Note: Descriptions are shown in the official language in which they were submitted.
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ANTIBODIES FOR PREVENTING AND TREATING
ATTACHING AND EFFACING ESCHERICHIA COLI (AEEC) ASSOCIATED
DISEASES
FIELD OF THE INVENTION
The present invention relates to antibodies immunologically specific for an
attaching and effacing Escherichia coli (AEEC) virulence-associated protein
and to
to the use thereof in the prevention of an AEEC infection in a mammal.
BACKGROUND OF THE INVENTION
Escherichia coli is associated with a wide variety of intestinal diseases in
human and in animals. Some pathogenic E. coli produces attaching and effacing
(A/E) lesions, characterized by intimate bacterial adherence to enterocytes
and
disruption of the underlying cytoskeleton. Such isolates have been termed A/E
E.coli
(AEEC).
Reports have shown that A/E lesions are characteristic of enteric pathogens
2 0 of humans such as enteropathogenic E. coli (EPEC) responsible for severe
childhood
diarrhea in the developing countries, and enterohemorrhagic E. coli (EHEC)
causing
hemorrhagic colitis and hemolytic uremic syndrome (HUS). A/E lesions have also
been associated with diarrhea in different animal species such as rabbits,
calves,
dogs, cats, Iambs, and pigs.
A/E lesions result from the intimate bacterial adherence to the apical surface
of the enterocytes and activation of several chromosomal gene products that
interact
with components of the host cell. Such gene products are commonly called AEEC
virulence-associated proteins. Examples of these virulence-associated proteins
are
the intimin (Eae) and the secreted proteins Tir (translocated intimin
receptor), EspA,
3 o EspD, and EspB.
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Currently, the only approach to the control and treatment of AEEC-associated
diseases is the use of antibiotics, an approach which is becoming less and
less
desirable due to problems of bacterial resistance and antibiotic residues.
Therefore,
alternative approaches to control post-weaning diarrhea must be sought.
s Alternatively, another approach known in the art suggests the use of AEEC
virulence-associated proteins as antigens to immunize an animal in order to
stimulate
antibody production against the related pathogen. However, no indication of
the
efficacy of this approach has been shown to prevent AEEC infection in a
mammal.
Examples of such an alternative approach are shown in US patent 6,204,004 and
in
to international patent applications WO 99/41614; WO 97/40063 and WO 99/24576.
Also known in the art is the use of avian antibodies (IgY) in passive
immunization. For instance, WO 00/52055 discloses the use of specific egg yolk
IgY
antibodies for immunotherapy in animal breeding and animal production. It also
discloses the use of IgY antibodies in kits for diagnostics.
15 US patent 5,932,250 is directed to the treatment of vascular disorders
particularly arteriosclerosis and atherosclerosis in warm-blooded animals.
More
specifically, this U.S. patent discloses methods of controlling cholesterol
levels, lipid
deposits, and the development of atheromatous lesions in warm-blooded animals
by
the ingestion of egg products containing IgY antibodies raised against
Escherichia coli
2 o proteins.
US patent 6,162,441 discloses a method for producing anti-E. coli 0157 IgY
antibodies in egg-laying hens.
The problem with WO 00/52055, 5,932,250 and 6,162,441 is that the in vivo
efficacy of the IgY produced has not been demonstrated. Indeed, none of these
2 s contain evidence with respect to the feasibility of an approach consisting
of
administering to a mammal, an antibody immunologically specific for an AEEC
virulence-associated protein for preventing an in vivo AEEC intestinal
infection.
Therefore, there is still a need for antibodies, egg products and methods for
the
prevention or treatment of AEEC-mediated diseases.
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SUMMARY OF THE INVENTION
The present invention relates to antibodies immunologically specific for an
attaching and effacing Escherichia coli (AEEC) virulence-associated protein
and to
s the use thereof in the prevention of an AEEC infection in a mammal.
According to a first aspect, the invention relates to an antibody
immunologically specific for an AEEC virulence-associated protein, the
antibody
being capable of preventing an in vivo AEEC intestinal infection when
administered
to a mammal.
to In a preferred embodiment, the antibody of the invention is resistant to
gastrointestinal digestion. More preferably, the antibody of the invention is
an IgY
antibody.
The antibody of the invention is preferably capable of preventing the adhesion
of the AEEC to the intestine of the mammal. Even more preferably, the antibody
of
15 the invention is capable of preventing the development of attaching and
effacing
(A/E) intestinal lesions associated with the AEEC. This aspect of the
invention is
achieved by particularly using antibodies immunologically specific for one or
more
AEEC virulence-associated proteins, such as Eae, Tir, EspA and Paa.
According to another aspect, the invention relates to a fowl egg and an
2 o isolated yolk of such egg containing an antibody as defined hereinabove.
According to a further aspect, the invention relates to a composition which
comprises a biologically acceptable vehicle or carrier and an antibody as
defined
above, a fowl egg as defined above; and/or its isolated yolk as defined above.
According to another aspect, the invention relates to a food additive which
2 s comprises an antibody as defined above, a fowl egg as defined above, a
yolk fraction
as defined above, and/or a composition as defined above.
According to a further aspect, the invention relates a process for obtaining
an
antibody as defined herein above, the process comprising the steps of:
a) actively immunizing a fowl hen for eliciting the production of antibodies
in
3 o an egg of the hen; and
b) recovering the antibodies from the egg.
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According to a further aspect, the invention relates to a method for
preventing
an attaching and effacing Escherichia coli (AEEC) infection in a mammal. The
method comprises the step of orally administering to the mammal an antibody as
defined above, a fowl egg as defined above and/or an isolated yolk as defined
above.
A major advantage of the invention is that it provides antibodies, products,
compositions and methods useful and efficient for the prevention of an AEEC
infection in a mammal.
Furthermore, it is particularly an advantage that the antibodies, products,
to compositions and methods of the invention prevent specifically the
development of
A/E intestinal lesions associated with the AEEC.
Other objects and advantages of the present invention will be apparent upon
reading the following non-restrictive description of several preferred
embodiments,
made with reference to the accompanying drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a bar graph showing that AEEC strains originating from the rabbit,
pig,
dog, bovine, and human (including non-0157 EHEC and EPEC), and producing Eae
of the a, Vii, b, or s subtypes, induced A/E lesions equally well on newborn
pig ileal
explants.
Figure 2 is a bar graph showing that a replacement of the Eae of the y subtype
by the
Eae of the a subtype in an 0157:H7 strain resulted in induction of A/E lesions
to a
2 5 similar extent as observed for the homologous porcine AEEC strain.
