and PT 21 ⁄ 28 (12Æ12%).
Conclusions: Escherichia coli O157:H7 is present in lambs at slaughter in Irish
abattoirs and the virulence profiles of these isolates reveals that they are poten-
tially harmful to humans.
Significance and Impact of the Study: The present study provides crucial infor-
mation indicating that sheep may be a significant contributing source to
human E. coli O157:H7 infection.

have previously been linked to two VTEC outbreaks, both

Introduction
Escherichia coli O157:H7 has emerged as a highly virulent
food-poisoning pathogen, which causes a wide range of
symptoms from mild nonbloody diarrhoea to haemor-
rhagic colitis (HC), thrombocytopenic purpura and hae-
molytic uraemic syndrome (HUS) (Griffin and Tauxe
1991). Pathogenicity of VTEC (verocytotoxigenic E. coli)
strains is mainly associated with formation of attaching-
and-effacing lesions (eaeA), production of one or both
verotoxins (vt1 and vt2) and production of haemolysin
(hlyA) (Paton and Paton 1998).
Cattle have been reported to be a major reservoir of
VTEC (Heuvelink et al. 1998; Chapman et al. 2001); how-
ever, recent studies suggest that other ruminants, especi-
ally sheep, may be important in contributing to human
clinical cases (Chapman et al. 2000, 2001). Sheep faeces
occurring in Scotland. The first (six cases) was related to
untreated drinking water contaminated with sheep faeces
(Licence et al. 2001). The second (20 cases) occurred at a
scout camp held at an agricultural showground that had
been grazed by approx. 300 sheep prior to the camp and
the following investigation revealed that clinical human
E. coli O157 isolates from the outbreak were indistin-
guishable by PFGE (pulsed field gel electrophoresis) from
those recovered from sheep (Ogden et al. 2002).
The presence of E. coli O157:H7 has been reported in
sheep and lamb products internationally. In the Nether-
lands, E. coli O157 strains were isolated from 3Æ8% of ewe
faeces and 4Æ1% of lamb faeces (Heuvelink et al. 1998).
Kudva et al. (1996) found that the incidence of VTEC
O157 in sheep in the USA was transient and ranged
from 31% in June to none in November. In the UK, the

