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

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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.

Introduction

Both porcine neonatal and post-weaning diarrhea (PWD) caused by enterotoxigenic Escherichia coli (ETEC) result in significant morbidity and mortality and are economically important diseases of pigs (Fairbrother et al., 2005, Nagy and Fekete, 2005). The colonization of ETEC in the small intestine is primarily mediated by fimbria which confer to ETEC the ability to attach to receptors on the enterocytes. Secretory diarrhea associated with ETEC infection is mediated by any of several enterotoxins 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 variant ETEC strain isolated from diarrheic pigs (Fairbrother 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 objective of this study was to examine the adherence of various strains of ETEC to two porcine intestinal epithelial cell lines IPEC-J2 and IPEC-1 and to compare their adherence to the human intestinal epithelial cell line INT-407. The findings of this study indicate that IPEC-J2 and IPEC-1 cell lines are superior to the human intestinal cell line (INT-407) in that they support the adherence of most porcine ETEC strains.

Section snippets

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 infusion base), except K12:K99 which was grown on Essential Salt Medium (Francis et al., 1982) supplemented with Eagle's essential amino acids and vitamins. The bacterial cultures were incubated for 18 h at 32 °C before use in the adherence studies.

Cell lines and culture conditions

The IPEC-J2 and IPEC-1 cell lines have been previously described (Lu et al., 2002, Schierack

Results

Both porcine small intestinal epithelial cell lines bound a range of ETEC, albeit to varying degrees. Representative photomicrographs indicating the degree of adherent bacteria are shown in Fig. 1A and 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

Discussion

In this study all three K88+ ETEC strains (K88ab, K88ac, and K88ad) adhered to both IPEC-J2 and 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 surprisingly, most porcine ETEC strains bound better to porcine epithelial cells than to INT-407

Acknowledgements

Financial support for this study was provided by the SDSU Research Support Fund 2005 and the SDSU Agricultural Experiment Station. Student support was from SDSU Center for Infectious Disease and Vaccinology (CIDRV) and partial funding was from South Dakota NSF EPSCoR program. This manuscript is published 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

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