Figure 3 is a bar graph showing that antibodies according to a preferred
embodiment
of the invention are able to significantly block the development of A/E
lesions in ileal
explants caused by the homologous strain 1390.
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S
Figure 4 is a bar graph showing that antibodies according to a preferred
embodiment
of the invention are able to significantly block the development of A/E
lesions in ileal
explants caused by various calf AEEC strains.
s Figures 5 and 6 are bar graphs showing that antibodies according to a
preferred
embodiment of the invention are able to significantly block the development of
A/E
lesions in ileal explants caused by various human AEEC strains.
Figure 7 is a bar graph showing that antibodies according to a preferred
embodiment
of the invention are able to significantly block the development of A/E
lesions in ileal
explants caused by various EPEC strains.
Figures 8A and 8B are bar graphs showing that oral administration of egg yolk
antibodies according to a preferred embodiment of the invention significantly
inhibit
the development of A/E lesions in the cecum and colon of piglets challenged
with an
homologous porcine AEEC strain.
Figures 9A and 9B are bar graphs showing that oral administration of egg yolk
antibodies according to a preferred embodiment of the invention inhibit the
2 o development of A/E lesions in the cecum and colon of piglets challenged
with an
0157:H7 AEEC strain.
DETAILED DESCRIPTION OF THE INVENTION
2 s As mentioned previously, the present invention relates to a new
alternative
approach as opposed to antibiotics for the prevention of an AEEC infection in
a
mammal.
More precisely, the present invention relates to antibodies immunologically
specific for an AEEC virulence-associated protein, these antibodies being
capable
30 of preventing an in vivo AEEC infection when administered in a mammal. More
particularly, the antibodies of the invention are capable of preventing the
adhesion
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of the AEEC to the intestine of the mammal, and even more preferably, they
prevent
the development of A/E intestinal lesions associated with the AEEC.
As used herein, the term "preventing" refers to a process by which the AEEC
infection is obstructed or delayed.
s As used herein, the term "mammal" refers to any mammal that has the
possibility of being infected by an AEEC. Among the mammals which are known to
be potentially infected by an AEEC, there are humans, pigs, bovines, ovines,
caprines, rabbits, dogs and cats. It will be apparent to one skilled in the
art that the
antibodies of the invention are intended to be immunologically specific to
virulence
1 o proteins isolated form a variety of AEEC strains that may cause intestinal
lesions,
such as those of enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli
(EHEC). Some examples of EPEC and EHEC strains are shown in Table I.
With respect to antibodies of the invention, the term "immunologically
specific" refers to antibodies that bind with a relatively high affinity to
one or more
15 epitopes of a protein of interest, but which do not substantially recognize
and bind
molecules other than the ones) of interest. As used herein, "antibody" and
"antibodies" include all of the possibilities mentioned hereinafter:
antibodies or
fragments thereof obtained by purification, proteolytic treatment or by
genetic
engineering, artificial constructs comprising antibodies or fragments thereof
and
2 o artificial constructs designed to mimic the binding of antibodies or
fragments thereof.
Such antibodies are discussed in Colcher et al. (Q J Nucl Med 1998; 42: 225-
241 ).
They include complete antibodies, F(ab')2 fragments, Fab fragments, Fv
fragments,
scFv fragments, other fragments, CDR peptides and mimetics. These can easily
be
obtained and prepared by those skilled in the art. For example, enzyme
digestion can
25 be used to obtain F(ab')2 and Fab fragments by subjecting an IgG molecule
to pepsin
or papain cleavage respectively. Recombinant antibodies are also covered by
the
present invention.
Alternatively, the antibody of the invention may be an antibody derivative.
Such an antibody may comprise an antigen-binding region linked or not to a non-
3 o immunoglobulin region. The antigen binding region is an antibody light
chain variable
domain or heavy chain variable domain. Typically, the antibody comprises both
light
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and heavy chain variable domains, that can be inserted in constructs such as
single
chain Fv (scFv) fragments, disulfide-stabilized Fv (dsFv) fragments,
multimeric scFv
fragments, diabodies, minibodies or other related forms (Colcher et al. Q J
Nucl Med
1998; 42: 225-241 ). Such a derivatized antibody may sometimes be preferable
since
s it is devoid of the Fc portion of the natural antibody that can bind to
several effectors
of the immune system and elicit an immune response when administered to a
human
or an animal. Indeed, derivatized antibody normally do not lead to immuno-
complex
disease and complement activation (type III hypersensitivity reaction).
Alternatively, a non-immunoglobulin region is fused to the antigen-binding
1 o region of the antibody of the invention. The non-immunoglobulin region is
typically a
non-immunoglobulin moiety and may be an enzyme, a region derived from a
protein
having known binding specificity, a region derived from a protein toxin or
indeed from
any protein expressed by a gene, or a chemical entity showing inhibitory or
blocking
activity(ies) against the AEEC virulence-associated proteins. The two regions
of that
15 modified antibody may be connected via a cleavable or a permanent linker
sequence.
Since the AEEC infection is generally localized in the intestinal tract, it is
highly
preferable that the antibody of the invention be resistant to gastrointestinal
digestion.
As used herein, the term "resistant" refers to an antibody that will
substantially retain
its immunological function even after being in contact with gastric acids for
a period
2 0 of time necessary to prevent an in vivo AEEC infection. Preferably, the
antibody of
the invention is an avian immunoglobulin, such as IgY. Indeed, it is well
known that
IgY antibodies show great acid and heat resistance. The antibody of the
invention
may be also a human or animal immunoglobulin such as IgG1, IgG2, IgG3, IgG4,
IgM, IgA, IgE or IgD carrying rat or mouse variable regions (chimeric) or CDRs
2 s (humanized or "animalized"). However, the invention is not restricted to
IgY antibodies
since it is conceivable that the resistance capacity of the antibody, if
lacking, can be
provided or increased by genetic engineering or by any other way known to one
skilled in the art. Furthermore, the antibody of the invention may also be
conjugated
to any suitable carrier known to one skilled in the art in order to increase
its resistance
3 o to gastric acids or to provide, for instance, a specific delivery and
prolonged retention
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of the antibody, either in a targeted local area, such as the intestine, or
for a systemic
application.
As mentioned above, the antibody of the invention is immunologically specific
to one or more AEEC virulence-associated proteins. The preferred AEEC
virulence-
s associated proteins contemplated by the invention are Eae, Tir, EspA, Paa
and
immunological derivatives thereof. As used herein, the term "immunological
derivative" refers to a protein/peptide that possesses an immunological
activity that
is substantially similar to the immunological activity of the whole
protein/peptide, and
such immunological activity refers to the capacity of stimulating the
production of
to antibodies immunologically specific to an AEEC virulence-associated protein
or
derivative thereof. The term "immunological derivative" therefore encompass
"fragments", "segments", "variants", or "analogs" of a protein/peptide.
