Porcine intestinal epithelial cell lines as a new in vitro model for studying adherence and pathogenesis of enterotoxigenic Escherichia coli.

Abstract

Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intestines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab, K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC pathogenesis.

Full text

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Veterinary and Biomedical Sciences, Department of
Papers in Veterinary and Biomedical Science
University of Nebraska - LincolnYear 
Porcine intestinal epithelial cell lines as a
new in vitro model for studying
adherence and pathogenesis of
enterotoxigenic Escherichia coli
Seung Y. Koh∗
Rodney A. Moxley∗∗
Sajan George†
David Francis††
Volker Brozel‡
Radhey Kaushik‡‡
∗South Dakota State University, Brookings, SD
†South Dakota State University, Brookings, SD
‡South Dakota State University, Brookings, SD
∗∗University of Nebraska - Lincoln, rmoxley1@unl.edu
††South Dakota State University, Brookings, SD
‡‡South Dakota State University, Brookings, SD
This paper is posted at DigitalCommons@University of Nebraska - Lincoln.
http://digitalcommons.unl.edu/vetscipapers/92

Page 2

Published in Veterinary Microbiology (2008); doi 10.1016/j.vetmic.2007.12.018
Copyright © 2007 Elsevier B.V. Used by permission.
Submitted August 22, 2007; revised December 8, 2007; accepted December 10, 2007; published online January 4, 2008.
short communication
Porcine intestinal epithelial cell lines as a new in vitro
model for studying adherence and pathogenesis of
enterotoxigenic Escherichia coli
Seung Y. Koh a, Sajan George b, c, Volker Brözel b, c, Rodney Moxley d,
David Francis a, c, and Radhey S. Kaushik a, b, c
a Department of Veterinary Science,
South Dakota State University, Brookings, SD 57007, USA
b Department of Biology and Microbiology,
South Dakota State University, Brookings, SD 57007, USA
c Center for Infectious Disease Research and Vaccinology (CIDRV),
South Dakota State University, Brookings, SD 57007, USA
d Department of Veterinary and Biomedical Sciences,
University of Nebraska–Lincoln, Lincoln, NE 68583, USA
Corresponding author: R. S. Kaushik, SNP 252A, P.O. Box 2140D,
Department of Biology and Microbiology, South Dakota State University,
Brookings, SD 57007, USA. Tel. 605 688-5501; fax 605 688-5624;
email radhey.kaushik@sdstate.edu
Abstract
Enterotoxigenic Escherichia coli (ETEC) infections result in large economic losses in the swine industry
worldwide. The organism causes diarrhea by adhering to and colonizing enterocytes in the small intes-
tines. While much progress has been made in understanding the pathogenesis of ETEC, no homologous
intestinal epithelial cultures suitable for studying porcine ETEC pathogenesis have been described prior
to this report. In the current study, we investigated the adherence of various porcine ETEC strains to two
porcine (IPEC-1 and IPEC-J2) and one human (INT-407) small intestinal epithelial cell lines. Each cell
line was assessed for its ability to support the adherence of E. coli expressing fimbrial adhesins K88ab,
K88ac, K88ad, K99, F41, 987P, and F18. Wild-type ETEC expressing K88ab, K88ac, and K88ad efficiently
bound to both IPEC-1 and IPEC-J2 cells. An ETEC strain expressing both K99 and F41 bound heavily to
both porcine cell lines but an E. coli strain expressing only K99 bound very poorly to these cells. E. coli
expressing F18 adhesin strongly bound to IPEC-1 cells but did not adhere to IPEC-J2 cells. The E. coli
strains G58-1 and 711 which express no fimbrial adhesins and those that express 987P fimbriae failed to
bind to either porcine cell line. Only strains B41 and K12:K99 bound in abundance to INT-407 cells. The
binding of porcine ETEC to IPEC-J2, IPEC-1 and INT-407 with varying affinities, together with lack of
binding of 987P ETEC and non-fimbriated E. coli strains, suggests strain-specific E. coli binding to these
cell lines. These findings suggest the potential usefulness of porcine intestinal cell lines for studying ETEC
pathogenesis.
Keywords: porcine, enterotoxigenic Escherichia coli, intestinal epithelial cells, adherence, pathogenesis
?

