<b>The quadruple gene mutant (</b><b><i>aer</i></b><b>A</b><b>-</b><b><i>ahh</i></b><b>1-</b><b><i>rtx</i></b><b>A</b><b>-</b><b><i>th</i></b><b>) of </b><b>A</b><b>eromonas dhakensis</b><b> shows reduced virulence and promising vaccine development potential</b>

<i>Aeromonas dhakensis</i> is a zoonotic pathogen responsible for severe infections in both humans and aquatic organisms. The increasing incidence of antibiotic resistance in <i>A. dhakensis</i> strains underscores the urgent need for alternative control strategies, such as vaccines. In this study, a quadruple gene deletion mutant of <i>A. dhakensis </i>△<i>aart</i> was generated by targeting the virulence genes: <i>aer</i>A, <i>ahh</i>1, <i>rtx</i>A, and <i>th</i>. The △<i>aart</i> exhibited significantly reduced virulence in hemolytic activity and cytotoxicity, whereas no impairment in bacterial growth. Using zebrafish model, the LD₅₀ of △<i>aart </i>was 136-fold higher than that of the wild-type strain. Following immunization, the peak expression of innate immune-related genes (<i>SOD</i>, <i>CAT</i>, <i>LZM</i>, <i>TNF-α</i>, <i>IL-6</i>, <i>IL-1β</i>) in the kidney was observed on day 3, followed by a decline; <i>MHC-I</i> peaked on day 7, and IL-10 on day 14, with similar trends noted in the intestine, while <i>MHC-I</i> did not show significant changes. Notably, IgM levels in the immunized zebrafish increased significantly at day 7 post-immunization, peaked at day 14, and remained at a high level at day 28. Furthermore, the △<i>aart</i> provided substantial protection against wild-type <i>A. dhakensis</i> infection, with a relative percent survival (RPS) of 56.67%, and less lesion was observed in intestines and kidney histopathology. These findings suggest that the △<i>aart</i> mutant holds promise as a safe and effective live-attenuated vaccine candidate for controlling <i>A. dhakensis</i> infections.达卡气单胞菌（Aeromonas dhakensis）是一种人畜共患病原菌，可引发人类与水生生物的重症感染。该菌株的抗生素耐药性发生率持续攀升，凸显了开发疫苗等替代防控策略的迫切需求。本研究通过靶向毒力基因aerA、ahh1、rtxA及th，成功构建达卡气单胞菌的四基因缺失突变株△aart。△aart的溶血活性与细胞毒性显著降低，但细菌生长未受任何影响。利用斑马鱼模型开展实验发现，△aart的半数致死量（LD₅₀）较野生型菌株提升136倍。免疫后，斑马鱼肾脏内的先天免疫相关基因超氧化物歧化酶（SOD）、过氧化氢酶（CAT）、溶菌酶（LZM）、肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）、白细胞介素-1β（IL-1β）的表达峰值出现在免疫后第3天，随后逐渐下降；主要组织相容性复合体I型（MHC-I）的表达峰值出现在第7天，白细胞介素-10（IL-10）则于第14天达到峰值，肠道组织中上述基因的表达趋势与之相似，但MHC-I未出现显著变化。值得注意的是，免疫斑马鱼体内的免疫球蛋白M（IgM）水平在免疫后第7天显著升高，于第14天达到峰值，并在第28天仍维持较高水平。进一步研究表明，△aart可对野生型达卡气单胞菌感染提供有效保护，相对保护率（RPS）达56.67%，且肠道与肾脏的组织病理学损伤明显减轻。上述研究结果显示，△aart突变株有望成为防控达卡气单胞菌感染的安全高效减毒活疫苗候选株。