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Previous Article | Next Article 
Clinical and Diagnostic Laboratory Immunology, July 1999, p. 610-614, Vol. 6, No. 4
1071-412X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Use of a Monoclonal Antibody against an
Escherichia coli O26 Surface Protein for Detection of
Enteropathogenic and Enterohemorrhagic Strains
Paul
Kerr,1,*
Hywel
Ball,1
Bernard
China,2
Jacques
Mainil,2
David
Finlay,1
David
Pollock,1
Ian
Wilson,3 and
Dermot
Mackie1
Department of Agriculture for Northern
Ireland, Veterinary Sciences Division, Belfast BT4
3SD,1 and Northern Ireland Public Health
Laboratory, Belfast City Hospital, Belfast BT9
7AB,3 Northern Ireland, and
Facutlé de Médecine Vétérinaire,
Université de Liège, Liege, Belgium2
Received 3 December 1998/Returned for modification 17 February
1999/Accepted 3 May 1999
 |
ABSTRACT |
A monoclonal antibody (MAb) was obtained from a mouse immunized
with solubilized outer membrane proteins extracted from a bovine
enterohemorrhagic strain of Escherichia coli (EHEC), O26. The MAb produced a strong immunoblot reaction at approximately 21 kDa
for an O26 strain containing the intimin gene (eae) and verocytotoxin (VT), but not with an O26 eae- and
VT-negative strain, or O157 eae- and VT-positive strains.
The MAb was used in a sandwich enzyme-linked immunosorbent assay
(ELISA) format to screen strains from animal and human sources, and all
reactive strains were characterized for the presence of eae
and the gene encoding VT factors by PCR. The antigen was detected in a
group of strains containing a high proportion of O26, the majority of
which were eae positive with or without VT; these were
isolated mostly from animal enteritis cases but included a small number
of human enteric isolates. Nonreactors included
eae-positive (with or without VT) O157 strains and one O26
strain. In a survey of mixed cultures from both animal and human
enteric disease, ELISA-positive reactions were obtained from 7.1 to
11.2% of samples from bovine, porcine, ovine, and human sources. The
two human O8 and ten animal O26 ELISA-reactive pure strains obtained
from these samples contained six eae- and/or VT-positive
strains; the other six strains lost their ELISA positivity following
storage at 
70°C, after which none were found to contain either
eae or VT factors. The association of the antigen detected by the MAb with significant enteropathogenic E. coli and
EHEC virulence factors in isolates from both animal and human enteric infections indicates a diagnostic potential for the assay developed.
| |
INTRODUCTION |
Enterohemorrhagic Escherichia
coli (EHEC) has been defined as a pathogenic group of strains
characterized by their intimate attachment to the mammalian gut wall,
leading to the production of attachment and effacement (a/e) lesions,
and by verocytotoxin (VT) production (19). Another
pathogenic group, the enteropathogenic E. coli (EPEC), is
also characterized by causing a/e lesions but differs from EHEC in that
it does not produce VTs. Strains from both these groups are important
causes of human enteric diseases (29). EHEC strains have
become prominent in recent years as causes of hemorrhagic enteritis and
the hemolytic uremic syndrome. The main serogroup implicated in human
disease caused by EHEC has been O157 (10), but other
serogroups, in particular O26, O103, O111, and O128, have also been
implicated in causing human disease (13, 22, 32).
EHEC and EPEC strains are also associated with enteric disease in
cattle (5, 6, 8, 20, 21, 25, 27, 31, 33, 37). The
significance of these pathogenic groups in bovine enteritis is probably
underestimated, possibly because of a lack of awareness of their
significance and a lack of appropriate assays for routine detection.
The widespread presence of VT-producing E. coli strains in
healthy cattle is also a complication (3, 8, 26, 35).
Demonstration of VT in cultures from bovine enteritis is not sufficient
to imply a causative association.
The object of the present study was to produce monoclonal antibodies
(MAbs) to EHEC surface adhesion antigens, and to investigate their
diagnostic application for the detection of EHEC in animal and human
enteric infections. Because of an association with both human and
bovine diseases, an EHEC strain of serotype O26 was selected for investigation.
| |
MATERIALS AND METHODS |
Preparation of antigens.
An outer membrane (OM) preparation
of E. coli O26 strain 4276 was prepared by the standard
sarcosine extraction method (11). This strain was isolated
from a calf enteritis case in Northern Ireland and was characterized as
intimin (encoded by gene eae) and VT positive. Briefly,
washed cells from an overnight broth culture, suspended in 0.01 M Tris
HCl-0.005 M EDTA buffer, pH 7.8, were disrupted by ultrasonication.
