For the F4ac ETEC infection, the responses of the host cells were characterized by great up regulations on immune, wound ing and inflammatory response. The findings herein pro vided a solid proof why ETEC with F4 may be more virulent compared to F18 which seems to elicit necessary milder effects, which further characterized and defined the gen etic mechanisms of responses to different ETEC colonization and adhesion in small intestine of piglets. Materials and Methods Cell culture The IPEC J2 cell line was grown in Dulbeccos modified eagle medium Hams F 12 medium supplemented with 5% fetal calf serum and was maintained in a 95% air 5% CO2 humidified atmosphere at 37 C, which were free of mycoplasma contamination. Bacterial strains F4ab ETEC strain 195 and F4ac ETEC strain 200 were removed from cryo storage and cultured in Ordin ary Broth Agar at 37 C for three generations.

ETEC strain 8813 was cultured in static Tryp tone Soya Agar medium at 37 C for 24 h, and then in static Tryptone Soya Broth medium at 37 C for two generations. For cell infection experiment, the E. coli strains were subcultured in shaking LB and TSB medium, respectively, at 37 C for 12 h, then centrifuged and washed with sterile PBS. Finally the bacterial suspension was prepared in PBS. Infection of the cell lines Monolayers of cells prepared in 24 well tissue culture plates were washed twice with PBS, then 0. 5 ml of DMEM was added. A total of 20ul of bacterial suspension was used for infection or the same volume of PBS as control. The cells were incubated at 37 C in a 95% air 5% CO2 air atmosphere for 3 h.

The adhesion values of the ETEC strains to IPEC J2 cells were checked by real time PCR with slightly modified procedures described by Candela et al. Twelve samples were prepared including nine with the three ETEC strains infection treatments and three samples as control. Total RNA isolation IPEC J2 cells infected with and without E. coli strains were washed twice with PBS, then lysed with TRIZOL Reagent directly in the culture dishes. Isolation of RNA was performed using TRIZOL Reagent following the manufacturers instructions and checked for a RIN number to inspect the RNA integration by an Agilent Bioanalyzer 2100. Qualified total RNA was further purified by RNeasy micro kit and RNase Free DNase Set.

Sample labeling and hybridization Total RNA was amplified and labelled by Low Input Quick Amp Labeling Kit, One Color, following the manu facturers instructions. The labeled cRNA was purified by RNeasy mini kit, then used for hybridization onto porcine oligo microarray slides containing 43,603 oligonucleotide probes at 65 C for 17 h. The hybri Batimastat dized microarray slides were washed according to the manufacturers instructions and were scanned by Agilent Microarray Scanner at 5 mm resolution. Raw data were normalized by Quantile algorithm, Gene Spring Soft ware 11. 0.