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E. coli O157:H7 in sheep
pathogen was recovered from 1Æ4% of 7200 sheep (Chap-
man et al. 2001) and was reported to be more frequently
isolated from lamb (2Æ9%) than from beef products
(1Æ1%) (Chapman et al. 2000).
As the incidence of VTEC in sheep has not previously
been examined in Ireland, the aim of the present study
was (i) to determine the prevalence of E. coli O157:H7 in
Irish lambs presented for slaughter by examining pre- and
postchill carcasses, faeces and fleece samples, (ii) to
investigate whether a seasonal pattern of shedding E. coli
O157:H7 existed and (iii) to determine the potential viru-
lence of the E. coli O157:H7 isolates obtained by investi-
gating their genetic profiles.
Materials and methods
Study design
Five sheep export abattoirs slaughtering 250 sheep per
hour were each visited four times over a 13-month period
between February 2005 and February 2006. Three of the
abattoirs slaughtered both cattle and sheep (A, C and E),
while the remaining two (B and D) slaughtered sheep
only. In each abattoir (n ¼ 5), animals were chosen at
random for sampling. At each visit (n ¼ 4), 20 pre- and
20 postchill carcass swabs, 20 faecal and 20 fleece samples
were taken to determine the presence of E. coli O157:H7.
A total of 1600 samples were examined over the course of
this study.
Sample collection
Carcass samples
Animals entering the abattoir from the lairage were elec-
trically stunned, exsanguinated by throat cutting and
allowed to bleed. After bleeding, the animals’ pelt was
removed, followed by evisceration and pluck removal.
Whole body prechill carcass swabs were taken from ani-
mals on the line immediately after evisceration and final
carcass washing with cold chlorinated water. Whole body
postchill carcass swabs were taken from a different set of
animals held in a chill for 24 h at a mean temperature of
4C. Carcasses were swabbed using a polyurethane sponge
(75 cm2) (Sydney Heath & Son Ltd, Stoke-on-Trent,
UK). Each sponge was placed in an autoclavable stoma-
cher bag (Sarstedt, Nümbrecht, Germany) to which 8 ml
of Maximum Recovery Diluent (Oxoid Ltd., Basingstoke,
UK) was added and autoclaved at 121C for 10 min. Each
sterile sponge was exposed by grasping through the auto-
clave bag and pulling the bag over the operative’s hand.
The entire outer surface of each side was swabbed with a
sterile sponge, starting from the hindquarter downward
to the forequarter (Lasta et al. 1992). The sponge was
2402 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 2401–2409
M. Lenahan et al.
then withdrawn into the stomacher bag and secured with
an elastic band. For recovery of E. coli O157:H7, separate
sponges were used to swab each side of the carcass. Swabs
from opposite sides of the same carcass were pooled
before analysis to give a whole body composite sample.
Faecal samples
Samples were obtained during evisceration by aseptically
slitting the distal colon with a sterile scalpel. Samples
were taken using wooden tongue depressors (Medical
Supply Co Ltd, Dublin, Ireland) that had been autoclaved
in stomacher bags at 121C for 10 min. The depressor
was grasped through the plastic bag exposed and used to
take a 1- to 2-g faecal sample. The faecal sample was
withdrawn into the stomacher bag and secured with an
elastic band. Samples were transported to the laboratory
in a cool box containing ice packs and stored in a chiller
(1C) overnight for analysis within 24 h.
Fleece samples
Samples were taken randomly from the rump area on line
before pelt removal. A sterile stomacher bag was inverted
aseptically over one hand, so that the inside of the bag
was exposed and could be used to grasp a handful of
fleece. A sharp scissors, which was sterilized between sam-
ples with alcoholic wipes (Look Sales Ltd, Dublin, Ire-
land), was used to cut the fleece sample from the pelt.
The sample (approx. 10 g) was withdrawn into the sterile
stomacher bag and secured with an elastic band. Fleece
samples were transported to laboratory in cool boxes con-
taining ice packs and stored in a chiller (1C) overnight
for analysis within 24 h.
Microbiological examination
A 90-ml volume of buffered peptone water (BPW)
(Oxoid) supplemented with 8 mg l–1 of vancomycin
(Oxoid), 0Æ05 mg l–1 cefixime (Oxoid) and 10 mg l–1
cefsulodin (Sigma-Aldrich, Steinheim, Germany) (BPW-
VCC) was added to each carcass swab (10 g) and fleece
sample (10 g) and stomached using a Model 400 stoma-
cher (Seward & Company Ltd, London, UK) for 30 s at
260 rpm. Sterile cotton-tipped swabs (Nuova Aptaca,
Canelli, Italy) were loaded with faeces (0Æ5 g) broken off
into 5-ml volumes of BPW-VCC and vortexed for 10 s
before incubation (Chapman et al. 2000). All samples
were incubated in BPW-VCC for 6 h at 37C.
Immunomagnetic separation (IMS) was used to recover
E. coli O157:H7 from enriched BPW-VCC samples. After
6 h incubation at 37C, 1 ml aliquots of the faecal, fleece
or carcass enrichment broth was added to 20 ll of immu-
nomagnetic beads coated with anti-E. coli O157 anti-
body (Dynabeads anti-E. coli O157, Dynal A.S., Oslo,
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M. Lenahan et al. E. coli O157:H7 in sheep