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2 s. Y. Koh et al. in Veterinary Microbiology (2008)
1. Introduction
Both porcine neonatal and post-weaning diarrhea
(PWD) caused by enterotoxigenic Escherichia coli
(ETEC) result in significant morbidity and mor-
tality and are economically important diseases of
pigs (Fairbrother et al., 2005; Nagy and Fekete,
2005). The colonization of ETEC in the small intes-
tine is primarily mediated by fimbria which confer
to ETEC the ability to attach to receptors on the en-
terocytes. Secretory diarrhea associated with ETEC
infection is mediated by any of several enterotox-
ins which include heat labile enterotoxin (LT),
heat stable enterotoxin-a (STa), and enterotoxin-
b (STb). The most common adhesins of porcine
ETEC include K88 (F4) (Jones and Rutter, 1972),
K99 (F5) (Moon et al., 1977), 987P (F6) (Isaacson
et al., 1978), F18 (Imberechts et al., 1996), and F41
(Morris et al., 1982). Three serological antigenic
variants of K88 fimbrae exist, K88ab, K88ac, and
K88ad (Gaastra and Pederson, 1986), and K88ac
is the most prevalent and clinically important vari-
ant ETEC strain isolated from diarrheic pigs (Fair-
brother et al., 2005; Francis et al., 1998; Nagy and
Fekete, 2005).
A number of cellular systems had been used to
study the adherence of ETEC, including erythrocytes
(Evans et al., 1979), primary enterocytes (Knutton et
al., 1984), brush border vesicles (Baker et al., 1997),
and human tumor cell lines (Roselli et al., 2006).
None of these cellular systems are highly suitable
for porcine ETEC pathogenesis studies. The objec-
tive of this study was to examine the adherence of
various strains of ETEC to two porcine intestinal ep-
ithelial cell lines IPEC-J2 and IPEC-1 and to com-
pare their adherence to the human intestinal epi-
thelial cell line INT-407. The findings of this study
indicate that IPEC-J2 and IPEC-1 cell lines are su-
perior to the human intestinal cell line (INT-407)
in that they support the adherence of most porcine
ETEC strains.
2. Materials and methods
2.1. Bacterial strains
All E. coli strains used in this study are listed, and
their phenotypes described in Table 1. These strains
were cultured on 5% sheep blood agar (brain heart
Table 1. Bacterial strains used for the cell adhesion assay
E. coli strains Relevant properties References
263
1476
F962
3030-2
K12:K88ac
F783 = F963
1836-2
2534-86
WAM 2317
MUN 285
MUN 287
Morris
K12:K88ad
F291
2134
K12:K99
B41
1194
G58-1
711
Wild-type O8:K87:K88ab ETEC, LT+, STb+
Laboratory construct K12:K88aba
K12: K88ab 5K, ampr, pFM205
Wild-type O157:K87:K88ac ETEC, LT+, STb+
Laboratory construct K12:K88aca
K12:K88ac C600 ampr, pDB88-102
Wild-type K88ac+; LT−, ST-, astA+
Wild-type O8:K87:NM: K88ac LT-I+, STb+
Nalidixic acid-resistant mutant of 2534-86, LT-I+, STb+
Mutant of WAM 2317, LT-I-
Nalr Kmr, LT-I+ complemented strain
Wild-type O8:K87:K88ad ETEC, LT+, STb+
Laboratory construct K12:K88ada
K12:K88ad 5K, ampr, pBad1
Wild-type, O157:H19:F18ac, 4P-, STa+, STb+
Laboratory construct K12:K99a
Wild-type, O101:K99; F41; STa+
Wild-type, O141:987P, STa+, STb+
Non-pathogenic wild-type, O101:K28:NM non-fimbriated, LT−
K12:K88-negative parent of laboratory constructs.
(Moon et al., 1968)
(Baker et al., 1997)
(Bakker et al., 1992)
(Francis and Willgohs, 1991)
(Baker et al., 1997)
(Bakker et al., 1992)
(Zhang et al., 2006)
(Moxley et al., 1998)
(Berberov et al., 2004)
(Berberov et al., 2004)
(Berberov et al., 2004)
(Baker et al., 1997)
(Baker et al., 1997)
(Gaastra and Pederson, 1986)
(Casey et al., 1992)
(Moon et al., 1977)
(Orskov et al., 1975)
(Mullaney et al., 1991)
(Francis et al., 1986)
(Baker et al., 1997)
a Wild-type plasmid was introduced by conjugation.

