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Chemokines are a superfamily of chemotactic cytokines that regulate the migration and immune responses of leukocytes. Depending on the arrangement of the first two cysteine residues, chemokines are divided into four groups: CXC (α), CC (β), C (γ), and CX3C (δ). Chemokine C-C motif ligand 34 (CCL34) is a member of the CC chemokine family and is known as a fish-specific CC chemokine. In this experiment, we analyzed the molecular cloning and characterization of the PoCCL34 gene in olive flounder (Paralichthys olivaceus), including CCL34a.3 (PoCCL34a.3) and CCL34b.3 (PoCCL34b.3). The amino acid sequence of PoCCL34 has four highly conserved cysteine residues and it has a C-C motif. Phylogenetic analysis revealed that PoCCL34 was phylogenetically clustered in the fish CCL34 subcluster. Recombinant PoCCL34 induced chemotaxis of head kidney leukocytes in a dose-dependent manner. Head kidney leukocytes stimulated with PoCCL34 also exhibited significant respiratory burst activity and increased expression of pro-inflammatory cytokines (IL-1β, IL-6, and CXCL8), but the overall expression of interferon-related genes (IFN-α/β, IFN-γ, Mx, and ISG15) did not increase. Olive flounder injected with recombinant PoCCL34 demonstrated increased expression of pro-inflammatory cytokines (IL-1β and IL-6) in the head kidney. However, there was no increase in the expression of interferon-related genes (IFN-α/β, IFN-γ, Mx, and ISG15). Additionally, recombinant PoCCL34 induced high lysozyme activity in the serum of the flounder. These results indicate that although PoCCL34 is not involved in the antiviral response, it may play a significant role in the overall immune response of the flounder, particularly in mediating the inflammatory response.

Viral hemorrhagic septicemia (VHS), caused by viral hemorrhagic septicemia virus (VHSV), is a viral disease affecting teleosts, and is the major cause of virus-related deaths in olive flounder (Paralichthys olivaceus). Research has focused on ways to control VHS, and recently, the use of polyinosinic-polycytidylic acid poly (I:C)-potentiated vaccination has been investigated, whereby fish are injected with poly (I:C) and then with live pathogenic virus, resulting in a significant decrease in VHSV-related mortality. T cell responses were investigated in the present study after vaccinating olive flounder with poly (I:C)-potentiated vaccination to understand the ability of poly (I:C) to induce T cell immunity. Stimulation of T cell responses with the poly (I:C)-potentiated vaccination was confirmed by examining levels of CD3+ T cells, CD4-1+ T cells and CD4-2+ T cells. Higher levels of CD4-2+ T cells were found in vaccinated fish than CD4-1+ T cells, believed to result from a synergistic effect between poly (I:C) administration and pathogenic VHSV immunization. More importantly, the role of CD4-2+ T cells in the antiviral response was clearly evident. The results of this study suggest that the outstanding protection obtained with the poly (I:C)-potentiated vaccination is due to the robust immune response initiated by the CD4-2+ T cells.

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder&rsquo, s CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.

In higher vertebrates, helper and cytotoxic T cells, referred to as CD4 and CD8 T lymphocytes, respectively, are mainly associated with adaptive immunity. The adaptive immune system in teleosts involves T cells equivalent to those found in mammals. We previously generated monoclonal antibodies (mAbs) against olive flounder (Paralichthys olivaceus) CD4 T cells, CD4-1 and CD4-2, and used these to describe the olive flounder’s CD4 Tcell response during a viral infection. In the present study, we successfully produced mAbs against CD8 T lymphocytes and their specificities were confirmed using immuno-blotting, immunofluorescence staining, flow cytometry analysis andreverse transcription polymerase chain reaction (RT-PCR). The results showed that these mAbs are specific for CD8 T lymphocytes. We also investigated variations in CD4 and CD8 T cells populations, and analyzed the expression of immune-related genes expressed by these cells in fish infected with nervous necrosis virus or immunized with thymus dependent and independent antigens. We found that both CD4 and CD8 T lymphocyte populations significantly increased in these fish and Th1-related genes were up-regulated compared to the control group. Collectively, these findings suggest that the CD4 and CD8 T lymphocytes in olive flounder are similar to the helper and cytotoxic T cells found in mammals, and Th1 and cytotoxic immune responses are primarily involved in the early adaptive immune response against extracellular antigens.

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3&epsilon, and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3&epsilon, T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

The presence of CD4 T lymphocytes has been described for several teleost species, while many of the main T cell subsets have not been characterized at a cellular level, because of a lack of suitable tools for their identification, e.g., monoclonal antibodies (mAbs) against cell markers. We previously described the tissue distribution and immune response related to CD3ε and CD4-1 T cells in olive flounder (Paralichthys oliveceus) in response to a viral infection. In the present study, we successfully produce an mAb against CD4-2 T lymphocytes from olive flounder and confirmed its specificity using immuno-blotting, immunofluorescence staining, flow cytometry analysis and reverse transcription polymerase chain reaction (RT-PCR). Using these mAbs, we were able to demonstrate that the CD3ε T cell populations contain both types of CD4+ cells, with the majority of the CD4 T cell subpopulations being CD4-1+/CD4-2+ cells, determined using two-color flow cytometry analysis. We also examined the functional activity of the CD4-1 and CD4-2 cells in vivo in response to a viral infection, with the numbers of both types of CD4 T cells increasing significantly during the virus infection. Collectively, these findings suggest that the CD4 T lymphocytes in olive flounder are equivalent to the helper T cells in mammals in terms of their properties and function, and it is the CD4-2 T lymphocytes rather than the CD4-1 T cells that play an important role in the Th1 immune response against viral infections in olive flounder.

