Characterization of the attaching and effacing mechanism of Escherichia coli in
the pig intestine using in vivo, organ culture, and tissue culture infection models
John M. Fairbrother, BVSc, PhD
OIE Reference Laboratory for Escherichia coli, Faculté de médecine vétérinaire,
Université de Montréal, St-Hyacinthe, Quebec, Canada
(john.morris.fairbrother@umontreal.ca)
Introduction
Enteropathogenic E. coli (EPEC) cause diarrhea in infants1, rabbits2, calves, pigs3,
lambs, and dogs4. Shiga toxin-producing E. coli (STEC), especially those of serotype
O157:H7, are important emerging pathogens causing food-borne infections leading to
bloody diarrhea and hemolytic uremic syndrome in humans1. Ruminants are an
important reservoir of STEC5. These STEC are carried at high frequency by cattle but
have not been implicated in natural disease, although they may be used to
experimentally reproduce disease in calves6.
EPEC, and certain STEC, including O157:H7, cause typical intestinal attaching and
effacing (AE) lesions which are characterized by intimate bacterial adherence to
intestinal epithelial cells, effacement of the brush border, F-actin rearrangement, and
formation of a pedestal of polymerized F-actin and other cytoskeletal elements
underneath the adherence site1. Collectively, these strains are called attaching and
effacing E. coli (AEEC). Most AE phenotype elements are encoded on a pathogenicity
island called locus of enterocytes effacement (LEE). The LEE contains genes encoding
an outer membrane adhesin termed intimin (eae gene), a type-III secretion system
machinery, chaperones, translocator (EspA, EspB and EspD) and effector (EspF, EspG,
Map) proteins as well as the translocated intimin receptor (Tir). In addition, the paa (for
porcine A/E associated) gene, located on the chromosome but outside the LEE, has
been found in AEEC, including O157:H77. Paa, a 27.6 kDa protein located on the
bacterial surface, appears to contribute to the AE process, and may be involved in the
initial bacterial adherence.
The roles of various virulence factors and the pathogenic mechanisms of human and
animal EPEC and STEC have been extensively studied using a largely molecular and
cellular approach. However, it is important to carry out studies in experimental infection
models in order to confirm the in vitro findings on pathogenic mechanisms in a more
realistic situation in vivo and to provide more appropriate models for the evaluation of
various prevention or treatment strategies.
In vivo models
We initially established a homologous infection model in which newborn colostrumdeprived pigs were challenged with an EPEC strain isolated from a pig with diarrhea3.
Histopathological lesions ranged from mild and scattered through the large and small
intestine, to severe and involving mostly the cecum and colon. They included light to
moderate inflammation of the lamina propria, enterocyte desquamation and some mild
ulceration, and light to moderate villus atrophy in the small intestine. Extensive
multifocal bacterial colonization of the surface epithelium by a thin layer of coccobacilli,
often oriented in a palisade pattern, was observed. Typical intimate attachment of
bacteria to intestinal epithelial cells and effacement of microvilli were observed on
electron microscopy. Thus, we demonstrated that a porcine E. coli strain could cause
lesions similar to those observed in naturally infected pigs and to those of human EPEC
strains in experimentally inoculated newborn pigs, making this an interesting model to
be further pursued.
We then more convincingly confirmed the role of intimin in the development of AE
lesions by a wide range of porcine EPEC strains in colostrum-deprived newborn
gnotobiotic piglets, where the absence of competing normal intestinal microflora
permitted a more precise study of the interaction between EPEC bacteria and the
intestinal mucosa8.
However, the animals challenged in these models are very young, and the results are
not necessarily applicable to older animals with a more mature immune system and
more complex intestinal microflora, in which natural disease usually occurs. Also, these
models are laborious and time consuming and difficult to adapt to study of prevention
strategies involving vaccination or in-feed administration. Frustratingly, we were unable
to induce the development of attaching and effacing lesions in challenge studies using
older, weaned animals. Several predisposing factors, such as a weaner diet containing
soybean and field peas or Porcine Reproductive and Respiratory Syndrome virus
infection, may enhance bacterial colonization and development of AE lesions. Host
immune status is an important factor in the development of AE lesions. Thus, colostrumdeprived piglets are more susceptible to infection than those having received colostrum.
Also, we had been unable to induce such lesions in older conventional pigs with a more
mature immune system.