Norway) and processed according to the manufacturer’s
instructions. Bead ⁄ sample suspensions were plated in
duplicate onto Sorbitol MacConkey agar (SMAC) (Ox-
oid) supplemented with Cefixime tellurite (Oxoid), and
incubated at 37C for 24 h.
Five nonpigmented nonsorbitol fermenting colonies
were selected from each duplicate plate, giving 10 colonies
in all, which were subjected to biochemical confirmation.
Colonies from CT-SMAC were streaked onto Nutrient
Agar (Oxoid) to obtain a purified culture and incubated
for 24 h at 37C. Colonies from the Nutrient Agar were
biochemically confirmed by streaking onto Levines Eosin
Methylene Blue agar (EMB) (Oxoid), Phenol Red Sorbitol
agar (Oxoid) supplemented with 4-methylumbelliferyl-B-
d-glucuronide (Oxoid) (PRS-MUG), inoculated into
Tryptone Tryptophane medium (TT) (Oxoid) and all
were incubated at 37C for 24 h. Colonies displaying a
green metallic sheen on EMB, no fluorescence on PRS-
MUG when illuminated with UV light, and which were
indole positive in TT medium on addition of Kovacs rea-
gent (Oxoid), were examined using a E. coli O157:H7
latex agglutination kit (Wellcolex, Merseyside, UK)
according to the manufacturer’s instructions to check for
the presence of the O157 antigen. All sorbitol nonfer-
menting, MUG-negative, indole-positive colonies forming
a visible antigen-antibody precipitate on latex test were
confirmed as presumptively E. coli O157 positive and
stored at )80C on ProtectTM Stock Culture Beads (Tech-
nical Service Consultants Ltd, Lancashire, UK) to enable
polymerase chain reaction (PCR) analysis to be carried
out at a later date. Isolates confirmed as E. coli O157:H7
by PCR were phage typed (Ahmed et al. 1987; Khakhria
et al. 1990) at the Laboratory of Enteric Pathogens, Cen-
tre of Infections, 61 Colindale Avenue, London NW9
5HT, UK.
Presumptive E. coli O157 isolates were subjected to
PCR analysis to confirm the presence of the O157 antigen
and H7 flagellum and to determine the presence or
absence of 11 other genes that encode proteins considered
to have a role in clinical illness caused by E. coli
O157:H7. The genes investigated and primers used are
detailed in Table 1. The formation of attaching and effa-
cing lesions, (eaeA intimin gene) where the pathogen
adheres tightly to the intestinal epithelial cells and pro-
duction of verotoxins 1 and 2, have been directly linked
to HUS and HC in human patients (Paton and Paton
1998), while the type III secretion system (TTSS) (TIR,
espA, espB, espF and espP genes) delivers several effector

Table 1 Oligonucleotide primer sequences

Amplification
Primer Sequence (5¢-3¢) Reference product size (bp)

vt1-F 5¢-ATAAATCGCCATTCGTTGACTAC-3¢ Paton and Paton (1998) 180

vt1-R 5¢-AGAACGCCCACTGAGATCATC-3¢
vt2-F 5¢-GGCACTGTCTGAAACTGCTCC-3¢ 255
vt2-R 5¢-TGCCCAGTTATCTGACATTCTG-3¢
eaeA-F 5¢-GACCCGGCACAAGCATAAGC-3¢ 384
eaeA-R 5¢-CCACCTGCAGCAACAAGAGG-3¢
hlyA-F 5¢-GCATCATCAAGCGTACGTTCC-3¢ 534
hlyA-R 5¢-AATGAGCCAAGCTGGTTAAGCT-3¢
rfbO157-F 5¢-CGGACATCCATGTGATATGG-3¢ 259
rfbO157-R 5¢-TTGCCTATGTACAGCTAATCC-3¢
fliCh7 -F 5¢-GCGCTGTCGAGTTCTATCGAGC-3¢ Fratamico et al. (2000) 625
fliCh7 -R 5¢-CAACGGTGACTTTATCGCCATTCC-3¢
TIR-F 5¢-GTGGCGCATTGATTCTTGG-3¢ Higgins et al. (2003) 53
TIR-R 5¢-CCGGCTGATTTTTTCGATGA-3¢
espA 5¢-CACGTCTTGAGGAAGTTTGG-3¢ McNally et al. (2001) 299
espA 5¢-CCGTTGTTAATGTGAGTGCG-3¢
espB 5¢-CGATGGTTAATTCCGCTTCG-3¢ 304
espB 5¢-GCCTGCTGAATCTGATACCT-3¢
espP 5¢-AAACAGCAGGCACTTGAACG-3¢ 301
espP 5¢-AGACAGTTCCAGCGACAACC-3¢
espF-F 5¢-CGGGATCCCCCTTTCTTCGATTGCTCATAG-3¢ Crane et al. (2001) 624
espF-R 5¢-CATGCCATGGTTAATGGAATTAGTAAC-3¢
etpD-F 5¢-CGTCAGGAGGATGTTCAG-3¢ Schmidt et al. (1999) 1062
etpD-R 5¢-CGACTGCACCTGTTCCTGATTA-3¢
katP-F 5¢-CTTCCTGTTCTGATTCTTCTGG-3¢ Brunder et al. (1996) 2125
katP-R 5¢-AACTTATTTCTCGCATCATCC-3¢