The T cell receptor (TCR) is the binding site of antigen and is responsible for specifically activating the adaptive immune response. CD3, an essential component of the CD3-TCR complex, is known to be composed of γδ and ε chains in teleost. However, there are few monoclonal antibodies (mAb) available to identify these molecules on T cells, so we aimed to produce a mAb against CD3ε to improve our understanding of T cell immune response in olive flounder (Paralichthys olivaceus). CD3ε recombinant protein was expressed in yeast, the expression of which was confirmed by SDS-PAGE, MALDI-TOF/TOF MS and Western blot analysis. A CD3ε-specific mAb 4B2 was selected, the specificity of which was examined by confocal microscopy, flow cytometry and RT-PCR, and the mAb was subsequently used to examine the CD3ε lymphocyte population in several different immune organs, with relatively high percentages of these cells seen in trunk-kidney and spleen, while lower percentages were seen in the liver and peripheral blood of olive flounder. During a viral hemorrhagic septicemia virus (VHSV) infection in olive flounder, the number of CD3ε lymphocytes was seen to gradually increase in the liver, spleen and trunk-kidney of infected fish until 7 days post infection (dpi). In peripheral blood, on the other hand, the increase in CD3ε lymphocyte numbers peaked by 3 dpi. These results suggest that CD3ε lymphocytes might be involved in the immune response against VHSV.

The cellular DEAD-box helicase DDX41, which functions as an initial sensor for cytoplasmic DNA, is involved in the activation of type I interferon (IFN-1) immune response in olive flounder. A plasmid encoding DDX41 (pEF-D) was introduced into flounder cells in vitro and in vivo. Immune responses induced by DDX41 were evaluated by relative quantification value (ΔΔCt) method of RT-qPCR using specific IFN-related gene primers. Results in in vitro, transcript levels of IFN-1, IRF-3, ISG-15 and IL-1β were significantly higher in pEF-D- than pEF-A-transfected cells, with 15-, 4-, 10- and 32-fold changes, respectively. In vivo, elevated expression of these genes was observed in the kidney of pEF-D group on days 1 and 3 post-treatment. The viral challenge test revealed higher survival rate in fish treated with pEF-D (67.5%) than controls, PBS- and pEF-A-treated fish (45%). Conclusively DDX41 elicits a robust IFN-mediated immune response, validating its adjuvant property.

Edwardsiella tarda is an important bacterial pathogen of olive flounder ( Paralichthys olivaceus ) and Japanese eel ( Anguilla japonica ). Here, a total of 66 strains of E. tarda isolated from olive flounder and Japanese eel were compared using biochemical tests, whole-cell protein analysis, lipopolysaccharide (LPS) profiling, and random amplified polymorphic DNA (RAPD) analysis to determine the host-related homogeneity of these pathogens. The E. tarda strains isolated from olive flounder were highly homogenous in the analyzed parameters, whereas the isolates from Japanese eel were highly heterogeneous. E. tarda isolated from Japanese eel did not demonstrate any pathogenic effect against olive flounder in challenge experiments. The phenotypic and genotypic homogeneity observed in the isolates from olive flounder may be helpful to develop an effective vaccine for edwardsiellosis in olive flounder.

The occurrence of CD4 helper T cells has already been established for a number of teleost species, though, it has not been possible to analyze these responses at a cellular level due to a large lack of appropriate monoclonal antibodies (mAbs). In the present study, we produced a mAb against olive flounder (Paralichthys olivaceus) CD4-1 lymphocyte to investigate the functional activity of the cells to improve our understanding of the T cell response in this species. This mAb is specifically able to detect CD4-1 lymphocytes in olive flounder proved by immunofluorescence staining and RT-PCR analysis. In flow cytometry analysis, the number of CD4-1-positive lymphocytes was observed to gradually increase from 3 days post infection (dpi) and then reach peak at 7 dpi against two viruses challenge. As a conclusion, both the basic properties of CD4-1 T cells and its response to viral infections in olive flounder are very similar to the helper T cells in terrestrial animals.

Streptococcus iniae causes severe mortalities among cultured marine species, especially in the olive flounder (Paralichthys olivaceus), which is economically important in Korea and Japan. Recently, there has been growing concern regarding the emergence of S. iniae as a zoonotic pathogen. Here, 89 S. iniae isolates obtained from diseased olive flounders collected from 2003 to 2008 in Jeju Island, South Korea, were characterized using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results were aligned both with the available Bruker Daltonics data-base and with a new set of S. iniae data entries developed in our laboratory, and the results were compared. When we used the Bruker Daltonics database, the 89 isolates yielded either "no reliable identification" or were incorrectly identified as Streptococcus pyogenes at the genus level. When we used the new data entries from our laboratory, in contrast, all of the isolates were correctly identified as S. iniae at the genus (100%) and species (96.6%) levels. We performed proteomic analysis, divided the 89 isolates into cluster I (51.7%), cluster II (20.2%), and cluster III (28.1%), and then used the MALDI Biotyper software to identify specific mass peaks that enabled discrimination between clusters and between Streptococcus species. Our results suggest that the use of MALDI TOF MS could outperform the conventional methods, proving easier, faster, cheaper and more efficient in properly identifying S. iniae. This strategy could facilitate the epidemiological and taxonomical study of this important fish pathogen.

Immunoglobulins (Ig) are heterodimeric proteins that play critical roles in the adaptive immune system of vertebrates. Because of their plasticity, teleostean Igs are more diverse, and thus do not conform to mammalian classifications. Because of this, mammalian-based Ig cell markers cannot be used successfully to study immune responses in fish. There is therefore a need to produce Ig-specific cell markers for fish. Here, we attempted to identify the specific isotype detected by an Ig light chain-specific monoclonal antibody (anti-olive flounder IgL-mAb: M7C3-4) that we had previously produced [11]. Three newly identified sequences of the Ig light chain from olive flounder were classified according to their isotypes. Subsequent analyses revealed that M7C3-4 was able to specifically detect lymphocytes expressing one of the κ chains (Igκ-a) in olive flounder. Interestingly, Igκ-a+ B cells were more abundant in spleen and trunk-kidney than in peripheral blood, indicating a distribution different from that of IgM+ B cells. Our work reveals interesting aspects of B cell distribution and differentiation, and may aid in the production of suitable and effective cell markers for olive flounder.