In a highly preferred embodiment, the present invention uses IgY antibodies
since and as mentioned above, they advantageously show gastric acid
resistance.
15 Furthermore, IgY antibodies have the advantage of not reacting with
mammalian
complement, Fc receptor, protein A or protein G. Also, IgY antibodies produced
in
eggs and their extraction from egg yolks can be performed on a large scale
without
costly investment.
In this connection and according to another aspect, the present invention is
2 o also directed to a process for obtaining the above mentioned antibody
immunologically specific for an AEEC virulence-associated protein. Although,
many
processes known in the art may be suitable to obtain the antibodies
contemplated by
the inventors, it is preferable that the process of the present invention
comprises the
steps of : a) actively immunizing a fowl hen for eliciting the production of
antibodies
2 s in an egg of the hen; and b) recovering the antibodies from the egg. A
person skilled
in the art will understand that the immunizing step is achieved by well known
methods. For instance, the AEEC-virulence protein may be given parenterally,
for
example intravenously, intramuscularly, subcutaneously: As used herein, the
term
"fowl" refers to any birds capable of being immunized. Among those, the common
3 o domesticated chicken is preferred. The process of the invention may also
comprises
a step of administering at least one booster of the proteins to maintain a
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hyperimmune state in the hen. Furthermore, the process of the invention
preferably
comprises a step of purifying the antibodies from a yolk fraction of the egg.
Again, the
purification step is achieved with methods well known to one skilled in the
art. Such
a person will also understand that the boosting step is not limited to being
done
s before the step of laying the egg. Indeed, a booster may also be given to
the same
hen even during or after the egg laying step.
Consequently, the present invention also relates to a fowl egg which contains
an antibody of the invention. The present invention further relates to an
isolated yolk
of the egg.
to The present invention also describes a method and compositions for the
prevention of AEEC-associated diseases. Many methods could be used to reduce
to
practice the present invention. However, it is particularly believed that a
method which
comprises the step of orally administering to a mammal an antibody of the
invention,
a fowl egg and/or an isolated yolk as previously defined is particularly
advantageous
15 from a commercial point of view. Nevertheless, methods that involve a
parenteral
administration of the antibody of the invention may be considered by one
skilled in
the art.
According to a preferred embodiment, the composition of the invention
comprises at least one element immunologically active against AEEC and a
2 o biologically acceptable vehicle or carrier. Such an element may either be
an antibody,
a fowl egg or an isolated yolk as defined above. For preparing the
compositions of
the present invention, methods well-known in the art may be used. As used
herein,
the term "immunologically active", or reference to the immunological activity
of an
element, such as an antibody of the invention, refers in that instance to the
ability of
2 s such antibody to prevent an AEEC infection in a mammal by binding to an
AEEC
virulence-associated protein. As used herein, the term "biologically
acceptable"
refers to a vehicle or a carrier that can be safely administered to a mammal,
particularly humans and animals, without overly negative or toxic side
effects. Such
vehicle or carrier may be used for various purposes, such as preserving
agents,
3 o solubilizing agents, stabilizing agents, wetting agents, emulsifiers,
sweeteners,
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colorants, odorants, salts, buffers, coating agents or antioxidants and the
like. They
may be readily prepared by those of skill in this art using well known
methods.
According to another embodiment, the composition of the invention is
formulated under the form of a pharmaceutical or a nutraceutical composition.
The
s present invention also provides a food additive comprising at least one of
the above
mentioned elements and further comprises a composition as defined above. It
will be
clear to one skilled in the art that although the compositions of the
invention are
preferably administered orally, they may be administered by any other suitable
route.
Indeed, it is conceivable that they could be given by other means. In the case
that the
1o compositions are given orally, they may be in the form of tablets,
capsules, powder,
syrups, etc.
The compositions of the invention may be used in conjunction with
pharmaceutical compositions known in the art. For instance, one may find it
advantageous to combine one of the elements of the composition of the
invention
with other active agents that may be used to treat or prevent diseases others
than
those induced by AEEC.
The amount of specific antibodies that is administered to a human or an
animal or that is present in the composition of the invention is a
therapeutically
effective amount. A therapeutically effective amount of antibody is that
amount
2 o necessary for obtaining beneficial results without causing overly negative
secondary
effects in the host to which the antibody or composition is administered.
Moreover,
an effective amount of an antibody for treating a particular disease is an
amount that
is sufficient to ameliorate, or in some manner reduce the symptoms associated
with
the disease. Such an amount may be administered as a single dosage or may be
2s administered according to a regimen, whereby it is effective. The amount
may cure
the disease but, typically, is administered in order to ameliorate the
symptoms of the
disease.
The exact amount of antibody or of each of the components in the composition
and amount of the composition to be administered will vary according to
factors such
3 o as the type of the condition to be treated, the other ingredients in the
composition, the
mode of administration, the age and weight of the mammal, etc. Without being
bound
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by any particular dosage, it is believed that for instance for oral
administration, a daily
dosage of about 100 to about 600 mg/kg of lyophilised egg yolk containing
antibodies immunologically specific for an AEEC virulence-associated protein
(usually
present as part of a composition as indicated above) may be suitable for
preventing
s a mammalian AEEC infection in a typical adult. This dosage may be repeated
as
often as appropriate. Typically, administration may be 1 to 21 times a week.
If side
effects develop, the amount and/or frequency of the dosage can be reduced.
EXAMPLE
to
The following example is illustrative of the wide range of applicability of
the
present invention and is not intended to limit its scope. Modifications and
variations
can be made therein without departing from the spirit and scope of the
invention.
Although any method and material similar or equivalent to those described
herein can
1 s be used in the practice for testing of the present invention, the
preferred methods and
materials are described.
Summary
2o The inventors have successfully produced egg yolk antibodies against
purified
fusion proteins of the known attaching and effacing virulence factors Eae,
EspA,
EspB, EspD, and Tir and of a new putative attaching and effacing virulence
factor
Paa. They have demonstrated that only the anti-Eae, anti-Tir, and anti-Paa
antibodies
were able to significantly block the development of attaching and effacing
(A/E)
2 s lesions due to the homologous porcine E, coli strain ex vivo in the pig
ileal organ
culture model. These antibodies were also able to block the development of A/E
lesions due to attaching and effacing E. coli originating from various other
animal
species such as the bovine and dog, and from humans, including both 0157:H7
and
non-0157:H7 E. coli. Finally, the inventors have demonstrated that the anti-
Eae
3 o antibodies were able to significantly reduce the development of A/E
lesions due to the
homologous pig and 0157:H7 human attaching and effacing E. coii in vivo in a
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newborn pig infection model. These results clearly demonstrate that chicken
egg yolk
antibodies specific for certain of the virulence factors involved in
attachment and
effacement are able to block bacterial infection and development of intestinal
lesions
in the pig and thus are a potential candidate for use in the treatment of
infections
s caused by attaching and effacing E. coli. They are also a potential
candidate as a
feed additive for oral administration to cattle in order to eliminate 0157:H7
and other
attaching and effacing E. coli from the intestine and prevent contamination of
meat
and consequent infection of humans.
