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Liu F.,Ocean University of China | Tang X.,Ocean University of China | Sheng X.,Ocean University of China | Xing J.,Ocean University of China | And 2 more authors.
International Journal of Molecular Sciences | Year: 2016

Outer membrane protein C of Edwardsiella tarda is a major cell surface antigen and it was identified to be an immunogenic protein by Western blot using flounder (Paralichthys olivaceus) anti-recombinant OmpC (rOmpC), and anti-E. tarda antibodies. rOmpC tested the immune protective effect against E. tarda challenge in a flounder model and produced a relative percentage of survival rate of 85%. The immune response of flounder induced by rOmpC was investigated, and the results showed that: (1) the levels of specific serum antibodies induced by rOmpC were significantly higher than the control group after the second week after immunization, and the peak level occurred at week five after immunization; (2) rOmpC could induce the proliferation of sIg+ lymphocytes, and the peak levels of sIg+ lymphocytes in blood, spleen, and pronephros occurred at 4–5 weeks after immunization; and (3) the MHCIIα, CD4-1, IL-1β, IL-6 and TNF-α genes were significantly induced after being injected with rOmpC. Taken together, these results demonstrated that rOmpC could evoke highly protective effects against E. tarda challenge and induce strong innate immune response and humoral immune response of flounder, which indicated that OmpC was a promising vaccine candidate against E. tarda infection. © 2016 by the authors; licensee MDPI, Basel, Switzerland.

Xu T.,Hong Kong Baptist University | Sun J.,Hong Kong Baptist University | Sun J.,Hong Kong University of Science and Technology | Lv J.,Ocean University of China | And 10 more authors.
Deep-Sea Research Part II: Topical Studies in Oceanography | Year: 2016

The present study aimed to generate genome-wide single nucleotide polymorphisms (SNPs) for the deep-sea mussel Bathymodiolus platifrons via a combination of genome survey sequencing and the type IIB endonuclease restriction-site associated DNA (2b-RAD) sequencing, assess the potential use of SNPs in detecting fine-sale population genetic structure and signatures of divergent selection, as well as their cross-species application in other bathymodioline mussels. Genome survey sequencing was conducted for one individual of B. platifrons. De novo assembly resulted in 781,720 sequences with a scaffold N50 of 2.9kb. Using these sequences as a reference, 9307 genome-wide SNPs were identified by 2b-RAD for 28 B. platifrons individuals collected from a seep and a vent population. Among these SNPs, 9 outliers showed significant evidence for divergent selection, and their positions in the genes or scaffolds were identified. The F ST estimated based on the putative neutral SNPs was low (0.0126) indicating the two B. platifrons populations having a high genetic connectivity. However, the permutation test detected significant differences (P<0.00001), indicating the two populations having clearly detectable genetic differentiation. The Bayesian clustering analyses and principle component analyses (PCA) performed based on either the putative neutral or outlier SNPs also showed that these two populations were genetically differentiated. In addition, 2b-RAD was also conducted to detect 10,199, 6429, and 3811 single nucleotide variants (SNVs) respectively in the bathymodioline mussels Bathymodiolus japonicus, Bathymodiolus aduloides and Idas sp. with different phylogenetic distances from B. platifrons. Overall, our study has demonstrated the feasibility and effectiveness of combining genome survey sequencing and 2b-RAD to rapidly generate genomic resources for use in fine-scale population genetic studies, and various cross-species applications. © 2016 Elsevier Ltd.

Li J.,Lake Superior State University | Li J.,CAS Qingdao Institute of Oceanology | Li J.,Laboratory for Marine Fisheries Science and Food Production Processes | Ma S.,CAS Qingdao Institute of Oceanology | Woo N.Y.S.,Chinese University of Hong Kong
International Journal of Molecular Sciences | Year: 2015

In order to develop more effective immunological strategies to prevent vibriosis of farmed marine fish in Hong Kong and southern China, various vaccine preparations including formalin-, phenol-, chloroform- and heat-killed whole cell bacterins and subcellular lipopolysaccharides (LPS), as well as different administration routes, were investigated. Fish immunized with the subcellular LPS exhibited the best protection [Relative Percent of Survival (RPS) = 100], while fish immunized with whole cell bacterins displayed varying degrees of protection (RPS ranged from 28 to 80), in descending order: formalin-killed > phenol-killed > heat-killed > chloroform-killed bacterins. Regarding various administration routes, fish immunized with two intraperitoneal (i.p.) injections exhibited the best protection, and the RPS values were 100 or 85 upon higher or lower doses of pathogenic V. alginolyticus challenges. Both oral vaccination and a combination of injection/immersion trial were also effective, which achieved relatively high protection (the RPS values ranged from 45 to 64.3). However, two hyperosmotic immersions could not confer satisfactory protection, especially when fish were exposed to the severe pathogenic bacteria challenge. Marked elevations of serum agglutinating antibody titer were detected in all immunized fish. Macrophage phagocytosis was enhanced significantly, especially in the fish immunized by formalin- and phenol-killed bacterins through various administration routes. Both adaptive (specific antibody) and innate (phagocytic activity) immunity elicited by different immunization strategies were in parallel with the degree of protection offered by each of them. Although all vaccination trials had no significant effect on the serum hematocrit and hemoglobin levels, the circulating lymphocyte counts were significantly elevated in the fish immunized with LPS, formalin- and phenol-killed bacterins. Serum cortisol levels appeared to be reduced in all immunized fish except the trial of hyperosmotic immersion, which indicated the stressful impact on vaccinated fish. © 2015 by the authors; licensee MDPI, Basel, Switzerland.