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E. coli O157:H7 in sheep
No. of positive samples 7
6
5
4
3
2 Figure 1 The number of fleece ( ), pre- ( )
1 and postchill ( ) carcass swabs samples
0 Escherichia coli O157:H7 was isolated from
F M A M J J A S
Month (Feb-05 to Feb-06)
proteins to the cytoplasm of the host epithelial cells that
subvert, inhibit or activate cellular processes (Garmendia
et al. 2005). Presumptive E. coli O157:H7 isolates stored
on Protect Beads were resuscitated in 10-ml volumes of
brain heart infusion broth (BHI; Oxoid) and incubated at
37C for 18 h. After incubation, genomic DNA was
extracted from a 1-ml portion of bacterial cells in BHI
using a DNeasy blood & tissue kit (QiagenTM, Crawley,
UK) as per the manufacturer’s instructions for Gram-neg-
ative bacteria. For each assay, 5 ll of genomic DNA was
used in each 50 ll PCR reaction. The concentration of
primers and reaction conditions for each gene investi-
gated were based on previous work which is stated in the
publications referenced in Table 1. An E. coli O157:H7
isolate, ATCC 43895, associated with the Oregon ⁄ Mich-
igan ground beef outbreak in the USA in 1982, obtained
from the Central Meat Control Laboratory, Department
of Agriculture, Abbotstown, Dublin, was included as a
positive control in each PCR assay, while sterile-filtered,
DNA- and RNA-free water (Sigma-Aldrich) was used as a
negative control. PCR reactions were replicated four times
for each isolate examined.
Results
Our survey detected E. coli O157:H7 in 5Æ75% (23 ⁄ 400)
of fleece samples, 1Æ5% (6 ⁄ 400) of pre- and in 1%
(4 ⁄ 400) of postchill carcass swabs. The pathogen was not
isolated from any of the 400 faecal samples examined in
this study. Escherichia coli O157:H7 was isolated from
fleece samples between the dates of 3 May 2005 and 30
January 2006, and from pre- and postchill carcass swabs
sporadically throughout the 13-month study period
(Fig. 1). The monthly prevalence of E. coli O157:H7 ran-
ged from 0% to 6Æ25% with none recovered during the
months of March, April and July 2005 and February
2006. The pathogen was recovered at four of the five
abattoirs visited, with abattoir B having the highest num-
ber of positive samples, while none were recovered at
abattoir D (Fig. 2). The pathogen was mostly isolated
from fleece at each abattoir, while the highest number of
positive carcasses was found at abattoir C.
2404 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 2401–2409
M. Lenahan et al.
O N D J F
each month. Escherichia coli O157:H7 was
not found in any faeces samples.
Table 2 details the abattoir isolates were recovered at,
the isolation dates and sample types positive for E. coli
O157:H7. Although genetic profiles were established for a
maximum of 10 nonsorbitol fermenting isolates from
CT-SMAC per sample (i.e. five per duplicate plate), a sin-
gle genetic profile was found to represent all isolates from
each positive sample. The virulence profiles were charac-
terized by determining the presence or absence of 13 dif-
ferent virulence-associated genes using PCR (Table 3).
Five isolates carried both the vt1 and vt2 genes typically
associated with human illness caused by E. coli O157:H7,
while 24 of the remaining isolates had the vt2 gene only
present. Four isolates did not have either verotoxin gene
present, while all 33 isolates were found to carry the eaeA
gene for intimin. The most frequently recovered PCR
profile (profile no. 2) contained all the genes investigated
except vt1 (15 isolates).
Phage typing of the E. coli O157:H7 isolates detected
four different subtypes. The majority of the isolates were
PT 32 (48Æ48%), followed by PT 8 (12Æ12%), PT 31
(12Æ12%) and PT 21 ⁄ 28 (12Æ12%). All isolates recovered
at a specific abattoir on a given date, regardless of sample
type, shared the same phage type, except on one occasion
at abattoir C on 5th December 2005, where a different
phage type was detected in the postchill carcass swab than
in the fleece samples. Isolates that shared the most com-
mon PCR profile (no. 2) did not all have the same phage
types, eight were PT 32, four were PT 31 and three were
PT21 ⁄ 28 (Table 3). The second most frequent PCR
40
Positive samples (%)
35
30
25
20
15
10
5
0
A B C D E
Abattoir
Figure 2 The percentage of fleece ( ), pre- ( ) and postchill ( )
carcass swabs found positive for Escherichia coli O157:H7 at each
abattoir. Escherichia coli O157:H7 was not found in any faeces samples.
ª 2007 The Authors
M. Lenahan et al. E. coli O157:H7 in sheep