Streptococcus iniae and S. parauberis types I and II are major bacterial pathogens affecting the olive flounder, Paralichthys olivaceus . With the introduction of formalin-killed vaccine for S. iniae , recent epidemiological studies have indicated that S. parauberis infections are becoming increasingly severe and frequent in the olive flounder farming industry. Here, the formalin-killed bacterins were used to develop a three-valent vaccine against three streptococcal infections, and its efficacy was assessed in laboratory- and field-challenge trials. In the laboratory-challenge test, it obtained 75%, 75% and 90% relative percent survival (RPS) values in vaccinated olive flounders challenged with S. iniae , S. parauberis type I, and S. parauberis type II, respectively. For the field-challenge trial, the three-valent vaccine was administered to over 100,000 olive flounders, which were compared to non-injected control fish. An RPS of 74% was observed when S. parauberis infection naturally occurred at 11 fish farms. Serum antibody measurements using olive flounder immunoglobulin-specific antibodies indicated that immunized fish had significantly higher serum antibody levels than control fish up to 6 months post-vaccination. These results demonstrate that the three-valent vaccine using formalin-killed bacterins could effectively protect olive flounders against three major streptococcal infections. Statement of relevance The olive flounder, P. olivaceus , is an economically important fish species in Northeast Asia and the most valuable fish species in South Korea, corresponding to 56.5% of the total mariculture production in 2010 (Baeck et al., 2006). However, streptococcal infections often break out among farmed olive flounders subjected to certain conditions (e.g., high stocking density, poor water quality and high water temperature), leading to mass mortality and severe economic losses (Baeck et al., 2006; Cho et al., 2008) Antibiotic treatments are usually recommended to address such outbreaks, but the prevalence of multi-drug-resistant bacteria means that antibiotic compounds are not always able to eradicate the infections (Creeper and Buller, 2006). The Gram-positive bacteria, Streptococcus iniae and S. parauberis , cause streptococcosis and have severe effects on aquaculture of the olive flounder, barramundi, channel catfish, European sea bass, rainbow trout, striped bass, tilapia, turbot and yellow tail (Gudmundsdottir and Bjornsdottir, 2007; Han et al., 2011). In the olive flounder, fish infected with S. iniae usually show severe pathological changes, such as darkening, exophthalmia, rectal hernia, abdominal distension, ascites and congestion of intestinal organs, whereas fish infected with S. parauberis rarely exhibited any remarkable pathological finding except for darkening of the skin (Kim et al., 2006). Some vaccines have been reported to prevent S. iniae infection, showing high relative percent survival (RPS) among olive flounders in laboratory trials; such vaccines are now commercially available (Cheng et al., 2010a). However, recent epidemiological studies have suggested that S. parauberis infection has increased and become more severe and frequent as much as infections of S. iniae , which was previously considered the major bacterial pathogen among olive flounder (Park et al., 2012; Park, 2009; Perera et al., 1998). S. parauberis can be divided into two serotypes (types I and II) based on serological testing of a distinctive capsular polysaccharide layer (Bromage and Owens, 2002; Plant and Lapatra, 2011). Since the two serotypes possess specific antigenic characteristics, a vaccine should be able to address both types in order to effectively prevent S. parauberis infection. The present study was undertaken to develop a three-valent vaccine against both serotypes of S. parauberis plus S. iniae using formalin-killed vaccine preparation method. The efficacy of this vaccine against the major streptococcal infections of olive flounder was calculated as RPS and conducted in both laboratory trials and large-scale field trial. This vaccine will be highly useful and more advantageous since the use of this does not require several antibiotics which means safer food production and more sustainable environment.

DDX41, a receptor belonging to the DExD family, functions as a DNA sensor in the mammalian cytoplasm and mediates the antiviral response in host cells. Here, the olive flounder DDX41 was found to have 2267-bp long and encodes a putative protein of 614 amino acid residues. The olive flounder DDX41 mRNA was presented in all tested tissues, and was distinctly expressed in fish naturally infected with LCDV. High expression levels were observed in the heart, liver, kidney and stomach. Furthermore, the olive flounder DDX41 mRNA expression increased significantly in adherent (monocyte-like) cells following stimulation with a DNA virus. Reporter assays showed that the transcriptional activity of the IFN-I promoter was enhanced in DDX41-overexpressing HINAE cells treated with C-di-GMP (dinucleotides). Overexpression of DDX41 also induced the antiviral and inflammatory cytokine gene expression through cytoplasmic C-di-GMP treatment. These results suggest that DDX41 functions as a cytosolic DNA sensor that is capable of inducing antiviral activity and inflammatory responses in the olive flounder.

The use of molecular adjuvants to improve the immunogenicity of DNA vaccines has been thoroughly studied in recent years. Glycoprotein (G)-based DNA vaccines had been proven to be effective in combating infection against Rhabdovirus (especially infectious hematopoietic necrosis virus, IHNV) in salmonids. DDX41 is a helicase known to induce antiviral and inflammatory responses by inducing a type I IFN innate immune response. To gain more information regarding G-based DNA vaccines in olive flounder (Paralicthys olivaceus), we tried to develop a more efficient G-based DNA vaccine by adding a molecular adjuvant, DDX41. We designed a DNA vaccine in which the VHSV glycoprotein (G-protein) and DDX41 were driven by the EF-1α and CMV promoters, respectively. Olive flounders were intramuscularly immunized with 1 μg of plasmids encoding the G-based DNA vaccine alone (pEF-G), the molecular adjuvant alone (pEF-D), or the vaccine-adjuvant construct (pEF-GD). At two different time points, 15 and 30 days later, the fish were intraperitoneally infected with VHSV (100 μL; 1 × 106 TCID50/mL). Our assays revealed that the plasmid constructs showed up-regulated expression of IFN-1 and its associated genes at day 3 post-vaccination in both kidney and spleen samples. Specifically, pEF-GD showed statistically higher expression of immune response genes than pEF-G and pEF-D treated group (p