We have now successfully reproduced extensive AE lesions in weaned pigs treated with
dexamethasone, used in immunosuppressive doses9. We demonstrated that cytokines
IL-1beta, IL-6, IL-8, and IL-12 are significantly upregulated in the ileum of challenged
pigs not treated with dexamethasone, whereas dexamethasone inhibits such
upregulation. Our results confirm that the host immune status is a key determinant
protecting against the development of AE lesions in weaned pigs, and it appears that IL1, IL-6, IL-8, and IL-12 are involved in the natural resistance to development of these
lesions.
Thus, we have shown that immunosuppression is required for development of AE
lesions in a post-weaning in vivo EPEC pig infection model9.The mechanisms by which
EPEC cause diarrhea and the role of co-infection with other pathogens are not known.
However, the similarity to human EPEC both in terms of the pattern of intestinal
colonization, the type of lesion produced and the presence of intimin on bacteria
strongly suggest that porcine and human EPEC share similar pathogenic mechanisms.
In vitro organ culture (IVOC) model
Study of pathogenic mechanisms
Although we succeeded in reproducing the AE lesions in older weaned pigs, this model
is very arduous and not very suitable for quantitative studies. We thus decided to use
the ileal in vitro organ culture (IVOC) model using tissues originating from colostrumdeprived newborn piglets to study the AE phenotype of porcine EPEC ex vivo.
We had previously demonstrated a role for the intimin protein in the development of AE
lesions following infection with pig EPEC strains, using pig ileal explants8, 10, 11. In
addition, we identified a novel bacterial factor, Paa, which appears to be involved in the
early steps of the adhesion mechanism of AEEC7. We then demonstrated that EPEC
and STEC strains from various animal species and humans induce the AE phenotype in
porcine ileal IVOC and that intimin subtype influences intestinal adherence and tropism
of these strains.
We also demonstrated that a tir mutant of an AEEC strain demonstrates close
adherence to the epithelial cells of porcine explants, with microvillous effacement but
with no evidence of actin polymerization and pedestal formation, and that intimin and
EspA, a constituent of the type III secretion apparatus, seem to be involved in this
phenotype12. This study provided further evidence for the existence of a host cellencoded intimin receptor, in addition to the bacterially encoded Tir.
We also used the pig ileal IVOC model to study the interaction with intestinal tissues of
an atypical sorbitol-fermenting, Shiga-toxin-negative E. coli O157:NM bovine isolate13. It
has been shown that sorbitol-fermenting E. coli O157 isolates are implicated in animal
and human diseases and may represent new emerging pathogens. This E. coli O157
isolate also possessed an intimin of type b, whereas the translocated intimin receptor
Tir and type III secretion system components EspA, EspB, and EspD were of type a. In
contrast, Shiga-toxin-positive O157:H7 isolates usually possess variants of type g. The
isolate did not present typical O157:H7 AE lesions in the newborn pig ileal IVOC model,
although extensive effacement and elongation of the microvilli were observed. Our
results suggested that such an SF, Shiga-toxin-negative O157:NM isolate found in
cattle may potentially cause disease, such as diarrhea without hemolytic uremic
syndrome, in humans.
Study of strategies for prevention of AEEC infections
Using the porcine ileal IVOC model, we have demonstrated that egg yolk-derived
antibodies specific for the AEEC virulence factors intimin and translocated intimin
receptor Tir, but not those specific for the AEEC-secreted proteins EspA, EspB and
EspD, significantly reduced the bacterial adherence of a porcine EPEC strain14.
Antibodies specific for intimin and Tir also significantly reduced bacterial adherence of
heterologous AEEC strains, including human, bovine and canine EPEC strains, as well
as of O157:H7 Shiga toxin-producing E. coli strains in this model. In addition, we
demonstrated that the oral administration of these anti-intimin antibodies significantly
reduced the extent of AE lesions found in the small intestine of weaned pigs challenged
with an EPEC strain. Overall, our results underline the potential use of specific egg yolkderived antibodies as a novel approach for the prevention of AEEC infections.
Tissue culture model
To date, adherence of porcine EPEC to the cell lines commonly used to study EPEC
has not been demonstrated. We have now found that porcine EPEC strains adhere and
induce AE lesions in a recently established porcine intestinal cell line IPEC-J2. Use of
this cell line has permitted us to study the pathogenesis of porcine EPEC infections,
particularly in the first 8 hours of infection.
Conclusion
Use of a combination of infection models has provided further insight into the
pathogenic mechanisms of attaching and effacing E. coli, the role of certain bacterial
virulence factors in the development of lesions, and at least one possible strategy for
preventing the development of these lesions.
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