Table 2 The abattoir, isolation date and sample type positive for 3Æ0% to 3Æ2% on prechilled beef carcasses (McEvoy et al.
Escherichia coli O157:H7 2003; Carney et al. 2006) while the current study detected
Isolate code Abattoir Isolation date Sample type the pathogen on 1Æ5% of prechill and 1Æ0% of postchill
lamb carcasses. In a previous study on Salmonella on Irish
1 A 8th February 2005 Prechill carcass swab cattle, it was established that the pathogen prevalence
2 A 3rd May 2005 Fleece
either decreased or increased after chilling (Kerr and
3 A 3rd May 2005 Postchill carcass swab
4 A 3rd May 2005 Fleece
Sheridan 2002). This arises because there is no specific
5 A 3rd May 2005 Fleece chilling regime that can be identified that will always give
6 E 18th May 2005 Fleece a reduction in bacterial numbers (Sheridan 2004).
7 E 18th May 2005 Fleece The current work did not isolate E. coli O157:H7 from
8 C 23rd May 2005 Fleece faecal samples; however, a number of other studies have
9 B 29th June 2005 Prechill carcass swab found the pathogen in sheep faeces (Kudva et al. 1996;
10 B 29th June 2005 Postchill carcass swab
Heuvelink et al. 1998; Chapman et al. 2001), but a Nor-
11 B 29th June 2005 Fleece
12 B 29th June 2005 Fleece
wegian study did not (Johnsen et al. 2001). The present
13 E 8th August 2005 Postchill carcass swab study examined faecal samples from the distal colon and
14 C 19th September 2005 Prechill carcass swab the lack of recovery in faeces found may result from the
15 C 19th September 2005 Prechill carcass swab difference in the specific location in colon where faeces
16 C 19th September 2005 Prechill carcass swab were taken, compared with other studies. Recently, it has
17 C 19th September 2005 Prechill carcass swab been reported that E. coli O157:H7 is more frequently iso-
18 B 4th October 2005 Fleece
lated at the rectoanal junction of cattle (Low et al. 2005),
19 B 4th October 2005 Fleece
20 B 4th October 2005 Fleece
and this may also be the case in sheep. Faeces from the
21 B 4th October 2005 Fleece rectoanal junction were not examined in the current
22 B 4th October 2005 Fleece study, and this may account for the low incidence of the
23 B 4th October 2005 Fleece pathogen found in the faeces.
24 E 7th November 2005 Fleece An alternative explanation for the absence of E. coli
25 C 5th December 2005 Fleece O157:H7 from faeces may be related to the methodology
26 C 5th December 2005 Fleece
used. The enrichment method used in the present study,
27 C 5th December 2005 Fleece
28 C 5th December 2005 Fleece
involving the use of BPW-VCC with incubation at 37C
29 C 5th December 2005 Postchill carcass for 6 h, has been used extensively in the isolation of E.
swab coli O157:H7 from faeces of cattle and sheep (Chapman
30 B 30th January 2006 Fleece et al. 1997, 2001; McEvoy et al. 2003; Rey et al. 2003;
31 B 30th January 2006 Fleece Ogden et al. 2005). The failure to isolate E. coli O157:H7
32 B 30th January 2006 Fleece from faeces in the present study was considered unusual,
33 B 30th January 2006 Fleece
especially as the pathogen was found on the fleece, as
well as on carcasses. While other studies involving the
profile (no. 3) contained all the genes investigated except same enrichment technique have failed to isolate this
vt1 and espP (6 isolates) and this group also shared the pathogen from sheep faeces (Johnsen et al. 2001), the
same phage type (PT 32). use of alternative methods have also been unsuccessful
(Dontorou et al. 2004). The isolation of the organism
from faeces presents problems in relation to the presence
Discussion
of a very high natural microflora in faeces. In conse-
While cattle are generally regarded as the main reservoir quence, antibiotics are used to assist in the isolation of
of E. coli O157:H7 infection, the results of the present specific pathogens. A wide variety of antibiotics and
study indicate that sheep may also be a significant contri- enrichment media are used in the isolation of E. coli
buting source. The current study found that E. coli O157:H7 from animal faeces, and it is difficult to deter-
O157:H7 was present on the fleece and pre- and postchill mine which is optimal (Chapman 2000; De Boer and
carcasses but not in the faeces, and there was no evidence Heuvelink 2000).
of seasonal variation. The majority of isolates were found Recently, it has been suggested that the isolation of
to have the vt2 and eaeA genes present and the most E. coil O157:H7 from faeces should be carried out by
common phage types were PT 32, PT 8, PT 31 and PT enrichment in a nonselective medium, such as tryptone
21 ⁄ 28. soya broth plus phosphate buffer (TSB+PO4) for 6 h at
Previous abattoir-based studies carried out in Ireland 42C, followed by IMS (Barkocy-Gallagher et al. 2002,
have reported an E. coli O157:H7 prevalence ranging from 2005). The basis of this and similar techniques is that the