The epidermal secretion of fish contains various non-specific immune substances that act as the first line of defense against invading pathogens. The present study investigated the level of mucosal antibodies, the activities of hemagglutinin and protease, and other enzymes in the skin mucus of farm reared olive flounder (Paralichthys olivaceus) for 1 year, in order to gain an insight into the relationship between these mucosal immune substances and their seasonal variation. These levels varied significantly during different months of sample collection. The present study showed a positive correlation between water temperature and the level of mucosal antibodies, and an inverse relationship between the level of mucosal antibodies and the activity of mucosal hemagglutinin and protease, but no relationship between lysozyme activity and other innate immune substances. This relationship is thought to be a compensatory response in olive flounder to protect itself against pathogenic microorganisms which are inherently present in the aquatic environment.

Interferon gamma (IFN-γ) is a cytokine that plays a very important role in defining Th1 immune response in all vertebrates. In this study, recombinant IFN-γ (rIFN-γ) from the olive flounder (Paralichthys olivaceus) was produced in an Escherichia coli system using a pET expression vector. Stimulation of whole kidney leukocytes (immune-related cells) in vitro with the resulting rIFN-γ significantly induced the gene expression of interleukin-1β (IL-1β), signal transducer and activator of transcription 1 (STAT1), CXCL13-like chemokine (CXCL13), and IFN-γ. rIFN-γ also weakly induced the expression of IL-1β, tumor necrosis factor-α (TNF-α), CXCL13, and IFN-γ in olive flounder-derived HINAE (non-immune) cells. The effects of rIFN-γ against Edwardsiella tarda infection in vivo were assessed by intraperitoneally injecting a mixture of rIFN-γ (100 ng) and E. tarda (1 × 105 CFU/ml) into the olive flounder. The survival rate in the rIFN-γ-injected group was 60% compared to 0% in the group treated with E. tarda only, demonstrating that olive flounder IFN-γ is effective in reinforcing immune responses and preventing against edwardsiellosis.

Type I interferon (IFN) induces the antiviral response in innate immunity. The type I IFN gene cloned from Japanese flounder ( Paralichthys olivaceus ) has a length of 1189 bp and consisting of 5 exons and 4 introns. In a phylogenetic tree of type I IFNs, Japanese flounder grouped with other Acanthopterygii. To gain insight into the transcriptional regulation of IFN gene, the 1.36 kb 5′-upstream region including numerous canonical motifs to bind transcription factors [for example, IFN regulatory factor (IRF)] was analyzed. In HINAE cells using a luciferase reporter assay, poly I:C-responsive transcriptional activity was found in the region from −634 to −179 bp. This region includes several IRF motifs. In the presence of poly I:C, overexpression of IRF3 and RLR strongly enhanced transcriptional activity. These results suggest that the transcriptional regulation of Japanese flounder type I IFN is regulated by IRF3 after triggering with dsRNA sensors.

The gene encoding nucleotide-binding oligomerization domain 1 (NOD1) was cloned from olive flounder (Paralichthys olivaceus) and the role played by NOD1 during Edwardsiella tarda infection was evaluated. The complete open reading frame of NOD1 was 2820 bp in length, encoding a 939-amino acid polypeptide. The NOD1 protein contains three conserved domain structures including C-terminal LRRs, a central NACHT motif, and an N-terminal CARD domain, which show similarities of 49-74% to those of other vertebrate counterpart proteins. NOD1 expression was observed in all fish tissues examined, and the levels increased in olive flounder infected with E. tarda, Streptococcus iniae, or viral hemorrhagic septicemia virus (VHSV). When hirame natural embryo (HINAE) cells over-expressing NOD1 were infected with E. tarda, bacterial growth was inhibited, and the IL-1β transcript level increased compared to that of the control. These findings imply that NOD1 plays an important role in response to E. tarda infection of olive flounder.

Pattern recognition receptors (PRRs) are involved in the effective innate defense against several microbes. Here, we identified a nucleotide-oligomerization domain (NOD)-like receptor subfamily C (NLRC) from Japanese flounder (Paralichthys olivaceus). Full-length transcript of JfNLRC is composed of 3976 bp encoding a protein of 1175 deduced amino acid residues. The presence of a signature nucleotide-binding domain (NACHT) and leucine-rich repeated domain (LRR) suggested that the protein is a member of the NLR family. Interestingly, its C-terminus presents an extra PRY/SPRY (B30.2) domain similar to fish in the Trim (finTrim) family. A phylogenic tree of JfNLRC revealed that full-length JfNLRC diverged from the NOD1 and NOD2 clusters, and the NACHT domain in JfNLRC was clustered within the NLRC3 group. Stimulation by formalin-killed Edwardsiella tarda, Streptococcus iniae, and lipopolysaccharide (LPS) showed that the JfNLRC expression was raised a few hours after stimulation, suggesting this novel protein is involved in the immediate response against both Gram-positive and Gram-negative bacteria. Furthermore, the IL-1β mRNA expression level in JfNLRC-over-expressing HINAE cells was significantly increased, when compared to a control, after LPS-stimulation and E. tarda infection. These results suggested that JfNLRC probably induced IL-1β gene expression mediated by LPS-stimulation.

Although the major capsid proteins (MCPs) of lymphocystis disease virus (LCDV) have been characterized, little is known about the host-derived immune response to MCPs and other LCDV antigenic proteins. To identify antigenic proteins of LCDV that could be used as vaccine candidates in olive flounder, Paralichthys olivaceus, we analysed the viral proteins responsible for its virulence by applying immuno-proteomics. LCDV proteins were separated by one-dimensional gel electrophoresis, transferred to polyvinylidene difluoride membrane, and probed with homogeneous P. olivaceus antisera elicited by LCDV natural infection and vaccination with formalin-killed LCDV. Four immune-reactive proteins were obtained at 68-, 51-, 41- and 21 kDa using antisera collected from natural infection while two proteins at 51- and 21 kDa exhibited response to antisera from vaccinated fish, indicating that the latter two proteins have vaccine potential. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nanoelectrospray MS/MS, the 51 and 21 kDa proteins were identified as MCP and an unknown protein, respectively.