After centrifugation at 15,000 × g for 30 min to
remove intact cells, the supernatant was mixed with a quarter volume of
2% (wt/vol) sodium n-lauroylsarcosine (Sigma) in Tris-EDTA
buffer at room temperature for 30 min and ultracentrifuged at
300,000 × g for 1 h. The resuspended pellet was
reextracted with an equal volume of 2% sarcosine for 1 h at room
temperature, repelleted, washed once in saline, and stored at 70°C.
Some of the washed OM was solubilized in a 6 M solution of the
chaotropic agent guanidine thiocyanate (Sigma) in Tris-EDTA. Insoluble
material was removed by ultracentrifugation, and the outer membrane
protein (OMP) solution was dialyzed against 100 volumes of 6 M urea in
Tris-EDTA buffer and stored at 70°C.
MAbs.
A BALB/c mouse was immunized intraperitoneally with
the solubilized OMP preparation of E. coli O26 strain 4276. Three inoculations of 100 µl, 50 µl, and 50 µl of OMP solution,
each mixed with 50 µl of adjuvant (125 µg of Quil A per ml)
(Superfos; DK-Vedbaek, Denmark), were given at 4-week intervals. Three
days after the final inoculation, the mouse spleen cells were fused
with the NSO myeloma cells at a ratio of 8:1 according to the protocol of Galfre and Milstein (12) with modifications by Teh and
Wong (34). The resulting hybridomas were maintained in RPMI
1640 medium (Gibco, Paisley, United Kingdom), supplemented with 20% gamma-globulin-free horse serum (Gibco).
The cell culture fluids from actively growing hybridomas were initially
screened by enzyme-linked immunosorbent assay (ELISA) in microtiter
plate wells (Dynatech, McLean, Va.) coated with OM preparations of
E. coli O26 strains 4276 (eae and VT positive) and 1045 (eae and VT negative). The hybridomas showing
specific reaction to strain 4276 antigen were cloned twice by limiting dilution.
Sandwich ELISA.
Ascites was produced by the intraperitoneal
inoculation of BALB/c mice with cloned hybridoma lines. The mice were
primed by intraperitoneal inoculation of Freund's incomplete adjuvant
2 days before cell inoculation (28). Ascites fluid was
removed from the mice approximately 10 days later and stored at
20°C. Immunoglobulin was purified from the ascites fluid by
caprylic acid precipitation (24).
The sandwich ELISA was performed on microtiter plates (Dynatech) as
previously described (2-4). Briefly, 100 µl of each
reagent was used per well. Optimum reagent dilutions were established by titration. The test samples were carried out in PTN (0.01 M phosphate-buffered saline [pH, 7.2] containing 0.04% [vol/vol] Tween 80 and additional NaCl [2%, vol/vol]). Between stages, the plate was washed six times with 0.01 M phosphate-buffered saline, pH
7.2, containing 0.05% (vol/vol) Tween 20. Purified MAb in 0.05 M
carbonate buffer, pH 9.5, was used to coat the wells either at 4°C
overnight or at 37°C for 1 h. The incubation stages thereafter were all 1 h at 37°C, except for the final substrate stage,
which was 10 min. The intervening sequential stages consisted of the test sample, the biotinylated MAb (16), and the
streptavidin-peroxidase (Sigma). The peroxidase substrate used was
3,3',5,5'-tetramethyl benzidine hydrochloride (Chemicon International,
Temecula, Calif.). The substrate reaction was stopped by the addition
of 50 µl of 2.5 M H2SO4 per well. The
absorbance was measured at 450 nm with an ELISA plate reader (Titertek
Mulitskan). The positive and negative controls consisted of E. coli strain 4276 and growth medium, respectively. Readings of
greater than three times the average negative-control value were taken
as positive reactions.
The sandwich ELISA sensitivity was determined by using dilutions of
strain 4276 after incubation at 37°C for 18 h on bovine blood
agar plates. Decimal dilutions were prepared in phosphate-buffered saline, and viable counts were determined on bovine blood agar after
overnight incubation.
A range of E. coli strains were used to evaluate the
sandwich ELISAs. These were selected as representative strains of
several pathogenic groups (Table 1).
Apart from the two rabbit O103 strains, which were obtained from A. Milon, Ecole Nationale Veterinaire, Toulouse, France, all were isolated
in Northern Ireland. In addition, the ELISAs were tested by using
representative strains from other Enterobacteriaceae genera
and assorted gram-negative species. Each strain tested was cultured
overnight at 37°C on bovine blood agar. A loopful of the colony was
mixed in 1 ml of PTN for application as a test sample to the sandwich
ELISA.
Test samples.