1 o Materials and Methods.
Bacterial strains, plasmids and media. E. coli M155(pREP4T"") (Qiagen) was
used
as the host strain for recombinant proteins. The porcine attaching and
effacing E. coli
(AEEC) P86-1390 (serogroup 045, streptomycin resistant, SmR) was isolated at
the
is Faculte de Medecine Veterinaire, Saint-Hyacinthe, Quebec, Canada from a 4-
week-
old pig with postweaning diarrhea. 045 strain P86-1390 induces typical
attaching and
effacing (A/E) lesions (Zhu et al., 1994 Infect Immun 62: 4153-4159; Zhu et
al., 1995
Can J Vet Res 59: 118-123) and its genomic DNA was used as template to amplify
the virulence factor genes carried on the locus of enterocyte effacement
(LEE).
2 o For explant model challenge (see Table 2 for strains characteristics),
bacteria
were grown overnight in Trypticase Soy Broth (TSB, Difco) with agitation (150
rpm)
at 37°C, then transferred in Dubelcco's Modified Eagle's Medium (DMEM,
GibcoBRL)
and grown to early exponential phase prior to use (ODsoo 0.7, corresponding to
approximately 2.0 X 10$ CFU, determined by use of specific growth curves). 1 %
final
2 s D-Mannose was added to each broth culture to minimize Type-1 fimbriae-
mediated
adherence prior to infection.
Construction of fusion genes. The 3' end (carboxy) or the entire (mature) eae
gene, espA, esp8, and espD genes, tir gene, and the 3' end (carboxy) or the
entire
3 0 (mature) paa gene were amplified by PCR using primer pairs listed in Table
3. The
amplicons were inserted into the pGEM-TT"" vector (Promega) and then
introduced
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into the pQE-30T"" expression vector (Qiagen) using the appropriate cloning
site
(between BamHl and Sall sites for eae carboxy, .espA, espB, espD, paa
carboxygenes, between BamHl and Sphl for eae mature gene, and between Hindlll
and Sacl for tir gene). Gene fusions were checked by sequencing.
Production and purification of fusion proteins. An overnight LB broth
preculture
was used to inoculate a 1 liter LB broth culture. Cells were grown at
37°C with
shaking until the ODsoor,m was 0.7-0.8. Isopropylthiogalactoside (IPTG) was
then
added to a final concentration of 1 mM. After incubation for about 4 hours,
cells were
1o harvested by centrifugation at 4000 x g for 10 minutes and resuspended in
two
volumes of buffer A (Qiagen). Samples can be centrifuged and the pellet stored
frozen at -70°C until use. Samples were thawed at room temperature and
resuspended into buffer A. One mg/ml of lysozyme and a final concentration of
100
pM of PMSF were added. The overall suspension was incubated 30 minutes on ice
and sonicated. For each ml of sample, 10 pg of Rnase A and 5 Ng of Dnase 1
were
added and after incubation for 15 minutes on ice, the sample was clarified by
centrifugation at 15000 x g for 20 minutes. The supernatant was mixed with a
50%
slurry of Ni-NTA resin (Qiagen) in buffer A (8 ml of slurry per liter of
cells). After gentle
mixing in a rotating tube for one night, the resin was added to a Qiagen
column,
2 o washed with buffer A until the OD28onm was less than 0.01, washed with
buffer B
(Qiagen) until the OD28onmwaS less than 0.02, washed with 0.1 M imidazole in
buffer
B (20 ml per liter of cells), and eluted with 0.25 imidazole in buffer B~ (10
ml per liter
of cells). Fractions (1-1.5 ml), collected upon strating the 0.25 M imidazole
elution,
were analyzed by SDS-PAGE through 15% acrylamide. Those containing proteins
2 s of interest were pooled, quantified by the Lowry method, and stored at -
70°C.
Western immunoblotting. His-tagged affinity column purified proteins were
mixed
with an equal volume of 2X Laemmli buffer, boiled for 5 minutes, applied to a
15%
SDS-PAGE, and electrotransferred onto 0.2 Nm nitrocellulose membrane (Bio
Rad).
3 o Blots were blocked and washed with 1 % bovine serum albumin (BSA) -0.1
TweenT"" 20 in Tris-buffered saline (TBS), and incubated overnight with
primary
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antibodies, including RGS-His antibody (Qiagen) and the anti-Eae, anti-EspA,
anti-
EspB, anti-EspD, and anti-Tir kindly provided by Dr. Gad Frankel and Dr. Frank
Ebel.
For chicken antibody (IgY) production analysis, purified IgY specificly
directed against
each of the LEE.virulence factors were used as primary antibodies. Filters
were then
s developed with secondary goat anti-rabbit (1/1000), goat anti-mouse (1/1000)
or
rabbit anti-chicken (1/5000) HRP-conjugated IgG and with H202-a-chloronaphtol
as
the substrate.
Immunization of animals and antibodies purification. Two to eight laying hens
1 o were immunized in their pectoral muscle at multiple sites with 50 Ng of
purified
proteins and emulsified in incomplete Freund's adjuvant on days 1, 14, 28, 42
and
56. M15 (pREP4 T"") total proteins was used as negative control for anti-
virulence
factor IgY production. Eggs were collected from day 28 and kept at 4°C
until
purification of antibodies. For the purification of antibodies, the yolks were
separated
is from the yolk membrane and egg white, pooled and one volume of PBS 1X was
added. The overall mixture was homogenized, mixed with one volume of
chloroform,
and centrifuged at 15000 x g for 10 minutes. An orange coloured solution
containing
the vitellus, a yellow semi-solid emulsion of the lipids in chloroform, and a
watery
phase containing chicken IgY were then observed from the bottom to the top of
the
2 o tube. Purified IgY were analyzed by SDS-PAGE stained with Coomassie blue
and by
Western immunoblotting as explained above.