Cytosolic pattern recognition receptors such as retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) play an important role in sensing viral RNAs. The receptor encoded by melanoma differentiation-associated gene 5 (MDA5), an RLR, recognizes viral RNA in the cytoplasm and enhances antiviral response in host cells. The full-length MDA5 gene in Japanese flounder, Paralichthys olivaceus was cloned and found to have 11,251 nucleotides. MDA5 transcript abundance was significantly increased in whole kidney infected with viral hemorrhagic septicemia virus (VHSV) as well as whole kidney and peripheral blood leukocytes stimulated with poly I:C in vitro. Hirame natural embryo (HINAE) cells overexpressing MDA5 showed a lower cytopathic effect (CPE) against VHSV, hirame rhabdovirus (HIRRV) and infectious pancreatic necrosis virus (IPNV) infection. When infected with VHSV, MDA5-overexpressing HINAE cells had 24-75 fold lower virus titer than normal HINAE cells. These results suggest that Japanese flounder MDA5 is involved in the induction of antiviral response.

Both PU.1 and C/EBPα transcription factors play important roles in myeloid development and inflammatory response. These transcripts were cloned from the Japanese flounder (Paralichthys olivaceus) and were highly conserved with those of other vertebrates. PU.1 mRNA was mainly expressed in lymphoid tissues while C/EBPα mRNA was widely expressed in all tissues examined. Higher levels of PU.1 mRNA were expressed in the IgM(+) cells of both PBL and KL, while C/EBPα expression was higher only in the IgM(-) cells of KL. The expression of C/EBPα mRNA was induced only in KL stimulated with LPS. Interestingly, PU.1 mRNA expression was induced by Edwardsiella tarda, whereas the expression of C/EBPα mRNA was induced by Streptococcus iniae infection. Both PU.1 and C/EBPα drove transcription from the LPS-responsive region of the Japanese flounder TNFα gene, suggesting that both PU.1 and C/EBPα induced by bacterial infection are involved in inflammation mediated through TNFα expression.

Molecular techniques are now essential for discovering new functional genes for the improvement of cultured marine organisms. Such techniques can be used to obtain expressed sequence tags (EST) and, through the use of microarrays, the entire transcriptome. For example, microarrays can be used to reveal biomarkers of health conditions, nutrient changes and immune response in fish and shellfish. EST-based microarray chips were constructed for cultured fish and shellfish species including Japanese flounder (Paralichthys olivaceus). Using the flounder microarray chip, the efficacy of two DNA vaccines derived from pathogenic viruses [hirame rhabdovirus (HRV) and viral hemorrhagic septicemia virus (VHSV)] was evaluated through gene expression profiles. The results suggest that both DNA vaccines were effective in protecting the flounder from HIRRV and VHSV. The flounder microarray was also used to compare gene expression patterns in fish that are susceptible and resistant to Edwardsiella tarda. At 3days post infection, the gene expression patterns between the two groups were dramatically changed. Thus, microarray analysis is a very powerful tool to understand gene expression profiles in fish.

LGP2 is an important intracellular receptor that recognizes viral RNAs in innate immunity. To understand the mechanism of viral RNA recognition, we cloned an LGP2 cDNA and gene in Japanese flounder (Paralichthys olivaceus). Viral hemorrhagic septicemia virus-induced expressions of LGP2 mRNA were evaluated in vivo and in vitro by quantitative real-time PCR (Q-PCR) using primers based on the clone sequences. The expression of LGP2 mRNA in the kidney dramatically increased at 3 d postinfection. The expression of LGP2 mRNA also increased in the head kidney leukocytes stimulated with artificial dsRNA (polyinosin-polycytidylic acid) in vitro. To evaluate the antiviral activity of the flounder LGP2, three expression constructs containing pcDNA4-LGP2 (full-length), pcDNA4-LGP2ΔRD (regulatory domain deleted), and pcDNA4-Empty (as a negative control) were transfected into the hirame (flounder) natural embryo (hirame natural embryo) cell line. Forty-eight hours after transfection, the transfected cells were infected with ssRNA viruses, viral hemorrhagic septicemia virus, or hirame rhabdovirus. The cytopathic effects of the viruses were delayed by the overexpression of Japanese flounder LGP2. The Q-PCR demonstrated that mRNA expression levels of type I IFN and IFN-inducible genes (Mx and ISG15) in the hirame natural embryo cells overexpressing LGP2 were increased by polyinosin-polycytidylic acid and viral infections. These results suggest that Japanese flounder LGP2 plays an important role in the recognition of both viral ssRNA and dsRNA to induce the antiviral activity by the production of IFN-stimulated proteins.

The mitochondrial adaptor, IFN-β promoter stimulator-1 (IPS-1), also known as MAVS/VISA/Cardif, plays a key role in the signal transduction of the RIG-1/MDA5 pathway to induce the production of interferons (IFNs) and other cytokines. In the present study, Japanese flounder (Paralichthys olivaceus) IPS-1 cDNA was cloned from Japanese flounder spleen using PCR-based methods. The full-length cDNA has 2235 nucleotides and encodes a polypeptide of 641 amino acids. The putative Japanese flounder IPS-1 protein contains an N-terminal CARD-like domain, a central proline-rich domain, a C-terminal transmembrane domain, and exhibits similarity to other teleost counterparts ranging from 20% to 34%. Semi-quantitative RT-PCR showed that Japanese flounder IPS-1 mRNA was expressed in all tissues examined. The expression level of flounder IPS-1 gene was unchanged in viral hemorrhagic septicemia virus (VHSV)-infected kidney as measured by quantitative real-time PCR (Q-PCR). In addition, Japanese flounder IPS-1-overexpressing cells were protected against hirame rhabdovirus (HIRRV) and VHSV infection as manifested by the delayed appearance of cytopathic effect (CPE) and decreased viral titers. Expression of IFN-inducible genes including Mx, ISG15 and IRF3 were also induced in the IPS-1-overexpressing cells. These results suggest that Japanese flounder IPS-1 is involved in the antiviral immunity as a one of the adaptors in fish IFN-activation pathway.