The MAb 2F3 sandwich ELISA was further applied
to a number of E. coli strains. These consisted of isolates
collected from field cases of animal enteritis in Northern Ireland,
from bovine enteritis cases in Belgium, and from healthy calves in
Belgium. A small number of bovine O157 strains previously isolated in
Northern Ireland (1) were also included, as were a small
number of isolates from cases of human diarrhea.
In addition, the assay was used to directly test mixed colony sweeps
from fresh overnight agar cultures of fecal samples, from animal and
human enteritis cases submitted to the Veterinary Sciences Division
laboratories in Belfast and the Northern Ireland Public Health
Laboratory, respectively. Six individual colonies from mixed agar
cultures, demonstrating a positive ELISA reaction, were purified and
tested individually. Purified, ELISA-positive reactants were
O-serotyped and tested for the presence of eae and VT as above.
PCR.
All of the ELISA-positive, and some of the
ELISA-negative, strains were tested for eae by PCR. A
proportion of these were similarly tested for the gene encoding VT. The
primers used in the procedure differed in the Northern Irish
(23) and the Belgian (7) laboratories.
Serogrouping.
All E. coli isolates obtained in
Northern Ireland were O-typed by slide agglutination (30),
by using a collection of 74 antisera, raised mainly against strains of
veterinary importance.
Immunoblotting.
Polyacrylamide gel electrophoresis was
carried out on whole-cell preparations of E. coli K-12 O26
(eae-positive and eae-negative strains) and O157
(eae-positive strains). NuPAGE 4 to 12% gels (Novex, San
Diego, Calif.) were used, in accordance with manufacturer's instructions. The bands were transferred onto nitrocellulose by overnight blotting at 30 mA. The nitrocellulose was blocked with 2%
(wt/vol) bovine serum albumin (Oxoid) in PTNE buffer (0.01 M PBS with
0.5% Tween 80 [vol/vol], 2 g of NaCl [wt/vol], 0.001 M EDTA),
pH 7.2. Following incubation with biotinylated MAb dilutions at 37°C
for 1 h, the nitrocellulose strips were washed in PTNE buffer
before the addition of streptavidin peroxidase for a further 1 h.
After another wash, the peroxidase substrate, 0.5 mg of
3,3'-diaminobenzidine tetrahydrochloride (Sigma) per ml in 0.02 M
Tris-HCl buffer, pH 7.2, with 0.3 µl of H2O2
(30% solution) per ml (vol/vol), was added. After incubation at room
temperature for 10 min, the strips were washed with distilled water,
which stopped any further reaction.
| |
RESULTS |
MAb-based sandwich ELISA.
Nine of the 430 hybridomas were
selected as being reactive to OM preparations of E. coli
strain 4276 and nonreactive to strain 1045. Three stable clones were
derived from these, one of which was no longer reactive with strain
4276. Ascites fluid was prepared with the remaining two lines, MAbs 2F3
and 6G5, and used to prepare capture and biotinylated MAb reagents for
use in sandwich ELISAs. Strain 4276 was used to optimize the assays.
Table 1 summarizes the sandwich ELISA results obtained with the
collection of E. coli strains initially examined. The MAb 6G5 sandwich ELISA reacted only with the strain used to immunize the
mouse for the hybridoma fusion. The MAb 2F3 sandwich ELISA reacted
positively with only three O26 strains containing eae and/or
the VT virulence factors. Neither of the assays reacted with
Salmonella arizonae, Salmonella kentucky,
Enterobacter spp., Klebsiella pneumoniae,
Shigella flexneri, Pseudomonas fluorescens, Pseudomonas putida, Hafnia alvei,
Serratia spp., Proteus vulgaris, Erwinia spp., Serratia liquefaciens, and
Citrobacter freundii.
The sensitivity of detection for the MAb 2F3 sandwich ELISA for strain
4276 was 105 CFU/ml.
Test samples.
The MAb 2F3 sandwich ELISA was used to screen
various groups of E. coli strains. Table
2 summarizes the characteristics of 46 ELISA-positive and 42 ELISA-negative strains; these were largely from a
collection of 216 strains isolated from animal enteritis cases in
Northern Ireland but included two ELISA-positive bovine O111 strains
from Belgium, 10 ELISA-negative Northern Ireland bovine O157 strains,
and ELISA-negative O118 (n = 5), O5 (n = 2)
O111 (n = 1), and O20 (n = 2) strains from
Belgian cattle. The majority of the ELISA-positive strains were O26,
although small numbers of other O-serotypes were evident. In addition, the majority of the ELISA-positive strains were either eae
or eae and VT positive, with low numbers of eae-
and VT-negative strains or strains only positive for VT. All of the O18
ELISA-negative and the majority of O26 ELISA-negative strains were
eae and VT negative, whereas both the ELISA-negative O111
strains were eae and VT positive. Included in Table 2 are
serotypes recognized as important causes of bovine enteritis: O5, O20,
O111, and O118 (10), and bovine isolates of O157, all of
which were positive for eae and/or VT.