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E. coli O157:H7 in sheep M. Lenahan et al.

Table 3 Virulence profiles and phage types of Escherichia coli O157:H7 isolates from fleece and pre- and postchill carcasses

Virulence genes
Polymerase
chain reaction No. of Phage
profile isolates Isolate code rfbO157 fliCh7 vt1 vt2 eaeA hlyA TIR katP espA espB espF espP etpD type

1 5 2, 3, 4, 5 + + + + + + + + + + + + + PT 8
6 PT 32
2 15 1, 7, 13, 17, 19, + + ) + + + + + + + + + + PT 32
20, 23, 24 PT 31
25, 26, 27, 28 PT 21 ⁄ 28
31, 32, 33
3 6 8, 15, 18, 21, 22, 29 + + ) + + + + + + + + ) + PT 32
4 1 14 + + ) ) + + + + + + + + + PT 32
5 1 16 + + ) + + + + + ) ) ) ) + NT
6 1 30 + + ) + + + ) ) ) ) + + ) PT 21 ⁄ 28
7 1 12 + + ) + + + + ) ) ) ) ) ) NT
8 1 10 + + ) ) + + + ) ) ) ) ) ) NT
9 1 9 + + ) ) + ) + ) ) ) ) ) ) NT
10 1 11 + ) ) ) + ) + ) ) ) ) ) ) NT

Gene present, +; Gene absent, ).

