State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, PR China.
Fish Shellfish Immunol. 2013 Sep;35(3):632-41. doi: 10.1016/j.fsi.2013.05.022. Epub 2013 May 31.
Edwardsiella tarda is an intractable Gram-negative pathogen in many fish species to cause edwardsiellosis. Its infection leads to extensive losses in a diverse array of commercially important fish. The type III secretion system (T3SS) has been considered as one of the major virulence factors and plays important roles in its intracellular lifestyle. In this study, an E. tarda EIB202 mutant WED with deletions in the T3SS genes for EseB, EseC, EseD and EscA, along with the aroC gene for the biosynthesis of chorismic acid, as well as the curing of endogenous plasmid pEIB202 was constructed by allelic exchange strategy. Compared to the wild-type EIB202 which was highly virulent towards turbot (Scophthamus maximus) via intraperitoneal (i.p.), intramuscular (i.m.) injection or immersion and caused systemic infection in turbot as well as the unexpected red mouth symptom when immersion challenged, WED was highly attenuated when inoculated into turbot via i.m., i.p. and immersion routes, and exhibited significantly impaired capacity to survive in fish tissues. WED showed 5700-fold higher 50% lethal dose (LD50) than that of the wild type when i.m. or i.p. challenged. Inoculation with WED by i.p. or immersion injection routes elicited significant protection against the challenge of the wild-type E. tarda after 5 weeks of vaccination. The vaccinated fish produced low while significant level of specific antibody and showed increased expression of immune-related factors including IL-1β, IFN-γ, MHC II, MHC-I and CD8, indicating that WED possesses significant immunoprotective potential. Furthermore, our data indicated that a single dose of i.p. and immersion vaccination with WED could produce significant protection as long as 12 and 6 months, respectively. These results demonstrated the feasibility of WED as a live attenuated vaccine in turbot against edwardsiellosis by immersion or i.p. injection routes.
迟缓爱德华氏菌是一种难以治疗的革兰氏阴性病原体，可引起爱德华氏菌病。它的感染导致了多种商业上重要鱼类的广泛损失。III 型分泌系统（T3SS）已被认为是主要毒力因子之一，在其细胞内生活方式中发挥重要作用。在这项研究中，通过等位基因交换策略构建了迟缓爱德华氏菌 EIB202 突变株 WED，该突变株缺失了 T3SS 基因 eseB、eseC、eseD 和 escA，以及 aroC 基因（用于合成分支酸），并消除了内源性质粒 pEIB202。与通过腹腔内（i.p.）、肌肉内（i.m.）注射或浸泡对大菱鲆（Scophthamus maximus）具有高致病性的野生型 EIB202 相比，WED 通过 i.m.、i.p.和浸泡途径接种到大菱鲆时高度减毒，并且在鱼组织中存活能力显著受损。与野生型相比，WED 通过 i.m.或 i.p.途径接种时的 50%致死剂量（LD50）高 5700 倍。通过 i.p.或浸泡途径接种 WED 后，在接种后 5 周可对野生型 E. tarda 的挑战产生显著保护作用。接种 WED 的鱼产生低而显著的特异性抗体水平，并表现出免疫相关因子（包括 IL-1β、IFN-γ、MHC II、MHC-I 和 CD8）的表达增加，表明 WED 具有显著的免疫保护潜力。此外，我们的数据表明，通过 i.p.和浸泡途径接种 WED 一次即可产生长达 12 个月和 6 个月的显著保护作用。这些结果表明，WED 作为一种通过浸泡或 i.p.注射途径对大菱鲆爱德华氏菌病的活减毒疫苗具有可行性。
