Lactic acid bacteria (LAB) strains modulate inflammatory genes in RTgutGC cells, and triploid Chinook salmon (Oncorhynchus tshawytscha) challenged with V. anguillarum

• The three Lactic Acid Bacteria strains did not have cytotoxic effects • The three Lactic Acid Bacteria strain did have differential effects om immune gene expression • The three Lactic Acid Bacteria strain did not improve growth or gut barrier function • The three Lactic Acid Bacteria strain did not improve survival or immune response Lactic acid bacteria (LAB) are known for their probiotic benefits, including enhancing fish growth, stress tolerance, and immune responses. While multi-strain probiotics have shown promise in improving survival and modulating inflammation in V. anguillarum -infected Chinook salmon, the effects of individual or dual-strain formulations on immune modulation and gut barrier integrity in less commonly farmed salmonids like Chinook salmon ( Oncorhynchus tshawytscha ) remain understudied. This research evaluated the potential of Limosilactobacillus reuteri LRE2, L. reuteri 830, and a mixture of L. reuteri RC-14 and L. rhamnosus GR-1 as oral immunostimulants. Using the RTgutGC intestinal epithelial cell line, cytotoxicity assays confirmed that none of the LAB strains affected cell viability at concentrations up to 10 ^7 CFU. Gene expression analysis revealed that L. reuteri LRE2 significantly downregulated il1b and il6 transcripts compared to L. reuteri 830 and the strain mixture, while tnfa expression was reduced in L. reuteri 830-treated cells. In vivo , juvenile Chinook salmon were supplemented with the LAB strains for four months, but no changes in weight were observed. Following V. anguillarum challenge, the probiotics did not improve survival. However, il8 expression increased significantly in the hindgut of fish supplemented with L. reuteri 830 and the strain mixture at 7 days post-infection. Tight junction gene expression remained unchanged across treatments. This study highlights strain-specific differences in immune modulation, though the tested LAB strains did not enhance survival during V. anguillarum infection. Further research is needed to optimize probiotic formulations for disease resistance in Chinook salmon.