Pimrawee Chaemlek

Bacterial diseases caused by Flavobacterium covae (Fc), Vibrio harveyi (Vh), Vibrio vulnificus (Vv) and Photobacterium damselae (Pd) seriously constrain Asian seabass aquaculture. Here we dissect the mucosal immune mechanisms engaged by a five-month sequential vaccination strategy that combines nanoemulsion immersion priming with multivalent oral hydrogel boosting. Juvenile seabass were vaccinated, then challenged with F. covae by freshwater immersion and with a Vibrio–Photobacterium (Vh/Vv/Pd) mix by immersion or intraperitoneal injection. Gills were sampled after immersion challenges and intestine after injection, and profiled by RNA sequencing and label-free quantitative proteomics, with selected genes validated by RT-qPCR. Principal component analysis showed clear separation of vaccinated and control fish in all tissues and challenges, indicating a strong and coherent transcriptional reprogramming. Vaccination markedly increased the number of upregulated genes, with Gene Ontology enrichment revealing dominant signatures of ribosome biogenesis, RNA processing, lysosomal organization and immune response. KEGG analysis highlighted cytokine receptor interaction; NOD and Toll-like receptor signaling; oxidative phosphorylation; and phagosome, lysosome and cell adhesion molecule pathways, consistent with heightened antimicrobial readiness. Volcano plots and focused heatmaps showed strong induction of interferon-stimulated genes, cytokines and chemokine receptors, complement components, macrophage mannose receptor, epithelial barrier mediators and numerous immunoglobulin transcripts, with tissue- and challenge-specific patterns. Proteomics corroborated these trends, demonstrating a higher abundance of immunoglobulin heavy chains, complement proteins, cathepsins, heat shock and redox chaperones, ribosomal proteins and cytoskeletal and adhesion regulators in vaccinated mucosae. Integrated pathway mapping linked endothelial adhesion molecules and leukocyte integrins with T cell costimulation networks and an intestinal immune network for immunoglobulin production, including enhanced pIgR-mediated transcytosis. Overall, the sequential vaccination regimen was associated with coordinated transcriptomic and proteomic signatures related to epithelial responses, innate immunity, and humoral immune functions across gill and intestinal tissues. These molecular patterns were accompanied by improved survival following bacterial challenge; however, the present data do not directly demonstrate the functional activity of the inferred immune mechanisms in Asian seabass.

Aquaculture production of Asian seabass is increasingly threatened by recurrent bacterial diseases, while practical vaccination strategies that provide protection against bacterial challenges involving multiple pathogens during extended grow-out periods remain limited. This study evaluated the immunological and protective effects of a sequential mucosal vaccination strategy in juvenile Asian seabass (Lates calcarifer; 200 fish per treatment, distributed among four replicate tanks of 50 fish) against four major bacterial pathogens: Flavobacterium covae, Vibrio harveyi, Vibrio vulnificus, and Photobacterium damselae. The vaccination regimen consisted of two nanoemulsion-based immersion vaccination events administered 14 days apart, followed by three oral chitosan–alginate hydrogel vaccination courses administered for 7, 5, and 3 consecutive days. Bacterial challenge experiments were conducted at three post-vaccination time points using immersion and intraperitoneal injection models. Growth performance and feed conversion were evaluated using replicate-tank means as the experimental units. Sequential vaccination did not significantly affect final body weight, weight gain, average daily gain, specific growth rate, or feed conversion ratio (p > 0.05). Vaccinated fish showed significantly higher antigen-specific IgM levels in skin mucus, intestinal mucus, and serum across the evaluated challenge conditions. Correspondingly, ighm, ighd, and ight expression was increased in the gills, skin, head kidney, and intestine, indicating enhanced immunoglobulin-associated responses in mucosal and lymphoid tissues. Full-length 16S rRNA gene sequencing showed vaccine-associated changes in gill and intestinal bacterial community composition, including a lower relative representation of several challenge-associated taxa and a higher relative representation of selected commensal-associated taxa. Vaccinated fish showed significantly higher cumulative survival following F. covae immersion, mixed V. harveyi/V. vulnificus/P. damselae immersion, and mixed-pathogen injection challenges (p < 0.05). These findings demonstrate that sequential nanoemulsion immersion priming and oral hydrogel boosting enhanced pathogen-specific humoral responses and increased immunoglobulin-gene expression. The sequential vaccination strategy improved survival following a separate Flavobacterium covae challenge and a combined Vibrio harveyi/Vibrio vulnificus/Photobacterium damselae challenge during the experimental period without adversely affecting growth. This needle-free strategy warrants further evaluation under commercial aquaculture conditions.