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Xiao J.,East China University of Science and Technology | Chen T.,East China University of Science and Technology | Liu B.,East China University of Science and Technology | Yang W.,East China University of Science and Technology | And 3 more authors.
Fish and Shellfish Immunology | Year: 2013

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 6months, respectively. These results demonstrated the feasibility of WED as a live attenuated vaccine in turbot against edwardsiellosis by immersion or i.p. injection routes. © 2013 Elsevier Ltd. Source


Gao Y.,East China University of Science and Technology | Wu H.,East China University of Science and Technology | Wang Q.,East China University of Science and Technology | Qu J.,Yantai Development Zone TianYuan Aquatic Products Co. | And 5 more authors.
Vaccine | Year: 2014

Edwardsiella tarda and Vibrio anguillarum are the two main pathogenic bacteria that cause edwardsiellosis and vibriosis in various species of fish raised in aquaculture. In our previous study, the live attenuated vaccines E. tarda WED and V. anguillarum MVAV6203 showed robust relative protection when vaccinated zebrafish or turbot were challenged with virulent E. tarda or V. anguillarum, respectively. Additionally, vaccinated fish processed the two vaccines through different pathways of antigen processing and presentation. Here, the immune protection of a combination vaccination consisting of E. tarda WED and V. anguillarum MVAV6203 was initially evaluated in zebrafish. After challenge with E. tarda and V. anguillarum at 1 month post-vaccination, the vaccinated zebrafish exhibited the relative protective survival of 70% and 90%, respectively. The expression of genes related to antigen recognition, processing and presentation were measured in the liver and spleen of vaccinated zebrafish. Gene expression profiling showed that more than one Toll-like receptor signaling pathway was activated and that both MHC I and II pathways of antigen processing and presentation were evoked. Later, the immune protection of the combination vaccine was evaluated in turbot and it showed similarly effective immune-mediated protection. By ELISA analysis, we found that the specific antibody levels in vaccinated turbot increased compared to those of fish vaccinated by a single vaccine during 2 months post-vaccination. Meanwhile, the expression levels of MHC I and II in the liver, spleen and kidney of vaccinated turbot were both up-regulated, suggesting that the MHC I and II pathways of antigen processing and presentation are activated in vaccinated turbot, similar to vaccinated zebrafish. In summary, a combination vaccine of live attenuated E. tarda WED and V. anguillarum MVAV6203 is effective and could be used widely in the future. © 2014 Elsevier Ltd. Source


Wang Y.,East China University of Science and Technology | Yang W.,East China University of Science and Technology | Wang Q.,East China University of Science and Technology | Qu J.,Yantai Development Zone TianYuan Aquatic Products Co. | Zhang Y.,East China University of Science and Technology
Journal of Biotechnology | Year: 2013

The twin-arginine translocation (Tat) system is a major pathway for transmembrane translocation of fully folded proteins. In this study, a multivalent vaccine to present foreign antigens on live attenuated vaccine Edwardsiella tarda WED using screened Tat signal peptide was constructed. Because the Tat system increases the yields of folded antigens in periplasmic space or extracellular milieu, it is expected to contribute to the production of conformational epitope-derived specific antibodies. E. tarda Tat signal peptides fused with the green fluorescent protein (GFP) was constructed under the control of an in vivo inducible dps promoter. The resulting plasmids were electroporated into WED and the subcellular localizations of GFP were analyzed with Western blotting. Eight signal peptides with optimized GFP translocation efficiency were further fused to a protective antigen glyceraldehyde-3-phosphate dehydrogenase (GapA) from a fish pathogen Aeromonas hydrophila. Signal peptides of DmsA, NapA, and SufI displayed high efficiency for GapA translocation. The relative percent survival (RPS) of turbot was measured with a co-infection of E. tarda and A. hydrophila, and the strain with DmsA signal peptide showed the maximal protection. This study demonstrated a new platform to construct multivalent vaccines using optimized Tat signal peptide in E. tarda. © 2013 Elsevier B.V. Source


Gao Y.,East China University of Science and Technology | Yi Y.,East China University of Science and Technology | Wu H.,East China University of Science and Technology | Wang Q.,East China University of Science and Technology | And 3 more authors.
Fish and Shellfish Immunology | Year: 2014

In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V-CH1-CH2-CH3-CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V.anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells. © 2014 Elsevier Ltd. Source

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