The etiological agents of streptococcosis were isolated from diseased olive flounder collected on the Jeju island of Korea. A total of 151 bacterial isolates were collected between 2003 and 2006. The isolates were examined using various phenotypic and proteomic analyses, including sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and glycoprotein assays. In addition, isolates were grown on blood agar to assess hemolytic activity, and biochemical assays were performed using the API20 Strep kit. Our results revealed that all isolates were nonmotile, Gram-positive cocci that displayed negative catalase and oxidase activities. Multiplex PCR assays revealed that 43% and 57% of the isolates were Streptococcus iniae and Streptococcus parauberis, respectively. These results were consistent with those of the SDS-PAGE and immunoblot analyses using whole-cell lysates of bacterial isolates. Significant differences were observed with respect to the Voges-Proskauer, pyrrodonyl arylamidase, alkaline phosphatase, and hemolytic activities of the S. iniae and S. parauberis isolates. Isolates of S. iniae displayed uniform profiles in the immunoblot and glycoprotein assays; however, immunoblot assays of S. parauberis isolates (using a chicken IgY antibody raised against a homologous isolate) revealed three distinct antigenic profiles. Our findings suggest that S. parauberis and S. iniae are endemic pathogens responsible for the development of streptococcosis in olive flounder.

The innate immune system, particularly the external body surface, plays a frontier role in protecting fish under intensive aquaculture and at prolonged low temperatures from relevant infections due to inadequate adaptive immune responses. In the present study we aimed to understand the mucosal immunity of an economically important mariculture fish, olive flounder ( Paralichthys olivaceus ) by evaluating the immune components from its skin mucus. The activities of lysozyme (233.33 ± 171.82 units mg −1 ), trypsin-like protease (42.84 ± 1.249 units mg −1 ), alkaline phosphatase (0.376 ± 0.005 units mg −1 ) and esterase (0.170 ± 0.006 units mg −1 ) were detected in the skin mucus. Transferrin was identified by MALDI-TOF/MS analysis. ELISA and immunoblot assays using anti-flounder IgM monoclonal antibody showed the presence of a significant level (1.80 ± 0.001, n = 3) of monomer immunoglobulin M (IgM) with approximate molecular weight of 160 and 25 kDa under non-denaturing and denaturing states, respectively. Skin mucus showed strong antibacterial activity against tested fish pathogenic bacteria. In addition, skin mucus successfully agglutinated (HA titre 2 8 ), but completely failed to haemolyse, rabbit erythrocytes. In conclusion, the major immune components of the skin mucus, identified in the present study, are possibly involved in the broad spectrum non-specific immunity of olive flounder.

Immunoglobulins (Igs) were purified from sera obtained from olive flounder Paralichthys olivaceus immunized with goat IgG using immunoaffinity and mannan-binding protein (MBP) affinity columns and designated IMMIg and MBPIg respectively. SDS-PAGE and two-dimensional gel electrophoresis (2-DE) analyses demonstrated that the olive flounder serum contained at least two different types of Ig in terms of molecular weight and pI of heavy chain. 2-DE separation of the Igs followed by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) revealed that these two Igs were IgM and IgM precursor of olive flounder. Monoclonal antibodies (MAbs) were raised against the IMMIg and MBPIg and proteome analysis of Lactococcus garvieae using these MAbs and olive flounder immune sera demonstrated that IMMIg and MBPIg recognized different antigens of L. garvieae. This suggested that different Igs are possibly involved in protecting olive flounder against L. garvieae infection.

The olive flounder (Paralichthys olivaceus) is a cultivated marine species that is economically important in Korea and Japan. Several bacterial pathogens have caused severe mortalities in farmed olive flounder, especially Streptococcus parauberis. We collected 145 S. parauberis isolates from diseased olive flounders from 2003 to 2008 in Jeju Island, South Korea and characterized them by Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI TOF MS) and by serology. The serological analysis divided the isolates into serotype I (62.1%) and serotype II (36.6%) and the proteome analysis divided the isolates into cluster 1 (43.4%) and cluster 2 (56.6%). All cluster 1 isolates had serotype I, but cluster 2 consisted of serotype I (32.9%), serotype II (64.6%), and others (2.5%). Further detailed analysis of the mass spectra led to identification of several specific m/z peaks that enabled discrimination between cluster 1 and 2 and between serotype I and II within cluster 2. Our results suggest that MALDI TOF MS analysis has potential as an alternative method for the rapid and reliable identification of the fish pathogen S. parauberis.

Recently, mass mortality of adult flounder showing ascites occurred at many aquaculture farms in Korea. While we investigated the cause of the mortality, marine birnavirus (MABV-F) and bacteria (Vibrio harveyi and Edwardsiella tarda) were isolated from diseased fish. By the intraperitoneal (IP) injection with MABV-F, the virus strain did not show pathogenicity to the flounder. When the fish co-infected with MABV-F (IP) and V. harveyi or E. tarda (immersion), the cumulative mortality did not show any significant difference with control group. In contrast, the case of co-infection with MABV-F and bacteria by IP, cumulative mortality reached more than 90%. Most of the moribund and dead fish except for the mock-infected fish displayed ascites, and the virus and bacteria were re-isolated from all the dead and moribund fish except for the control fish. The results indicate that the cause of mass mortality of the adult fish may be due to bacterial infection. Higher mortalities may occur due to co-infection of MABV-F and bacteria, although MABV-F alone did not cause direct mortality.

