Zhang Fengli, Li Ji-Tong, Zhang Tengyuan, Wang Xiao, Yao Yuanyuan, Zhou Zhigang
Department of Aquaculture, Zhejiang Ocean University, Zhoushan, 316000, China.
Health Science Center, Ningbo University, Ningbo, 315211, Zhejiang, China.
Sci Rep. 2025 Jul 2;15(1):23666. doi: 10.1038/s41598-025-08150-8.
Micropterus salmoides, adaptable and fast-growing, is a major farmed fish in China. Intensive farming causes multiple diseases, especially hard-to-treat nocardiosis. Vaccines are an effective and safe prevention and control strategy against fish diseases. In this experiment, the highly antigenic HRP1 gene of Nocardia seriolae (N. seriolae) was displayed on the surface of Bacillus subtilis (B. subtilis), and an oral vaccine (HRP1-cotC-B. subtilis) was successfully developed. Immune responses in inoculated fish were tested intermittently over 5 weeks post-administration. Immune protection was evaluated via challenge test. Serological parameters testing showed that the activity of lysozyme and Glutathione Peroxidase (GSH-Px) were significantly increased at the 7th and 21st day post-immunization, and the activity of Alkaline Phosphatase was extremely increased compared to the control. qRT-PCR detection found that oral vaccine could significantly boost the IgM expression of spleen and head-kidney. The expression of spleen major histocompatibility complex (MHC) was enhanced, MHCII at the 7th, 21st day and MHCI at 35th day post-immunization respectively. MHCI has an increasing trend in the head-kidney. CD8 increased in both spleen and head-kidney at different stages of immunization. The inflammatory cytokine il-1β and anti-inflammatory cytokine TGF-β significantly increased in head-kidney at 35th day after immunization. The live bacterial vaccine altered the composition of the intestinal flora, demonstrated a decline in Firmicutes and an increase in Fusobacteria, and a significant decrease in Clostridium replaced by Cetobacterium at the genus level. Largemouth bass immunized with HRP1-cotC-B. subtilis spores exhibited a 18.18% relative survival rate after N. seriolae infection. In conclusion, this study developed a novel oral vaccine against N. seriolae in largemouth bass using B. subtilis spore surface display technology. Oral vaccination improves nonspecific immunity, induces innate and cellular immunity, strengthens bacterial resistance, and increases survival after pathogen infection. This offers an effective strategy for controlling fish nocardiosis in aquaculture.
大口黑鲈适应性强、生长迅速，是中国主要的养殖鱼类。集约化养殖导致多种疾病，尤其是难以治疗的诺卡氏菌病。疫苗是防治鱼类疾病的一种有效且安全的策略。在本实验中，将高抗原性的鰤诺卡氏菌（N. seriolae）HRP1基因展示在枯草芽孢杆菌（B. subtilis）表面，成功研制出一种口服疫苗（HRP1-cotC-B. subtilis）。在接种后5周内对受试鱼的免疫反应进行了间歇性检测。通过攻毒试验评估免疫保护效果。血清学参数检测显示，免疫后第7天和第21天，溶菌酶和谷胱甘肽过氧化物酶（GSH-Px）活性显著升高，碱性磷酸酶活性与对照组相比极度升高。qRT-PCR检测发现，口服疫苗可显著提高脾脏和头肾中IgM的表达。脾脏主要组织相容性复合体（MHC）的表达增强，免疫后第7天、第21天的MHCII以及第35天的MHCI分别增强。头肾中MHCI有上升趋势。免疫不同阶段，脾脏和头肾中的CD8均增加。免疫后第35天，头肾中炎性细胞因子il-1β和抗炎细胞因子TGF-β显著增加。活菌疫苗改变了肠道菌群组成，厚壁菌门减少，梭杆菌门增加，属水平上梭菌属显著减少，被鲸杆菌属取代。用HRP1-cotC-B. subtilis孢子免疫的大口黑鲈在感染鰤诺卡氏菌后相对存活率为18.18%。综上所述，本研究利用枯草芽孢杆菌孢子表面展示技术研制出一种新型大口黑鲈抗鰤诺卡氏菌口服疫苗。口服疫苗可提高非特异性免疫力，诱导先天性和细胞免疫，增强细菌抵抗力，并提高病原体感染后的存活率。这为水产养殖中控制鱼类诺卡氏菌病提供了一种有效策略。