ELISA. Anti-virulence factor IgY titers in yolk eggs were determined using
microtiter
plates (Immulon 2HB, Dynec) precoated with 100 ng of purified proteins in
carbonate
2 s buffer (pH 9.6). Purified IgY serially diluted in PBS 9pH 7.4) containing
1 % BSA and
0.05% Tween 20 was added to the wells and incubated for 2 hours at
37°C. After
washing thrice with PBS containing 0.05% Tween T"" 20, bound antibodies were
detected by adding rabbit anti-chicken IgG (1/25000) conjugated to peroxidase
(Jackson Immuno Research Laboratories, Inc). After a 10 minutes enzyme-
substrate
3 o reaction, the absorbance at 405 nm was read and antibody titers were
expressed as
the logo of the reciprocal dilution. To monitor non-specific reactions,
absorbances
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measured with IgY from chickens immunized with the total protein extract from
the
M14 (pREP4T"") strain were subtracted from absorbances obtained with test
samples.
Antibody extraction from egg yolks. Virulence factor-specific IgY was
extracted
5 from egg yolks by a method described by [Akita and Nakai, (1993) Immunol.
Methods
18: 162(e): 155-164; 1993 Immunol. Methods 2: 160(2): 207-214], with some
modifications. Briefly, egg yolks were separated from albumin then placed onto
a
towel. Egg yolks were gently rolled onto the towel to removed albumin
residues, then
punctured to aspirate the yolk without the vitellus. An equal volume of
Phosphate
1 o Buffered Saline (PBS) was added to the yolks, then homogenized by Vortex
agitation.
An equal volume of chloroform was added to the solution, then mixed until
there was
a solid homogenate. The preparation was centrifuged for 5 minutes at 14 000
rpm,
and the supernatant containing IgY was removed. The supernatant was
lyophilised
prior to use.
Collection and culture of explant tissues. The explant culture technique was
derived from Zhu et aL, 1994 (supra). Briefly, mucosal tissues from ileum were
obtained from newborn colostrum-deprived piglets from a conventional herd.
Piglets
were tranquilized with ketamin hydrochloride before being euthanized with a
2 o pentobarbital overdose. The time lapse between death and initiation of
explant
cultures was approximately 1 h and each strain was tested in at least 2
piglets. Upon
collection, the serosa was carefully removed and mucus was gently discarded
with
steril swab. Tissues were immersed in sterile complete RPMI 1640 T"" media
(GibcoBRL), transported to the laboratory on ice, and placed on a rocking
platform
for 30 minutes. Prior to culture, tissues were cut into 5 X 5 mm pieces and
placed
mucosal side up onto biopsy foam pads (Curtin Matheson Scientific, Inc.) in
multidish
four-well Nunclon Delta T"" Surface tissue culture plates (Nalge Nunc
International).
One tissue (now called an explant) was placed on each sponge with 1 sponge per
well. Complete RPMI 1640 medium was added to wells without submerging the
3 o explants. Plates were incubated at 37°C on a rocker (position 2,5)
in 95% 02 and 5%
C02 atmosphere.
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Inoculation and examination of explants. The explants were infected three
times
at hourly intervals with 50 p,l of broth culture (approximately 1 X 10' CFU)
applied to
the mucosal surface and incubated for 8 hours. To prevent bacterial overgrowth
and
s acidic pH, hourly changes with sterile fresh complete RPMI 1640 T"" medium
were
carried out during culture starting 2 hours after initial explant infection.
Treatment with antibodies in the explant model. Broth cultures were incubated
at
37°C with an equal volume of lyophilised antibody preparation,
previously
1 o reconstituted with PBS, for 30 minutes prior to explant infection.
Light microscopy. After culture, explants were fixed in 10% buffered formalin
for
microscopic examination. The same protocol was used for sections from in vivo
experimentally infected newborn piglets. Formalin-fixed sections were further
placed
is into Nylon T"" tissue biopsy bags (Shandon Inc.), processed, paraffin-
embedded,
sectioned at 5 ~.m, and stained with hematoxylin, phloxine, and safranine
(HPS)
according to standard techniques. Sections stained with HPS were examined by
light
microscopy for epithelial intimate-adherent bacteria. Each intact villus was
examined
for the presence of intimate-adherent bacteria, and evaluation of mean
epithelial
2 o surface covered with intimate-adherent bacteria was performed, according
to a scale
from 0 to 100%.
Immunofluorescence. Sections on glass slides were deparaffined into xylene for
5
minutes before rehydration into progressive ethanol solutions. Sections were
rinsed
2 s in PBS then antigenic binding sites were blocked in PBS-1 % BSA-0.1 %
Tween20
solution at 37°C for 20 minutes. Thereafter, sections were rinsed in
PSB and
incubated at 37°C for 20 minutes with 1:50 dilution of primary
antibodies (Escherichia
coli Serotyping Laboratory, Canada) according to the specific strain serotype.
After
rinsing out into PSB, sections were incubated at 37°C with 1:200
dilution of goat anti-
3 o rabbit FITC-conjugated secondary antibody (Jackson ImmunoResearch
Laboratories
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inc., USA) for 20 minutes. Sections were finally counterstained with 0.2%
Evan's Blue
TM (Fisher Scientific Company, USA). Mounted sections were examined with a
Leitz
Diaplan T"" microscope equipped with epifluorescence.
Transmission electron microscopy (TEM). Small ileum, cecum, or colon sections
(3mm x 3mm) were fixed for 2 hours at room temperature in 2.5% (v/v)
glutaraldehyde, then rinsed in cacodylate buffer (0.1 M cacodylate, pH 7.3)
for 1.5
hours with regular changes. Thereafter, tissues were post-fixed for 1 hour at
room
temperature into 2% osmium tetroxide (Os04), then rinsed into water for 1.5
hours
to with regular changes, dehydrated in graded ethanol series, and finally
embedded in
Spurr resin (Marivac, Nova Scotia, Canada). Thin sections were mounted on
copper
grids, stained with uranyl acetate and lead citrate, and examined for AE
lesions with
a Philips 420 T"" transmission electron microscope at 80kV (Philips
Electronics, The
Netherlands).
Piglet infection and antibody challenges in vivo. 22 newborn piglets from a
conventional herd were used to assay effects of anti-eaeM antibodies on
bacterial
adherence in vivo. 12 piglets were colostrum-deprived prior to infection with
porcine
strain 1390, while the other 10 piglets were colostrum-fed prior to infection
with
2 o human EHEC strain 85-170. All piglets were kept in cages, and fed with
evaporated
milk during the experiment. Group 1 (1390 infection) piglets were infected
with
1x10'° CFU of 1390 EPEC strain in 2 ml of TSB broth for 2 days, and
received twice
a day egg yolks from hens immunized with sonicate of PREp15 strain as a
adherence
positive control (n=6), or egg yolks from hens immunized with EaeM fusion
protein
2 5 (n=6). Group 2 (85-170 infection) piglets were infected with 1 x10°
CFU of 85-170
EHEC strain in 2 ml of TSB broth for 2 days, and received three times a day,
lyophilized egg yolks reconstituted in milk from hens immunized with a
sonicate of
PREp4 strain as an adherence positive control (n=5), or lyophilized egg yolks
reconstituted in milk from hens immunized with EaeM fusion protein (n=5).