Streptococcus parauberis (stain SNUFPC-050803), isolated from diseased olive flounder Paralichthys olivaceus in Jeju Island, Korea, was evaluated for its pathogenicity to healthy juvenile flounder (29.3 g in average body weight). When challenged with the isolate by intraperitoneal injection with tenfold serial dilutions of 4.5 × 10-106 CFU/fish, the cumulative mortality ranged from 10% to 80% within 14 days except for 4.5 × 10 CFU/fish and control with no mortality. Disease signs were hemorrhage around the mouth, eyes and pectoral fins, pale and friable liver with hepatomegaly and ascitic fluid in the peritoneal cavity. These signs were similar to those of naturally affected fish. S. parauberis was reisolated and identified by PCR method, which confirmed the pathogenicity of the bacterium to olive flounder.

The olive flounder, Paralichthys olivaceus, is an economically important food fish in Japan and Korea. Scuticociliatosis is a major parasitic disease, and fatal infection with scuticociliates, or mixed infections with scuticociliates and other pathogenic agents (e.g., Vibrio spp.) cause severe mortalities in farmed olive flounders. To date, however, effective chemotherapeutic treatment of scuticociliatosis has only been reported at the in vitro level. In this study, we employed combination treatment, using benzalkonium chloride (to remove excess mucus from the body surface) and bronopol (to kill the parasites), to overcome the protective effect of mucus by some medicine to the scuticociliates. In the presence of the mucus mixture, the higher dose of bronopol (156 ppm) yielded morphologies and motilities similar to those of ciliates treated with the lower dose of bronopol (80 ppm) in the absence of mucus. We also investigated the in vivo effects of this treatment in field trials involving a total of 15,025 naturally infected flounders. We observed that short-term bath treatments with benzalkonium chloride (50 ppm) followed by bronopol (500 ppm) were effective, assessed by the relative percentage mortality (RPS) value. Thus, this study provides a notable therapeutic strategy by removing the mucus to treat scuticociliatosis in olive flounders at the aquaculture field level.

A multiplex PCR protocol was established to simultaneously detect major bacterial pathogens in olive flounder (Paralichthys olivaceus) including Edwardsiella (E.) tarda, Streptococcus (S.) parauberis, and S. iniae. The PCR assay was able to detect 0.01 ng of E. tarda, 0.1 ng of S. parauberis, and 1 ng of S. iniae genomic DNA. Furthermore, this technique was found to have high specificity when tested with related bacterial species. This method represents a cheaper, faster, and reliable alternative for identifying major bacterial pathogens in olive flounder, the most important farmed fish in Korea.

T cell activation is initiated by the binding of antigen to the specific T cell receptor (TCR) that triggers the formation of CD3-signal transduction complex and ends in proliferation and differentiation of antigenspecific T cells. CD3, an essential component of the CD3-TCR complex, has been classified into γ, δ, e, and ζ chains. The CD3γ, CD3δ, and CD3e chains are highly related to cell surface proteins of the immunoglobulin superfamily containing a single extracellular immunoglobulin domain. In mammals, T cells were characterized by detecting one of the CD3 molecules through the use of specific antibody for CD3e. This suggests that CD3 molecules can be used as markers in identifying T cells in teleost fish; hence, it is essential to produce CD3-specific antibodies that could serve as T cell markers in fish. Here, we produced a CD3e-specific monoclonal antibody. Western blot result showed a distinct band at approximately 15 kDa detecting the CD3e antibody while MALDI-TOF analysis, clearly identified the band as olive flounder CD3e. Once a monoclonal antibody has been produced, it can be used to detect the presence of a substance specific to it. The results from this study revealed that the CD3e monoclonal antibody produced could detect CD3e in olive flounder. However, cell markers for T cell differentiation in olive flounder were not fully investigated yet so we hope that this antibody could be useful in elucidating T cell differentiation in teleosts.

In innate immunity, LGP2 (laboratory of genetics and physiology 2) plays a very important role in the production of type I interferon (IFN) through recognition of cytosolic viral RNA. Although viral infection or stimulation with double-strand RNA dramatically induces expression of the LGP2 gene, the underlying transcriptional mechanism has never been studied. Here, we cloned and characterized the 5'-upstream region (-1,337 bp) of the LGP2 gene in olive flounder (Paralichthys olivaceus). Numerous canonical motifs for IFN-regulatory factors (IRFs) were found in this region, and reporter assays identified a poly I: C-responsive promoter region (-506 to -398) that regulated LGP2 transcription. Transcriptional activity of the LGP2 promoter was strongly enhanced by IRF3, which bound to IRF3 motif #3 (-480). The LGP2 promoter was also responsive to viral infection in vitro. These results suggest that LGP2 transcriptional control is crucially involved to regulated by IRF3 function after viral infection or stimulation with poly I:C.

Heat shock proteins (HSPs) have been observed in cells exposed to a variety of stresses, including infectious pathogens. This study used a label-free, quantitative proteomic approach and transcriptional gene expression analysis to investigate infection-related HSP proteins and their encoding genes in whole kidneys from olive flounder (Paralichthys olivaceus). During Streptococcus parauberis infection in the flounder, the genes encoding Hsp10, Hsp40A4, Hsp40B6, Hsp40B11, Hsp60, Hsp70, glucose regulated protein 78 (Grp78), Hsp90α, Hsp90β and Grp94 were induced, and the protein levels of Hsp60, Hsp70, Hsp90α, Hsp90β and Grp94 were differentially regulated over time. Subsequent results also revealed that Hsp60, Hsp70, Hsp90α, Hsp90β and Grp94 appear to be the dominant and critical HSPs in olive flounder during bacterial infection. This is the first estimation of the differential involvement of HSPs in the immune response of olive flounder exposed to bacterial infection.

