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Abstract: Edwardsiella tarda is a Gram-negative bacterial pathogen and the etiological agent of a systematic fish disease called edwardsiellosis, which affects a wide range of marine and freshwater fish. E. tarda vaccines in various forms have been reported by a number of research groups; however, comparative studies on the immune mechanisms of these vaccines are lacking. In this report, we identified a new E. tarda vaccine candidate, Eta2, and analyzed in a comparative manner the immune response induced by Eta2 in two different forms: purified recombinant subunit vaccine and DNA vaccine. Eta2 is a protein of 178 residues and shares high levels of sequence identities with the OmpH family of outer membrane protein chaperones of several bacterial species. Recombinant Eta2 (rEta2) purified from Escherichia coli was highly protective against E. tarda challenge in a Japanese flounder (Paralichthys olivaceus) model and produced relative percent of survival rates of 83% and 78%, respectively, at 4- and 8-week post-vaccination (p.v.). Eta2 as a DNA vaccine in the form of plasmid pCEta2 also induced strong protective immunity at 4- and 8-week p.v. Immunological analysis indicated that (i) rEta2 and pCEta2 enhanced head kidney macrophage activation at 1- and, for pCEta2, 7-day p.v.; (ii) rEta2 and pCEta2 induced similar patterns of serum antibody production, however, the antibodies induced by rEta2 were of much higher levels and afforded stronger passive immunoprotection upon naïve flounder than those induced by pCEta2; (iii) both rEta2 and pCEta2 upregulated the expression of specific and nonspecific immune factors which include, in the case of pCEta2 but not rEta2, interferon, interferon-induced Mx protein, and CD8α; however, the induction patterns effected by rEta2 and pCEta2 were different. While high levels of interleukin 1β (IL-1β), natural killer cell enhancing factor, Mx, MHC Iα, and IgM inductions were observed in pCEta2-vaccinated fish, only IL-1β, complement C3, and IgM inductions were highly induced in rEta2-vaccinated fish. Taken together, these results indicate that both rEta2 and pCEta2 induce specific and nonspecific immunities, however, pCEta2 induces both B cell and T cell responses, whereas rEta2 induces mainly humoral response. [Copyright &y& Elsevier]

Abstract: Oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs in certain contexts are known to be immunostimulatory in vertebrate systems. CpG ODNs with immune effects have been identified for many fish species but, to our knowledge, not for turbot. In this study, a turbot-effective CpG ODN, ODN 205, was identified and a plasmid, pCN5, was constructed which contains the CpG motif of ODN 205. When administered into turbot via intraperitoneal (i.p.) injection, both ODN 205 and pCN5 could (i) inhibit bacterial dissemination in blood in dose and time dependent manners, and (ii) protect against lethal bacterial challenge. Immunological analyses showed that in vitro treatment with ODN 205 stimulated peripheral blood leukocyte proliferation, while i.p. injection with ODN 205 enhanced the respiratory burst activity, chemiluminescence response, and acid phosphatase activity of turbot head kidney macrophages. pCN5 treatment-induced immune responses similar to those induced by ODN 205 treatment except that pCN5 could also enhance serum bactericidal activity in a calcium-independent manner. To examine whether ODN 205 and pCN5 had any effect on specific immunity, ODN 205 and pCN5 were co-administered into turbot with a Vibrio harveyi subunit vaccine, DegQ. The results showed that pCN5, but not ODN 205, significantly increased the immunoprotective efficacy of DegQ and enhanced the production of specific serum antibodies in the vaccinated fish. Further analysis indicated that vaccination with DegQ in the presence of pCN5 upregulated the expression of the genes encoding MHC class IIα, IgM, Mx, and IL-8 receptor. Taken together, these results demonstrate that ODN 205 and pCN5 can stimulate the immune system of turbot and induce protection against bacterial challenge. In addition, pCN5 also possesses adjuvant property and can potentiate vaccine-induced specific immunity. [Copyright &y& Elsevier]