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Porcine intestinal ePithelial cell lines as a new in vitro model for enterotoxigenic e. coli ?
infusion base), except K12:K99 which was grown
on Essential Salt Medium (Francis et al., 1982)
supplemented with Eagle’s essential amino ac-
ids and vitamins. The bacterial cultures were incu-
bated for 18 h at 32 °C before use in the adherence
studies.
2.2. Cell lines and culture conditions
The IPEC-J2 and IPEC-1 cell lines have been pre-
viously described (Lu et al., 2002; Schierack et al.,
2006). These undifferentiated porcine intestinal
epithelial cell lines were derived from jejunum and
small intestine respectively, from un-suckled 1-day-
old piglets. Neither of these cell lines was immor-
talized and therefore they represent a better model
of normal porcine intestinal epithelium than trans-
formed cell lines. Both IPEC-J2 and IPEC-1 cells
were seeded on plastic cell culture flasks (25 cm2,
Corning, NY). These cell lines were cultured and
maintained in Dulbecco’s Modified Eagle Medium
(DMEM)-Hank F12 (GIBCO, Invitrogen Corpora-
tion, Grand Island, NY) supplemented with 5% fetal
calf serum (FCS; Atlanta Biologicals, Lawrenceville,
GA), penicillin (100 IU/ml), streptomycin (100 μg/
ml) (Invitrogen), insulin (5 μg/ml), transferrin
(5 μg/ml), selenium (5 ng/ml) (ITS Premix; Sigma,
St. Louis, MI), and 5 ng/ml of epidermal growth fac-
tor (EGF; Sigma), hereafter, referred as IPEC me-
dia. The stock culture of INT-407 (ATCC, CCL-6), a
non-transformed human embryonic intestinal epi-
thelial cell line, was obtained from American Type
Culture Collection (ATCC, Manassas, VA). INT-
407 cells were cultured and maintained in DMEM
(GIBCO, Invitrogen) supplemented with 10% FCS,
l-glutamine (2 μM), penicillin (100 IU/ml), strep-
tomycin (100 μg/ml), and amphotericin (25 μg/
ml) (Mediatech, Herndon, VA), hereafter, referred
as DMEM-10. The cultures were maintained in a
humidified incubator in an atmosphere of 5% CO2
and 95% air at 37 °C. After 4 to 5 days of culturing,
all the three types of cultured cells became conflu-
ent. Cell monolayers were washed with phosphate
buffered saline (PBS) and trypsinized with 1× tryp-
sin-EDTA (Mediatech). The detached cells were
pelleted at 200 × g for 5 min, re-suspended in anti-
biotic-free medium, and used for the adherence as-
says. The continuous cultures of epithelial cell lines
were maintained by seeding culture flasks at 1:3 ra-
tios at each passage.
2.3. Polarized IPEC-J2 cells
The IPEC-J2 cells were differentiated and polar-
ized on collagen-coated permeable supports in se-
rum-free medium. Two million IPEC-J2 cells har-
vested from confluent culture flasks (25 cm2) were
seeded on a 24-mm diameter (growth area 4.7 cm2)
collagen-coated transwell permeable support fil-
ter (3.0 μm pore size). Two ml of IPEC medium was
added to the upper chamber while 3 ml of the me-
dium was added to the bottom of wells in a 6-well
transwell culture plate (Corning). The cells were
maintained in serum-containing medium for 48 h
and then switched to the same medium containing
10−7 M dexamethasone (Sigma) without FCS to min-
imize cell growth and enhance differentiation. The
medium was changed every second day until cells
showed features of polarization as judged by a sig-
nificant increase in trans-epithelial electrical resis-
tance (TER) (Schierack et al., 2006). Cell cultures
showing TER values between 2000 and 5500 Ω cm2
were used for adherence assays.
