Abstract
Post-weaning diarrhoea is а striking issue of the pig industry and its onset is primarily associated with Escherichia coli К88 (Е. coli). То assess the effectiveness of bioactives in preventing the damages exerted bу the pathogen, the aim of this study was to set-up an in vitro model to mimic an Е. coli challenge on intestinal cells.
Сасо-2 and IPEC-J2 cells were differentiated on 3.0 µm-pore Transwell inserts while cultured in DMEM+10% FBS (basal medium, ВМ) in 5% СО2 at 37°С. Then, cells were infected with Е. coli 5х10^7 (Сасо-2) or 5х10^6 (IPEC-J2) CFU/mL and treated with ВМ (CTR-), ВМ + Е. coli (CTR + ), or ВМ + Е. coli + colistin 4 ppm (COL + ). After 2h and 4h, trans-epithelial electrical resistance (TER), paracellular permeability (РСР) with fluorescein, and bacterial translocation (ВТ) were measured. For the latter, aliquots of basolateral media were counted on agar plates. Moreover, both cell lines were differentiated on 24-wells and challenged with Е. coli 5х10^7 (Сасо-2) or 5х10^6 (IPEC-J2) CFU/mL with or without colistin 4 ppm (CTR+ and COL+, respectively). After 1h, cells were washed, lysed, and seeded on agar plates to quantify adhered Е. coli. Data were analysed with One-way (ВТ, РСР, adhesion test) or Two-way (ТЕR) ANOVA, with each treatment having 6 replicates.
For both cellular lines, CTR + decreased TER (at 4h: -86% of CTR- for Сасо-2; -68% of CTR- for IPEC-J2) and increased РСР and ВТ at 2h and 4h (Р < 0.05). Colistin avoided the drop in TER and kept РСР and ВТ values in line with CTR-. COL+ minimized Е. coli counts during adhesion (-100% compared to CTR+, Р<0.05).
In conclusion, an effective in vitro model to mimic an Е. coli intestinal challenge was successfully developed. Future studies will employ this model to explore host-pathogen interactions and how bioactive compounds reduce Е. coli-mediated damages.For more information: marketing@vetagro.com