Piglets
3 o were monitored daily for any clinical signs of diarrhea, and were
necropsied at 48
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18
hours after the initial infection. Piglets were tranquilized with ketamine
hydrochloride,
then euthanized with an overdose of pentobarbital solution.
Results and Discussion
a) Production of fusion proteins
It has clearly been demonstrated in the literature that the intimin (Eae) and
the
secreted proteins Tir (Translocated intimin receptor), EspA, EspD and EspB are
virulence factors playing an important role in the pathogenesis of various
AEEC
to infections and could elicit an antibody response. Furthermore, the
inventors have
discovered a new protein called Paa (for Porcine attaching and effacing
associated)
that is also involved in AEEC adhesion to host cells (see section d). These
different
virulence factors were thus considered as good candidates for protective
immunity
and/or as markers in a diagnostic test. Also, it was considered that it would
be
is important to produce a fusion protein by using only the C-terminal end of
intimin (Eae
carboxy) which is involved in receptor recognition. Thus, seven (7) fusion
proteins
were produced corresponding to the listed proteins in the pQE30 expression
vector
which links in frame a His6 tag at the N-terminal end of the proteins. Primer
pairs
specific for each of the virulence factors were chosen to amplify by PCR the
entire
2 0 (Eae, EspA, EspB, EspD, Paa, Tir) or a part (Eae carboxy) of the proteins
from
genomic DNA of enteropathogenic E. coii porcine strain 1390. The fusion
proteins
were detected by Western blot analysis revealed with anti-histidine antibodies
and
with antibodies specifically directed against each of the virulence factors.
However,
the His-Paa protein was only detected in low quantity. Since Paa is predicted
to be
2s unstable by the EXPASY program, a fusion with the stable C-terminal end of
Paa
(Paa carboxy) was carried out.
All fusion proteins were then produced on a large scale and purified on a
nickel
affinity column in sufficient quantities for immunization of chickens and for
use as
antigens in the ELISA. The purified His-Eae carboxy, His-Eae, His-EspA, His-
EspB,
3 o His-EspD, His-Paa and His-Tir proteins were obtained.
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b) Production and purification of the eaa yolk antibodies
The immunization of chickens and the antibody purification technique were
carried out from modified techniques as described in the Materials and
Methods.
SDS-PAGE analysis demonstrated production of IgY as early as 28 days after
initial
s immunization with proteins His-Eae carboxy, His-Eae, His-EspA, His-EspB, His-
EspD, His-Paa and His-Tir.
c) Sensitivity and the specificity of the eg~a yolk antibodies against each of
the
roteins
to Western blot analysis showed that purified IgY recognized homologous fusion
proteins. The titer of the specific IgY for each protein was then measured by
ELISA
using homologous purified proteins as the antigen. The ELISA test conditions
were
determined for each antigen and results showed a high titer (>1/25000) for
each
specific IgY after 42 days. The capacity of each antibody to detect the
corresponding
is native antigen in the homologous strain 1390 and in AEEC strains from
different
animal species was also examined. However it appeared that the tested
conditions
and/or the antigen presentation did not favor the detection by ELISA of some
virulence factors such as Eae or Paa in wild type bacteria.
2 o d) Capacity of specific antibodies to prevent the development of A/E
lesions
in vitro in the pig ileal organ culture model
~ Elaboration of the model
The first step of this work was to determine the explant culture conditions
which
2s would allow AEEC adherence to ileal and cecal epithelial cells of weaned
piglets.
Different conditions were tested and a rapid technique for microscopic
analysis of
tissue sections by light microscopy and confirmation by electron microscopy,
was set
up (see Materials and Methods). A greater and more consistent adherence of the
1390 strain was observed on ileal explants from newborn piglets than on
explants
from weaned pigs. Hence, the newborn pig expl~ant model was used in subsequent
experiments.
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The inventors have demonstrated that AEEC strains originating from the rabbit,
pig, dog, bovine, and human (including non-0157 EHEC and EPEC), and producing
Eae of the a, ~3, b, or s subtypes, induced A/E lesions equally well on
newborn pig
ileal explants (Figure 1 ). More specifically, figure 1 shows the mean
percentage of
s intact villi showing bacterial adherence on ileal explant sections. Strains
from various
animal species and from humans show a similar percentage adherence, when
compared to the homologous porcine 1390 AEEC strain, except those from 0157:H7
serotype (EC505, 43888, STJ348, 85-170, 43895, STJ854, and STJ919 on the
graph). All except 0157:H7 strains are significantly different from eae-
mutant strain
to ICC-170 (negative control). These data validate our model for the study of
the
attaching and effacing phenotype expression for both homologous and
heterologous
strains, except for those belonging to 0157:H7 serotype. Results are presented
as
the mean ~ the standard deviation of the mean.
On the other hand, 0157:H7 AEEC strains producing Eae of the y subtype
15 adhered to a much lesser extent to the ileal epithelium. However,
replacement of the
Eae of the y subtype by the Eae of the a subtype in an 0157:H7 strain resulted
in
induction of A/E lesions to a similar extent as observed for the homologous
porcine
AEEC strain (Figure 2). More specifically, Figure 2 shows the effects of
intimin
subtype switch from gamma to alpha on the mean percentage of intact villi
showing
2 o bacterial adherence of the human 0157 :H7 strain 85-170 on ileal explant
sections.
Complemented double-mutant strain PCVD-438 (intimin alpha from human E2348/69
EPEC strain) shows similar adherence as homologous porcine strain 1390. PICC-
55
is a gamma-intimin subtype complemented mutant strain (similar to the wild
strain
85-170). These data confirm the problem of gamma-intimin subtype in adhering
to
2 s the ileal explant model. Results are presented as the mean ~ the standard
deviation
of the mean. These results also suggest that Eae of the y subtype of 0157:H7
strains
recognizes receptors on porcine ileal epithelial cells less well than the Eae
of the
other known subtypes. Nevertheless, these results confirm that pig ileal
explants are
an appropriate model for the examination of the effect of specific antibodies
on the
3 o formation of A/E lesions by AEEC of diverse origin.