Tang X.,Ocean University of China | Li W.,Ocean University of China | Xing J.,Ocean University of China | Sheng X.,Ocean University of China | And 2 more authors.
PLoS ONE | Year: 2016

In previous work, small ubiquitin-like modifier (SUMO) in hemocytes of Chinese shrimp Fenneropenaeus chinensis was found to be up-regulated post-white spot syndrome virus (WSSV) infection using proteomic approach. However, the role of SUMO in viral infection is still unclear. In the present work, full length cDNAs of SUMO (FcSUMO) and SUMO-conjugating enzyme E2 UBC9 (FcUBC9) were cloned from F. chinensis using rapid amplification of cDNA ends approach. The open reading frame (ORF) of FcSUMO encoded a 93 amino acids peptide with the predicted molecular weight (M.W) of 10.55 kDa, and the UBC9 ORF encoded a 160 amino acids peptide with the predicted M.W of 18.35 kDa. By quantitative real-time RT-PCR, higher mRNA transcription levels of FcSUMO and FcUBC9 were detected in hemocytes and ovary of F. chinensis, and the two genes were significantly upregulated post WSSV infection. Subsequently, the recombinant proteins of FcSUMO and FcUBC9 were expressed in Escherichia coli BL21 (DE3), and employed as immunogens for the production of polyclonal antibody (PAb). Indirect immunofluorescence assay revealed that the FcSUMO and UBC9 proteins were mainly located in the hemocytes nuclei. By western blotting, a 13.5 kDa protein and a 18.7 kDa protein in hemocytes were recognized by the PAb against SUMO or UBC9 respectively. Furthermore, gene silencing of FcSUMO and FcUBC9 were performed using RNA interference, and the results showed that the number of WSSV copies and the viral gene expressions were inhibited by knockdown of either SUMO or UBC9, and the mortalities of shrimp were also reduced. These results indicated that FcSUMO and FcUBC9 played important roles in WSSV infection. © 2016 Tang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Gao Y.,Ocean University of China | Tang X.,Ocean University of China | Sheng X.,Ocean University of China | Xing J.,Ocean University of China | And 2 more authors.
Fish and Shellfish Immunology | Year: 2016

Antigen uptake is a critical process for activation of the immune system, and therefore the ability to enhance antigen uptake is a primary consideration in the development of an immersion vaccination of fish. In the present work, flounders (Paralichthys olivaceus) were immersed in three hyperosmotic solutions with 40, 50 and 60‰ salinities, then transferred into seawater of normal salinity (i.e. 30‰) containing formalin-inactivated Edwardsiella tarda for 30 min. The antigen uptake in vaccinated flounder was determined using an absolute quantitative PCR (qPCR). The results showed significantly higher antigen uptake in the tissues of flounders immersed in solutions with 50‰ and 60‰ salinity compared to the control group directly immersed in vaccine (DI) (P < 0.05), and the highest amount of antigen was detected in flounders immersed in the 50‰ salinity solution, whereas there was no significant difference in antigen uptake between the 40‰ salinity group and the DI group (P > 0.05). A rapid and significant increase in antigen uptake was detected in the mucosal-associated tissues including the gill, skin and intestine (P < 0.05) compared with the spleen, kidney and liver. Antigen uptake in the gill and skin both peaked at 30 min post immersion, which was significantly higher than the levels of uptake measured in the other tissues (P < 0.05), and then quickly declined. In contrast, antigen uptake in the spleen, kidney and liver gradually increased 3 h post immersion (hpi). The expression profiles of four antigen presentation-related immune genes (MHC Iα, MHC IIα, CD4-1 and CD8α) were investigated after immersion. These four genes showed a significantly stronger response in the immersed flounders exposed to 50‰ salinity compared with the DI group (P < 0.05). In the mucosal-associated tissues, the expression of MHC Iα and CD8α genes peaked at 24 hpi, while the expression of MHC IIα and CD4-1 genes showed up-regulation in the gill and skin and reached the peak in these tissues at 48 hpi. The expressions of the four genes were also up-regulated in spleen, kidney and liver, but reached peak expression in these tissues at 48–72 hpi. The results demonstrated that hyperosmotic immersion, notably 50‰ salinity significantly enhanced antigen uptake and the expression of selected genes associated with antigen presentation, providing evidence for an enhanced immune activation of the fish's immune response by the hyperosmotic immersion treatment prior to vaccination. © 2016 Elsevier Ltd

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