2.4. Bacterial adherence assays
Fimbriae-mediated binding specificity of various E.
coli strains was determined by a cell adhesion as-
say. The cell lines IPEC-J2 (passage 42–52), IPEC-1
(passage 25–30), and INT-407 (passage 12–16) were
used for this study. The adherence assay procedure
was described previously (Baker et al., 1997). Briefly,
E. coli were cultured overnight on 5% sheep blood
agar plates at 32 °C. Bacterial cells were suspended
in sterile PBS to achieve an optical density of approx-
imately 1.0 at 520 nm. Fifty microliters (50 μl) of E.
coli suspension was added to a plate well followed
by addition of a suspension of porcine/human intes-
tinal epithelial cells (~5 × 104 cells) in PBS contain-
ing 4% of D-Mannose (to prevent binding by Type I
fimbriae if present). These suspensions were mixed
for 20 min with rotational agitation at 140 rpm and
then incubated for an additional 4 h at 4 °C. ETEC
binding to epithelial cells was observed by phase
contrast microscopy and bright field microscopy un-
der oil (1000× magnification), and extent of bacte-
rial binding to the epithelial cells was determined.
The relative number of bacteria binding to epithelial
cells was judged and rated (− = no adherent bacte-
ria; + = few bound bacteria, ++ = several to a mod-
erate number of bacteria bound; +++ = large num-

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4 s. Y. Koh et al. in Veterinary Microbiology (2008)
bers of bacteria bound). Each adherence assay was
repeated three times or more with each cell line (8×
with IPEC-J2, 3× with IPEC-1, and 4× with INT-
407) to confirm the consistency of results. In some
assays, in order to ascertain the viability of adherent
ETEC, parallel bacterial suspensions were stained
with Live/Dead® BacLight™ stain (Invitrogen, CA,
USA) with live cells fluorescing green and dead cells
fluorescing red. Bacterial adherence was observed
by fluorescence microscopy using an AX70 fluores-
cence microscope (Olympus).
2.5. Green fluorescence protein-tagged E. coli
To enhance the visualization of bacterial adherence
and confirm the findings obtained using unlabelled
ETEC, the confluently binding strain B41 was trans-
formed to express eGFP from plasmid pGLO (Bio-
Rad, Hercules, CA). The eGFP gene is under the
control of an arabinose-inducible promoter. Strain
B41 was made chemically competent by standard
methods (Sambrook et al., 2001) and transformed
by heat shock with pGLO as per the manufacturer’s
instructions. Transformants were selected by plating
on an LB agar plate containing ampicillin (50 μg/
ml) and arabinose (6 mg/ml), and incubated over-
night at 37 °C. Successfully transformed colonies
fluoresced green upon UV excitation. Transformants
to be used in adherence assays were cultured over-
night on sheep blood agar amended with arabinose
(6 mg/ml). The adherence assay was performed
as described above. Cells were viewed by confocal
scanning laser microscopy (CSLM) using an Olym-
pus Fluoview FV300 Laser Scanning Confocal Mi-
croscope System interfaced with an inverted-micro-
scope (Olympus IX70) using Blue Argon (488 nm)
and Green Helium Neon (543 nm) excitation lasers.
3. Results
Both porcine small intestinal epithelial cell lines
bound a range of ETEC, albeit to varying degrees.
Representative photomicrographs indicating the de-
gree of adherent bacteria are shown in Figure 1A and
Figure 1. Adherence of E. coli strains to IPEC-J2 and IPEC-1 cells. (A) Shows the degree of E. coli
binding to porcine intestinal epithelial cells. Final magnifications 1000×. −: No binding of bacte-
ria; +: few bacteria bound per cell; ++: several bacteria bound per cell and +++: large numbers of
bacteria bound per cell. GFP-transformed bacteria and BacLight dye were used to enhance the bac-
terial visualization. (B) The confocal image showed GFP-transformed bacterial (ETEC strain B41)
attachment to IPEC-J2 cells. (C) ETEC F18 strain 2134 intensively bound to IPEC-1 cells. All bacte-
rial cells attached to the IPEC-1 cell were live cells.

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Porcine intestinal ePithelial cell lines as a new in vitro model for enterotoxigenic e. coli ?
the results of adherence assays are summarized in
Table 2. Wild-type E. coli strains 263, 3030-2, and
Morris expressing K88ab, K88ac, or K88ad fimbriae
strongly bound to both IPEC-J2 and IPEC-1 cells. E.
coli strain B41 expressing both K99 and F41 fimbria
heavily bound to all three epithelial cell lines (IPEC-
1, IPEC-J2, and INT-407). The laboratory construct
K12:K99 bound poorly to either IPEC-J2 or IPEC-
1 cells when compared to the binding of the B41
strain, but heavily bound to INT-407.
