Evaluation of Immunoprotective Efficacy of Recombinant Pseudomonas plecoglossicida TolC Protein(rPpTolC)in Zebrafish

Pseudomonas plecoglossicida infection induces rapid mass mortality in fish; the outer membrane protein TolC from Gram-negative bacteria typically exhibits potent immunogenicity, and this study focuses on the immunoprotective efficacy of TolC from this specific pathogen in zebrafish(Danio rerio). The PptolC gene was successfully cloned, and its B-cell antigenic epitopes and sequence conservation were analyzed. Zebrafish were immunized with rPpTolC to assess its capacity to induce non-specific immune responses and immunoglobulin production, as well as its protective efficacy against challenges with heterologous pathogens. Results showed that PpTolC encodes a 457-amino acid protein with high sequence conservation and five high-confidence B-cell antigenic epitopes. Zebrafish were immunized with rPpTolC to assess its capacity to induce non-specific immune responses(such as myd88, tnf-α and tgf-β1)and immunoglobulin production(IgM, IgD, IgZ)in gill and intestinal tissues. Following separate challenges with P. plecoglossicida and Vibrio parahaemolyticus(each at 2×LD50), immunized zebrafish exhibited survival rates exceeding 60% against both pathogens individually. Concurrently, immunized fish showed significantly reduced intestinal ROS and MPO levels, alongside decreased pathogen loads in respective challenge groups. Collectively, rPpTolC demonstrates robust cross-protective immunity in zebrafish and represents a promising candidate for subunit vaccine development.