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Abstract Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli isolated from cattle
were studied by DNA colony hybridization to subtype their intimin-encoding (eae) gene with probes derived
from the variable parts of the eae gene of the human EPEC strain E2348/69, the eae gene of the human
O157:H7 EHEC strain ATCC43888, and the eae gene of the bovine O26:H- EHEC strain 193, whose eae
gene was first cloned and sequenced during this work. The EPEC and EHEC had been isolated from
diarrhoeic calves (143 EPEC and 48 EHEC) and from healthy animals at the slaughterhouse (10 EPEC and 34
EHEC). The 191 bovine EPEC and EHEC isolated from diseased calves were positive with the Eae probe (55
and 27% respectively) and with the Eae probe (9 and 73% respectively), whereas 52 EPEC (36%) were
negative with the Eae, Eae, and Eae probes. The results were different for the 44 bovine EPEC and EHEC
isolated from healthy cattle at slaughterhouses: most tested positive with the Eae probe (80 and 82%
respectively) and the remaining (20 and 18% respectively) with the Eae probe. Nine O26 human EHEC tested
positive with the Eae probe and seven O111 with the Eae probe. The bovine and human EPEC and EHEC
belonging to these two serogroups gave identical results: the 18 bovine and human O26 isolates tested
positive with the Eae probe, whereas the 13 O111 isolates were positive with the Eae probe. In contrast, the
isolates belonging to other serogroups (O5, O15, O18, O20, and O118) gave more variable results. The eae and
eae, but not the eae, variants were thus distributed amongst bovine EPEC and EHEC. The eae variant
seemed to be more frequently associated with the presence of clinical signs in calves, but one third of EPEC
from diarrhoeic calves carried an eae gene variant other than the , , or variants. In addition, the use of these
gene probes did not enable differentiation between bovine and human EHEC belonging to the same O
serogroup. Elsevier, Paris
eae gene / Escherichia coli / locus of enterocyte effacement / cloning / gene probe
1. Introduction
Enteropathogenic (EPEC) and enterohaemorrhagic (EHEC) Escherichia coli cause enteritis
* Correspondence and reprints
Tel.: 32 4 366 4090
Fax: 32 4 366 4056
jg.mainil@ulg.ac.be
Abbreviations: AE, attaching/effacing; AEEC, attaching/
effacing E. coli; BEHEC, bovine EHEC; BEPEC, bovine
EPEC; DEPEC, dog EPEC; EHEC, enterohaemorrhagic
E. coli; EPEC, enteropathogenic E. coli; HEHEC, human
EHEC; HEPEC, human EPEC; IPTG, isopropyl-betathiogalactoside; LEE, locus of enterocyte effacement; PEPEC, pig EPEC; REPEC, rabbit EPEC.
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China et al.
EPEC and EHEC strains isolated from diarrhoeic calves, healthy adult cattle, and humans.
The eae gene was cloned from the O26:K-:HBEHEC strain 193 originally isolated in 1962
from a diarrhoeic calf in the USA [22]. Collections of E. coli strains were subsequently studied by colony hybridization to determine the
variants of the eae gene present: 143 BEPEC and
48 BEHEC strains isolated from 2- to 8-week-old
calves with clinical diarrhoea [11, 23]; 10 BEPEC
and 34 BEHEC strains isolated from healthy
cattle at slaughterhouses [11]; and 16 HEHEC
strains received from Dr D. Pirard (AZ-VUB,
Brussels, Belgium). The definition of EPEC
must, however, be taken with some caution, as
EHEC can lose their stx genes in vitro or in
vivo [18, 19, 23]. Twenty-one BEPEC and BEHEC strains from diarrhoeic calves and 18 from
healthy cattle had been serotyped previously:
they belonged to the O5, O15, O18, O20, O26,
O111, and O118 serogroups [23] (unpublished
data). The HEHEC strains belonged to O26
(nine isolates) or O111 (seven isolates) serogoups (Pirard, personal communication).
Three strains were used as positive controls
for the gene probes: the HEPEC strain
E2348/69, carrying an eae gene, the REPEC
strain RDEC1, carrying an eae gene, and the
O157:H7 HEHEC strain ATCC43888 strain, carrying an eae gene [2]. The BEHEC strain 193
carrying a still uncharacterized eae gene was the
fourth control strain. Strain HS was the negative
control for all probes [22, 23, 28].
2.2. Cloning and sequencing of the eae gene
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3. Results
3.1. Cloning of the eae gene
from O26:K-:H-BEHEC strain 193
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China et al.
Figure 1. Restriction map and subclones of the pCUR3 recombinant plasmid DNA insert carrying the eae gene of the O26 BEHEC
strain 193 (1 = start codon <ATG>; 939 = stop codon <TAA>) and derivation of the Eae probe. Restriction sites above the line of
pCUR3 were conserved compared to the restriction maps of the eae gene of the HEPEC strain E2348/69 and of the eae gene of
O157:H7 HEHEC strains; restriction sites below the line were not. The pCUR25H fragment was used as an Eae probe; the Eae
(amplification with primers B75 and B76 and restriction by HinfI) and Eae (amplification with primers B73 and B74) probe fragments
are also presented.
327
Table I. Identity between the eae gene of the O26 BEHEC strain 193 and the eae [17], eae [3], eae [6, 40], and eae [4] gene
variants.
% Identity with the respective eae variant
eae Gene
eae
eae
eae
eae
Strain
E2348/69 (HEHEC)
RDEC1 (REPEC)
EDL933 (HEHEC)
4221 (DEPEC)
Overall
(2.8 kb)
Proximal
(1.8 kb)
Distal
(1.0 kb)
83 %
99.9 %
83 %
NA
94 %
100 %
93 %
NA
62 %
98 %
65 %
77 %
Figure 2. Phylogenetic tree representation of the sequences of the distal third (1.0 kb) of the different eae gene variants. Gene bank
access numbers: RDEC1 = U60002; 193 = AF043226; 4221 = U66102; E2348/69 = AF022236; O157:H7 = AF071034.
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China et al.
Table II. Colony hybridization results of bovine and human EPEC and EHEC with gene probes derived from the variable 3 ends of the
eae genes coding for intimins (Eae probe), (Eae probe), and (Eae probe).
Type of isolate
(no. isolates)
Origin
Pathotype
Eae
Eae
Eae
(%)
EPEC (143)
EHEC (48)
78 (55)
13 (27)
13 (9)
35 (73)
52 (36)
EPEC (10)
EHEC (34)
2 (20)
6 (18)
8 (80)
28 (82)
Humans (16)
EHEC (16)
9 (55)
7 (45)
4. Discussion
AEEC, either enterohaemorrhagic (EHEC) or
enteropathogenic (EPEC), cause enteric diseases
Table III. Correlation between serogroup O of bovine and human AEEC strains and probe hybridization results (strains are EHEC
unless otherwise stated).
Serogroup O
O5
O15
O18
O20
O26
O111
O118
a
Eae probe
Healthy cattle
Humans
4/4
2/2
2/3a
1/3
6/6b
1/1
6/6
1/4
3/4
1/2
1/2
3/3
5/5
4/4
9/9
7/7
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Eae
Both are EPEC. bFive are EPEC and EHEC is strain 193.
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China et al.
Acknowledgments
their serotypes. This work was financially supported by the Ministre fdral des classes
moyennes et de lagriculture, Brussels, Belgium
(research contract 5740), the Fonds national de
la recherche scientifique (Crdit aux chercheurs,
1995), and the University of Lige (Crdit du
conseil de la recherche, 1996).
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