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TABLE 2.
Characterization of the E. coli strains
demonstrating positive and negative reactions in the MAb 2F3
sandwich ELISA
|
|
Table 3 summarizes the ELISA results
obtained with the remaining Belgian bovine strains examined, none of
which was serotyped, but all of which were characterized for the
presence of eae, and some for the presence of VT. Twenty-one
of the 56 strains isolated from 0- to 10-week-old calves that had died
with enteritis were ELISA-positive, and 35 were ELISA negative; both
groups of these strains were entirely eae positive, with or
without VT. Twenty-three out of 67 E. coli strains isolated
from the intestinal contents of healthy 6-month-old calves, sampled at
an abattoir, were ELISA positive and all contained one or both of the
eae and VT virulence factors; 33 of the 44 ELISA-negative
strains also contained these factors. The third set of Belgian strains
tested were 190 eae-positive E. coli strains
isolated from two ~8-week-old calves with enteritis; out of these,
115 were ELISA positive and 78 were ELISA negative.
From six E. coli strains isolated from children <6 months
old, five were ELISA positive; these consisted of four O26 strains and
one O111 strain, all of which contained the eae and VT
virulence factors, as did the one ELISA-negative O111 strain.
The results of the field survey of mixed cultures from human and animal
diarrhea cases are summarized in Table 4.
ELISA-positive reactions were obtained with 7.1 to 11.2% of the
cultures tested from bovine, porcine, ovine, and human origins; single
cultures were also recorded positive for the 8 canine and 31 avian
strains examined. Pure ELISA-positive strains were obtained from 12 of these mixed cultures; these consisted of two O8 strains, from human
samples, and 10 O26 strains, one from an ovine sample and nine from
bovine samples. Only six of these (one O8 and five O26 strains)
retained their ELISA-positive activity on retesting following storage
at 70°C. The single O8 strain and three of the O26 strains were PCR
positive for both eae and the gene encoding VT, and the other two O26 strains were positive for only eae or only the
gene encoding VT. The six strains that had lost their ELISA activity were PCR negative for these virulence factors.
Immunoblotting.
A strong immunoblot reaction was demonstrated
at approximately 21 kDa for strain 4276 (O26, eae and VT
positive), with MAb dilutions of up to 1:10,000 (Fig.
1). No immunoblot reactions were observed
for K-12 or for strain 1045 (O26, eae and VT negative), S784
(O157, eae and VT positive), or 3680 (O157, eae
positive and VT negative).
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FIG. 1.
Nitrocellulose immunoblot with MAb 2F3 with whole-cell
preparations of E. coli strains K-12 (A), 1045 (O26;
eae and VT negative) (B), 4276 (O26; eae and VT
positive) (C), S784 (O157; eae and VT positive) (D), and
3680 (O157; eae positive and VT negative) (E).
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| |
DISCUSSION |
The MAb 2F3 produced in this study demonstrated a high level of
specificity for a group of E. coli strains, in particular, strains of serotype O26, with the potential to express the EHEC and
EPEC virulence factors of eae and VT. eae is the
gene for the expression of intimin, which is regarded as a significant virulence factor in both EHEC and EPEC strains. If it is assumed that
all strains with eae are potentially pathogenic, the
application of MAb 2F3 in a sandwich ELISA format enables the rapid
detection of a group of pathogenic strains from within these groups.
The identity of the antigen detected by MAb 2F3 is not clear.
Immunoblotting demonstrated a strong reaction at 21 kDa with only the
eae- and VT-positive O26 antigen used. From the molecular weight and surface presence of this protein, it is possible that it is
fimbriae (14) or the recently described EspA protein
(18). The former is indicated by the loss of antigen from
six strains following storage, possibly from plasmid loss. Fimbriae
implicated in early host cell adhesion of EPEC strains, named the
bundle forming pili (bfp), have been defined as plasmid located
(14). Giron et al. (15), using a molecular probe,
demonstrated that bfp were only present in the EPEC strains of the
human pathogenic E. coli strains that were examined. China
et al. (9) failed to find bfp in animal EPEC or EHEC strains
by using this human EPEC probe. Wieler et al. (36)
demonstrated a significant increase in cell attachment of bovine EHEC
O118 strains on fetal calf lung cells (90.5%) compared with human
HEp-2 cells (52.4%) by using the fluorescent actin staining test
(17). These studies indicate differences in adhesins between
EPEC and EHEC strains. It is possible that the antigen detected in the
present study is an alternative to bfp for preliminary cell attachment.