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~ Blocking of AlE lesions
The capacity of the specific antibodies to prevent the development of A/E
lesions
ex vivo in the pig explant culture model and in vivo in the newborn pig
infection
s model was tested with the homologous porcine strain 1390 and different
heterologous AEEC strains. The results showed that the antibodies specific for
the
mature and carboxy terminal of Eae, Tir and Paa, in the case of the homologous
porcine strain 1390, are able to significantly block the development of A/E
lesions in
ileal explants, for the homologous strain 1390 (Figure 3). More specifically,
Figure
3 shows the effect of antibodies on mean percentage of intact villi showing
adherence when infected by porcine strain 1390. 1390 anti-T(-) is a positive
control
for adherence. Results show a significant decrease in bacterial adherence (*
on top
of column) with anti-EaeC, anti-EaeM, anti-Tir, and anti-PaaM. Results are
presented
as the mean ~ the standard deviation of the mean. A Kruskal-Wallis test was
is performed with commercially available software, and post hoc 2-by-2
comparisons
were done to assess differences between the groups; P < 0.0001 was taken to be
significant.
The results show that the antibodies specific for the mature and carboxy
terminal
of Eae, Tir and Paa, in the case of the homologous porcine strain 1390, are
able to
2 o significantly block (* on top of column) the development of A/E lesions in
ileal
explants for all of the tested AEEC strains from the calf (Figure 4), and
human, the
latter including both 0157:H7 EHEC (Figures 5 and 6) and EPEC strains (Figure
7).
The anti-EspA, anti-EspB, and anti-EspD antibodies did not affect the
development
of A/E lesions by any of the tested AEEC strains, with the exception of a
human
2s EPEC strain, for which the anti-EspA antibodies did significantly block the
development of A/E lesions (Figure 7). Anti-Paa antibodies did not block the
development of A/E lesions due to this human EPEC strain which produced Eae
but
not Paa. Hence, the anti-Eae mature, anti-Tir, and anti-Paa antibodies, and
possibly
the anti-EspA antibodies, were considered as potential candidates for the
blocking
30 of development of A/E lesions in vivo.
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More specifically, Figure 4 shows the effect of antibodies on mean percentage
of
intact villi showing adherence when infected by bovine strain B00-H854. B00-
H854
represents the strain alone, whereas B00-H854 anti-T(-) is a positive control
for
adherence. Results show a significant decrease in bacterial adherence (*on top
of
s column) with anti-EaeC, anti-EaeM, and anti-Tir, and, to a lesser extent,
with anti-
PaaM. Results are presented as the mean ~ the standard deviation of the mean.
A
Kruskal-Wallis test was performed with commercially available software, and
post
hoc 2-by-2 comparisons were done to assess difference between the groups; P <
0.0001 was taken to be significant.
to Figure 5 shows the effect of antibodies on mean percentage of intact villi
showing adherence when infected by human 0157:H7 EHEC strain STJ348. STJ348
represents the strain alone, whereas STJ348 anti-T(-) is a positive control
for
adherence, and ICC-170 is an eae- mutant strain used as a negative control.
Results
show a significant decrease in bacterial adherence (*on top of column) with
anti-Tir,
15 anti-EaeM, and, to a lesser extent, with anti-PaaC and anti-EaeC. Results
are
presented as the mean ~ the standard deviation of the mean. A Kruskal-Wallis
test
was performed with commercially available software, and post hoc 2-by-2
comparisons were done to assess difference between the groups; P < 0.0001 was
taken to be significant.
2 o Figure 6 shows the effect of antibodies on mean percentage of intact villi
showing adherence when infected by human 0157:H7 EHEC strain 85-170. 85-170
represents the strain alone, whereas 85-170 anti-T(-) is a positive control
for
adherence, and ICC-170 is an eae- mutant strain used as a negative control.
Results
show a significant decrease in bacterial adherence (*on top of column) with
anti-
2 s EaeM, anti-Tir, and, to a lesser extent, with anti-EaeC and anti-PaaM.
Results are
presented as the mean ~ the standard deviation of the mean. A Kruskal-Wallis
test
was performed with commercially available software, and post hoc 2-by-2
comparisons were done to assess difference between the groups; P < 0.0001 was
taken to be significant.
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Moreover, Figure 7 shows the effect of antibodies on mean percentage of intact
villi showing adherence when infected by human EPEC strain E2348/69. E2348/69
represents the strain alone, whereas E2348/69 anti-T(-) is a positive control
for
adherence. Results show a significant decrease in bacterial adherence (*on top
of
s column) with anti-EspA, anti-EaeC, anti-Tir, and anti-EaeM. Results are
presented
as the mean ~ the standard deviation of the mean. A Kruskal-Wallis test was
performed with commercially available software, and post hoc 2-by-2
comparisons
were done to assess difference between the groups; P < 0.0001 was taken to be
significant.
e) Ability of appropriate ega yolk antibodies to prevent the development of
intestinal colonization and diarrhea in vivo in the newborn and weaned pia
experimental infection models
Since in vivo challenge experiments in pigs are costly and time-consuming, the
1 s inventors have decided to focus on the evaluation of the effect of the
anti-Eae
antibodies on the development of A/E lesions in vivo. Using the colostrum-
deprived
newborn piglet model, it was demonstrated that oral administration of the
purified anti-
Eae egg yolk antibodies significantly inhibited the development of A/E lesions
in the
cecum and colon of piglets challenged with the homologous porcine AEEC strain
2 0 (Figures 8A and 8B). More specifically, Figure 8A shows antibody effects
in the
cecum, whereas Figure 8B shows antibody effects in the colon. Anti-T(-)
results are
from piglets (n=6) that received egg yolks from non-immunized hens, and
represent
a positive control for adherence, whereas anti-EaeM represent piglets (n=6)
that
received egg yolks containing anti-EaeM antibodies directed against mature
intimin.
2s Results show a significant decrease in bacterial adherence with anti-EaeM,
when
compared with anti-T(-). Results are presented as the mean ~ the standard
deviation
of the mean. Statistical analysis is still in progress.
Since 0157:H7 AEEC strains are an important cause of problems in humans,
it was decided to also focus on the evaluation of the effect of the anti-Eae
antibodies
3 0 on the development of A/E lesions in vivo in an 0157:H7 pig challenge
model. It has
been previously shown that 0157:H7 strains induce A/E lesions to a greater
extent
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in colostrum-fed rather than in colostrum-deprived piglets. Using a colostrum-
fed 3-
day-old pig model, the inventors have demonstrated that oral administration of
the
purified anti-Eae egg yolk antibodies also inhibited the development of A/E
lesions
in the cecum and colon of piglets challenged with an 0157:H7 AEEC strain
(Figures
s 9a and 9B). More specifically, Figure 9A shows antibody effects in the
cecum,
whereas Figure 9B shows antibody effects in the colon. Anti-T(-) results are
from
piglets (n=5) that received lyophilised egg yolks from non-immunized hens, and
represent a positive control for adherence, whereas anti-EaeM represents
piglets
(n=5) that received lyophilised egg yolks containing anti-EaeM antibodies
directed
to against mature intimin. Results show a decrease in bacterial adherence with
anti-
EaeM, when compared with anti-T(-). Results are presented as the mean ~ the
standard deviation of the mean. Statistical analysis is still in progress.