Isolate was not phage typed.

sensitivity of the enrichment procedure is improved and
that injured and stressed pathogen cells are capable of
survival and growth in these conditions, which may not
occur in the presence of different cocktails of antibiotics,
such as those used in the present study. Low levels of
injured E. coli O157:H7 cells have recently been detected
in bovine faeces (Scott et al. 2006). In addition, it appears
that the prevalence of this pathogen is generally more
common in bovine than in sheep faeces, making the
detection of injured cells very important in indicating the
presence of this organism in sheep (Paiba et al. 2002;
Keen et al. 2006). The enrichment method used in the
present study may not therefore have been sufficiently
sensitive to detect the low levels of the organism and, in
particular, to detect the injured cells.
The majority of VTEC isolates recovered were from the
fleece (5Æ75%), and on visual inspection most of the fleece
samples examined were heavily soiled with faeces. As the
current work did not detect any E. coli O157:H7 in faeces,
it implies that the source of contamination in fleece may
be environmental. Ireland’s sheep industry is the third
largest farm enterprise in the country and most farmers
that keep sheep also have cattle. These animal husbandry
practices may contribute to the survival and recycling of
E. coli O157:H7 within the grazing herds (Heuvelink et al.
1998; Kudva et al. 1998; Cornick et al. 2000). Other
potential reservoirs of E. coli O157:H7 on mixed farms
include feed, water troughs, feedlot pens and the applica-
tion of manure to agricultural land (Hancock et al. 2001;
McGee et al. 2004; Scott et al. 2006).
Even if sheep and cattle have been farmed separately,
there is potential for transmission of VTEC to occur
between animals during transport and in abattoir holding
pens (Childs et al. 2006). Previous studies have indicated
a link between cattle hides with high levels of E. coli
O157:H7 present and carcass contamination (Tutenel
et al. 2003; Fegan et al. 2005), and it is possible that this
is also the case for sheep. Cattle remain an important res-
ervoir of the pathogen through faecal shedding (McGee
et al. 2004) and 7Æ3% of bovine hides at an Irish beef
abattoir were contaminated with E. coli O157 (O’Brien
et al. 2005). Similarly, fleece contaminated with E. coli
O157:H7 may be a source of carcass contamination for
sheep. A survey on the prevalence of E. coli O157:H7 in
sheep lairages by Small et al. (2002) identified critical
control points (e.g. unloading ramp, pen floor and water
trough) that can come in intimate contact with every sin-
gle animal passing through the slaughtering process,
which results in an ‘indirect mixing’ of animals from dif-
ferent groups or farms. The critical control points identi-
fied above may contribute to the pathogen’s persistence
in the abattoirs in the present study.
There was no evidence of seasonal variation of the
pathogen in the present study. This was not surprising
given that the majority of studies on seasonal variation
are based on the presence of the organism in animal fae-
ces (Chapman et al. 2000; McEvoy et al. 2003; Kudva
et al. 1996; Chapman et al. 2001). Escherichia coli
O157:H7 was isolated at four of the five abattoirs exam-
ined. The pathogen was not recovered at abattoir D, while
the percentage recovered in the other four abattoirs var-
ied. The absence at abattoir D was the most unusual and
a satisfactory explanation for this could not be found.
This difference may be attributed to a number of factors