The E. coli strain 2134 expressing F18 bound
only to IPEC-1 cells. However, E. coli that expressed
987P failed to bind any of the cell lines. In addition,
non-fimbriated E. coli strains G58-1 and 711 failed
to bind cells of any of the three cell lines we exam-
ined. Polarization of IPEC-J2 cells did not result
in the appearance of binding patterns that differed
from those of non-polarized cells (data not shown).
Wild-type E. coli K88ac strain 3030-2 bound in
greater abundance to IPEC-J2 cells than did labo-
ratory constructs K12:K88ac and F783, which ex-
pressed the same fimbrial antigens. The K88ac-
positive but toxin-negative
1836-2 showed very limited binding to both IPEC-1
and IPEC-J2 cells. However, wild-type K88ac strain
2534-86 and its nalidixic acid-resistant (NalR) mu-
wild-type strain
tant (WAM2317) and LT− mutant derivative of
WAM2317 (MUN285) mutants adhered to both por-
cine cell types (IPEC-J2 and IPEC-1) and human cell
line INT-407 with similar efficiencies (Table 2). Wild-
type strains expressing K88ab and K88ad bound in
abundance to IPEC-J2, but K88ab and K88ad lab-
oratory constructs bound in small numbers to these
cells. However, these wild-type strains and labora-
tory constructs bound equally to IPEC-1 cells.
Adherence assays with GFP-tagged bacteria (B41)
were also conducted and their adherence to all the
three cell lines was observed using confocal scan-
ning laser microscopy (Figure 1B). Similar adher-
ence patterns as observed with GFP-negative bac-
teria (Table 2) were detected. BacLight staining was
used in the adherence assays with ETEC F18 strain
2134 to enhance the visualization of bacterial adher-
ence and differentiate between live and dead bacte-
ria. All the attached bacterial cells fluoresced green
indicating that they were live (Figure 1C).
4. Discussion
In this study all three K88+ ETEC strains (K88ab,
K88ac, and K88ad) adhered to both IPEC-J2 and
Table 2. Results of adherence assays with pig intestinal epithelial cell lines using ETEC and laboratory constructs
Strains Adhesin IPEC-J2 cells IPEC-1 cells INT-407 cells
263a
K12:K88ab
F962
3030-2a
K12:K88ac
F783
1836-2a
2534-86a
WAM 2317
MUN 287
MUN 285
Morrisa
K12:K88ad
F291
1194a
2134a
B41a
K12:K99
G58-1a
711
K88ab
K88ab
K88ab
K88ac
K88ac
K88ac
K88ac
K88ac
K88ac
K88ac
K88ac
K88ad
K88ad
K88ad
987P
F18
K99, F41
K99
None
None
++ or +++
+++
+ or ++
+++
+
+
+
++ or +++
++
++ or +++
++ or +++
+++
+ or ++
+ or +++
−
−
+++
+
−
−
+++
+++
+++
++ or +++
++ or +++
++
++
++ or +++
++ or +++
++ or +++
+++
+++
+++
+++
−
+++
+++
+
−
−
+
+
+
++
+
−
−
++ or +++
++ or +++
++ or +++
++ or +++
++ or +++
−
−
−
−
+++
+++
−
−
a Wild-type strains are indicated in bold.

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6 s. Y. Koh et al. in Veterinary Microbiology (2008)
IPEC-1 cells, and the degree of ETEC adherence to
these cells was strain-specific. Adhesin-negative E.
coli strains G-58-1 and 711 did not adhere to any of
three cell lines tested in this study. Furthermore,
ETEC strains expressing K99 and 987P fimbria did
not adhere to IPEC-J2 and IPEC-1 cells. Not sur-
prisingly, most porcine ETEC strains bound better
to porcine epithelial cells than to INT-407 cells. The
K99+ ETEC strain was the exception to this general
finding as it bound extensively to INT-407 and not
to either porcine cell line. Overall, IPEC-1 cells were
superior to IPEC-J2 cells in ETEC binding, both in
terms of the types of adhesins that bound to these
cells and the intensity of bacterial binding (Table
2). In general, the observations of this study suggest
that IPEC-J2 and IPEC-1 strongly support the adhe-
sion of porcine ETEC and may be a suitable model
for studying some aspects of ETEC pathogenesis.