Conclusion
is Hence, the inventors have~shown, for the first time, that orally
administered
antibodies specific for AEEC virulence-associated protein, such as the adhesin
Eae,
are able to inhibit the development of A/E lesions in the live animal. In
addition, they
have showed that antibodies specific for the Eae of the ~i subtype and
produced by
an AEEC of pig origin are able to inhibit the development of A/E lesions due
to an
20 0157:H7 AEEC of human origin that produces Eae of the y subtype.
Altogether,
these ex vivo and in vivo results show that antibodies specific for the Eae
and Tir of
the porcine AEEC are useful for the inhibition of development of A/E lesions
due to
AEEC infections in the various animal species and in humans. Antibodies
specific
for Paa are useful, at least, for the inhibition of development of A/E lesions
due to the
2 s AEEC homologous to the strain used for preparation of the Paa antibodies.
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Table 1. Serogroups and serotypes of AEEC
Human EHEC
Sero rou 0157, 0111, 026
Serotype 04.H-; 05.H-; 016.H6; 026.H11; 026.H21; 026.H32;
046.H31; 048.H21; 055.H7; 091.H10; 091.H21; 098.H-;
0111.H2; 0111.H8; 0111.H-; 0113.H21; 0117.H14;
0118.H12; 0119.H6; 0125.H-; 0126.H8; 0128.H2;
0145.H-;
0157.H7; 0157.H-; 0172.H-.
Human SPEC
Sero rou 026, 055, 0111, 0119, 0125, 0126, 0127, and 0128
Serotype 018a,c.H7; 020.H26; 020.H34; 025.H1; 026.H11;
026.H-;
044.H34; 055.H6; 055.H7; 055.H-; 086.H27; 086.H34;
086.H-; 091.H7; 091.H-; 0111.H2; 0111.H12; 0111.H-;
0114.H10; 0114.H32; 0119.H6; 0119.H-; 0125.H21;
0125.H-; 0126.H2; 0126.H-; 0127.H9; 0127.H21;
0127.H40;
0127.H-; 0128.H2; 0128.H7; 0128.H8; 0128.H12;
0128.H-;
0142.H6; 0158.H23.
Rabbit EPEC
Newborn rabbit 02, 08, 015, 0103, 0109
sero rou
Weaned rabbit 015, 020, 025, 0103, 0109, 0128, 0132
sero rou
Pi EPEC
Sero rou 045. 0103, 0108, NT
Cattle EPEC
Sero rou 05, 026, 011, 0118
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Table 2. Bacterial strains, and characteristics
BacterialDescription Serotype Genetic
characteristics
strains and origin
P86-1390Porcine EPEC 045 eae/3+,espA+,tir+,paa+
C89-4221Canine EPEC 0112ab eaea+, espA+,tir+,paa+
C86-4225Canine EPEC 049 eae8+, espA+,tir+,paa+
RDEC-1 Rabbit EPEC 015 :NM eae/3+,espA+,tir+,paa+
E22 Rabbit EPEC 0103: eae~3+,espA+,tir+,paa+
H2
97-5899-Rabbit EPEC Non- eae~+, espA+,tir+,paa-
175 typable
96-1744-Rabbit EPEC Non- eaelj+,espA+,tir+,paa+
174 specific
typing
97-1746-Rabbit EPEC 055 eaeR+, espA+,tir+,paa+
175
E2348/69Human EPEC 0127: eaea+, espA+,tir+,paa-
H6
STJ348 EHEC 0157: eaey+, espA+,tir+,paa+
H7
EC505 EHEC 0157: eaey+, espA+,tir+,paa+
H7
STJ919 EHEC 0157: eaey+, espA+,tir+,paa+
H7
STJ854 EHEC 0157: eaey+, espA+,tir+,paa+
H7
43888 EHEC, 0157: eaey+, espA+,tir+,paa+
H7
negative for
vt genes
43895 EHEC 0157: eaey+, espA+,tir+,paa+
H7
85-170 EHEC isolate 0157:H7 eaey+, dvt-,espA+,tir+,
from United paa+
States, cured
of phage
encoding
verotoxin
ICC-170 EHEC 0157:H7 deae-, dvt-, espA+,tir+,
paa+
PCVD-438EHEC 0157:H7 eaea+',dvt-,
espA+, tir+,
paa+
PICC-55 EHEC 0157:H7 eaey+',dvt-, espA+,tir+,
paa+
B00-H854Bovine EPEC 045 eaeE+, espA+,tir+, paa+
B00-5999Bovine EPEC 026 eae/j+,espA+,tir+, paa+
FH1299 Non-0157 EHEC026 eaelj+,espA+,tir+, paa+
FH894 Non-0157 EHEC045 eaee+, espA+,tir+,paa+
FH303 Non-0157 EHEC0103 eaeE+, espA+,tir+,paa+
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Table 3. Primers and PCR products
Sequence of primers Amplicon
Genes 5,_3, size (bp)
Eae F GGATCCGCAACAACCGATCAGAAT
carboxy 702
R CTCGAGTTTTACACAAACAGGAAA
Eae F GGATCCAATGGTGAAAAT 2712
matureR AAGCTTTTTTACACAAACAGG
EspA F GGATCCATGGATACATCAACTGCA 585
R CTCGAGTTTACCAAGGGATA
EspB F GGATCCATGAATACTATTGATTAT 954
R CTCGAGACCAGCTAAGCGAACCGA
EspD F GGATCCATGCTTAATGTAAATAGC 1152
R CTCGAGAACTCGACCACTAACAAT
Tir F GAGCTCATGCCTATTGGTAAT 1626
R AAGCTTAACGAAACGTGCGG
Paa F GGATCCCTTTATCTGCGAAAAA 488
carboxyR CTCGAGAGTGCCTTTCCTGG
Paa F GGATCCATGAGGAACATAA 765
R CTCGAGAGTGCCTTTCCTGG
Although preferred embodiments of the present invention have been described in
detail herein and illustrated in the accompanying drawings, it is to be
understood that
the invention is not limited to these precise embodiments and that various
changes
and modifications may be effected therein without departing from the scope or
spirit
of the present invention as defined in the claims.
