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