In this study, higher numbers of K88ac bacte-
ria of wild-type strain 3030-2 bound to IPEC-J2
cells when compared to laboratory constructs. The
reason for such a difference in binding was unclear
and was not investigated. However, it is possible
that fimbria are expressed in greater abundance on
the wild-type strains compared to laboratory con-
structs. Another possibility is that there are other
fimbriae or non-fimbrial adhesins involved in bind-
ing that vary in their presence among laboratory
constructs and wild-type strains. Two different wild-
type K88ac strains, 3030-2 and 2534-86, bound to
all the three cell lines efficiently, supporting the re-
producibility of the adherence assays and cell cul-
ture models. Recent studies from our laboratory
have suggested that LT contributes to the coloniza-
tion of K88+ strains in the intestines of piglets (Ber-
berov et al., 2004; Zhang et al., 2006). ETEC K88ac
strain 1836-2 which lacks LT and ST did not adhere
efficiently to IPEC-J2 cells when compared to wild-
type strain 3030-2. In contrast, ETEC 3030-2 and
1836-2 bound equally to IPEC-1 cells. ETEC K88ac
strain 2534-86 and its LT− mutant (MUN285)
bound to both porcine cell types with equal efficien-
cies. Therefore, further investigations are required
to clarify the role of LT, if any in bacterial adher-
ence. In this study, F18 ETEC strain 2134 bound to
IPEC-1 cells but not to IPEC-J2 cells. This indicated
that IPEC-1 cells might represent a suitable in vitro
cellular model for studying the pathogenesis of F18
ETEC. To our knowledge, no other porcine intesti-
nal epithelial cell line susceptible to ETEC F18 has
been described. When polarized IPEC-J2 cells were
used, the same E. coli binding patterns and intensi-
ties were displayed as observed with non-polarized
cells suggesting that both non-polarized and polar-
ized porcine intestinal cells are equally suitable for
in vitro ETEC studies.
Each antigenic variant of K88, namely K88ab,
K88ac and K88ad, exhibits a unique specificity to
host receptors and various sugars are involved in the
binding of different K88 variants to host receptors
(Francis et al., 1999; Jin and Zhao, 2000). Based
on the phenotypic diversity of binding of three an-
tigenic variants of K88 ETEC, six different pheno-
types (A-E) of pigs have been described (Baker et
al., 1997; Bijlsma et al., 1982). As both IPEC-J2 and
IPEC-1 cells bound to all three antigenic variants of
K88, both cell lines were probably derived from phe-
notype A pigs.
In summary, this study demonstrates that various
porcine ETEC strains bind to IPEC-J2 and IPEC-1 in
greater abundance than to INT-407 cells and both
IPEC-J2 and IPEC-1 cells provide a biologically rel-
evant in vitro model system for studying porcine
ETEC-host intestinal epithelial cell interactions.
Acknowledgments
Financial support for this study was provided by the
SDSU Research Support Fund 2005 and the SDSU Ag-
ricultural Experiment Station. Student support was
from SDSU Center for Infectious Disease and Vaccin-
ology (CIDRV) and partial funding was from South Da-
kota NSF EPSCoR program. This manuscript is pub-
lished as South Dakota Agricultural Experiment Station
(AES) Journal series number 3614. We thank Dr. Bruce
D. Schultz, Kansas State University, and Dr. Anthony
Blikslager, North Carolina State University, for provid-
ing IPEC-J2 and IPEC-1 cells respectively for this study
and Dong He for assistance with microscopy.
References
Baker et al., 1997 — D. R. Baker, L. O. Billey, and D. H.
Francis, Distribution of K88 Escherichia coli-adhesive
and nonadhesive phenotypes among pigs of four breeds,
Vet. Microbiol. 54 (1997), pp. 123–132.
Bakker et al., 1992 — D. Bakker, P. T. Willemsen, L. H. Si-
mons, F. G. van Zijderveld, and F. K. de Graaf, Charac-
terization of the antigenic and adhesive properties of
FaeG, the major subunit of K88 fimbriae, Mol. Micro-
biol. 6 (1992), pp. 247–255.
Berberov et al., 2004 — E. M. Berberov, Y. Zhou, D. H.
Francis, M. A. Scott, S. D. Kachman, and R. ,A. Moxley,
Relative importance of heat-labile enterotoxin in the cau-
sation of severe diarrheal disease in the gnotobiotic pig-
let model by a strain of enterotoxigenic Escherichia coli
that produces multiple enterotoxins, Infect. Immun. 72
(2004), pp. 3914–3924.
