Galectin-9 is a β-galactoside-binding lectin which could modulate a variety of biological functions including recognition, aggregation and clearance of pathogen. In this study, one Galectin-9 (named PoGalectin-9) was identified from Japanese flounder Paralichthys olivaceus. PoGalectin-9 belongs to the tandem-repeat type, containing one 127-amino acids CRD domain within N terminal and one 122-amino acids CRD domain within C-terminal. The open reading frame of PoGalectin-9 cDNA was 921 bp encoding 306 amino acids. Sequence similarity comparison confirmed that PoGalectin-9 shared high homology with other Galectin-9. The tissue distribution and expression profiles after bacterial infection were also investigated. PoGalectin-9 was widely distributed in all of the examined tissues of Japanese flounder but was predominantly expressed in the spleen, kidney and intestine. After Edwardsiella tarda challenge, the expression of PoGalectin-9 was up-regulated in spleen and down regulated in kidney. ELISA experiment showed that recombinant PoGalectin-9 (rPoGalectin-9) exhibit binding capacity to lipopolysaccharide (LPS) and peptidoglycan (PGN), which is significantly correlated with the concentration of rPoGalectin-9. Meanwhile, the rPoGalectin-9 protein showed strong agglutinating activities against both Gram-negative bacteria and Gram-positive bacteria. Bacterial binding experiments showed that rPoGalectin-9 could bind all examined bacteria. In conclusion, the present study indicate that PoGalectin-9 might play important roles during the immune responses of Japanese flounder against bacterial pathogens.

Cytokines have the potential as adjuvants for the application of vaccines in mammals. However, the adjuvant potential of teleost cytokines was limited. In the present work, the adjuvant effects of four recombinant cytokines including IL-1β, IL-8, TNF-α and G-CSF on E. tarda subunit vaccine rOmpV were comparatively investigated in flounder (Paralichthys olivaceus). Compared with control, the levels of specific serum antibodies and IgM + B lymphocytes were significantly enhanced by rIL-1β, rIL-8 and rG-CSF, whereas rIL-1β and rIL-8 induced significantly higher levels than rG-CSF. All four cytokines enhanced the expression of genes involved in humoral and/or cellular immunities, whereas rIL-1β and rIL-8 induced highest levels of genes involved in humoral immunities and cellular immunities, respectively. Compared to the relative percent survivals (RPS) of control group (40%) and rOmpV plus rG-CSF group (54%), rOmpV plus rIL-1β or rIL-8 produced higher RPS of 75% and 68%, respectively. Our results indicated that rIL-1β and rIL-8 are promising adjuvants for subunit vaccines against E. tarda.

To obtain effective vaccine candidates for Edwardsiella tarda, six outer membrane proteins (OMPs) of E. tarda were selected and recombinantly expressed. Western blotting analysis showed that flounder anti-E. tarda serum could recognize rOmpV, rOmpK, rOmpX and rOmpI, but not rOmpH and rOmpR. Immunization of flounder with different rOMPs resulted in high levels of protection following the E. tarda challenge with relative percent survivals (RPS) of 92.1% (rOmpI), 84.2% (rOmpX), 68.4% (rOmpK), 60.5% (rOmpV), 57.9% (rOmpH) and 39.5% (rOmpR). Meanwhile, the immune response of flounder induced by rOMPs was investigated, and the results showed that: (1) The levels of specific antibodies against E. tarda induced by rOmpI and rOmpX were significantly higher at weeks 3 and 4 post-vaccination than those of the other rOMPs; (2) all six rOMPs could induce the proliferation of sIg+ lymphocytes, and the peaks of sIg+ lymphocytes in the blood and spleen of rOmpI-, rOmpX- and rOmpH-vaccinated fish were significantly higher than those of other rOMPs; (3) all six rOMPs could induce up-regulation of the MHC IIα, CD4-1, CD4-2, CD8α, CD8β, IL-1β, TNF-α and IFN-γ genes. Among them, the mRNA levels of the CD4-1 and CD4-2 genes induced by rOmpI and rOmpX were generally higher than those induced by the other rOMPs, while there was no significant difference in the expression of TLR2, TLR5M and MHC Iα between the rOMPs-vaccinated group and control group. These results demonstrate that rOmpI and rOmpX could produce a RPS of over 80% against the challenge of E. tarda and induce stronger immune responses in flounder, which indicates that OmpI and OmpX are promising vaccine candidates against E. tarda infection.

