Escherichia coli in the pig intestine using in vivo, organ culture, and tissue culture infection models

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 ([e-mail])
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 colostrum-deprived 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, colostrum-deprived 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 IL-1, 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 colostrum-deprived 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 explants^{8, 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 cell-encoded 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 yolk-derived 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.

References:

1. 
   Nataro JP and Kaper JB. Diarrheagenic Escherichia coli. Clin Microbiol Rev 11:142-201,1998.
   
2. 
   Cantey JR and Blake RK. Diarrhea due to Escherichia coli in the rabbit: a novel mechanism.
   J Infect Dis 135:454-462, 1977.
   
3. 
   Helie P, Morin M, Jacques M, Fairbrother JM. Experimental infection of newborn pigs with an attaching and effacing Escherichia coli O45:K"E65" strain. Infect Immun 59: 814-821, 1991.
   
4. 
   Janke BH, Francis DH, Collins JE, Libal MC, Zeman DH, Johnson DD. Attaching and effacing Escherichia coli infections in calves, pigs, lambs, and dogs. J Vet Diagn Invest 1:6-11, 1989.
   
5. 
   Stevens MP, van Diemen PM, Dziva F, Jones PW, Wallis TS. Options for the control of enterohaemorrhagic Escherichia coli in ruminants. Microbiol 148:3767-3778, 2002.
   
6. 
   Dean-Nystrom EA, Bosworth BT, Cray WC Jr, Moon HW. Pathogenicity of Escherichia coli O157:H7 in the intestines of neonatal calves. Infect Immun 65:1842-1848, 1997.
   
7. 
   Batisson I, Guimond MP, Girard F, An H, Zhu C, Oswald E, Fairbrother JM, Jacques M, Harel J. Characterization of the novel factor paa involved in the early steps of the adhesion mechanism of attaching and effacing Escherichia coli. Infect Immun 71: 4516–4525, 2003.
   
8. 
   Zhu C, Harel J, Jacques M, Desautels C, Donnenberg MS, Beaudry M, Fairbrother JM. Virulence properties and attaching-effacing activity of Escherichia coli O45 associated from swine post weaning diarrhea. Infect Immun 62: 4153-4159, 1994.
   
9. 
   Girard F, Oswald I, Taranu I, Hélie P, Appleyard G, Harel J and Fairbrother JM. Host Immune Status Influences the Development of Attaching and Effacing Lesions in Weaned Pigs. Infect Immun 73: 5514-5523, 2005.
   
10. 
    Zhu C, Ménard S, Dubreuil JD and Fairbrother JM. Detection and localization of the EaeA protein of attaching and effacing Escherichia coli O45 from pigs using a monoclonal antibody. Microb Pathog 21: 205-213, 1996.
    
11. 
    Zhu C, Harel J, Jacques M and Fairbrother JM. Interaction with pig ileal explants of Escherichia coli from swine with post weaning diarrhea. Can J Vet Res
    59: 118-123, 1995.
    
12. 
    Girard F, Batisson I, Frankel G, Harel J, Fairbrother JM. Interaction of Enteropathogenic and Shiga-Toxin Producing Escherichia coli with Porcine Intestinal Mucosa: Role of Intimin and Tir in Adherence. Infect Immun 73: 6005-6016, 2005.
    
13. 
    Lefebvre B, Diarra MS, Fairbrother JM, Nadeau E, Dubois M Jr, Malouin F. Intestinal mucosa adherence and cytotoxicity of a sorbitol-fermenting, Shiga-toxin-negative Escherichia coli O157:NM isolate with an atypical type III secretion system. Foodborne Pathog Dis 7: 985-990, 2010.
    
14. 
    Girard F, Batisson I, Martinez G, Breton C, Harel J and Fairbrother JM. Use of virulence factor-specific egg yolk-derived immunoglobulins as a promising alternative to antibiotics for prevention of attaching and effacing Escherichia coli infections. FEMS Immunol Med Microbiol 46: 340-350, 2006.