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M. Lenahan et al.
that varied among the five abattoirs. Samples were taken
from three abattoirs that slaughtered both the cattle and
sheep (A, C and E), and the remaining two abattoirs
slaughtered only the sheep (B and D). Other differences
between abattoirs included geographic location, animal
origin (e.g. from mixed or nonmixed farms), animal
handling, transport of animals to the factories, lairage
design, cleaning and disinfection practices.
The predominant VTEC O157 PCR profile causing
clinical infections in Ireland has the toxin genotype of vt2
alone, with smaller percentages carrying genes for both
vt1 and vt2 (Carroll et al. 2005; Garvey et al. 2006).
Examination of the E. coli O157:H7 profiles from the pre-
sent study revealed that six of the profiles were similar to
those outlined above in relation to clinical infections,
while in the remaining four profiles the vt1 and vt2 genes
were missing. The isolates from these latter four profiles
may have contained the vt1 and vt2 genes previously, but
lost these during culturing or as a result of passage
through the animal (Karch et al. 1992; McCleery and
Rowe 2002). In addition, three of the four profiles also
have all of the TTSS genes investigated missing (i.e. espA,
espB, espD and espP).
The most common E. coli O157:H7 phage type recov-
ered in human isolates in Ireland in 2004 was PT 32
(63Æ5%), the second was PT 8 (10Æ5%) with only a small
number of PT 21 ⁄ 28 (4%) and PT 31 (1Æ9%) detected
(Carroll et al. 2005). The majority of isolates from the
present study were PT 32 (48Æ5%), followed by PT 8
(12Æ1%), PT 21 ⁄ 28 (12Æ1%) and PT 31 (12Æ1%). These
data suggest that the most common phage types found in
sheep in Ireland are also associated with clinical disease
in humans.
Of the VTEC isolates found, it was noted that six did
not possess the espA, espB, espF and espP genes required
as part of the organism’s TTSS. The TTSS apparatus plays
a critical role in the adherence of E. coli O157:H7 to
intestinal epithelial cells by directly translocating virulence
factors from the bacterium into the targeted host cells in
a single step (Garmendia et al. 2005). This allows the
pathogen to deliver several effector proteins to the cyto-
plasm of host epithelial cell that can subvert, inhibit, or
activate cellular processes beneficial for the bacterium
while allowing it to remain extracellular (Zhang et al.
2004; Garmendia et al. 2005).
The TTSS machinery comprises the products of
approx. 20 genes, and central to its development is the
EspA filament, a polymer of the translocator protein EspA
(Garmendia et al. 2005). Evidence suggests that EspA is
the major component of this filamentous structure, while
EspD, in complex with a second translocator protein,
EspB, is predicted to form a translocation pore in the
host cell membrane that facilitates subsequent entry of
ª 2007 The Authors
Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 2401–2409
E. coli O157:H7 in sheep
effector proteins, including TIR and EspF (Dahan et al.
2004). The EspA filament establishes a crucial transient
link between the bacterium and the host cell which facili-
tates the necessary intimate bacterial attachment through
intimin–TIR interactions (Garmendia et al. 2005).
Of the six isolates that did not possess the esp genes
investigated, three carried the vt2 gene, which has been
shown to be strongly associated with human illness
(Boerlin et al. 1999). However, without the esp genes pre-
sent, these three isolates are unlikely to be pathogenic, as
they would be unable to attach successfully to the intesti-
nal epithelium, and deliver toxin into the host cell. Simi-
larly, the three remaining isolates that are negative for
vt1, vt2, espA, espB, espF and espP are considered to be
nonpathogenic to humans.
In conclusion, results from the present study indicate
that the fleece and carcasses of sheep presented for
slaughter in Ireland are a source of E. coli O157:H7. The
absence of the pathogen from faeces was considered unu-
sual and may have been related to the isolation methodo-
logy used. PCR profiling showed that 87Æ9% of the E. coli
O157:H7 isolates possessed the vt2 gene, suggesting that
they could potentially cause human illness. However,
some of the VTEC isolates recovered may be more patho-
genic to humans than others. Previous research into the
role played by the type three secretion system suggests
that the isolates recovered in the present study missing
the TTSS genes would not be as efficient at attaching to
the host epithelial intestinal cell and therefore would be
less pathogenic to humans. Further investigation of these
isolates is required to determine if the absence of these
TTSS genes can significantly affect the virulence potential
of these isolates to humans or their ability to colonize the
host animal.
Acknowledgements
The authors would like to thank the Laboratory of
Enteric Pathogens, Colindale, London, UK for carrying
out phage typing of isolates. We acknowledge, with gratit-
ude, funding from the Department of Agriculture and
Food under the Food Institutional Research Measure
(FIRM), Project no: 01 ⁄ R&D ⁄ D ⁄ 170.
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