Edwardsiella tarda is an enteric opportunistic pathogen that causes a great loss in aquaculture. This species has been described as a phenotypical homogeneous group; in contrast, serological studies and molecular typing revealed a wide heterogeneity. In this work, a proteomic study of differential expression of a virulent isolate from turbot cultured in the Norwest of Spain in comparison with an avirulent collection strain was performed in order to recognize proteins involved in virulence. One hundred and three proteins that presented different abundance were successfully identified and classified into 11 functional categories according to their biological processes: amino acid, carbohydrate and lipid metabolism, tricarboxylic cycle, stress response and protein fate, protein synthesis, biogenesis of cellular components, cell rescue defence and virulence, cell membrane and transport, signal transduction and purine and pyrimidine metabolism. Twenty three protein spots detected only in turbot isolate were identified. It was shown that the same proteins appeared in different spots in the two isolates. Mass spectra obtained by MALDITOF/TOF of some of these proteins and DNA sequencing explained the changes as a result of different amino acid sequences. Several proteins related with the virulence of E. tarda (FliC, ArnA or FeSODI) were only detected in the turbot European isolate. This article is part of a Special Issue entitled: HUPO 2014.

OBJECTIVE: The immunotoxicities of oil and its components on fish immunities have been investigated, but there is little literature on the recovery of the fish from the immune suppression. Therefore, the recovery of Japanese flounder Paralichthys olivaceus from an immunosuppressive effect due to heavy oil (HO) exposure was investigated in this study.
METHODS: Fish were exposed to HO at a concentration of 0.385 g/L for 2 days, while control fish received no exposure. Seven fish were sampled at 0, 3, 7, and 14 days post-exposure. The respiratory rate was measured everyday as an indicator of the acute effect of HO exposure. Fish serum was collected and used for antibacterial activity assay against Edwardsiella tarda. Expression changes of respiratory and immune-related genes were evaluated by real-time PCR.
CONCLUSIONS: The respiratory rate was significantly increased in the HO-exposed group until 4 days post-exposure. A respiratory-related gene, β-hemoglobin, was also significantly downregulated in the spleen both at 0 and 7 days post-exposure and kidney at 3 days post-exposure in HO-exposed fish. Immunotoxicity, including suppression of antibacterial activities and downregulation of the IgM gene, was observed in HO-exposed fish until 3 days post-exposure, but not after that time. From these results, we conclude that the fish likely return to normal status around 1 week.

Edwardsiella tarda and Vibrio harveyi are Gram-negative bacterial pathogens that affect a wide range of cultured fish. In previous studies, we have reported an E. tarda live vaccine ATCC15947 and a V. harveyi subunit vaccine DegQ. On the basis of these studies, in the present study we developed a cross protective vaccine against both E. tarda and V. harveyi by constructing a recombinant ATCC15947, Et15VhD, that expresses and secrets V. harveyi DegQ as a soluble antigen. Laboratory studies in a turbot (Scophthalmus maximus) model showed that Et15VhD elicited significant protections against E. tarda and V. harveyi when administered via intraperitoneal injection, oral feeding, immersion, and oral plus immersion, respectively. Microbiological analysis indicated dissemination and transient colonization of Et15VhD in fish tissues following vaccination. Since, compared to injection, oral plus immersion is a practically more acceptable vaccination procedure in aquaculture, we conducted a mock field trial to further examine the potential of Et15VhD as an oral plus immersion vaccine. The results showed that during the period before artificial bacterial challenge, mortality was observed in both the vaccinated group and the control group; however, the mortality of Et15VhD-vaccinated fish was significantly lower than that of the control fish. Following experimental challenge with E. tarda and V. harveyi at one and two months post-vaccination, Et15VhD-vaccinated fish exhibited dramatically increased survival rates compared to control fish. Serum antibody analysis indicated specific antibody production in Et15VhD-vaccinated fish. Taken together, these results demonstrate that Et15VhD induces strong protective immunity against E. tarda and V. harveyi under both laboratory and mock field conditions, which suggests a potential for Et15VhD to be used in aquaculture.

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.