If this is confirmed, since it was demonstrated in both human- and
animal-isolated strains, it must be concluded that either there is a
common host receptor or that these strains possess more than a single
host cell attachment mechanism.
The loss of the antigen detected by the ELISA in six strains which did
not possess either the eae or the VT factor and its presence
in a small number of VT-positive and eae-negative isolates demonstrate its occurrence in non-EPEC and non-EHEC strains. It can be
speculated that the presence of eae and/or VT provides some
plasmid stability, but whether these strains are of any pathological significance is unknown and requires further investigation.
Nonpathogenic strains, such as many VT-producing strains, can express a
virulence factor(s). It is recognized that virulence is the result of a combination of factors which, individually, have limited pathogenic effect. Apart from experimental infection, the significance of these
factors in bacterial strains is determined by their presence in
combination with other factors, and by their more-common occurrence in
strains isolated from diseased animals or humans. The association of
the vast majority of E. coli strains that reacted with MAb 2F3 to the presence of the gene for intimin, which is regarded as a
virulence factor of notable significance, is a strong indication of the
importance of the antigen to which it reacts.
The association of MAb 2F3 with eae was in the presence
or absence of VT factors.
Because MAb 2F3 associated with
eae in the presence and absence of VT factors, the assay
developed could not distinguish between EHEC and EPEC strains. This
result indicates a close relationship between the strains detected from
the two pathogroups. Whether the virulence differences of these groups
are associated with the presence or absence of VT requires further investigation.
A number of eae-positive strains with or without VT did not
react with MAb 2F3. These include a number of strains of recognized bovine (O118 and O5), rabbit (O103), and human (O157) pathogenicity. In
addition, of the 10 eae- or eae- and VT-positive
O111 strains examined, only eight were ELISA reactive. If the antigen
targeted by this MAb is confirmed as making a significant contribution to the virulence of EHEC and EPEC strains, it could be concluded that
these nonreactive strains possess antigenic variations of this factor.
The high prevalence of O26 strains amongst the positive reactants to
this MAb indicates the probable significance of this serotype in animal
enteritis in Northern Ireland. A number of O26 strains were also
present in the ELISA-negative group, the majority of these being
eae and VT negative. This indicates that other pathogroups
of this serotype are of probable significance in this condition.
Although only a small number of strains from human diarrhea were
tested, the demonstration, by ELISA, of a common antigen in bovine and
human isolates could indicate a zoonotic risk of bovine strains to
humans. The commonality of the MAb-detected antigen was also
demonstrated in the results obtained with isolates from the field
survey of human and animal enteritis (Table 4). The fact that strains
from the same serotypes have been implicated in both bovine and human
diseases (O26, O111, and O18) also supports these findings. The
presence of a high percentage of EHEC and EPEC strains in both
ELISA-positive and -negative groups isolated from healthy calves
sampled at an abattoir (Table 4) indicates a significant potential for
infection of susceptible cattle and for zoonotic transfer to humans.
The results of the survey conducted with nearly 1,000 animal and human
enteric isolates demonstrated a significant presence of strains
expressing the targeted antigen (Table 4), in particular from bovine,
human, ovine, and porcine samples. Although only five EHEC-EPEC strains
were purified from these samples by the limited method employed, the
presence of a virulence-associated antigen was demonstrated in a high
proportion of the mixed cultures. This finding indicates a significant
pathogenic role of EHEC and EPEC strains in both human and animal
enteric diseases and highlights the diagnostic potential of the assay
developed. Further studies to develop MAbs to surface antigens of the
eae-positive strains that were nonreactive to the O26 MAb in
this study would clarify the significance of the antigens in terms of
virulence and virulent-strain detection.
| |
ACKNOWLEDGMENTS |
The technical assistance of Neill Brice is gratefully
acknowledged, as is the help of our colleagues from the institute's diagnostic laboratory. In addition, we are indebted to Vinciane Pirson
for the work carried out at Liège.
The Department of Health (under grant DH Code 246) financially
supported this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Veterinary
Sciences Division, Stoney Rd., Stormont, Belfast, Northern Ireland BT4
3SD. Phone: 44 (0) 1232 525694. Fax: 44 (0) 1232 525745. E-mail:
pgkerr{at}qub.ac.uk.
| |
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Clinical and Diagnostic Laboratory Immunology, July 1999, p. 610-614, Vol. 6, No. 4
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Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
