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Fish were administered an experimental vaccine via intraperitoneal injection, consisting of formalin\u2010inactivated IHNV formulated with Montanide ISA 763A VG. A 2\u2009\u00d7\u20092 factorial design was employed to compare two injection volumes (50\u2009\u03bcL or 150\u2009\u03bcL) and two immunisation regimens (single versus double injection). Serum neutralising antibody titres, splenic expression of six immune\u2010related genes (\n                    <jats:italic>TLR7<\/jats:italic>\n                    ,\n                    <jats:italic>MyD88<\/jats:italic>\n                    ,\n                    <jats:italic>IRF7<\/jats:italic>\n                    ,\n                    <jats:italic>IFN\u2010\u03b1<\/jats:italic>\n                    ,\n                    <jats:italic>IgM<\/jats:italic>\n                    ,\n                    <jats:italic>Mx\u20101<\/jats:italic>\n                    ), and gut microbiota composition were assessed before and after IHNV challenge. A double immunisation regimen with 50\u2009\u03bcL elicited the highest antibody titres and the most robust post\u2010challenge upregulation of all target genes, whereas administration of 150\u2009\u03bcL alone yielded inferior outcomes. 16S rRNA gene sequencing revealed Proteobacteria and Cyanobacteria as the predominant bacterial phyla across all treatment groups. Immunisation significantly reduced the abundance of potentially pathogenic taxa (e.g.,\n                    <jats:italic>Flavobacterium<\/jats:italic>\n                    ,\n                    <jats:italic>Vibrio<\/jats:italic>\n                    ) and increased that of\n                    <jats:italic>Pseudomonas<\/jats:italic>\n                    and\n                    <jats:italic>Acinetobacter<\/jats:italic>\n                    , indicating enhanced intestinal homeostasis and antiviral gene expression. Collectively, a two\u2010dose regimen with 50\u2009\u03bcL maximises systemic immunity, strengthens mucosal defence and supports a balanced gut microbiota, providing a practical and effective strategy for IHNV control in rainbow trout aquaculture.\n                  <\/jats:p>","DOI":"10.1111\/jfd.70131","type":"journal-article","created":{"date-parts":[[2026,1,28]],"date-time":"2026-01-28T18:11:32Z","timestamp":1769623892000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Immune Efficacy and Intestinal Microbiota of Rainbow Trout (\n                    <i>Oncorhynchus mykiss<\/i>\n                    ) After Inactivated\n                    <scp>IHNV<\/scp>\n                    Vaccination via Distinct Injection 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