Toll-like receptor (TLR) 5 is responsible for the bacterial flagellin recognition in vertebrates. Synergistic role of TLR 5 membrane form (TLR 5M) and TLR 5 soluble form (TLR 5S) have been reported from the study on rainbow trout. This system is regarded as the unique system in teleost fish. However, systemic response of TLR 5 genes in teleost fish has not been fully understood. Hence, we cloned Japanese flounder (Paralichthys olivaseus) TLR 5M and TLR 5S genes and their expressions were analyzed. The coding region of Japanese founder TLR 5M and TLR 5S cDNA were 2670 bp and 1923 bp, encoding 889 and 640 amino acid residues, respectively. The Japanese flounder TLR 5M was composed of an extracellular leucine rich repeats (LRRs), a transmembrane and an intracellular Toll/interleukin-1 receptor (TIR) domains, whereas TLR 5S possessed only the LRR domain. TLR 5M was highly expressed in the gill, head kidney, heart and liver. TLR 5S was highly expressed in the brain, head kidney and heart. Flagellin stimulation (1 and 5 microg/ml) led to strong gene expression of TLR 5S in peripheral blood leukocytes (PBLs) and liver cells. In contrast to TLR 5S, TLR 5M was down-regulated until 3 h after flagellin stimulation in PBLs and liver cells. The flagellin stimulation also resulted in the production of the flounder IL-1beta and IL-6 from the liver cells and PBLs. The gene expression of TLR 5M was highly induced in the liver, while TLR 5S gene expression was drastically increased in the intestine following challenge with Edwardsiella tarda. Increased number of TLR 5M- and 5S-expressing cell populations were detected by in situ hybridization in the lamina propria of the intestine and liver after E. tarda infection, respectively. These results imply that the expression of these TLR 5 paralogs in Japanese flounder are differently regulated in the whole body and play important roles in the immune response against bacterial pathogens.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many different fish species cultured worldwide. Et49 is a major E. tarda antigen with weak immunoprotective potential. In this study, using Et49 as an example vaccine, the adjuvanticity of Freund\'s incomplete adjuvant (FIA), aluminum hydroxide, and aluminum phosphate adjuvant were evaluated in a Japanese flounder model. The results showed that the presence of FIA, aluminum hydroxide, and aluminum phosphate adjuvant increased the relative percent of survival of Et49-vaccinated fish by 47%, 19%, and 35%, respectively. Fish vaccinated with FIA-adjuvanted Et49 exhibited longer persistence of vaccine at the injection site and more severe intra-abdominal lesions than fish vaccinated with aluminum-adjuvanted Et49. Both aluminum adjuvants and, to a lesser degree, FIA augmented the production of specific serum antibodies, which reached the highest levels at 6 and 7 weeks post-vaccination. Passive immunization of Japanese flounder with sera from fish vaccinated with aluminum- and FIA-adjuvanted Et49 induced no protection against lethal E. tarda challenge. Examination of the transcription profile of immune-related genes showed that vaccination with aluminum-adjuvanted Et49 significantly enhanced the expression of the genes that are associated mainly with humoral immunity, whereas vaccination with FIA-adjuvanted Et49 induced the expression of a much broader spectrum of genes that are likely to be involved in humoral and innate cellular immunity. These results provide new insights to the action mechanisms of FIA and aluminum adjuvants in Japanese flounder and may be useful for the selection of adjuvant for vaccine formulations intended for Japanese flounder.

It is difficult to use tissue culture assays to investigate adherence and other properties of Edwardsiella tarda because the organism is invasive and produces a potent hemolysin. We therefore relied on polymerase chain reaction (PCR) to determine the occurrence of genes for enterotoxins (LT-I, EAST-1), Shiga toxin (Stx-1, Stx-2), cytotoxic necrotizing factors (CNF-1, CNF-2), aerobactin, invasion plasmid of enteroinvasive Escherichia coli, EPEC adherence factor (EAF), intimin (Eae), enterohemolysin (EntHly) and hemolysin (Hly) in 53 isolates of E. tarda from humans and fish from several countries. All isolates were negative for all genes investigated by PCR. Adhesion to and invasion of HeLa cells were determined by using the unusually short incubation time of 1h or 30 min. All isolates adhered and invaded in these tests. Finally, a random amplified polymorphic DNA (RAPD) test distinguished, with a few exceptions, isolates of human and fish origin.