Bijlsma et al., 1982 — I. G. Bijlsma, A. de Nijs, C. van der
Meer, and J. F. Frik, Different pig phenotypes affect ad-
herence of Escherichia coli to jejunal brush borders by

Page 8

Porcine intestinal ePithelial cell lines as a new in vitro model for enterotoxigenic e. coli ?
K88ab, K88ac, or K88ad antigen, Infect. Immun. 37
(1982), pp. 891–894.
Casey et al., 1992 — T. A. Casey, B. Nagy, and H. W. Moon,
Pathogenicity of porcine enterotoxigenic Escherichia coli
that do not express K88, K99, F41, or 987P adhesins,
Am. J. Vet. Res. 53 (1992), pp. 1488–1492.
Evans et al., 1979 — D. J. Evans Jr., D. G. Evans, and H. L.
DuPont, Hemagglutination patterns of enterotoxigenic
and enteropathogenic Escherichia coli determined with
human, bovine, chicken, and guinea pig erythrocytes in
the presence and absence of mannose, Infect. Immun.
23 (1979), pp. 336–346.
Fairbrother et al., 2005 — J. M. Fairbrother, E. Nadeau,
and C. L. Gyles, Escherichia coli in postweaning diarrhea
in pigs: an update on bacterial types, pathogenesis, and
prevention strategies, Anim. Health Res. Rev. 6 (2005),
pp. 17–39.
Francis et al., 1986 — D. H. Francis, J. E. Collins, and J.
R. Duimstra, Infection of gnotobiotic pigs with an Esch-
erichia coli O157:H7 strain associated with an outbreak
of hemorrhagic colitis, Infect. Immun. 51 (1986), pp.
953–956.
Francis et al., 1999 — D. H. Francis, A. K. Erickson, and P.
A. Grange, K88 adhesins of enterotoxigenic Escherichia
coli and their porcine enterocyte receptors, Adv. Exp.
Med. Biol. 473 (1999), pp. 147–154.
Francis et al., 1998 — D. H. Francis, P. A. Grange, D. H.
Zeman, D. R. Baker, R. Sun, and A. K. Erickson, Expres-
sion of mucin-type glycoprotein K88 receptors strongly
correlates with piglet susceptibility to K88(+) enterotoxi-
genic Escherichia coli, but adhesion of this bacterium to
brush borders does not, Infect. Immun. 66 (1998), pp.
4050–4055.
Francis et al., 1982 — D. H. Francis, G. A. Remmers, and
P. S. DeZeeuw, Production of K88, K99, and 987P anti-
gens by Escherichia coli cultured on synthetic and com-
plex media, J. Clin. Microbiol. 15 (1982), pp. 181–183.
Francis and Willgohs, 1991 — D. H. Francis and J. A.
Willgohs, Evaluation of a live avirulent Escherichia coli
vaccine for K88+, LT+ enterotoxigenic colibacillosis in
weaned pigs, Am. J. Vet. Res. 52 (1991), pp. 1051–1055.
Gaastra and Pederson, 1986 — W. Gaastra and P. A. Ped-
erson, Serologic variants of the K88 antigen. In: D. L.
Lark and S. Normark, Editors, Protein–Carbohydrate
Interactions in Biological Systems, Academic Press. Inc.,
London (1986).
Imberechts et al., 1996 — H. Imberechts, P. Wild, G. Char-
lier, H. De Greve, P. Lintermans, and P. Pohl, Character-
ization of F18 fimbrial genes fedE and fedF involved in
adhesion and length of enterotoxemic Escherichia coli
strain 107/86, Microb. Pathog. 21 (1996), pp. 183–192.
Isaacson et al., 1978 — R. E. Isaacson, P. C. Fusco, C. C.
Brinton, and H. W. Moon, In vitro adhesion of Esch-
erichia coli to porcine small intestinal epithelial cells:
pili as adhesive factors, Infect. Immun. 21 (1978), pp.
392–397.
Jin and Zhao, 2000 — L. Z. Jin and X. Zhao, Intestinal re-
ceptors for adhesive fimbriae of enterotoxigenic Esche-
richia coli (ETEC) K88 in swine—a review, Appl. Micro-
biol. Biotechnol. 54 (2000), pp. 311–318.
Jones and Rutter, 1972 — G. W. Jones and J. M. Rutter,
Role of the K88 antigen in the pathogenesis of neonatal
diarrhea caused by Escherichia coli in piglets, Infect. Im-
mun. 6 (1972), pp. 918–927.