Abstract: Vibrio harveyi is an important aquaculture pathogen that can infect a number of fish species and marine invertebrates. A putative protease, Vhp1, was identified from a pathogenic V. harveyi strain isolated from diseased fish as a protein with secretion capacity. Vhp1 is 530 amino acids in length and shares high sequence identities with several extracellular serine proteases of the Vibrio species. In silico analysis identified a protease domain in Vhp1, which is preceded by a subtilisin-N domain and followed by a bacterial pre-peptidase C-terminal domain. Purified recombinant protein corresponding to the protease domain of Vhp1 exhibited apparent proteolytic activity that was relatively heat-stable and reached maximum at pH 8.0 and 50°C. The activity of purified recombinant Vhp1 protease was enhanced by Ca2+ and inhibited by Mn2+ and ethylenedinitrilotetraacetic acid. Cytotoxicity analyses indicated that recombinant Vhp1 protease was toxic to cultured Japanese flounder cells and could cause complete cell lysis. Immunoprotective analysis using Japanese flounder as an animal model showed that purified recombinant Vhp1 in the form of a denatured and proteolytically inactive protein was an effective subunit vaccine. To improve the vaccine potential of Vhp1, an Escherichia coli strain that expresses and secrets a cytotoxically impaired Vhp1 was constructed, which, when used as a live vaccine, afforded a high level of protection upon the vaccinated fish against lethal V. harveyi challenge. Taken together, these results demonstrate that Vhp1 is a cytotoxic protease and an effective vaccine candidate against V. harveyi infection. [Copyright &y& Elsevier]

Abstract: Edwardsiella tarda is an opportunistic pathogen that can infect humans, animal, and fish. Two E. tarda antigens, Eta6 and FliC, which are homologues to an ecotin precursor and the FliC flagellin, respectively, were identified by in vivo-induced antigen technology from a pathogenic E. tarda strain isolated from diseased fish. When used as a subunit vaccine, purified recombinant Eta6 was moderately protective against lethal challenge of E. tarda in a Japanese flounder model, whereas purified recombinant FliC showed no apparent immunoprotectivity. Similarly, DNA vaccines based on eta6 and fliC in the form of plasmids pEta6 and pFliC induced, respectively, moderate and marginal protection against E. tarda infection. To improve the vaccine efficacy of eta6, a chimeric DNA vaccine, pCE6, was constructed, which encodes Eta6 fused in-frame to FliC. pCE6 was found to induce significantly higher level of protection than pEta6. Likewise, another chimeric DNA vaccine, pCE18, which expresses FliC fused to a previously identified E. tarda antigen Et18, elicited significantly stronger protective immunity than the DNA vaccine based on et18 alone. Fish immunized with pEta6 and pCE6 produced specific serum antibodies and exhibited significantly enhanced expression of the genes encoding elements that are involved in both innate and adaptive immune responses. Furthermore, the induction magnitudes of most of these genes were significantly higher in pCE6-vaccinated fish than in pEta6-vaccinated fish. [Copyright &y& Elsevier]

Abstract: VhhP2 is an outer membrane protein identified in a pathogenic Vibrio harveyi strain, T4, isolated from diseased fish. When used as a subunit vaccine, purified recombinant VhhP2 affords high level of protection upon Japanese flounder against V. harveyi challenge. Vaccination with VhhP2 induced the expression of a number of immune-related genes, especially those encoding immunoglobulin M (IgM) and major histocompatibility complex (MHC) IIα. A VhhP2 surface display system, in the form of the fish commensal strain PF3 harboring the vhhP2-expressing plasmid pJVP, was constructed. PF3/pJVP is able to produce and present recombinant VhhP2 on cell surface. Vaccination of fish with live PF3/pJVP via intraperitoneal injection elicited strong immunoprotection. Vaccination of fish orally with live PF3/pJVP embedded in alginate microspheres also induced effective immunoprotection. In addition, a VhhP2-based surface display system was created, in which VhhP2 serves as a carrier for the surface delivery of a heterologous Edwardsiella tarda immunogen, Et18, that is fused in-frame to VhhP2. DH5α/pJVP18, which expresses and surface-displays the VhhP2–Et18 chimera, proved to be an effective vaccine that can protect fish against infections by V. harveyi and E. tarda to the extents comparable to those produced by vaccination with purified recombinant VhhP2 and Et18, respectively. These data suggest that VhhP2 may be applied as a vaccine and a vaccine carrier against infections by V. harveyi and other pathogens such as E. tarda. [Copyright &y& Elsevier]