Cell-Free Culture Supernatant of AG01 and subsp. AG02 Reduces the Pathogenicity of NetB-Positive in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by . This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of and to investigate the mode of action of cell-free supernatants (CFS) from probiotic AG01 and subsp. AG02 in reducing the pathogenicity of . The cell adhesion, permeability and cytotoxicity were assessed under challenge with four strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from subsp. AG02 but not AG01 reduced the cell adhesion of three out of four strains (by 77-85% vs. the control, < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by and reveal a beneficial mode of action of subsp. AG02 in reducing cell adhesion and, together with AG01, in reducing cytotoxicity.