Knutton et al., 1984 — S. Knutton, D. R. Lloyd, D. C.
Candy, and A. S. McNeish, In vitro adhesion of entero-
toxigenic Escherichia coli to human intestinal epithelial
cells from mucosal biopsies, Infect. Immun. 44 (1984),
pp. 514–518.
Lu et al., 2002 — S. Lu, Y. Yao, S. Meng, X. Cheng, and D. D.
Black, Overexpression of apolipoprotein A-IV enhances
lipid transport in newborn swine intestinal epithelial
cells, J. Biol. Chem. 277 (2002), pp. 31929–31937.
Moon et al., 1977 — H. W. Moon, B. Nagy, R. E. Isaacson,
and I. Orskov, Occurrence of K99 antigen on Escherichia
coli isolated from pigs and colonization of pig ileum by
K99+ enterotoxigenic E. coli from calves and pigs, Infect.
Immun. 15 (1977), pp. 614–620.
Moon et al., 1968 — H. W. Moon, D. K. Sorensen. and J.
H. Sautter, Experimental enteric colibacillosis in piglets,
Can. J. Comp. Med. 32 (1968), pp. 493–497.
Morris et al., 1982 — J. A. Morris, C. Thorns, A. C. Scott,
W. J. Sojka. and G. A. Wells, Adhesion in vitro and in
vivo associated with an adhesive antigen (F41) produced
by a K99 mutant of the reference strain Escherichia coli
B41, Infect. Immun. 36 (1982), pp. 1146–1153.
Moxley et al., 1998 — R. A. Moxley, E. M. Berberov, D. H.
Francis, J. Xing, M. Moayeri, R. A. Welch, D. R. Baker.
and R. G. Barletta, Pathogenicity of an enterotoxigenic
Escherichia coli hemolysin (hlyA) mutant in gnotobiotic
piglets, Infect. Immun. 66 (1998), pp. 5031–5035.
Mullaney et al., 1991 — C. D. Mullaney, D. H. Francis. and
J. A. Willgohs, Comparison of seroagglutination, ELISA,
and indirect fluorescent antibody staining for the detec-
tion of K99, K88, and 987P pilus antigens of Escherichia
coli, J. Vet. Diagn. Invest. 3 (1991), pp. 115–118.
Nagy and Fekete, 2005 — B. Nagy and P. Z. Fekete, En-
terotoxigenic Escherichia coli in veterinary medicine,
Int. J. Med. Microbiol. 295 (2005), pp. 443–454.
Orskov et al., 1975 — I. Orskov, F. Orskov, H. W. Smith,
and W. J. Sojka, The establishment of K99, a thermo-
labile, transmissible Escherichia coli K antigen, previ-
ously called “Kco”, possessed by calf and lamb entero-
pathogenic strains, Acta Pathol. Microbiol. Scand. [B]
83 (1975), pp. 31–36.
Roselli et al., 2006 — M. Roselli, A. Finamore, M. S. Britti,
and E. Mengheri, Probiotic bacteria Bifidobacterium an-
imalis MB5 and Lactobacillus rhamnosus GG protect in-
testinal Caco-2 cells from the inflammation-associated
response induced by enterotoxigenic Escherichia coli
K88, Br. J. Nutr. 95 (2006), pp. 1177–1184.
Sambrook et al., 2001 — J. Sambrook, P. MacCallum, and
D. Russell, Molecular Cloning: A Laboratory Manual
(third ed.), Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, USA (2001).
Schierack et al., 2006 — P. Schierack, M. Nordhoff, M.
Pollmann, K. D. Weyrauch, S. Amasheh, U. Lodemann,
J. Jores, B. Tachu, S. Kleta, A. Blikslager, K. Tedin, and
L. H. Wieler, Characterization of a porcine intestinal ep-
ithelial cell line for in vitro studies of microbial patho-
genesis in swine, Histochem. Cell. Biol. 125 (2006), pp.
293–305.
Zhang et al., 2006 — W. Zhang, E. M. Berberov, J. Freel-
ing, D. He, R. A. Moxley, and D. H. Francis, Significance
of heat-stable and heat-labile enterotoxins in porcine co-
libacillosis in an additive model for pathogenicity stud-
ies, Infect. Immun. 74 (2006), pp. 3107–3114.