Cathepsin activities are responsible for mediating various pathways involved in immune response, including the apoptosis pathway, toll-like receptor (TLR) signaling, cytokine induction and activation of granule serine proteases. In the present study, we investigated cathepsin responses in the kidneys of olive flounder infected with Streptococcus parauberis, analyzing cathepsin expression using a label-free, quantitative proteomic approach in conjunction with quantitative real-time polymerase chain reaction (qRT-PCR). In proteomic analyses, we detected cathepsin B, D, L and S proteins, noting significant decreases and increases in cathepsins B and L, respectively, with infection. Taken together with an evaluation of cathepsin B, D, F, K, L, S and X gene expression in normal and infected kidneys by qRT-PCR, our results indicate that cathepsins B, D, L and S are the dominant lysosomal proteases in the immune system of the teleostei, olive flounder. Cathepsins F, K and X were regarded as minor cathepsins.

Although Streptococcus parauberis is known as a bacterial pathogen associated with bovine udder mastitis, it has recently become one of the major causative agents of olive flounder ( Paralichthys olivaceus ) streptococcosis in northeast Asia, causing massive mortality resulting in severe economic losses. S. parauberis contains two serotypes, and it is likely that capsular polysaccharide antigens serve to differentiate the serotypes. In the present study, the complete genome sequence of S. parauberis (serotype I) was determined using the GS-FLX system to investigate its phylogeny, virulence factors, and antigenic proteins. S. parauberis possesses a single chromosome of 2,143,887 bp containing 1,868 predicted coding sequences (CDSs), with an average GC content of 35.6%. Whole-genome dot plot analysis and phylogenetic analysis of a 60-kDa chaperonin-encoding gene and the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-encoding gene showed that the strain was evolutionarily closely related to Streptococcus uberis. S. parauberis antigenic proteins were analyzed using an immunoproteomic technique. Twenty-one antigenic protein spots were identified in S. parauberis , by reaction with an antiserum obtained from S. parauberis -challenged olive flounder. This work provides the foundation needed to understand more clearly the relationship between pathogen and host and develops new approaches toward prophylactic and therapeutic strategies to deal with streptococcosis in fish. The work also provides a better understanding of the physiology and evolution of a significant representative of the Streptococcaceae .

Lactococcus garvieae is an important etiological agent of lactococcosis in various fish species including olive flounder (Paralichthys olivaceus). In this study, proteomic and immunoproteomic analyses were employed to compare the antigenic profiles of strains KG9408, MS93003, and NSS9310 strains of L. garvieae. Proteomic analysis using two-dimensional gel electrophoresis (2-DE) revealed differences in five protein spots among the different L. garvieae strains. In immunoproteomic analysis, there was a significant difference in the 2-DE immunoblot profiles of the L. garvieae strains using sera collected from fish surviving infection with either L. garvieae strains KG9408 or NSS9310. These sera reacted with 8 and 7 unique antigenic protein spots, respectively. Heat shock protein (HSP) 70 and DNA-directed RNA polymerase were among the specific antigens recognized by the anti-NSS9310 serum. In addition, the anti-NSS9310 and anti-KG9408 olive flounder sera reacted with 25 common antigenic protein spots of all the L. garvieae strains, which included elongation factor (EF)-Tu, arginine deiminase (AD), inosine-5'-monophosphate dehydrogenase (IMPD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphomannomutase (PMM), L-lactate dehydrogenase (L-LDH), 6-phosphofructokinase and UDP-galactose 4-epimerase (UDP-galactose). Based on the present results, the 8 antigens recognized by the anti-KG9408 serum and the 25 common antigens recognized by both sera may serve as potential markers for developing an effective vaccine against this bacterium.

Streptococcus iniae is the major etiological agent of streptococcosis, which is responsible for hemorrhagic septicemia in fish, particularly olive flounder (Paralichthys olivaceus). In the present study, we sought to understand the pathogenicity and immunogenicity of S. iniae in order to develop a vaccine for streptococcosis. Immunoproteomics, a technique involving two-dimensional gel electrophoresis (2-DE) followed by immunoblotting, was employed to investigate the pathogenicity and immunogenicity of two S. iniae isolates, Jeju-13 and Jeju-45, in olive flounder. The virulence of Jeju-13 was moderate whereas that of Jeju-45 was high. A vaccination trial with formalin-killed Jeju-45 demonstrated relatively low protection against the homologous isolate compared with the heterologous isolate. A significant difference in the secretion of extracellular products (ECPs) was noticed between the two S. iniae isolates. ECP antigens were highly immunogenic compared to those from whole cell lysates as determined by 2-DE immunoblot assay of Jeju-13 and Jeju-45 anti-sera collected from post-challenge survival fish. Furthermore, there were differences in the appearance of antigenic spots on 2-DE immunoblot profiles of ECPs of the respective sera. Interestingly, the mixture of killed-cells and concentrated ECPs from Jeju-45 led to significant protection against the homologous isolate of S. iniae in olive flounder. The present study demonstrates the usefulness of immunoproteomics in understanding the pathogenicity of S. iniae to aid the development of a vaccine for fish streptococcosis.

Infection with Edwardsiella tarda, a gram-negative bacterium, causes high morbidity and mortality in both marine and freshwater fish. Outer membrane vesicles (OMVs) released from gram-negative bacteria are known to play important roles in bacterial pathogenesis and host immune responses, but no such roles for E. tarda OMVs have yet been described. In the present study, we investigated the proteomic composition of OMVs and the immunostimulatory effect of OMVs in a natural host, as well as the efficacy of OMVs when used as a vaccine against E. tarda infection. A total of 74 proteins, from diverse subcellular fractions, were identified in OMVs. These included a variety of important virulence factors, such as hemolysin, OmpA, porin, GAPDH, EseB, EseC, EseD, EvpC, EvpP, lipoprotein, flagellin, and fimbrial protein. When OMVs were administrated to olive flounder, significant induction of mRNAs encoding IL-1β, IL-6, TNFα, and IFNγ was observed, compared with the levels seen in fish injected with formalin-killed E. tarda. In a vaccine trial, olive flounder given OMVs were more effectively protected (p