Abstract: 【Objective】 Porcine enterotoxigenic Escherichia coli (ETEC) is a worldwide cause of bacteria induced diarrhoea in piglets. In the veterinary practices of pig production, serological identification of ETEC shows that F4ac is the most common serological type expressed in ETEC strains isolated from diarrheic piglets. So far, the mechanism by which ETEC produces diarrhoea in piglets has been clearly elucidated. However, the immunostimulation of ETEC-culture to host target cells has not been studied or described. In the present study, the immunostimulation of the supernatant of F4ac ETEC-culture to IPEC-J2 cells was studied. 【Method】 The culture of F4ac ETEC strain 200 was collected and centrifuged (4℃, 4 000 r/min for 15 min) after 12 hours in culture, and the supernatant was sterilized by passing it through a 0.22 μm filter. The solution combined the sterilized supernatant with equivalent DMEM/F12 medium was used to challenge IPEC-J2 cells for 3 hours. The IPEC-J2 cells co-cultured with fresh LB medium and equivalent DMEM/F12 medium were as the control group. For both treatments, each experiment was repeated three times. Total RNAs of the stimulated and control IPEC-J2 cells were extracted using TRIZOL Reagent following the manufacturer’s instructions. According to the manufacturer’s instructions, complementary DNA (cDNA) was synthesized from RNA using the Prime Script® RT reagent Kit with gDNA Eraser (Perfect Real Time). The cDNA samples were then analyzed with real time RT-PCR using a LightCycler® 480 Real-Time PCR System. The real time RT-PCR reactions were performed in a final volume of 20 μL with the Roche SYBR Green PCR Kit according to the manufacturer’s instructions. The pig house-keeping gene β-actin was used as the internal standards to correct the input of cDNA. Triplicate qRT-PCRs were performed on each cDNA and the average Ct was used for further analysis. The relative quantification values were calculated using the 2-ΔΔCt. Differential mRNA expression profiles of IL8、TNF-α, CXCL2, IL6, IL1A, TLR4, SLPI, PLAU and MUC13 between the stimulated and control IPEC-J2 cell groups were tested by amplification using documented and self-designed primers in the presence of SYBR Green. 【Result】 Compared with the control group, the mRNA expression of three important cytokines IL8, TNF-α and CXCL2 were up-regulated in the stimulated treatment group with the fold-change of IL8 being 3.24 (P<0.05), the fold-change of TNF-α also being 3.24 (P<0.01) and that of CXCL2 being 1.65 (P<0.001). For the rest six genes (IL6, IL1A, TLR4, SLPI, PLAU and MUC13), no statistically significant difference was observed in the mRNA expression levels between the two groups. 【Conclusion】 In this study, the IPEC-J2 cells were challenged with the supernatant of F4ac ETEC-culture for 3 hours, and it was found that this stimulation increased the mRNA expression levels of three key pro-inflammatory genes IL8, TNF-α and CXCL2, which proved that the supernatant of F4ac ETEC-culture showed immunostimulation to the IPEC-J2 cells. The discovery in this study provided insights into the immunostimulation of enterotoxin and/or adhesion of F4ac ETEC to porcine intestinal epithelial cells.

Key words: F4ac ETEC , culture supernatant , IPEC-J2 cells , FQ-PCR , immunostimulation