Comparative Transcriptomic Analysis Underlies the Differential Virulence of Vibrio harveyi and Vibrio vulnificus in American Eels (Anguilla rostrata)

<i>Vibrio harveyi</i> (Vh) and <i>Vibrio vulnificus</i> (Vv) are major bacterial pathogens affecting farmed eels, but their comparative virulence mechanisms remain poorly characterized. This study combined histopathology and transcriptomic profiling to investigate organ-specific damage and host responses in American eels (<i>Anguilla rostrata</i>, 20 g per fish, for a total of 60 fish) following experimental infection with LD₅₀ doses of Vh (strain HA_1, 7.5 × 10⁴ CFU/fish) and Vv (strain FJ_4, 5.0 × 10⁵ CFU/fish). Tissue samples from liver, kidney, and spleen were collected at 0, 36, and 60 h post-infection (hpi). Histopathological analysis revealed distinct injury patterns: Vh induced severe hepatic edema and necrosis, whereas Vv caused vacuolar degeneration and vascular congestion in the liver. In the kidney, Vv triggered acute necrosis and vacuolization by 36 hpi, while Vh-induced renal damage was delayed until 60 hpi. Transcriptomic analysis of spleen tissue identified 4779 and 1215 differentially expressed genes (DEGs) in the Vh_36 vs. Vv_36 and Vh_60 vs. Vv_60 comparisons, respectively. Functional enrichment analysis associated these DEGs with 109 Gene Ontology (GO) terms-mainly catalytic activity, biological regulation, and binding-and 51 KEGG pathways, including "tuberculosis" and "pathways in cancer". Differential alternative splicing (DAS) analysis further uncovered 1579 and 1214 DAS events originating from 12,482 and 12,316 splicing genes in the two comparisons. These were enriched in GO categories such as "binding", "cellular process", and "cell part", as well as KEGG pathways related to "signal transduction", "infectious diseases", and "immune system." Protein-protein interaction network analysis identified 119 cross-DAS-encoded proteins, including 8 that were predicted as key regulators of virulence differences. In summary, this work presents the first integrative study comparing the pathogenicity and host transcriptional dynamics of Vh and Vv in American eels, providing new molecular insights into their distinct virulence strategies.